AGEs-RAGE通路在2型糖尿病肠道L细胞早期损伤及凋亡中的机制研究
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摘要
研究背景：
随着人们生活水平的提高及生活方式的改变,2型糖尿病(type2diabetes mellitus, T2DM)的发病率在我国呈现出高速发展的趋势。据2009年流行病学数据显示,我国T2DM的发病率已达到9.7%,总患病人数9240万,跃居世界第一位。因此,探索糖尿病的发病机制一直是糖尿病领域研究的热点及重点。肠促胰岛素效应是机体调控餐后胰岛素分泌及餐后血糖的重要机制,该效应的发现使2型糖尿病在发病机制和治疗上取得了突破性的进展。胰高血糖素样肽1(glucagon-like peptide-1, GLP-1)则是肠道L细胞分泌的一种重要的肠肽激素,可通过其受体葡萄糖依赖性的促进胰岛素的分泌,并能促进胰岛p细胞增殖和抑制其凋亡。诸多研究证实,2型糖尿病患者中肠促胰岛素效应明显受损,主要表现为内源性GLP-1的产生和分泌不足,而肠道L细胞的早期损伤或凋亡可能是GLP-1分泌减少的主要原因并参与了2型糖尿病的发生和发展。
目前研究多集中在不同药物对肠道内源性GLP-1分泌的调节作用及L细胞分泌GLP-1的可能通路方面。很少有针对肠道L细胞早期损伤及凋亡方面开展的研究,特别是糖尿病糖基化终末产物(advanced glycation end-products, AGEs)对L细胞功能的可能影响方面未见研究报道,AGEs及其受体(RAGE)通路是导致糖尿病多种组织细胞损伤的重要机制,因此,探明AGEs-RAGE是否会导致肠道L细胞的损伤以及损伤的可能机制和信号通路对于深入理解和完善2型糖尿病的发病机制有着重要意义。
糖基化终末产物(AGEs)是长期高血糖与多种蛋白质发生糖基化作用,形成的具有毒性而不可逆的化合物,大量的不可逆的AGEs的产生和积聚是细胞损伤及糖尿病慢性进展的重要机制。AGEs与其受体RAGE (Receptor for AGE)结合可以上调炎症反应并导致组织细胞的破坏,因此AGEs被认为是一类能激活细胞并能促进氧化应激反应的前炎症介质。诸多研究发现,AGEs-RAGE通路不仅介导了多种细胞的损伤,而且导致胰岛等内分泌细胞凋亡及功能损伤,而2013年最新的研究也得出类似的研究结果：高浓度的AGEs可以明显上调胰岛细胞表面RAGE受体的表达,AGEs与其受体RAGE结合后可激活NADPH氧化酶导致细胞内活性氧产生增加,促进炎症核转录因子NF-κB表达及上调Bax/Bcl-2比例,最终导致胰岛细胞凋亡率的明显增加,利用NADPH氧化酶的抑制剂可改善AGEs诱导的胰岛细胞凋亡。由此可见,AGEs-RAGE通路通过激活NADPH氧化酶导致细胞内ROS增加,进而促进炎症核转录因子NF-κB表达以及Bax/Bcl-2比例上调,这是导致胰岛细胞凋亡及胰岛素分泌减少的一个重要机制。有意思的是,本科室其他同事已完成的脂多糖(LPS)对肠道L细胞凋亡及分泌功能影响的实验研究,发现LPS体外可通过促进NF-κB的表达和释放以及下调抗凋亡基因Bcl-2水平诱导体外培养的L细胞发生凋亡,进而减少内源性GLP-1的分泌水平,这种损伤具有时间及剂量的依赖性,而LPS和AGEs均可通过RAGE受体及其下游通路介导细胞的损伤。这表明L细胞凋亡可能是体内GLP-1分泌减少的原因之一,而RAGE起着重要的作用。
因此,本课题以L细胞株(GLUTag细胞)为研究对象,拟从以下几个方面进行初步探讨：1、观察AGEs对肠道L细胞株(GLUTag细胞)早期损伤、凋亡及GLP-1分泌水平的影响；2、探讨AGEs是否通过AGEs-RAGE通路对GLUTag细胞产生损伤、凋亡及其相关机制。为进一步阐明2型糖尿病体内GLP-1减少的原因以及探索新的2型糖尿病治疗方案提供新的线索。
第一部分AGEs对L细胞株(GLUTag)早期损伤、凋亡及GLP-1分泌水平的影响
目的：
观察AGEs对肠道L细胞株GLUTag细胞早期损伤、凋亡及GLP-1分泌水平的影响。
方法：
(1)体外制备、鉴定糖基化终产物
将胎牛血清白蛋白(BSA)和D-葡萄糖溶于PBS中,避光孵育12周制备AGE-BSA。以荧光分光光度计鉴定AGE-BSA。
(2)传代培养GLUTag细胞
用低糖DMEM培养基,加入10%的胎牛血清,100IM/ml青霉素和100IM/ml链霉素所配置的细胞完全培养基传代培养GLUTag细胞。
(3)实验分组：
不同浓度AGEs的影响：空白对照组,即不加入干预因素；BSA对照组；AGEs100μg/ml组; AGEs200μg/ml组；AGEs300μg/ml组,分别作用细胞24h。AGEs作用不同时间的影响：200μg/mL AGEs分别作用GLUTag细胞0、12、24、48h。
(4) AGEs对GLUTag细胞凋亡的影响
分组及培养条件同上,各组细胞干预结束后利用Hoechst33258荧光染色观察细胞形态学变化。Annexin V-FITC/PI染色检测细胞凋亡率。
(5) AGEs对GLUTag细胞活性的影响
分组及培养条件同上,各组均利用CCK-8法检测GLUTag细胞增殖活性。
(6) AGEs对GLUTag细胞GLP-1分泌的影响
分组及培养条件同上,收集细胞上清液,利用ELISA检测GLUTag细胞GLP-1分泌水平。
统计学处理：
采用SPSS13.0进行统计分析,实验数据计量资料以均数±标准差(i±s)表示,采用单向方差分析(One-Way ANOVA)检验,两两比较采用LSD法。凋亡率采用析因设计的方差分析。P<0.050具有统计学意义。
结果：
(1)不同浓度AGEs对GLUTag细胞形态学的影响
各组经药物干预24h后,Hoechst33258染色后荧光显微镜下观察：空白对照组及BSA对照组GLUTag细胞的细胞核呈均匀荧光,为浅蓝色,且结构正常；AGEs100μg/ml组GLUTag细胞大部分细胞核均匀浅蓝色染色,偶见部分细胞核固缩,核染色致密呈亮蓝色或裂解呈颗粒状；AGEs200μg/ml组GLUTag细胞质固缩、偏移,核致密浓染,核呈颗粒状或碎片状,核染色质边集;AGEs300μg/ml组GLUTag细胞大片浓染,呈大面积亮蓝色,凋亡小体、核固缩及核碎裂的细胞较AGEs200μg/ml组明显增多。
(2)比较AGEs不同作用时间对GLUTag细胞形态学的影响
200μg/ml AGEs作用时间(0h)后的GLUTag细胞的细胞核呈均匀淡染；200μg/ml AGEs作用时间(12h)后的GLUTag细胞的细胞核大部分呈均匀浅染,偶见部分细胞核染色致密呈亮蓝色；200μg/ml AGEs作用时间(24h)后的GLUTag细胞的细胞质固缩,核致密浓染或裂解呈颗粒状,核染色质边集；200μg/ml AGEs作用时间(48h)后的GLUTag细胞的细胞核大面积浓染,细胞皱缩,核固缩、偏移及碎裂的细胞较作用时间为24h明显增多。
(3)不同浓度AGEs处理24h对GLUTag细胞凋亡率的影响
各组细胞经药物干预24h后,100μg/ml AGEs组的凋亡细胞百分率为(15.30±2.89)%,显著高于空白对照组(6.43±1.03)%和BSA对照组(7.37±1.50)%(P=0.000);200μg/ml AGEs组的凋亡细胞百分率为(24.10±2.95)%,显著高于空白对照组和BSA对照组(P=0.000),同时亦显著高于100μg/ml AGEs组(P=0.000);300μg/ml AGEs组的凋亡细胞百分率为(35.19±3.76)%,显著高于空白对照组、BSA对照组及100μg/ml AGEs组(P=0.000),同时亦显著高于200μg/ml AGEs组(P<0.05)。
(4)比较AGEs不同作用时间对GLUTag细胞凋亡的影响
200μg/ml AGEs作用时间(12h)后的凋亡细胞百分率为(12.61±1.94)%,显著高于阴性对照组(6.30±2.23)%(P=0.000)；200μg/ml AGEs作用时间(24h)后的凋亡细胞百分率为(21.77±3.41)%,显著高于阴性对照组及作用12h组(P=0.00);200μg/ml AGEs作用时间(48h)后的凋亡细胞百分率为(29.52±4.42)%,显著高于阴性对照组及作用12h组(P=0.000),同时亦显著高于作用24h组(P<0.05)。
(5)不同浓度AGEs处理24h对GLUTag细胞活性的影响
100、200.300μg/ml AGEs作用GLUTag细胞24h后,OD值分别为0.8±0.05,0.66±0.05,0.4±0.06,均显著低于空白对照组(control)(1.21±0.05)和BSA对照组(200μg/ml BSA)(1.19±0.03)(P=0.000)。而且,200μg/ml AGEs组显著低于100μg/ml AGEs组(P=0.000),300μg/ml AGEs组显著低于100μg/ml AGEs组(P=0.000)及200μg/ml AGEs组(P<0.05)。
(6)比较AGEs不同作用时间对GLUTag细胞活性的影响
200μg/ml AGEs作用时间(12h)后的细胞OD值为0.87±0.05,显著低于阴性对照组(作用时间Oh)(1.110±0.05)(P=0.000);200μg/ml AGEs作用时间(24h)后的细胞OD值为0.6±0.04,显著低于阴性对照组和作用12h组(P=0.000)；200μg/ml AGEs作用时间(48h)后的细胞OD值为0.3±0.04,显著低于阴性对照组和作用12h组(P=0.000),同时亦显著低于作用24h组(P<0.05)。
(7)不同浓度AGEs处理24h对GLUTag细胞GLP-1分泌水平的影响
100、200、300μg/ml AGEs作用GLUTag细胞24h后,细胞分泌GLP-1浓度(pmol/L)分别为69.97±4.30,55.27±6.20,37.13±5.55,均显著低于空白对照组(control)(91.27±5.23)和BSA对照组(200μg/ml BSA)(86.63±6.71)(P=0.000)。而且,200μg/ml AGEs组显著低于100μg/ml AGEs组(P=0.000),300μg/ml AGEs组显著低于100μg/ml AGEs组(P=0.000)及200μg/ml AGEs组(P<0.05)。
(8)比较AGEs不同作用时间对GLUTag细胞GLP-1分泌水平的影响
200μg/ml AGEs作用时间(12h)后的细胞分泌GLP-1浓度为71.10±4.75,显著低于阴性对照组(作用时间Oh)(83.97±7.28)(P=0.000);200μg/ml AGEs作用时间(24h)后的细胞分泌GLP-1浓度为55.27±6.20,显著低于阴性对照组和作用12h组(P=0.000);200μg/ml AGEs作用时间(48h)后的细胞分泌GLP-1浓度为33.93±6.01,显著低于阴性对照组和作用12h组(P=0.000),同时亦显著低于作用24h组(P<0.05)。
结论：
肠道L细胞株GLUTag细胞的凋亡在相同作用时间下随着AGEs的浓度增高而增多,且在相同剂量下随着AGEs的作用时间延长而增多。我们首次发现AGEs可呈剂量、时间依赖性诱导肠道L细胞凋亡,并显著降低L细胞活力以及分泌GLP-1的能力。
第二部分探讨AGEs是否通过AGEs-RAGE通路对GLUTag细胞产生损伤、凋亡及其相关机制
第一节AGEs对GLUTag细胞的凋亡及其相关通路的影响
目的：
探讨AGEs-RAGE及其下游分子信号通路在AGEs诱导的GLUTag细胞凋亡中的作用。
方法：
(1)实验分组：
探讨不同浓度AGEs对GLUTag细胞中RAGE表达的影响时按以下分组：空白对照组,即不加入干预因素；BSA对照组;AGEs100μg/ml组;AGEs200μg/ml组；AGEs300μg/ml组,分别作用细胞24h。探讨AGEs对RAGE及其下游分子信号通路的影响时按照以下分组：空白对照组；BSA对照组；AGEs200μg/ml组;AGEs+siRNA-RAGE组：即利用siRNA-RAGE转染靶细胞后加入AGEs共培养细胞。AGEs+apocynin组：即加入AGEs和600μM的apocynin (NADPH氧化酶阻断剂)培养细胞。
(2) AGEs对GLUTag细胞中RAGE表达的影响
传代培养GLUTag细胞(方法同第一部分),并制备AGEs-BSA,采用已确定的AGEs浓度及时间。利用RT-PCR及Western blot检测各组细胞RAGE mRNA及RAGE蛋白表达水平。
(3) AGEs对GLUTag细胞ROS水平及NADPH氧化酶亚单位表达的影响
分组及培养条件同上,Western blot检测细胞中NADPH氧化酶亚单位gp22phox、p47phox的磷酸化,荧光探针检测细胞内活性氧ROS的水平。
(4) AGEs对GLUTag细胞中p53、Bax因子的影响
分组及培养条件同上,Western-blot法检测P53、Bax因子的变化。
(5) AGEs对GLUTag细胞中caspase-3及-9的活性
分组及培养条件同上,用酶联免疫法测定caspase-3及-9的活性。
统计学处理：
采用SPSS13.0进行统计分析,实验数据计量资料以均数±标准差(x±s)表示,采用单向方差分析(One-Way ANOVA)检验,两两比较采用LSD法。P<0.050具有统计学意义。
结果：
(1)不同浓度的AGEs对GLUTag细胞RAGE mRNA的影响
100、200、300μg/ml AGEs作用GLUTag细胞24h后,RAGE mRNA表达量分别为1.45±0.13,2.14±0.010,2.45±0.32,均显著高于空白对照组(1.01±0.04)和BSA对照组(1.09±0.03)(P=0.000)。而且,200μg/ml AGEs组显著高于100μg/ml AGEs组(P=0.000),300μg/ml AGEs组显著高于AGEs组(P=0.000)及200μg/ml AGEs组(P<0.05)。
(2)不同浓度的AGEs对GLUTag细胞内RAGE蛋白表达水平的影响
100.200、300μg/ml AGEs作用GLUTag细胞24h后,RAGE蛋白表达水平分别为0.36±0.04,0.56±0.009,0.72±0.06,均显著高于空白对照组(0.144±0.04)和BSA对照组(0.18±0.03)(P=0.000)。而且,200μg/ml AGEs组显著高于100μg/ml AGEs组(P=0.000),300μg/ml AGEs组显著高于100μg/ml AGEs组(P=0.000)及200μg/ml AGEs组(P<0.05)。
(3) AGEs对GLUTag细胞中NADPH氧化酶亚单位表达的影响
各组细胞经干预24h后,200μg/ml AGEs组的gp22phox、p47phox蛋白表达水平分别为0.66±0.08,0.76±0.09,均显著高于BSA对照组(0.27±0.06,0.30±0.06)(P<0.05); AGEs+apocynin组的gp22phox、p47phox蛋白表达水平分别为0.40±±0.08,0.4±0.09,均显著高于BSA对照组(P<0.05),而且显著低于200μg/ml AGEs组(P<0.05)；AGEs+siRNA-RAGE组的gp22phox、p47phox蛋白表达水平分别为0.32±0.04,0.344±0.056,均显著低于200μg/ml AGEs组(P<0.05)。
(4) AGEs对GLUTag细胞内活性氧水平的影响
各组细胞经干预24h后,200μg/ml AGEs组的荧光强度为57.58±±4.69,显著高于BSA对照组(23.16±3.47)(P<0.05)；AGEs+apocynin组的荧光强度为34.97±±6.42,显著高于BSA对照组(P<0.05),而且显著低于200μg/ml AGEs组(P<0.05); AGEs+siRNA-RAGE组的荧光强度为29.444±5.27,显著低于200μg/ml AGEs组(P<0.05)。
(5) AGEs对GLUTag细胞凋亡相关蛋白p53及Bax表达的影响
各组细胞经干预24h后,200μg/ml AGEs组的p53蛋白表达水平为0.42±±0.05,显著低于BSA对照组(0.73±±0.05)(P<0.05),而Bax蛋白表达水平为0.66±±0.08,显著高于BSA对照组(0.27±0.08)(P<0.05)；AGEs+apocynin组和AGEs+siRNA-RAGE组的p53蛋白表达水平分别为0.644±0.05,0.71±±0.06,均显著高于200μg/ml AGEs组(P<0.05),而Bax蛋白表达水平分别为0.40±0.08,0.32±0.04,均显著低于200μg/ml AGEs组(P<0.05)。
(6) AGEs对GLUTag细胞凋亡蛋白酶caspase-3及-9活性的影响
各组细胞经干预24h后,200μg/ml AGEs组的caspase-3及-9活性分别为1.03±0.10,1.22±0.05,均显著高于BSA对照组(0.43±0.05,0.47±0.02)(P<0.05); AGEs+apocynin组的caspase-3及-9活性分别为0.65±0.07,0.68±0.09,均显著高于BSA对照组(P<0.05),而且显著低于200μg/ml AGEs组(P<0.05);AGEs+siRNA-RAGE组的caspase-3及-9活性分别为0.55±0.08,0.57±0.07,均显著低于200μg/ml AGEs组(P<0.05)。
结论：
随着AGEs干预浓度的升高,GLUTag细胞RAGE mRNA表达量和RAGE蛋白表达水平显著上升,提示AGEs可通过上调RAGE受体表达增强GLUTag细胞的氧化应激损伤。AGEs作用于GLUTag细胞24h后,NADPH氧化酶亚单位gp22phox、p47phox的表达增多,细胞内ROS水平升高,p53表达下调,Bax表达上调,凋亡蛋白酶caspase-3及-9的活性增加,细胞凋亡增加。提示NADPH氧化酶、ROS、p53/Bax及caspase-3及-9是AGEs-RAGE导致GLUTag细胞凋亡的主要下游通路。
第二节AGEs对GLUTag细胞炎症损伤及p38MAPK/NF-κB通路的影响
目的：
观察AGEs对肠道L细胞株GLUTag细胞中炎症损伤及相关机制的影响。
方法：
(1)AGEs对GLUTag细胞中p38MAPK磷酸化水平的影响
传代培养GLUTag细胞(方法同第一部分),并制备AGEs-BSA,采用已确定的AGEs作用时间。
实验分组：空白对照组,即不加入干预因素；
BSA对照组,即加入BSA培养细胞作为阳性对照；
100μg/ml AGEs组,即加入100μg/ml AGEs培养细胞；
200μg/ml AGEs组,即加入200μg/ml AGEs培养细胞；
300μg/ml AGEs组,即加入300μg/ml AGEs培养细胞；
AGEs+apocynin组：即加入AGEs和600μM的apocynin培养细胞。
各组分别进行下列处理,利用Western blot检测GLUTag细胞内p38MAPK磷酸化水平。
(2) AGEs对GLUTag细胞中NF-κB因子的影响
分组及培养条件同上,Western blot检测NF-κB蛋白表达的变化。
(3) AGEs对GLUTag细胞中炎症因子的影响
分组及培养条件同上,ELISA法检测炎症因子TNF-α、IL-1、IL-6的变化。
统计学处理：
采用SPSS13.0进行统计分析,实验数据计量资料以均数±标准差(x±s)表示,采用单向方差分析(One-Way ANOVA)检验,两两比较采用LSD法。P<0.050具有统计学意义。
结果：
(1) AGEs对GLUTag细胞中p38MAPK磷酸化水平的影响
100.200、300μg/ml AGEs作用GLUTag细胞24h后,p-p38MAPK的相对表达量分别为0.41±0.05,0.61±0.05,0.82±0.04,均显著高于空白对照组(0.22±0.03)和BSA对照组(0.24±0.04)(P=0.000)。而且,200μg/ml AGEs组显著高于100μg/ml AGEs组(P=0.000),300μg/ml AGEs组显著高于100μg/mlAGEs组及200μg/ml AGEs组(P=0.000)。 AGEs+apocynin组的p-p38MAPK相对表达量为0.28±0.03,与200μg/ml AGEs组相比显著降低(P=0.000)。
(2) AGEs对GLUTag细胞中NF-κB因子表达的影响
100、200、300μg/ml AGEs作用GLUTag细胞24h后,NF-κB表达量分别为0.36±0.04,0.55±0.08,0.67±0.05,均显著高于空白对照组(0.18±0.02)和BSA对照组(0.20±±0.02)(P=0.000)。而且,200μg/ml AGEs组显著高于100μg/ml AGEs组(P=0.000),300μg/ml AGEs组显著高于100μg/ml AGEs组及200μg/ml AGEs组(P=0.000)。AGEs+apocynin组的NF-κB表达量为0.22±0.03,与200μg/ml AGEs组相比显著降低(P=0.000)。
(3) AGEs对GLUTag细胞中炎症因子TNF-α、IL-1、IL-6表达的影响
100、200、300μg/ml AGEs作用GLUTag细胞24h后,TNF-α的相对含量分别为27.23±±4.25,38.93±±3.62,54.47±±5.85,均显著高于空白对照组(15.30±±1.71)和BSA对照组(16.10±±2.15)(P=0.000)。而且,200μg/ml AGEs组显著高于100μg/ml AGEs组(P=0.000),300μg/ml AGEs组显著高于100μg/ml AGEs组及200μg/ml AGEs组(P=0.000). AGEs+apocynin组的TNF-a相对含量为18.40±±3.75,与200μg/ml AGEs组相比显著降低(P=0.000)。
100.200.300μg/ml AGEs作用GLUTag细胞24h后,IL-1的相对含量分别为136.50±±13.05,191.17±±12.01,287.7±±13.75,均显著高于空白对照组(50.67±4.99)和BSA对照组(55.33±6.06)(P=0.000)。而且,200μg/ml AGEs组显著高于100μg/ml AGEs组(P=0.000),300μg/ml AGEs组显著高于100μg/ml AGEs组及200μg/ml AGEs组(P=0.000)。AGEs+apocynin组的IL-1相对含量为68.17±8.72,与200μg/ml AGEs组相比显著降低(P=0.000)。
100、200、300μg/ml AGEs作用GLUTag细胞24h后,IL-6的相对含量分别为95.93±12.29,177.93±9.26,234.27±19.74,均显著高于空白对照组(43.80±5.94)和BSA对照组(50.40±5.64)(P=0.000)。而且,200μg/ml AGEs组显著高于100μg/ml AGEs组(P=0.000),300μg/ml AGEs组显著高于100μg/ml AGEs组及200μg/ml AGEs组(P=0.000)。AGEs+apocynin组的IL-6相对含量为63.50±7.62,与200μg/ml AGEs组相比显著降低(P=0.000)。
结论：
随着AGEs浓度的增加,GLUTag细胞内p38MAPK磷酸化水平上升,NF-κB因子活化增多及炎症因子TNF-α、IL-1、IL-6表达增多。利用apocynin抑制NADPH氧化酶,可调节AGEs诱导的GLUTag细胞内p38MAPK、NF-κB、TNF-α、 IL-1、IL-6等表达水平。本部分实验证明AGEs可至少部分通过激活p38MAPK/NF-κB炎症通路导致肠道L细胞的炎症损伤。
Background:
With the improvement of people's living standard and the changes of life style, the morbidity of type2diabetes (T2DM) presents a tendency of rapid increasing in our country. According to the latest data in2009, the morbidity of T2DM has reached9.7%in our country, and the total number of patients was92.40million which has climbed atop the first in the world. Therefore, to explore the mechanisms of diabetes mellitus has been a hotspot and focus in the field of diabetes. The effect of incretin is an important mechanism for regulating and controlling postprandial insulin secretion and postprandial glucose, which is a breakthrough for the pathogenesis and treatment of T2DM. Glucagon-like peptide-1(GLP-1) is produced in L cells with the functions of glucose-dependent insulin secretion and improving the proliferation and apoptosis of p-cells. Many studies suggest that the effect of incretin is significantly impaired in patients with T2DM, favoring the hyposecretion of GLP-1. Therefore we propose that the early damage and apoptosis of intestinal L cells may play a central role in the hyposecretion of GLP-1and the development of T2DM.
Present studies mostly focus on the regulating effect of different medicine in secretion of GLP-1and its possible pathway, whereas few studies aim at the early damage and apoptosis of intestinal L cells, especially the effects of advanced glycation end-products (AGEs) in controlling the function of L cells. The pathway of AGEs and the receptor for advanced glycation end products (RAGE) is an important mechanism in the damage of various histocyte with diabetes. So to explore whether AGEs-RAGE result in the damage of L cells and its mechanism plays a central role in understanding and improving the pathogenesis of T2DM.
AGEs are toxic and irreversible compounds that are formed by the glycosylation between long-term hyperglycemia and various proteins. The production and accumulation of massive irreversible AGEs is an important mechanism in cellular damage and chronic progress of diabetes. AGEs combining with RAGE can up-regulate the inflammatory response and lead to the destruction of histiocyte, so AGEs may be a pre-inflammatory mediator that can activate cells and promote the response to oxidative stress. Many studies suggest that the pathway of AGEs-RAGE not only mediates damage of various cells but also leads to apoptosis and dysfunction in islet cells. The similar results are also obtained by the latest study in2013:high concentration of AGEs can obviously up-regulate the expression of RAGE on the surface of islet cells. Moreover, the combination between AGEs and RAGE activates NADPH oxidase to increase the production of intracellular reactive oxygen species (ROS), then promotes the expression of nuclear factor kappa (NF-κB) and up-regulates the ratio of Bax/Bcl-2, which finally obviously increases the apoptosis rates of islet cells. Therefore, using the inhibitor of NADPH oxidase can improve the apoptosis of islet cells induced by AGEs. It follows that the pathway of AGEs-RAGE that activates NADPH oxidase to increase the production of intracellular ROS, then promotes the expression of NF-κB and up-regulates the ratio of Bax/Bcl-2is an important mechanism for the apoptosis of inlet cells and the reduced secretion of insulin. Interestingly, our colleagues conducted a study about the effect of lipopolysaccharide (LPS) in the apoptpsis and secretion punction of L cells, and confirmed that lipopolysaccharide (LPS) could induced the apoptpsis of L cells and reduced the secretion of GLP-1through promoting the expression of NF-κB and the downregulation of Bcl-2. This damage was a time-dependent and dose-dependent effect. Moreover, LPS, as well as AGEs, could induced the damage of cells through RAGE and its related pathway. It indicated that the apoptosis of L cells was a cause for reduction of GLP-1, and RAGE played an important role in it.
Therefore, this program focus on L cell line (GLUTag cells), and discuss from the following aspects:1. we observe the effect of AGEs in the early damage and apoptosis of GLUTag cells and the secretion of GLP-1.2. we observe the effect of AGEs-RAGE and its related mechanism with monoclonal antibody interfering RAGE in the damage and apoptosis of GLUTag cells. The study will further explain the cause why GLP-1decrease secretion in patients with T2DM and provide new foundation to explore the new therapy in T2DM.
Part1The effects of AGEs on the early damage and apoptosis of GLUTag and the secretion of GLP-1
Objective:
To study the effect of AGEs in the early damage and apoptosis of GLUTag cells and the secretion of GLP-1.
Methods:
(1)Preparation of AGEs-BSA in vitro
Briefly, BSA was dissolved in phosphate-buffered saline (PBS; pH=7.4) with D-glucose, and incubated for12weeks in the dark. AGE-BSA was identified using a fluorescence spectrophotometer.
(2)Culture of GLUTag cells
GLUTag cells were subcultured with low glucose DMEM containing10%fetal bovine serum,100IM/ml penicillin and100IM/ml streptomycin.
(3)Groups:
The effect of different concentration of AGEs in L cells:The blank control group was cultured with no intervention factor. The BSA control group was cultured with BSA as the positive control. The100μg/ml AGEs group was cultured with100μg/ml AGEs. The200μg/ml AGEs group was cultured with200μg/ml AGEs. The300μg/ml AGEs group was cultured with300μg/ml AGEs.
The effect of AGEs in L cells for different times:200μg/ml AGEs incubated with cells for0,12,24,48h.
(4) The effects of AGEs in the apoptosis of GLUTag cells
The cells were divided into5groups as above. The morphological change of apoptosis were detected with Hoechst33258fluorescence staining.The apoptosis rate was detected with AnnexinⅤ-FITC/PI method.
(5) The effects of AGEs in the proliferation of GLUTag cells
The cells were divided into5groups as above. The proliferation of GLUTag cells was tested with CCK-8method.
(6)The effects of AGEs in the secretion of GLP-1in GLUTag cells
The cells were divided into5groups as above and the supernate of cells were collected. The secretion level of GLP-1in GLUTag cells was tested with ELISA method.
Statistical Analysis:
All analyses were carried out with SPSS13.0software. Data are expressed as mean±standard deviation (SD). Differences between groups were tested by one-way ANOVA followed by a LSD test. Apoptotic rate was determined by the factorial design ANOVA. Statistical significance was defined as two-sided p<0.05.
Results:
(1)The effect of different concentration of AGEs in the morphological change of GLUTag cells
All the groups were observed with fluorescence microscope by Hoechst33258staining after intervention for24h. The apoptotic cells in100μg/ml AGEs group showed little morphological change. The apoptotic cells in200ug/ml AGEs group and300μg/ml AGEs group showed topic morphological changes. Apoptotic cells were observed as intact round nuclei and fragmented (or condensed) nuclei.
(2)The effect of200μg/ml AGEs in the morphological change of GLUTag cells for different time
The apoptotic cells in12h group showed little morphological change. The apoptotic cells in24h group and48h group showed topic morphological changes were observed with Hoechst33258staining. Apoptptic cells were observed as intact round nuclei and fragmented (or condensed) nuclei.
(3) The effect of different concentration of AGEs in the apoptosis of GLUTag cells
The results obtained from FCM showed that the apoptotic rate in100μg/ml AGEs group was (15.30±2.89)%, which significantly higher than that of control group (6.43±1.03)%and BSA group (7.37±1.50)%(P=0.000). Apoptptic rate in200μg/ml AGEs group was (24.10±2.95)%, which significantly higher than that of control group, BSA group and100μg/ml AGEs group (P=0.000). Apoptptic rate in300μg/ml AGEs group was (35.19±3.76)%, which significantly higher than that of control group, BSA group and100μg/ml AGEs group (P=0.000), and higher than 200μg/ml AGEs group (P<0.05).
(4) The effect of200μg/ml AGEs in the apoptpsis of GLUTag cells for different time
The results obtained from FCM showed that the apoptotic rate in12h group was (12.61±1.94)%, which significantly higher than that of control group (6.30±2.23)%(P=0.000). Apoptptic rate in24h group was (21.77±3.41)%, which significantly higher than that of control group and12h group (P=0.000). Apoptptic rate in48h group was (29.52±4.42)%, which significantly higher than that of control group and12h group (P=0.000), and higher than24h group (P<0.05).
(5) The effect of different concentration of AGEs in the activity of GLUTag cells
After treatment with100,200,300μg/mL AGEs for24h, the OD value were0.86±0.05,0.66±0.05,0.46±0.06, respectively, and significantly lower than control group (1.21±0.05) and the BSA group (1.19±0.03)(P=0.000). Moreover, the OD value of200μg/mL AGEs group significantly lower than that of100μg/mL AGEs group (P=0.000). The OD value of300μg/mL AGEs group significantly lower than that of100μg/mL AGEs group (P=0.000) and200μg/mL AGEs group (P<0.05).
(6) The effect of200μg/ml AGEs in the activity of GLUTag cells for different time
After treatment with200μg/mL AGEs for12h, the OD value was0.87±0.05, which significantly lower than control group (Oh,1.110±0.05)(P=0.000). The OD value of24h group was0.66±0.04, which significantly lower than control group and12h group (P=0.000). The OD value of48h group was0.39±0.04, which significantly lower than control group and12h group (P=0.000), and also significantly lower than24h group (P<0.05).
(7) The effect of different concentration of AGEs in GLP-1secretion of GLUTag cells
After treatment with100,200,300μg/mL AGEs for24h, the concentration of GLP-1from the GLUTag cells were69.97±4.30,55.27±6.20,37.13±5.55, respectively, and significantly lower than control group (91.27±5.23) and the BSA group (86.63±6.71)(P=0.000). Moreover, the concentration of GLP-1in200μg/mL AGEs group significantly lower than that of100μg/mL AGEs group (P=0.000). The concentration of GLP-1in300μg/mL AGEs group significantly lower than that of100μg/mL AGEs group (P=0.000) and200μg/mL AGEs group (P<0.05).
(8) The effect of200μg/ml AGEs in the GLP-1secretion of GLUTag cells for different time
After treatment with200μg/mL AGEs for12h, the concentration of GLP-1was71.10±4.75, which significantly lower than control group (Oh,83.97±7.28)(P=0.000). The concentration of GLP-1in24h group was55.27±6.20, which significantly lower than control group and12h group (P=0.000). The concentration of GLP-1in48h group was33.93±6.01, which significantly lower than control group and12h group (P=0.000), and also significantly lower than24h group (P<0.05).
Conclusions:
The apoptosis of GLUTag cells became severer with both the concentration of AGEs increasing for the same aging time and the longer time of exposure to AGEs in the same does. We found for the first time that the apoptpsis of L cells were induced by AGEs in a dose, time-dependent manner and the GLP-1secretion from L cells decreased.
Part2The effect of AGEs-RAGE and its related mechanism in the damage and apoptosis of GLUTag cells
Section1The effect of AGEs-RAGE in the apoptosis of GLUTag cells and its related machanism
Objective:
To investigate the effect of AGEs-RAGE and its related pathway in the apoptosis of GLUTag cells induced by AGEs
Methods:
(1) Groups
The effect of different concentration of AGEs in the expression of RAGE of GLUTag cells:The blank control group was cultured with no intervention factor. The BSA control group was cultured with BSA as the positive control. The100μg/ml AGEs group was cultured with100μg/ml AGEs. The200μg/ml AGEs group was cultured with200μg/ml AGEs. The300μg/ml AGEs group was cultured with300μg/ml AGEs. All the groups were incubated for24h. The effect of AGEs in the expression of RAGE and its related pathway:The blank control group was cultured with no intervention factor. The BSA control group was cultured with BSA as the positive control. The AGEs group was cultured with200μg/ml AGEs. The AGEs+siRNA-RAGE group was cultured with AGEs after the transfection of siRNA-RAGE. The AGEs+apocynin group was cultured with AGEs and apocynin.
(2) The effect of AGEs in the expression of RAGE of GLUTag cells
GLUTag cells were subcultured (the method as part1), and AGEs-BSA was prepared. The concentration and time of AGEs were adopted the identified one. The expression of RAGE mRNA was detected with RT-PCR. The expression of protein for RAGE was detected with Western blot.
(3) The effects of AGEs in the level of ROS and the expression of NADPH oxidase in GLUTag cells
The cells were divided into5groups as above. The expression of gp22phox and p47phox was detected with Western blot.The change of intracellular ROX was tested with fluorescent probe method.
(4) The effects of AGEs in the expression of P53and Bax in GLUTag cells
The cells were divided into5groups as above.The change of P53and Bax was detected with Western bolt.
(5) The effects of AGEs in the activity of caspase-3,-9in GLUTag cells
The cells were divided into5groups as above. The activity of caspase-3,-9was detected with ELISA method.
Statistical Analysis:
All analyses were carried out with SPSS13.0software. Data are expressed as mean±standard deviation (SD). Differences between groups were tested by one-way ANOVA followed by a LSD test. Statistical significance was defined as two-sided p<0.05.
Results:
(1) The effect of different concentration of AGEs in the expression of RAGE mRNA in GLUTag cells
After treatment with100,200,300μg/mL AGEs for24h, the expression of RAGE mRNA were1.45±0.13,2.14±0.010,2.45±0.32, respectively, and significantly higher than control group (1.01±0.04) and the BSA group (1.09±0.03)(P=0.000). Moreover, the expression of RAGE mRNA in200μg/mL AGEs group significantly higher than that of100μg/mL AGEs group (P=0.000). The expression of RAGE mRNA in300μg/mL AGEs group significantly higher than that of100μg/mL AGEs group (P=0.000) and200μg/mL AGEs group (P<0.05).
(2) The effect of different concentration of AGEs in the protein level of RAGE in GLUTag cells
After treatment with100,200,300μg/mL AGEs for24h, the protein level of RAGE were0.36±0.04,0.56±0.009,0.72±0.06, respectively, and significantly higher than control group (0.14±0.04) and the BSA group (0.18±0.03)(P=0.000). Moreover, the protein level of RAGE in200μg/mL AGEs group significantly higher than that of100μg/mL AGEs group (P=0.000). The protein level of RAGE in300μg/mL AGEs group significantly higher than that of100μg/mL AGEs group (P=0.000) and200μg/mL AGEs group (P<0.05).
(3) The effect of AGEs in the expression of NADPH oxidase subunit in GLUTag cells
Cells in groups were treated with indicated treatment for24h. The expressions of gp22phox and p47phox in200μg/mL AGEs group were0.66±0.08and0.76±0.09, which significantly higher than that of the BSA group (0.27±0.06and0.30±0.06)(P<0.05). The expressions of gp22phox and p47phox in AGEs+apocynin group were0.40±0.08and0.47±0.09, which significantly higher than that of the BSA group (P<0.05) and lower than that of the200μg/mL AGEs group (P<0.05). The expressions of gp22phox and p47phox in AGEs+siRNA-RAGE group were0.32±0.04and0.34±0.056, which significantly lower than that of the200μg/mL AGEs group (P<0.05).
(4)The effect of AGEs in the activity of ROS in GLUTag cells
Cells in groups were treated with indicated treatment for24h. The fluorescence intnesity of200μg/mL AGEs group was57.58±4.69, which significantly higher than that of the BSA group (23.16±3.47)(P<0.05). The fluorescence intnesity of AGEs+apocynin group was34.97±0.08, which significantly higher than that of the BSA group (P<0.05) and lower than that of the200μg/mL AGEs group (P<0.05). The fluorescence intnesity of AGEs+siRNA-RAGE group was29.44±5.27, which significantly lower than that of the200μg/mL AGEs group (P<0.05).
(6) The effect of AGEs in the expression of p53and Bax in GLUTag cells
Cells in groups were treated with indicated treatment for24h. The expression of p53in200μg/mL AGEs group was0.42±0.05, which significantly lower than that of the BSA group (0.73±0.05)(P<0.05), wherase the expression of Bax in this group was0.66±0.08, which significantly higher than that of the BSA group (0.27±0.08)(P<0.05). The expression of p53in AGEs+apocynin group and AGEs+siRNA-RAGE group were0.64±0.05and0.71±0.06, which significantly higher than that of the200μg/mL AGEs group (P<0.05), wherase the expression of Bax in these group were0.40±0.08and0.32±0.04, which significantly lower than that of the200μg/mL AGEs group (P<0.05).
(7) The effect of AGEs in the activity of caspase-3and-9in GLUTag cells
Cells in groups were treated with indicated treatment for24h. The activity of caspase-3and-9in200μg/mL AGEs group were1.03±0.10and1.22±0.05, which significantly higher than that of the BSA group (0.43±0.05and0.47±0.02)(P<0.05). The activity of caspase-3and-9in AGEs+apocynin group were0.65±0.07and0.68±0.09, which significantly higher than that of the BSA group (P<0.05) and lower than that of the200μg/mL AGEs group (P<0.05). The activity of caspase-3and-9in AGEs+siRNA-RAGE group were0.55±0.08and0.57±0.07, which significantly lower than that of the200μg/mL AGEs group (P<0.05).
Conclusions:
The expression of RAGE mRNA and the protein level of RAGE in GLUTag cells increased with the concentration of AGEs increasing for the same aging time, which suggested AGEs could enhance the oxidative stress of GLUTag cell via the increasing the expression of RAGE. After indicated treatment for24h, the expression of gp22phox and p47phox and the activity of ROS increased, resulting to the downregulation of p53and the upregulation of Bax, as well as increasing the activity of caspase-3and-9, which finally increased the apoptpsis of L cells. That meant that NADPH oxidase, ROS, p53/Bax and caspase-3,-9were the majoy pathway of the apoptosis in GLUTag cells induced by AGEs-RAGE.
Section2The effect of AGEs-RAGE in the inflammation of GLUTag cells and its related machanism
Objective:
To observe the effect of AGEs on the inflammation of GLUTag cells and the pathway of p38MAPK/NF-κB
Methods:
(1) The effect of AGEs in the phosphorylation of p38MAPK in GLUTag cells
GLUTag cells were subcultured (the method as part1), and AGEs-BSA was prepared. The time of AGEs were adopted the identified one.
Groups:
The blank control group was cultured with no intervention factor;
The BSA control group was cultured with BSA as the positive control;
The100g/ml AGEs group was cultured with100μg/ml AGEs;
The200μg/ml AGEs group was cultured with200μg/ml AGEs;
The300μg/ml AGEs group was cultured with300μg/ml AGEs;
The AGEs+apocynin group was cultured with AGEs and apocynin.
All the groups were accepted the following tests. The phosphorylation of p38MAPK was detected with Western blot.
(2) The effect of AGEs in the activity of NF-κB in GLUTag cells
The cells were divided into6groups as above.The activity of NF-κB was detected with Western blot.
(3) The effect of AGEs in the inflammatory factor of GLUTag cells
The cells were divided into6groups as above. The change of inflammatory factor TNF-α、IL-1、IL-6were detected with ELISA method.
Statistical Analysis:
All analyses were carried out with SPSS13.0software. Data are expressed as mean±standard deviation (SD). Differences between groups were tested by one-way ANOVA followed by a LSD test. Statistical significance was defined as two-sided p<0.05.
Results:
(1) The effect of AGEs in the phosphorylation of p38MAPK in GLUTag cells
After treatment with100,200,300μg/mL AGEs for24h, the expression of p-p38MAPK were0.41±0.05,0.61±0.05,0.82±0.04, respectively, and significantly higher than control group (0.22±0.03) and the BSA group (0.24±0.04)(P=0.000). Moreover, the expression of p-p38MAPK in200μg/mL AGEs group significantly higher than that of100μg/mL AGEs group (P=0.000). The expression of p-p38MAPK in300μg/mL AGEs group significantly higher than that of1000μg/mL AGEs group and200μg/mL AGEs group (P=0.000). The expression of p-p38MAPK in AGEs+apocynin group was0.28±0.03, which significantly lower than that of200μg/mL AGEs group (P=0.000).
(2) The effect of AGEs in the expression of NF-κB in GLUTag cells
After treatment with100,200,300μg/mL AGEs for24h, the expression of NF-κB were0.36±0.04,0.55±0.08,0.67±0.05, respectively, and significantly higher than control group (0.18±0.02) and the BSA group (0.20±0.02)(P=0.000). Moreover, the expression of NF-κB in200μg/mL AGEs group significantly higher than that of100μg/mL AGEs group (P=0.000). The expression of NF-κB in300μg/mL AGEs group significantly higher than that of100μg/mL AGEs group and200μg/mL AGEs group (P=0.000). The expression of NF-κB in AGEs+apocynin group was0.22±0.03, which significantly lower than that of200μg/mL AGEs group (P=0.000).
(3)The effect of AGEs in the inflammatory factor of GLUTag cells
After treatment with100,200,300μg/mL AGEs for24h, the content of TNF-a were27.23±4.25,38.93±3.62,54.47±5.85, respectively, and significantly higher than control group (15.30±1.71) and the BSA group (16.10±2.15)(P=0.000). Moreover, the content of TNF-α in200μg/mL AGEs group significantly higher than that of100μg/mL AGEs group (P=0.000). The content of TNF-α in300μg/mL AGEs group significantly higher than that of100μg/mL AGEs group and200μg/mL AGEs group (P=0.000). The content of TNF-α in AGEs+apocynin group was18.40±3.75, which significantly lower than that of200μg/mL AGEs group (P=0.000).
After treatment with100,200,300μg/mL AGEs for24h, the content of IL-1were136.50±13.05,191.17±12.01,287.7±13.75, respectively, and significantly higher than control group (50.67±4.99) and the BSA group (55.33±6.06)(P=0.000). Moreover, the content of IL-1in200μg/mL AGEs group significantly higher than that of100μg/mL AGEs group (P=0.000). The content of IL-1in300μg/mL AGEs group significantly higher than that of100μg/mL AGEs group and200μg/mL AGEs group (P=0.000). The content of IL-1in AGEs+apocynin group was68.17±8.72, which significantly lower than that of200μg/mL AGEs group (P=0.000).
After treatment with100,200,300μg/mL AGEs for24h, the content of IL-6were95.93±12.29,177.93±9.26,234.27±19.74, respectively, and significantly higher than control group (43.80±5.94) and the BSA group (50.40±5.64)(P=0.000). Moreover, the content of IL-6in200μg/mL AGEs group significantly higher than that of100μg/mL AGEs group (P=0.000). The content of IL-6in300μg/mL AGEs group significantly higher than that of100μg/mL AGEs group and200μg/mL AGEs group (P=0.000). The content of IL-6in AGEs+apocynin group was63.50±7.62, which significantly lower than that of200μg/mL AGEs group (P=0.000).
Conclusions:
The phosphorylation of p38MAPK and the expression of NF-κB, TNF-α, IL-1, IL-6increased with the concentration of AGEs increasing for the same aging time. The NADPH oxidase inhibitor apocynin could regulate the expression of p38MAPK, NF-κB, TNF-α, IL-1, IL-6in GLUTag cells induced by AGEs. This part suggested that AGEs could induce the inflammatory injury of L cells through the pathway of p38MAPK/NF-κB.
随着人们生活水平的提高及生活方式的改变,2型糖尿病(type2diabetes mellitus, T2DM)的发病率在我国呈现出高速发展的趋势。据2009年流行病学数据显示,我国T2DM的发病率已达到9.7%,总患病人数9240万,跃居世界第一位。因此,探索糖尿病的发病机制一直是糖尿病领域研究的热点及重点。肠促胰岛素效应是机体调控餐后胰岛素分泌及餐后血糖的重要机制,该效应的发现使2型糖尿病在发病机制和治疗上取得了突破性的进展。胰高血糖素样肽1(glucagon-like peptide-1, GLP-1)则是肠道L细胞分泌的一种重要的肠肽激素,可通过其受体葡萄糖依赖性的促进胰岛素的分泌,并能促进胰岛p细胞增殖和抑制其凋亡。诸多研究证实,2型糖尿病患者中肠促胰岛素效应明显受损,主要表现为内源性GLP-1的产生和分泌不足,而肠道L细胞的早期损伤或凋亡可能是GLP-1分泌减少的主要原因并参与了2型糖尿病的发生和发展。
目前研究多集中在不同药物对肠道内源性GLP-1分泌的调节作用及L细胞分泌GLP-1的可能通路方面。很少有针对肠道L细胞早期损伤及凋亡方面开展的研究,特别是糖尿病糖基化终末产物(advanced glycation end-products, AGEs)对L细胞功能的可能影响方面未见研究报道,AGEs及其受体(RAGE)通路是导致糖尿病多种组织细胞损伤的重要机制,因此,探明AGEs-RAGE是否会导致肠道L细胞的损伤以及损伤的可能机制和信号通路对于深入理解和完善2型糖尿病的发病机制有着重要意义。
糖基化终末产物(AGEs)是长期高血糖与多种蛋白质发生糖基化作用,形成的具有毒性而不可逆的化合物,大量的不可逆的AGEs的产生和积聚是细胞损伤及糖尿病慢性进展的重要机制。AGEs与其受体RAGE (Receptor for AGE)结合可以上调炎症反应并导致组织细胞的破坏,因此AGEs被认为是一类能激活细胞并能促进氧化应激反应的前炎症介质。诸多研究发现,AGEs-RAGE通路不仅介导了多种细胞的损伤,而且导致胰岛等内分泌细胞凋亡及功能损伤,而2013年最新的研究也得出类似的研究结果：高浓度的AGEs可以明显上调胰岛细胞表面RAGE受体的表达,AGEs与其受体RAGE结合后可激活NADPH氧化酶导致细胞内活性氧产生增加,促进炎症核转录因子NF-κB表达及上调Bax/Bcl-2比例,最终导致胰岛细胞凋亡率的明显增加,利用NADPH氧化酶的抑制剂可改善AGEs诱导的胰岛细胞凋亡。由此可见,AGEs-RAGE通路通过激活NADPH氧化酶导致细胞内ROS增加,进而促进炎症核转录因子NF-κB表达以及Bax/Bcl-2比例上调,这是导致胰岛细胞凋亡及胰岛素分泌减少的一个重要机制。有意思的是,本科室其他同事已完成的脂多糖(LPS)对肠道L细胞凋亡及分泌功能影响的实验研究,发现LPS体外可通过促进NF-κB的表达和释放以及下调抗凋亡基因Bcl-2水平诱导体外培养的L细胞发生凋亡,进而减少内源性GLP-1的分泌水平,这种损伤具有时间及剂量的依赖性,而LPS和AGEs均可通过RAGE受体及其下游通路介导细胞的损伤。这表明L细胞凋亡可能是体内GLP-1分泌减少的原因之一,而RAGE起着重要的作用。
因此,本课题以L细胞株(GLUTag细胞)为研究对象,拟从以下几个方面进行初步探讨：1、观察AGEs对肠道L细胞株(GLUTag细胞)早期损伤、凋亡及GLP-1分泌水平的影响；2、探讨AGEs是否通过AGEs-RAGE通路对GLUTag细胞产生损伤、凋亡及其相关机制。为进一步阐明2型糖尿病体内GLP-1减少的原因以及探索新的2型糖尿病治疗方案提供新的线索。
第一部分AGEs对L细胞株(GLUTag)早期损伤、凋亡及GLP-1分泌水平的影响
目的：
观察AGEs对肠道L细胞株GLUTag细胞早期损伤、凋亡及GLP-1分泌水平的影响。
方法：
(1)体外制备、鉴定糖基化终产物
将胎牛血清白蛋白(BSA)和D-葡萄糖溶于PBS中,避光孵育12周制备AGE-BSA。以荧光分光光度计鉴定AGE-BSA。
(2)传代培养GLUTag细胞
用低糖DMEM培养基,加入10%的胎牛血清,100IM/ml青霉素和100IM/ml链霉素所配置的细胞完全培养基传代培养GLUTag细胞。
(3)实验分组：
不同浓度AGEs的影响：空白对照组,即不加入干预因素；BSA对照组；AGEs100μg/ml组; AGEs200μg/ml组；AGEs300μg/ml组,分别作用细胞24h。AGEs作用不同时间的影响：200μg/mL AGEs分别作用GLUTag细胞0、12、24、48h。
(4) AGEs对GLUTag细胞凋亡的影响
分组及培养条件同上,各组细胞干预结束后利用Hoechst33258荧光染色观察细胞形态学变化。Annexin V-FITC/PI染色检测细胞凋亡率。
(5) AGEs对GLUTag细胞活性的影响
分组及培养条件同上,各组均利用CCK-8法检测GLUTag细胞增殖活性。
(6) AGEs对GLUTag细胞GLP-1分泌的影响
分组及培养条件同上,收集细胞上清液,利用ELISA检测GLUTag细胞GLP-1分泌水平。
统计学处理：
采用SPSS13.0进行统计分析,实验数据计量资料以均数±标准差(i±s)表示,采用单向方差分析(One-Way ANOVA)检验,两两比较采用LSD法。凋亡率采用析因设计的方差分析。P<0.050具有统计学意义。
结果：
(1)不同浓度AGEs对GLUTag细胞形态学的影响
各组经药物干预24h后,Hoechst33258染色后荧光显微镜下观察：空白对照组及BSA对照组GLUTag细胞的细胞核呈均匀荧光,为浅蓝色,且结构正常；AGEs100μg/ml组GLUTag细胞大部分细胞核均匀浅蓝色染色,偶见部分细胞核固缩,核染色致密呈亮蓝色或裂解呈颗粒状；AGEs200μg/ml组GLUTag细胞质固缩、偏移,核致密浓染,核呈颗粒状或碎片状,核染色质边集;AGEs300μg/ml组GLUTag细胞大片浓染,呈大面积亮蓝色,凋亡小体、核固缩及核碎裂的细胞较AGEs200μg/ml组明显增多。
(2)比较AGEs不同作用时间对GLUTag细胞形态学的影响
200μg/ml AGEs作用时间(0h)后的GLUTag细胞的细胞核呈均匀淡染；200μg/ml AGEs作用时间(12h)后的GLUTag细胞的细胞核大部分呈均匀浅染,偶见部分细胞核染色致密呈亮蓝色；200μg/ml AGEs作用时间(24h)后的GLUTag细胞的细胞质固缩,核致密浓染或裂解呈颗粒状,核染色质边集；200μg/ml AGEs作用时间(48h)后的GLUTag细胞的细胞核大面积浓染,细胞皱缩,核固缩、偏移及碎裂的细胞较作用时间为24h明显增多。
(3)不同浓度AGEs处理24h对GLUTag细胞凋亡率的影响
各组细胞经药物干预24h后,100μg/ml AGEs组的凋亡细胞百分率为(15.30±2.89)%,显著高于空白对照组(6.43±1.03)%和BSA对照组(7.37±1.50)%(P=0.000);200μg/ml AGEs组的凋亡细胞百分率为(24.10±2.95)%,显著高于空白对照组和BSA对照组(P=0.000),同时亦显著高于100μg/ml AGEs组(P=0.000);300μg/ml AGEs组的凋亡细胞百分率为(35.19±3.76)%,显著高于空白对照组、BSA对照组及100μg/ml AGEs组(P=0.000),同时亦显著高于200μg/ml AGEs组(P<0.05)。
(4)比较AGEs不同作用时间对GLUTag细胞凋亡的影响
200μg/ml AGEs作用时间(12h)后的凋亡细胞百分率为(12.61±1.94)%,显著高于阴性对照组(6.30±2.23)%(P=0.000)；200μg/ml AGEs作用时间(24h)后的凋亡细胞百分率为(21.77±3.41)%,显著高于阴性对照组及作用12h组(P=0.00);200μg/ml AGEs作用时间(48h)后的凋亡细胞百分率为(29.52±4.42)%,显著高于阴性对照组及作用12h组(P=0.000),同时亦显著高于作用24h组(P<0.05)。
(5)不同浓度AGEs处理24h对GLUTag细胞活性的影响
100、200.300μg/ml AGEs作用GLUTag细胞24h后,OD值分别为0.8±0.05,0.66±0.05,0.4±0.06,均显著低于空白对照组(control)(1.21±0.05)和BSA对照组(200μg/ml BSA)(1.19±0.03)(P=0.000)。而且,200μg/ml AGEs组显著低于100μg/ml AGEs组(P=0.000),300μg/ml AGEs组显著低于100μg/ml AGEs组(P=0.000)及200μg/ml AGEs组(P<0.05)。
(6)比较AGEs不同作用时间对GLUTag细胞活性的影响
200μg/ml AGEs作用时间(12h)后的细胞OD值为0.87±0.05,显著低于阴性对照组(作用时间Oh)(1.110±0.05)(P=0.000);200μg/ml AGEs作用时间(24h)后的细胞OD值为0.6±0.04,显著低于阴性对照组和作用12h组(P=0.000)；200μg/ml AGEs作用时间(48h)后的细胞OD值为0.3±0.04,显著低于阴性对照组和作用12h组(P=0.000),同时亦显著低于作用24h组(P<0.05)。
(7)不同浓度AGEs处理24h对GLUTag细胞GLP-1分泌水平的影响
100、200、300μg/ml AGEs作用GLUTag细胞24h后,细胞分泌GLP-1浓度(pmol/L)分别为69.97±4.30,55.27±6.20,37.13±5.55,均显著低于空白对照组(control)(91.27±5.23)和BSA对照组(200μg/ml BSA)(86.63±6.71)(P=0.000)。而且,200μg/ml AGEs组显著低于100μg/ml AGEs组(P=0.000),300μg/ml AGEs组显著低于100μg/ml AGEs组(P=0.000)及200μg/ml AGEs组(P<0.05)。
(8)比较AGEs不同作用时间对GLUTag细胞GLP-1分泌水平的影响
200μg/ml AGEs作用时间(12h)后的细胞分泌GLP-1浓度为71.10±4.75,显著低于阴性对照组(作用时间Oh)(83.97±7.28)(P=0.000);200μg/ml AGEs作用时间(24h)后的细胞分泌GLP-1浓度为55.27±6.20,显著低于阴性对照组和作用12h组(P=0.000);200μg/ml AGEs作用时间(48h)后的细胞分泌GLP-1浓度为33.93±6.01,显著低于阴性对照组和作用12h组(P=0.000),同时亦显著低于作用24h组(P<0.05)。
结论：
肠道L细胞株GLUTag细胞的凋亡在相同作用时间下随着AGEs的浓度增高而增多,且在相同剂量下随着AGEs的作用时间延长而增多。我们首次发现AGEs可呈剂量、时间依赖性诱导肠道L细胞凋亡,并显著降低L细胞活力以及分泌GLP-1的能力。
第二部分探讨AGEs是否通过AGEs-RAGE通路对GLUTag细胞产生损伤、凋亡及其相关机制
第一节AGEs对GLUTag细胞的凋亡及其相关通路的影响
目的：
探讨AGEs-RAGE及其下游分子信号通路在AGEs诱导的GLUTag细胞凋亡中的作用。
方法：
(1)实验分组：
探讨不同浓度AGEs对GLUTag细胞中RAGE表达的影响时按以下分组：空白对照组,即不加入干预因素；BSA对照组;AGEs100μg/ml组;AGEs200μg/ml组；AGEs300μg/ml组,分别作用细胞24h。探讨AGEs对RAGE及其下游分子信号通路的影响时按照以下分组：空白对照组；BSA对照组；AGEs200μg/ml组;AGEs+siRNA-RAGE组：即利用siRNA-RAGE转染靶细胞后加入AGEs共培养细胞。AGEs+apocynin组：即加入AGEs和600μM的apocynin (NADPH氧化酶阻断剂)培养细胞。
(2) AGEs对GLUTag细胞中RAGE表达的影响
传代培养GLUTag细胞(方法同第一部分),并制备AGEs-BSA,采用已确定的AGEs浓度及时间。利用RT-PCR及Western blot检测各组细胞RAGE mRNA及RAGE蛋白表达水平。
(3) AGEs对GLUTag细胞ROS水平及NADPH氧化酶亚单位表达的影响
分组及培养条件同上,Western blot检测细胞中NADPH氧化酶亚单位gp22phox、p47phox的磷酸化,荧光探针检测细胞内活性氧ROS的水平。
(4) AGEs对GLUTag细胞中p53、Bax因子的影响
分组及培养条件同上,Western-blot法检测P53、Bax因子的变化。
(5) AGEs对GLUTag细胞中caspase-3及-9的活性
分组及培养条件同上,用酶联免疫法测定caspase-3及-9的活性。
统计学处理：
采用SPSS13.0进行统计分析,实验数据计量资料以均数±标准差(x±s)表示,采用单向方差分析(One-Way ANOVA)检验,两两比较采用LSD法。P<0.050具有统计学意义。
结果：
(1)不同浓度的AGEs对GLUTag细胞RAGE mRNA的影响
100、200、300μg/ml AGEs作用GLUTag细胞24h后,RAGE mRNA表达量分别为1.45±0.13,2.14±0.010,2.45±0.32,均显著高于空白对照组(1.01±0.04)和BSA对照组(1.09±0.03)(P=0.000)。而且,200μg/ml AGEs组显著高于100μg/ml AGEs组(P=0.000),300μg/ml AGEs组显著高于AGEs组(P=0.000)及200μg/ml AGEs组(P<0.05)。
(2)不同浓度的AGEs对GLUTag细胞内RAGE蛋白表达水平的影响
100.200、300μg/ml AGEs作用GLUTag细胞24h后,RAGE蛋白表达水平分别为0.36±0.04,0.56±0.009,0.72±0.06,均显著高于空白对照组(0.144±0.04)和BSA对照组(0.18±0.03)(P=0.000)。而且,200μg/ml AGEs组显著高于100μg/ml AGEs组(P=0.000),300μg/ml AGEs组显著高于100μg/ml AGEs组(P=0.000)及200μg/ml AGEs组(P<0.05)。
(3) AGEs对GLUTag细胞中NADPH氧化酶亚单位表达的影响
各组细胞经干预24h后,200μg/ml AGEs组的gp22phox、p47phox蛋白表达水平分别为0.66±0.08,0.76±0.09,均显著高于BSA对照组(0.27±0.06,0.30±0.06)(P<0.05); AGEs+apocynin组的gp22phox、p47phox蛋白表达水平分别为0.40±±0.08,0.4±0.09,均显著高于BSA对照组(P<0.05),而且显著低于200μg/ml AGEs组(P<0.05)；AGEs+siRNA-RAGE组的gp22phox、p47phox蛋白表达水平分别为0.32±0.04,0.344±0.056,均显著低于200μg/ml AGEs组(P<0.05)。
(4) AGEs对GLUTag细胞内活性氧水平的影响
各组细胞经干预24h后,200μg/ml AGEs组的荧光强度为57.58±±4.69,显著高于BSA对照组(23.16±3.47)(P<0.05)；AGEs+apocynin组的荧光强度为34.97±±6.42,显著高于BSA对照组(P<0.05),而且显著低于200μg/ml AGEs组(P<0.05); AGEs+siRNA-RAGE组的荧光强度为29.444±5.27,显著低于200μg/ml AGEs组(P<0.05)。
(5) AGEs对GLUTag细胞凋亡相关蛋白p53及Bax表达的影响
各组细胞经干预24h后,200μg/ml AGEs组的p53蛋白表达水平为0.42±±0.05,显著低于BSA对照组(0.73±±0.05)(P<0.05),而Bax蛋白表达水平为0.66±±0.08,显著高于BSA对照组(0.27±0.08)(P<0.05)；AGEs+apocynin组和AGEs+siRNA-RAGE组的p53蛋白表达水平分别为0.644±0.05,0.71±±0.06,均显著高于200μg/ml AGEs组(P<0.05),而Bax蛋白表达水平分别为0.40±0.08,0.32±0.04,均显著低于200μg/ml AGEs组(P<0.05)。
(6) AGEs对GLUTag细胞凋亡蛋白酶caspase-3及-9活性的影响
各组细胞经干预24h后,200μg/ml AGEs组的caspase-3及-9活性分别为1.03±0.10,1.22±0.05,均显著高于BSA对照组(0.43±0.05,0.47±0.02)(P<0.05); AGEs+apocynin组的caspase-3及-9活性分别为0.65±0.07,0.68±0.09,均显著高于BSA对照组(P<0.05),而且显著低于200μg/ml AGEs组(P<0.05);AGEs+siRNA-RAGE组的caspase-3及-9活性分别为0.55±0.08,0.57±0.07,均显著低于200μg/ml AGEs组(P<0.05)。
结论：
随着AGEs干预浓度的升高,GLUTag细胞RAGE mRNA表达量和RAGE蛋白表达水平显著上升,提示AGEs可通过上调RAGE受体表达增强GLUTag细胞的氧化应激损伤。AGEs作用于GLUTag细胞24h后,NADPH氧化酶亚单位gp22phox、p47phox的表达增多,细胞内ROS水平升高,p53表达下调,Bax表达上调,凋亡蛋白酶caspase-3及-9的活性增加,细胞凋亡增加。提示NADPH氧化酶、ROS、p53/Bax及caspase-3及-9是AGEs-RAGE导致GLUTag细胞凋亡的主要下游通路。
第二节AGEs对GLUTag细胞炎症损伤及p38MAPK/NF-κB通路的影响
目的：
观察AGEs对肠道L细胞株GLUTag细胞中炎症损伤及相关机制的影响。
方法：
(1)AGEs对GLUTag细胞中p38MAPK磷酸化水平的影响
传代培养GLUTag细胞(方法同第一部分),并制备AGEs-BSA,采用已确定的AGEs作用时间。
实验分组：空白对照组,即不加入干预因素；
BSA对照组,即加入BSA培养细胞作为阳性对照；
100μg/ml AGEs组,即加入100μg/ml AGEs培养细胞；
200μg/ml AGEs组,即加入200μg/ml AGEs培养细胞；
300μg/ml AGEs组,即加入300μg/ml AGEs培养细胞；
AGEs+apocynin组：即加入AGEs和600μM的apocynin培养细胞。
各组分别进行下列处理,利用Western blot检测GLUTag细胞内p38MAPK磷酸化水平。
(2) AGEs对GLUTag细胞中NF-κB因子的影响
分组及培养条件同上,Western blot检测NF-κB蛋白表达的变化。
(3) AGEs对GLUTag细胞中炎症因子的影响
分组及培养条件同上,ELISA法检测炎症因子TNF-α、IL-1、IL-6的变化。
统计学处理：
采用SPSS13.0进行统计分析,实验数据计量资料以均数±标准差(x±s)表示,采用单向方差分析(One-Way ANOVA)检验,两两比较采用LSD法。P<0.050具有统计学意义。
结果：
(1) AGEs对GLUTag细胞中p38MAPK磷酸化水平的影响
100.200、300μg/ml AGEs作用GLUTag细胞24h后,p-p38MAPK的相对表达量分别为0.41±0.05,0.61±0.05,0.82±0.04,均显著高于空白对照组(0.22±0.03)和BSA对照组(0.24±0.04)(P=0.000)。而且,200μg/ml AGEs组显著高于100μg/ml AGEs组(P=0.000),300μg/ml AGEs组显著高于100μg/mlAGEs组及200μg/ml AGEs组(P=0.000)。 AGEs+apocynin组的p-p38MAPK相对表达量为0.28±0.03,与200μg/ml AGEs组相比显著降低(P=0.000)。
(2) AGEs对GLUTag细胞中NF-κB因子表达的影响
100、200、300μg/ml AGEs作用GLUTag细胞24h后,NF-κB表达量分别为0.36±0.04,0.55±0.08,0.67±0.05,均显著高于空白对照组(0.18±0.02)和BSA对照组(0.20±±0.02)(P=0.000)。而且,200μg/ml AGEs组显著高于100μg/ml AGEs组(P=0.000),300μg/ml AGEs组显著高于100μg/ml AGEs组及200μg/ml AGEs组(P=0.000)。AGEs+apocynin组的NF-κB表达量为0.22±0.03,与200μg/ml AGEs组相比显著降低(P=0.000)。
(3) AGEs对GLUTag细胞中炎症因子TNF-α、IL-1、IL-6表达的影响
100、200、300μg/ml AGEs作用GLUTag细胞24h后,TNF-α的相对含量分别为27.23±±4.25,38.93±±3.62,54.47±±5.85,均显著高于空白对照组(15.30±±1.71)和BSA对照组(16.10±±2.15)(P=0.000)。而且,200μg/ml AGEs组显著高于100μg/ml AGEs组(P=0.000),300μg/ml AGEs组显著高于100μg/ml AGEs组及200μg/ml AGEs组(P=0.000). AGEs+apocynin组的TNF-a相对含量为18.40±±3.75,与200μg/ml AGEs组相比显著降低(P=0.000)。
100.200.300μg/ml AGEs作用GLUTag细胞24h后,IL-1的相对含量分别为136.50±±13.05,191.17±±12.01,287.7±±13.75,均显著高于空白对照组(50.67±4.99)和BSA对照组(55.33±6.06)(P=0.000)。而且,200μg/ml AGEs组显著高于100μg/ml AGEs组(P=0.000),300μg/ml AGEs组显著高于100μg/ml AGEs组及200μg/ml AGEs组(P=0.000)。AGEs+apocynin组的IL-1相对含量为68.17±8.72,与200μg/ml AGEs组相比显著降低(P=0.000)。
100、200、300μg/ml AGEs作用GLUTag细胞24h后,IL-6的相对含量分别为95.93±12.29,177.93±9.26,234.27±19.74,均显著高于空白对照组(43.80±5.94)和BSA对照组(50.40±5.64)(P=0.000)。而且,200μg/ml AGEs组显著高于100μg/ml AGEs组(P=0.000),300μg/ml AGEs组显著高于100μg/ml AGEs组及200μg/ml AGEs组(P=0.000)。AGEs+apocynin组的IL-6相对含量为63.50±7.62,与200μg/ml AGEs组相比显著降低(P=0.000)。
结论：
随着AGEs浓度的增加,GLUTag细胞内p38MAPK磷酸化水平上升,NF-κB因子活化增多及炎症因子TNF-α、IL-1、IL-6表达增多。利用apocynin抑制NADPH氧化酶,可调节AGEs诱导的GLUTag细胞内p38MAPK、NF-κB、TNF-α、 IL-1、IL-6等表达水平。本部分实验证明AGEs可至少部分通过激活p38MAPK/NF-κB炎症通路导致肠道L细胞的炎症损伤。
Background:
With the improvement of people's living standard and the changes of life style, the morbidity of type2diabetes (T2DM) presents a tendency of rapid increasing in our country. According to the latest data in2009, the morbidity of T2DM has reached9.7%in our country, and the total number of patients was92.40million which has climbed atop the first in the world. Therefore, to explore the mechanisms of diabetes mellitus has been a hotspot and focus in the field of diabetes. The effect of incretin is an important mechanism for regulating and controlling postprandial insulin secretion and postprandial glucose, which is a breakthrough for the pathogenesis and treatment of T2DM. Glucagon-like peptide-1(GLP-1) is produced in L cells with the functions of glucose-dependent insulin secretion and improving the proliferation and apoptosis of p-cells. Many studies suggest that the effect of incretin is significantly impaired in patients with T2DM, favoring the hyposecretion of GLP-1. Therefore we propose that the early damage and apoptosis of intestinal L cells may play a central role in the hyposecretion of GLP-1and the development of T2DM.
Present studies mostly focus on the regulating effect of different medicine in secretion of GLP-1and its possible pathway, whereas few studies aim at the early damage and apoptosis of intestinal L cells, especially the effects of advanced glycation end-products (AGEs) in controlling the function of L cells. The pathway of AGEs and the receptor for advanced glycation end products (RAGE) is an important mechanism in the damage of various histocyte with diabetes. So to explore whether AGEs-RAGE result in the damage of L cells and its mechanism plays a central role in understanding and improving the pathogenesis of T2DM.
AGEs are toxic and irreversible compounds that are formed by the glycosylation between long-term hyperglycemia and various proteins. The production and accumulation of massive irreversible AGEs is an important mechanism in cellular damage and chronic progress of diabetes. AGEs combining with RAGE can up-regulate the inflammatory response and lead to the destruction of histiocyte, so AGEs may be a pre-inflammatory mediator that can activate cells and promote the response to oxidative stress. Many studies suggest that the pathway of AGEs-RAGE not only mediates damage of various cells but also leads to apoptosis and dysfunction in islet cells. The similar results are also obtained by the latest study in2013:high concentration of AGEs can obviously up-regulate the expression of RAGE on the surface of islet cells. Moreover, the combination between AGEs and RAGE activates NADPH oxidase to increase the production of intracellular reactive oxygen species (ROS), then promotes the expression of nuclear factor kappa (NF-κB) and up-regulates the ratio of Bax/Bcl-2, which finally obviously increases the apoptosis rates of islet cells. Therefore, using the inhibitor of NADPH oxidase can improve the apoptosis of islet cells induced by AGEs. It follows that the pathway of AGEs-RAGE that activates NADPH oxidase to increase the production of intracellular ROS, then promotes the expression of NF-κB and up-regulates the ratio of Bax/Bcl-2is an important mechanism for the apoptosis of inlet cells and the reduced secretion of insulin. Interestingly, our colleagues conducted a study about the effect of lipopolysaccharide (LPS) in the apoptpsis and secretion punction of L cells, and confirmed that lipopolysaccharide (LPS) could induced the apoptpsis of L cells and reduced the secretion of GLP-1through promoting the expression of NF-κB and the downregulation of Bcl-2. This damage was a time-dependent and dose-dependent effect. Moreover, LPS, as well as AGEs, could induced the damage of cells through RAGE and its related pathway. It indicated that the apoptosis of L cells was a cause for reduction of GLP-1, and RAGE played an important role in it.
Therefore, this program focus on L cell line (GLUTag cells), and discuss from the following aspects:1. we observe the effect of AGEs in the early damage and apoptosis of GLUTag cells and the secretion of GLP-1.2. we observe the effect of AGEs-RAGE and its related mechanism with monoclonal antibody interfering RAGE in the damage and apoptosis of GLUTag cells. The study will further explain the cause why GLP-1decrease secretion in patients with T2DM and provide new foundation to explore the new therapy in T2DM.
Part1The effects of AGEs on the early damage and apoptosis of GLUTag and the secretion of GLP-1
Objective:
To study the effect of AGEs in the early damage and apoptosis of GLUTag cells and the secretion of GLP-1.
Methods:
(1)Preparation of AGEs-BSA in vitro
Briefly, BSA was dissolved in phosphate-buffered saline (PBS; pH=7.4) with D-glucose, and incubated for12weeks in the dark. AGE-BSA was identified using a fluorescence spectrophotometer.
(2)Culture of GLUTag cells
GLUTag cells were subcultured with low glucose DMEM containing10%fetal bovine serum,100IM/ml penicillin and100IM/ml streptomycin.
(3)Groups:
The effect of different concentration of AGEs in L cells:The blank control group was cultured with no intervention factor. The BSA control group was cultured with BSA as the positive control. The100μg/ml AGEs group was cultured with100μg/ml AGEs. The200μg/ml AGEs group was cultured with200μg/ml AGEs. The300μg/ml AGEs group was cultured with300μg/ml AGEs.
The effect of AGEs in L cells for different times:200μg/ml AGEs incubated with cells for0,12,24,48h.
(4) The effects of AGEs in the apoptosis of GLUTag cells
The cells were divided into5groups as above. The morphological change of apoptosis were detected with Hoechst33258fluorescence staining.The apoptosis rate was detected with AnnexinⅤ-FITC/PI method.
(5) The effects of AGEs in the proliferation of GLUTag cells
The cells were divided into5groups as above. The proliferation of GLUTag cells was tested with CCK-8method.
(6)The effects of AGEs in the secretion of GLP-1in GLUTag cells
The cells were divided into5groups as above and the supernate of cells were collected. The secretion level of GLP-1in GLUTag cells was tested with ELISA method.
Statistical Analysis:
All analyses were carried out with SPSS13.0software. Data are expressed as mean±standard deviation (SD). Differences between groups were tested by one-way ANOVA followed by a LSD test. Apoptotic rate was determined by the factorial design ANOVA. Statistical significance was defined as two-sided p<0.05.
Results:
(1)The effect of different concentration of AGEs in the morphological change of GLUTag cells
All the groups were observed with fluorescence microscope by Hoechst33258staining after intervention for24h. The apoptotic cells in100μg/ml AGEs group showed little morphological change. The apoptotic cells in200ug/ml AGEs group and300μg/ml AGEs group showed topic morphological changes. Apoptotic cells were observed as intact round nuclei and fragmented (or condensed) nuclei.
(2)The effect of200μg/ml AGEs in the morphological change of GLUTag cells for different time
The apoptotic cells in12h group showed little morphological change. The apoptotic cells in24h group and48h group showed topic morphological changes were observed with Hoechst33258staining. Apoptptic cells were observed as intact round nuclei and fragmented (or condensed) nuclei.
(3) The effect of different concentration of AGEs in the apoptosis of GLUTag cells
The results obtained from FCM showed that the apoptotic rate in100μg/ml AGEs group was (15.30±2.89)%, which significantly higher than that of control group (6.43±1.03)%and BSA group (7.37±1.50)%(P=0.000). Apoptptic rate in200μg/ml AGEs group was (24.10±2.95)%, which significantly higher than that of control group, BSA group and100μg/ml AGEs group (P=0.000). Apoptptic rate in300μg/ml AGEs group was (35.19±3.76)%, which significantly higher than that of control group, BSA group and100μg/ml AGEs group (P=0.000), and higher than 200μg/ml AGEs group (P<0.05).
(4) The effect of200μg/ml AGEs in the apoptpsis of GLUTag cells for different time
The results obtained from FCM showed that the apoptotic rate in12h group was (12.61±1.94)%, which significantly higher than that of control group (6.30±2.23)%(P=0.000). Apoptptic rate in24h group was (21.77±3.41)%, which significantly higher than that of control group and12h group (P=0.000). Apoptptic rate in48h group was (29.52±4.42)%, which significantly higher than that of control group and12h group (P=0.000), and higher than24h group (P<0.05).
(5) The effect of different concentration of AGEs in the activity of GLUTag cells
After treatment with100,200,300μg/mL AGEs for24h, the OD value were0.86±0.05,0.66±0.05,0.46±0.06, respectively, and significantly lower than control group (1.21±0.05) and the BSA group (1.19±0.03)(P=0.000). Moreover, the OD value of200μg/mL AGEs group significantly lower than that of100μg/mL AGEs group (P=0.000). The OD value of300μg/mL AGEs group significantly lower than that of100μg/mL AGEs group (P=0.000) and200μg/mL AGEs group (P<0.05).
(6) The effect of200μg/ml AGEs in the activity of GLUTag cells for different time
After treatment with200μg/mL AGEs for12h, the OD value was0.87±0.05, which significantly lower than control group (Oh,1.110±0.05)(P=0.000). The OD value of24h group was0.66±0.04, which significantly lower than control group and12h group (P=0.000). The OD value of48h group was0.39±0.04, which significantly lower than control group and12h group (P=0.000), and also significantly lower than24h group (P<0.05).
(7) The effect of different concentration of AGEs in GLP-1secretion of GLUTag cells
After treatment with100,200,300μg/mL AGEs for24h, the concentration of GLP-1from the GLUTag cells were69.97±4.30,55.27±6.20,37.13±5.55, respectively, and significantly lower than control group (91.27±5.23) and the BSA group (86.63±6.71)(P=0.000). Moreover, the concentration of GLP-1in200μg/mL AGEs group significantly lower than that of100μg/mL AGEs group (P=0.000). The concentration of GLP-1in300μg/mL AGEs group significantly lower than that of100μg/mL AGEs group (P=0.000) and200μg/mL AGEs group (P<0.05).
(8) The effect of200μg/ml AGEs in the GLP-1secretion of GLUTag cells for different time
After treatment with200μg/mL AGEs for12h, the concentration of GLP-1was71.10±4.75, which significantly lower than control group (Oh,83.97±7.28)(P=0.000). The concentration of GLP-1in24h group was55.27±6.20, which significantly lower than control group and12h group (P=0.000). The concentration of GLP-1in48h group was33.93±6.01, which significantly lower than control group and12h group (P=0.000), and also significantly lower than24h group (P<0.05).
Conclusions:
The apoptosis of GLUTag cells became severer with both the concentration of AGEs increasing for the same aging time and the longer time of exposure to AGEs in the same does. We found for the first time that the apoptpsis of L cells were induced by AGEs in a dose, time-dependent manner and the GLP-1secretion from L cells decreased.
Part2The effect of AGEs-RAGE and its related mechanism in the damage and apoptosis of GLUTag cells
Section1The effect of AGEs-RAGE in the apoptosis of GLUTag cells and its related machanism
Objective:
To investigate the effect of AGEs-RAGE and its related pathway in the apoptosis of GLUTag cells induced by AGEs
Methods:
(1) Groups
The effect of different concentration of AGEs in the expression of RAGE of GLUTag cells:The blank control group was cultured with no intervention factor. The BSA control group was cultured with BSA as the positive control. The100μg/ml AGEs group was cultured with100μg/ml AGEs. The200μg/ml AGEs group was cultured with200μg/ml AGEs. The300μg/ml AGEs group was cultured with300μg/ml AGEs. All the groups were incubated for24h. The effect of AGEs in the expression of RAGE and its related pathway:The blank control group was cultured with no intervention factor. The BSA control group was cultured with BSA as the positive control. The AGEs group was cultured with200μg/ml AGEs. The AGEs+siRNA-RAGE group was cultured with AGEs after the transfection of siRNA-RAGE. The AGEs+apocynin group was cultured with AGEs and apocynin.
(2) The effect of AGEs in the expression of RAGE of GLUTag cells
GLUTag cells were subcultured (the method as part1), and AGEs-BSA was prepared. The concentration and time of AGEs were adopted the identified one. The expression of RAGE mRNA was detected with RT-PCR. The expression of protein for RAGE was detected with Western blot.
(3) The effects of AGEs in the level of ROS and the expression of NADPH oxidase in GLUTag cells
The cells were divided into5groups as above. The expression of gp22phox and p47phox was detected with Western blot.The change of intracellular ROX was tested with fluorescent probe method.
(4) The effects of AGEs in the expression of P53and Bax in GLUTag cells
The cells were divided into5groups as above.The change of P53and Bax was detected with Western bolt.
(5) The effects of AGEs in the activity of caspase-3,-9in GLUTag cells
The cells were divided into5groups as above. The activity of caspase-3,-9was detected with ELISA method.
Statistical Analysis:
All analyses were carried out with SPSS13.0software. Data are expressed as mean±standard deviation (SD). Differences between groups were tested by one-way ANOVA followed by a LSD test. Statistical significance was defined as two-sided p<0.05.
Results:
(1) The effect of different concentration of AGEs in the expression of RAGE mRNA in GLUTag cells
After treatment with100,200,300μg/mL AGEs for24h, the expression of RAGE mRNA were1.45±0.13,2.14±0.010,2.45±0.32, respectively, and significantly higher than control group (1.01±0.04) and the BSA group (1.09±0.03)(P=0.000). Moreover, the expression of RAGE mRNA in200μg/mL AGEs group significantly higher than that of100μg/mL AGEs group (P=0.000). The expression of RAGE mRNA in300μg/mL AGEs group significantly higher than that of100μg/mL AGEs group (P=0.000) and200μg/mL AGEs group (P<0.05).
(2) The effect of different concentration of AGEs in the protein level of RAGE in GLUTag cells
After treatment with100,200,300μg/mL AGEs for24h, the protein level of RAGE were0.36±0.04,0.56±0.009,0.72±0.06, respectively, and significantly higher than control group (0.14±0.04) and the BSA group (0.18±0.03)(P=0.000). Moreover, the protein level of RAGE in200μg/mL AGEs group significantly higher than that of100μg/mL AGEs group (P=0.000). The protein level of RAGE in300μg/mL AGEs group significantly higher than that of100μg/mL AGEs group (P=0.000) and200μg/mL AGEs group (P<0.05).
(3) The effect of AGEs in the expression of NADPH oxidase subunit in GLUTag cells
Cells in groups were treated with indicated treatment for24h. The expressions of gp22phox and p47phox in200μg/mL AGEs group were0.66±0.08and0.76±0.09, which significantly higher than that of the BSA group (0.27±0.06and0.30±0.06)(P<0.05). The expressions of gp22phox and p47phox in AGEs+apocynin group were0.40±0.08and0.47±0.09, which significantly higher than that of the BSA group (P<0.05) and lower than that of the200μg/mL AGEs group (P<0.05). The expressions of gp22phox and p47phox in AGEs+siRNA-RAGE group were0.32±0.04and0.34±0.056, which significantly lower than that of the200μg/mL AGEs group (P<0.05).
(4)The effect of AGEs in the activity of ROS in GLUTag cells
Cells in groups were treated with indicated treatment for24h. The fluorescence intnesity of200μg/mL AGEs group was57.58±4.69, which significantly higher than that of the BSA group (23.16±3.47)(P<0.05). The fluorescence intnesity of AGEs+apocynin group was34.97±0.08, which significantly higher than that of the BSA group (P<0.05) and lower than that of the200μg/mL AGEs group (P<0.05). The fluorescence intnesity of AGEs+siRNA-RAGE group was29.44±5.27, which significantly lower than that of the200μg/mL AGEs group (P<0.05).
(6) The effect of AGEs in the expression of p53and Bax in GLUTag cells
Cells in groups were treated with indicated treatment for24h. The expression of p53in200μg/mL AGEs group was0.42±0.05, which significantly lower than that of the BSA group (0.73±0.05)(P<0.05), wherase the expression of Bax in this group was0.66±0.08, which significantly higher than that of the BSA group (0.27±0.08)(P<0.05). The expression of p53in AGEs+apocynin group and AGEs+siRNA-RAGE group were0.64±0.05and0.71±0.06, which significantly higher than that of the200μg/mL AGEs group (P<0.05), wherase the expression of Bax in these group were0.40±0.08and0.32±0.04, which significantly lower than that of the200μg/mL AGEs group (P<0.05).
(7) The effect of AGEs in the activity of caspase-3and-9in GLUTag cells
Cells in groups were treated with indicated treatment for24h. The activity of caspase-3and-9in200μg/mL AGEs group were1.03±0.10and1.22±0.05, which significantly higher than that of the BSA group (0.43±0.05and0.47±0.02)(P<0.05). The activity of caspase-3and-9in AGEs+apocynin group were0.65±0.07and0.68±0.09, which significantly higher than that of the BSA group (P<0.05) and lower than that of the200μg/mL AGEs group (P<0.05). The activity of caspase-3and-9in AGEs+siRNA-RAGE group were0.55±0.08and0.57±0.07, which significantly lower than that of the200μg/mL AGEs group (P<0.05).
Conclusions:
The expression of RAGE mRNA and the protein level of RAGE in GLUTag cells increased with the concentration of AGEs increasing for the same aging time, which suggested AGEs could enhance the oxidative stress of GLUTag cell via the increasing the expression of RAGE. After indicated treatment for24h, the expression of gp22phox and p47phox and the activity of ROS increased, resulting to the downregulation of p53and the upregulation of Bax, as well as increasing the activity of caspase-3and-9, which finally increased the apoptpsis of L cells. That meant that NADPH oxidase, ROS, p53/Bax and caspase-3,-9were the majoy pathway of the apoptosis in GLUTag cells induced by AGEs-RAGE.
Section2The effect of AGEs-RAGE in the inflammation of GLUTag cells and its related machanism
Objective:
To observe the effect of AGEs on the inflammation of GLUTag cells and the pathway of p38MAPK/NF-κB
Methods:
(1) The effect of AGEs in the phosphorylation of p38MAPK in GLUTag cells
GLUTag cells were subcultured (the method as part1), and AGEs-BSA was prepared. The time of AGEs were adopted the identified one.
Groups:
The blank control group was cultured with no intervention factor;
The BSA control group was cultured with BSA as the positive control;
The100g/ml AGEs group was cultured with100μg/ml AGEs;
The200μg/ml AGEs group was cultured with200μg/ml AGEs;
The300μg/ml AGEs group was cultured with300μg/ml AGEs;
The AGEs+apocynin group was cultured with AGEs and apocynin.
All the groups were accepted the following tests. The phosphorylation of p38MAPK was detected with Western blot.
(2) The effect of AGEs in the activity of NF-κB in GLUTag cells
The cells were divided into6groups as above.The activity of NF-κB was detected with Western blot.
(3) The effect of AGEs in the inflammatory factor of GLUTag cells
The cells were divided into6groups as above. The change of inflammatory factor TNF-α、IL-1、IL-6were detected with ELISA method.
Statistical Analysis:
All analyses were carried out with SPSS13.0software. Data are expressed as mean±standard deviation (SD). Differences between groups were tested by one-way ANOVA followed by a LSD test. Statistical significance was defined as two-sided p<0.05.
Results:
(1) The effect of AGEs in the phosphorylation of p38MAPK in GLUTag cells
After treatment with100,200,300μg/mL AGEs for24h, the expression of p-p38MAPK were0.41±0.05,0.61±0.05,0.82±0.04, respectively, and significantly higher than control group (0.22±0.03) and the BSA group (0.24±0.04)(P=0.000). Moreover, the expression of p-p38MAPK in200μg/mL AGEs group significantly higher than that of100μg/mL AGEs group (P=0.000). The expression of p-p38MAPK in300μg/mL AGEs group significantly higher than that of1000μg/mL AGEs group and200μg/mL AGEs group (P=0.000). The expression of p-p38MAPK in AGEs+apocynin group was0.28±0.03, which significantly lower than that of200μg/mL AGEs group (P=0.000).
(2) The effect of AGEs in the expression of NF-κB in GLUTag cells
After treatment with100,200,300μg/mL AGEs for24h, the expression of NF-κB were0.36±0.04,0.55±0.08,0.67±0.05, respectively, and significantly higher than control group (0.18±0.02) and the BSA group (0.20±0.02)(P=0.000). Moreover, the expression of NF-κB in200μg/mL AGEs group significantly higher than that of100μg/mL AGEs group (P=0.000). The expression of NF-κB in300μg/mL AGEs group significantly higher than that of100μg/mL AGEs group and200μg/mL AGEs group (P=0.000). The expression of NF-κB in AGEs+apocynin group was0.22±0.03, which significantly lower than that of200μg/mL AGEs group (P=0.000).
(3)The effect of AGEs in the inflammatory factor of GLUTag cells
After treatment with100,200,300μg/mL AGEs for24h, the content of TNF-a were27.23±4.25,38.93±3.62,54.47±5.85, respectively, and significantly higher than control group (15.30±1.71) and the BSA group (16.10±2.15)(P=0.000). Moreover, the content of TNF-α in200μg/mL AGEs group significantly higher than that of100μg/mL AGEs group (P=0.000). The content of TNF-α in300μg/mL AGEs group significantly higher than that of100μg/mL AGEs group and200μg/mL AGEs group (P=0.000). The content of TNF-α in AGEs+apocynin group was18.40±3.75, which significantly lower than that of200μg/mL AGEs group (P=0.000).
After treatment with100,200,300μg/mL AGEs for24h, the content of IL-1were136.50±13.05,191.17±12.01,287.7±13.75, respectively, and significantly higher than control group (50.67±4.99) and the BSA group (55.33±6.06)(P=0.000). Moreover, the content of IL-1in200μg/mL AGEs group significantly higher than that of100μg/mL AGEs group (P=0.000). The content of IL-1in300μg/mL AGEs group significantly higher than that of100μg/mL AGEs group and200μg/mL AGEs group (P=0.000). The content of IL-1in AGEs+apocynin group was68.17±8.72, which significantly lower than that of200μg/mL AGEs group (P=0.000).
After treatment with100,200,300μg/mL AGEs for24h, the content of IL-6were95.93±12.29,177.93±9.26,234.27±19.74, respectively, and significantly higher than control group (43.80±5.94) and the BSA group (50.40±5.64)(P=0.000). Moreover, the content of IL-6in200μg/mL AGEs group significantly higher than that of100μg/mL AGEs group (P=0.000). The content of IL-6in300μg/mL AGEs group significantly higher than that of100μg/mL AGEs group and200μg/mL AGEs group (P=0.000). The content of IL-6in AGEs+apocynin group was63.50±7.62, which significantly lower than that of200μg/mL AGEs group (P=0.000).
Conclusions:
The phosphorylation of p38MAPK and the expression of NF-κB, TNF-α, IL-1, IL-6increased with the concentration of AGEs increasing for the same aging time. The NADPH oxidase inhibitor apocynin could regulate the expression of p38MAPK, NF-κB, TNF-α, IL-1, IL-6in GLUTag cells induced by AGEs. This part suggested that AGEs could induce the inflammatory injury of L cells through the pathway of p38MAPK/NF-κB.
引文
[1]Yang W, Lu J, Weng J, et al. Prevalence of diabetes among men and women in China[J]. N Engl J Med,2010,362(12):1090-1101.
[2]Xu Y, Wang L, He J, et al. Prevalence and control of diabetes in Chinese adults[J]. JAMA,2013,310(9):948-959.
[3]Intensive blood-glucose control with sulphonylureas or insulin compared with conventional treatment and risk of complications in patients with type 2 diabetes (UKPDS 33). UK Prospective Diabetes Study (UKPDS) Group[J]. Lancet,1998,352(9131):837-853.
[4]Effect of intensive blood-glucose control with metformin on complications in overweight patients with type 2 diabetes (UKPDS 34). UK Prospective Diabetes Study (UKPDS) Group[J]. Lancet,1998,352(9131):854-865.
[5]Baggio L L, Drucker D J. Biology of incretins:GLP-1 and GIP[J]. Gastroenterology,2007,132(6):2131-2157.
[6]Nauck M A. Incretin-based therapies for type 2 diabetes mellitus:properties, functions, and clinical implications[J]. Am J Med,2011,124(1 Suppl):S3-S18.
[7]Liu H, Hu Y, Simpson R W, et al. Glucagon-like peptide-1 attenuates tumour necrosis factor-alpha-mediated induction of plasminogen [corrected] activator inhibitor-1 expression[J]. J Endocrinol,2008,196(1):57-65.
[8]Singh D K, Winocour P, Farrington K. Oxidative stress in early diabetic nephropathy:fueling the fire[J]. Nat Rev Endocrinol,2011,7(3):176-184.
[9]Park C W, Kim H W, Ko S H, et al. Long-term treatment of glucagon-like peptide-1 analog exendin-4 ameliorates diabetic nephropathy through improving metabolic anomalies in db/db mice[J]. J Am Soc Nephrol,2007,18(4):1227-1238.
[10]Perfetti R, Merkel P. Glucagon-like peptide-1:a major regulator of pancreatic beta-cell function[J]. Eur J Endocrinol,2000,143(6):717-725.
[11]Freeman J S. The pathophysiologic role of incretins[J]. J Am Osteopath Assoc,2007,107 Suppl:S6-S9.
[12]Ranganath L R. Incretins:pathophysiological and therapeutic implications of glucose-dependent insulinotropic polypeptide and glucagon-like peptide-1 [J]. J Clin Pathol,2008,61 (4):401-409.
[13]Nauck M, Stockmann F, Ebert R, et al. Reduced incretin effect in type 2 (non-insulin-dependent) diabetes[J].Diabetologia,1986,29(1):46-52.
[14]Nauck M A, Heimesaat M M, Orskov C, et al. Preserved incretin activity of glucagon-like peptide 1 [7-36 amide] but not of synthetic human gastric inhibitory polypeptide in patients with type-2 diabetes mellitus[J]. J Clin Invest,1993,91(1):301-307.
[15]Mannucci E, Ognibene A, Cremasco F, et al. Glucagon-like peptide (GLP)-l and leptin concentrations in obese patients with Type 2 diabetes mellitus[J]. Diabet Med,2000,17(10):713-719.
[16]Vilsboll T, Agerso H, Krarup T, et al. Similar elimination rates of glucagon-like peptide-1 in obese type 2 diabetic patients and healthy subjects[J]. J Clin Endocrinol Metab,2003,88(1):220-224.
[17]Aaboe K, Knop F K, Vilsboll T, et al. KATP channel closure ameliorates the impaired insulinotropic effect of glucose-dependent insulinotropic polypeptide in patients with type 2 diabetes[J]. J Clin Endocrinol Metab,2009,94(2):603-608.
[18]Gautier J F, Choukem S P, Girard J. Physiology of incretins (GIP and GLP-1) and abnormalities in type 2 diabetes[J]. Diabetes Metab,2008,34 Suppl 2:S65-S72.
[19]Muscelli E, Mari A, Casolaro A, et al. Separate impact of obesity and glucose tolerance on the incretin effect in normal subjects and type 2 diabetic patients[J]. Diabetes,2008,57(5):1340-1348.
[20]Daoudi M, Hennuyer N, Borland M G, et al. PPARbeta/delta activation induces enteroendocrine L cell GLP-1 production [J]. Gastroenterology,2011,140(5):1564-1574.
[21]Holst J J, Mcgill M A. Potential new approaches to modifying intestinal GLP-1 secretion in patients with type 2 diabetes mellitus:focus on bile acid sequestrants[J]. Clin Drug Investig,2012,32(1):1-14.
[22]Singh R, Barden A, Mori T, et al. Advanced glycation end-products:a review[J]. Diabetologia,2001,44(2):129-146.
[23]Yan S F, D'Agati V, Schmidt A M, et al. Receptor for Advanced Glycation Endproducts (RAGE):a formidable force in the pathogenesis of the cardiovascular complications of diabetes & aging[J]. Curr Mol Med,2007,7(8):699-710.
[24]Bierhaus A, Hofmann M A, Ziegler R, et al. AGEs and their interaction with AGE-receptors in vascular disease and diabetes mellitus. I. The AGE concept[J]. Cardiovasc Res,1998,37(3):586-600.
[25]Schmidt A M, Hori O, Cao R, et al. RAGE:a novel cellular receptor for advanced glycation end products[J]. Diabetes,1996,45 Suppl 3:S77-S80.
[26]Brownlee M. Biochemistry and molecular cell biology of diabetic complications[J]. Nature,2001,414(6865):813-820.
[27]Singh R, Barden A, Mori T, et al. Advanced glycation end-products:a review[J]. Diabetologia,2001,44(2):129-146.
[28]Chen J, Jin J, Song M, et al. C-reactive protein down-regulates endothelial nitric oxide synthase expression and promotes apoptosis in endothelial progenitor cells through receptor for advanced glycation end-products[J]. Gene,2012,496(2):128-135.
[29]Wendt T M, Tanji N, Guo J, et al. RAGE drives the development of glomerulosclerosis and implicates podocyte activation in the pathogenesis of diabetic nephropathy[J]. Am J Pathol,2003,162(4):1123-1137.
[30]Li Z, Xu J, Xu P, et al. Wnt/beta-catenin signalling pathway mediates high glucose induced cell injury through activation of TRPC6 in podocytes[J]. Cell Prolif,2013,46(1):76-85.
[31]Reiniger N, Lau K, Mccalla D, et al. Deletion of the receptor for advanced glycation end products reduces glomerulosclerosis and preserves renal function in the diabetic OVE26 mouse[J]. Diabetes,2010,59(8):2043-2054.
[32]Bondeva T, Wojciech S, Wolf G. Advanced glycation end products inhibit adhesion ability of differentiated podocytes in a neuropilin-1-dependent manner[J]. Am J Physiol Renal Physiol,2011,301 (4):F852-F870.
[33]Waanders F, van den Berg E, Nagai R, et al. Renoprotective effects of the AGE-inhibitor pyridoxamine in experimental chronic allograft nephropathy in rats[J]. Nephrol Dial Transplant,2008,23(2):518-524.
[34]Cheng C L, Tang Y, Zheng Z, et al. Advanced glycation end-products activate the renin-angiotensin system through the RAGE/PI3-K signaling pathway in podocytes[J]. Clin Invest Med,2012,35(5):E282.
[35]Zhou L L, Cao W, Xie C, et al. The receptor of advanced glycation end products plays a central role in advanced oxidation protein products-induced podocyte apoptosis[J]. Kidney Int,2012,82(7):759-770.
[36]Alikhani M, Maclellan C M, Raptis M, et al. Advanced glycation end products induce apoptosis in fibroblasts through activation of ROS, MAP kinases, and the FOXO1 transcription factor[J]. Am J Physiol Cell Physiol,2007,292(2):C850-C856.
[37]Smith W W, Norton D D, Gorospe M, et al. Phosphorylation of p66Shc and forkhead proteins mediates Abeta toxicity[J]. J Cell Biol,2005,169(2):331-339.
[38]Chuang P Y, Yu Q, Fang W, et al. Advanced glycation endproducts induce podocyte apoptosis by activation of the FOXO4 transcription factor [J]. Kidney Int,2007,72(8):965-976.
[39]Hirose A, Tanikawa T, Mori H, et al. Advanced glycation end products increase endothelial permeability through the RAGE/Rho signaling pathway[J]. FEBS Lett,2010,584(1):61-66.
[40]Yan S F, Ramasamy R, Schmidt A M. The RAGE axis:a fundamental mechanism signaling danger to the vulnerable vasculature[J]. Circ Res,2010,106(5):842-853.
[41]Takuma K, Fang F, Zhang W, et al. RAGE-mediated signaling contributes to intraneuronal transport of amyloid-beta and neuronal dysfunction [J]. Proc Natl Acad Sci U S A,2009,106(47):20021-20026.
[42]Lee B W, Chae H Y, Kwon S J, et al. RAGE ligands induce apoptotic cell death of pancreatic beta-cells via oxidative stress[J]. Int J Mol Med,2010,26(6):813-818.
[43]Zhu Y, Shu T, Lin Y, et al. Inhibition of the receptor for advanced glycation endproducts (RAGE) protects pancreatic beta-cells[J]. Biochem Biophys Res Commun,2011,404(1):159-165.
[44]Costal F, Oliveira E, Raposo A, et al. Dual effect of advanced glycation end products in pancreatic islet apoptosis[J]. Diabetes Metab Res Rev,2013,29(4):296-307.
[1]Elrick H, Stimmler L, Hlad C J, et al. PLASMA INSULIN RESPONSE TO ORAL AND INTRAVENOUS GLUCOSE ADMINISTRATION[J]. J Clin Endocrinol Metab,1964,24:1076-1082.
[2]Vilsboll T, Krarup T, Sonne J, et al. Incretin secretion in relation to meal size and body weight in healthy subjects and people with type 1 and type 2 diabetes mellitus[J]. J Clin Endocrinol Metab,2003,88(6):2706-2713.
[3]李强笃,李斌,张倩,等.糖尿病患者42例心理健康状况调查分析[J].中国煤炭工业医学杂志,2008,11(5)：748-749.
[4]李秀钧,董砚虎,程丽霞.糖尿病研究进展——第16届国际糖尿病联盟大会纪要[J].中华内分泌代谢杂志,1998(2)：72.
[5]李玫,许瑞珍,孙涛,等.糖尿病患者医院社区互动护理模式的建立及运行[J].中华护理杂志,2009,44(8)：703-704.
[6]纪立农.糖尿病患病形势严峻,“狼”真的来了[J].糖尿病天地·文摘(中旬),2012(1)：插3.
[7]Meier J J, Nauck M A. Incretins and the development of type 2 diabetes[J]. Curr Diab Rep,2006,6(3):194-201.
[8]Edie E S. Further Observations on the Treatment of Diabetes Mellitus by Acid Extract of Duodenal Mucous Membrane[J]. Biochem J,1906,1(8-9):446-454.
[9]Elrick H, Stimmler L, Hlad C J, et al. PLASMA INSULIN RESPONSE TO ORAL AND INTRAVENOUS GLUCOSE ADMINISTRATION [J]. J Clin Endocrinol Metab,1964,24:1076-1082.
[10]Freeman J S. The pathophysiologic role of incretins[J]. J Am Osteopath Assoc,2007,107 Suppl:S6-S9.
[11]Ranganath L R. Incretins:pathophysiological and therapeutic implications of glucose-dependent insulinotropic polypeptide and glucagon-like peptide-1[J]. J Clin Pathol.2008,61 (4):401-409.
[12]Knop F K, Vilsboll T, Hojberg P V, et al. Reduced incretin effect in type 2 diabetes:cause or consequence of the diabetic state?[J]. Diabetes,2007,56(8):1951-1959.
[13]Toft-Nielsen M B, Damholt M B, Madsbad S, et al. Determinants of the impaired secretion of glucagon-like peptide-1 in type 2 diabetic patients[J]. J Clin Endocrinol Metab,2001,86(8):3717-3723.
[14]Nauck M A, Heimesaat M M, Orskov C, et al. Preserved incretin activity of glucagon-like peptide 1 [7-36 amide] but not of synthetic human gastric inhibitory polypeptide in patients with type-2 diabetes mellitus[J]. J Clin Invest,1993,91(1):301-307.
[15]Willms B, Werner J, Holst J J, et al. Gastric emptying, glucose responses, and insulin secretion after a liquid test meal:effects of exogenous glucagon-like peptide-1 (GLP-1)-(7-36) amide in type 2 (noninsulin-dependent) diabetic patients[J]. J Clin Endocrinol Metab,1996,81(1):327-332.
[16]Lim G E, Huang G J, Flora N, et al. Insulin regulates glucagon-like peptide-1 secretion from the enteroendocrine L cell[J]. Endocrinology,2009,150(2):580-591.
[17]Daoudi M, Hennuyer N, Borland M G, et al. PPARbeta/delta activation induces enteroendocrine L cell GLP-1 production[J]. Gastroenterology,2011,140(5):1564-1574.
[18]Holst J J, Mcgill M A. Potential new approaches to modifying intestinal GLP-1 secretion in patients with type 2 diabetes mellitus:focus on bile acid sequestrants[J]. Clin Drug Investig,2012,32(1):1-14.
[19]Singh R, Barden A, Mori T, et al. Advanced glycation end-products:a review[J]. Diabetologia,2001,44(2):129-146.
[20]G H A, G R J, H V. Diabetes and advanced glyeoxidation end products[J]. 2006,29:1420-1432.
[21]Schmidt A M, Yan S D, Yan S F, et al. The multiligand receptor RAGE as a progression factor amplifying immune and inflammatory responses[J]. J Clin Invest,2001,108(7):949-955.
[22]Hj H, C F, H R. Receptor for advanced glycation end products (RAGE)-mediated neurite outgrowth and activation of NF-kappaB require the cytoplasmic domain of the receptor but different downstream signaling pathways[J].1999,274:19919-19924.
[23]Vlassara H, Bucala R. Recent progress in advanced glycation and diabetic vascular disease: role of advanced glycation end product receptors[J]. Diabetes,1996,45 Suppl 3:S65-S66.
[24]Y C W, H V, Peten E P. Advanced glycation endproducts up-regulate gene expression found in diabetic glomerular disease[J].1994,91:9436-9440.
[25]Sd. Y, J. F, M. C, et al. RAGE AND AMYLOID-BETA PEPTIDE NEUROTOXICITY IN ALZHEIMERS DISEASE[J].1996,382(6593):685-691.
[26]Y S, Y S, S Y, et al. Upregulation of retinal vascular endothelial growth factor mRNAs in spontaneously diabetic rats without ophthalmoscopic retinopathy.A possible participation of advanced glycation end products in the development of the early phase of diabetic retinopathy[J]. 1998,30:333-339.
[27]Zhu Y, Shu T, Lin Y, et al. Inhibition of the receptor for advanced glycation endproducts (RAGE) protects pancreatic beta-cells[J]. Biochem Biophys Res Commun,2011,404(1):159-165.
[28]Rakonczay Z J, Jarmay K, Kaszaki J, et al. NF-kappaB activation is detrimental in arginine-induced acute pancreatitis[J]. Free Radic Biol Med,2003,34(6):696-709.
[29]Costal F, Oliveira E, Raposo A, et al. Dual effect of advanced glycation end products in pancreatic islet apoptosis[J]. Diabetes Metab Res Rev,2013,29(4):296-307.
[30]Brubaker P L, Schloos J, Drucker D J. Regulation of glucagon-like peptide-1 synthesis and secretion in the GLUTag enteroendocrine cell line[J]. Endocrinology,1998,139(10):4108-4114.
[31]Se S, M G, Ab L. Minireview:Development and differentiation of gut endocrine cells[J].2004,145:2639-2644.
[32]孙慧琳,刘珍珍,李琼,等.胰高血糖素样肽-1对糖基化终末产物诱导的血管内皮细胞损伤的保护作用[J].实用医学杂志,2013,29(3)：496-498.
[33]陈瑜,刘翠萍,茅晓东,等.糖基化终产物诱导小鼠足细胞内质网应激和细胞凋亡的研究[J].医学研究生学报,2013,26(11)：1129-1133.
[34]汪峻岭,刘瑶,郑杰.糖基化终产物对体外培养兔视网膜Muller细胞的影响[J].现代医学,2006,34(4)：229-233.
[1]张玉峰,党中勤,李鲜.慢性胃炎细胞凋亡指数与中医证型相关性研究[J].光明中医,2008,23(5)：557-559.
[2]Zhou L L, Cao W, Xie C, et al. The receptor of advanced glycation end products plays a central role in advanced oxidation protein products-induced podocyte apoptosis[J]. Kidney Int,2012,82(7):759-770.
[3]Costal F, Oliveira E, Raposo A, et al. Dual effect of advanced glycation end products in pancreatic islet apoptosis[J]. Diabetes Metab Res Rev,2013,29(4):296-307.
[4]Neeper M, Schmidt A M, Brett J, et al. Cloning and expression of a cell surface receptor for advanced glycosylation end products of proteins[J]. J Biol Chem,1992,267(21):14998-15004.
[5]Schmidt A M, Yan S D, Yan S F, et al. The multiligand receptor RAGE as a progression factor amplifying immune and inflammatory responses[J]. J Clin Invest,2001,108(7):949-955.
[6]Haslbeck K M, Schleicher E, Bierhaus A, et al. The AGE/RAGE/NF-(kappa)B pathway may contribute to the pathogenesis of polyneuropathy in impaired glucose tolerance (IGT)[J]. Exp Clin Endocrinol Diabetes,2005,113(5):288-291.
[7]Li J, Schmidt A M. Characterization and functional analysis of the promoter of RAGE, the receptor for advanced glycation end products[J]. J Biol Chem,1997,272(26):16498-16506.
[8]Lin L. RAGE on the Toll Road?[J]. Cell Mol Immunol,2006,3(5):351-358.
[9]Py C, Q Y, W F. Advanced glycation endproducts induce podocyte apoptosis by activation of the FOXO4 transcription factor[J].2007,72:965-976.
[10]于青,袁伟杰,刘智辉,等.晚期糖基化终末产物与其受体相互作用对足细胞凋亡的影响[J].中华肾脏病杂志,2008,24(11)：804-809.
[11]游捷,朱明理,彭伟,等AGEs通过RAGE促进Jurkat细胞分泌肿瘤坏死因子-a[J].中国免疫学杂志,2012,28(7)：594-598.
[12]Gd F, S E, Ed M. The role of reactive oxygen species in insulin signaling in the vasculature.[J].2005,7(7/8):1053-1061.
[13]Yamada Y, Ishibashi K, Ishibashi K, et al. The expression of advanced glycation endproduct receptors in rpe cells associated with basal deposits in human maculas[J]. Exp Eye Res,2006,82(5):840-848.
[14]Van Houten B, Woshner V, Santos J H. Role of mitochondrial DNA in toxic responses to oxidative stress[J]. DNA Repair (Amst),2006,5(2):145-152.
[15]Dworakowski R, Alom-Ruiz S P, Shah A M. NADPH oxidase-derived reactive oxygen species in the regulation of endothelial phenotype.[J].2008,60(l):21-28.
[16]Nabeebaccus A, Zhang M, Shah A M. NADPH oxidases and cardiac remodelling[J]. Heart Fail Rev,2011,16(1):5-12.
[17]Zhou Y J, Yang H W, Wang X G, et al. Hepatocyte growth factor prevents advanced glycation end products-induced injury and oxidative stress through a PI3K/Akt-dependent pathway in human endothelial cells[J]. Life Sci,2009,85(19-20):670-677.
[18]Ishibashi Y, Matsui T, Takeuchi M, et al. Glucagon-like peptide-1 (GLP-1) inhibits advanced glycation end product (AGE)-induced up-regulation of VCAM-1 mRNA levels in endothelial cells by suppressing AGE receptor (RAGE) expression[J]. Biochem Biophys Res Commun,2010,391(3):1405-1408.
[19]Wautier M P, Chappey O, Corda S, et al. Activation of NADPH oxidase by AGE links oxidant stress to altered gene expression via RAGE[J]. Am J Physiol Endocrinol Metab,2001,280(5):E685-E694.
[20]Du X,Fu X, Hao Q中国肿瘤临床(英文版),2007,4(5)：343-346.
[21]Xanthis A, Hatzitolios A, Fidani S, et al. Receptor of advanced glycation end products (RAGE) positively regulates CD36 expression and reactive oxygen species production in human monocytes in diabetes[J]. Angiology,2009,60(6):772-779.
[22]Zn O, Ci M, Sj K. Bel-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed all death[J].1993,74:609.
[23]朱国萍,娄阳,徐冲.Bcl-2蛋白家族与细胞凋亡[J].细胞生物学杂志,2001,23(1)：20-23.
[24]曹智刚,袁守军.抑制Bax基因表达与细胞保护的研究进展[J].中国临床药理学与治疗学,2003,8(3)：245-248.
[25]Padanilam B J. Cell death induced by acute renal injury:a perspective on the contributions of apoptosis and necrosis[J]. Am J Physiol Renal Physiol,2003,284(4):F608-F627.
[26]Kumar S, Vaux D L. Apoptosis. A cinderella caspase takes center stage[J]. Science,2002,297(5585):1290-1291.
[27]Wang J, Lenardo M J. Roles of caspases in apoptosis, development, and cytokine maturation revealed by homozygous gene deficiencies[J]. J Cell Sci,2000,113 (Pt 5):753-757.
[28]Lo A C, Woo T T, Wong R L, et al. Apoptosis and other cell death mechanisms after retinal detachment:implications for photoreceptor rescue[J]. Ophthalmologica,2011,226 Suppl 1:10-17.
[29]Le Bras M, Rouy I, Brenner C. The modulation of inter-organelle cross-talk to control apoptosis[J]. Med Chem,2006,2(1):1-12.
[30]Kuranaga E. Beyond apoptosis:caspase regulatory mechanisms and functions in vivo[J]. Genes Cells,2012,17(2):83-97.
[31]Choudhuri T, Pal S, Agwarwal M L, et al. Curcumin induces apoptosis in human breast cancer cells through p53-dependent Bax induction[J]. FEBS Lett,2002,512(1-3):334-340.
[32]Hoffmann S, Podlich D, Hahnel B, et al. Angiotensin II type 1 receptor overexpression in podocytes induces glomerulosclerosis in transgenic rats[J]. J Am Soc Nephrol,2004,15(6):1475-1487.
[33]Ding G, Reddy K, Kapasi A A, et al. Angiotensin II induces apoptosis in rat glomerular epithelial cells[J]. Am J Physiol Renal Physiol,2002,283(1):F173-F180.
[34]Zhou Y J, Wang J H, Zhang J. Hepatocyte growth factor protects human endothelial cells against advanced glycation end products-induced apoptosis [J]. Biochem Biophys Res Commun,2006,344(2):658-666.
[1]Vaccaro O, Eberly L E, Neaton J D, et al. Impact of diabetes and previous myocardial infarction on long-term survival:25-year mortality follow-up of primary screenees of the Multiple Risk Factor Intervention Trial[J]. Arch Intern Med,2004,164(13):1438-1443.
[2]Vlassara H, Bucala R, Striker L. Pathogenic effects of advanced glycosylation:biochemical, biologic, and clinical implications for diabetes and aging[J]. Lab Invest,1994,70(2):138-151.
[3]Scivittaro V, Ganz M B, Weiss M F. AGEs induce oxidative stress and activate protein kinase C-beta(II) in neonatal mesangial cells[J]. Am J Physiol Renal Physiol,2000,278(4):F676-F683.
[4]Yang C W, Vlassara H, Peten E P, et al. Advanced glycation end products up-regulate gene expression found in diabetic glomerular disease[J]. Proc Natl Acad Sci U S A,1994,91 (20):9436-9440.
[5]Haddad E B, Birrell M, Mccluskie K, et al. Role of p38 MAP kinase in LPS-induced airway inflammation in the rat[J]. Br J Pharmacol,2001,132(8):1715-1724.
[6]Haslbeck K M, Schleicher E, Bierhaus A, et al. The AGE/RAGE/NF-(kappa)B pathway may contribute to the pathogenesis of polyneuropathy in impaired glucose tolerance (IGT)[J]. Exp Clin Endocrinol Diabetes,2005,113(5):288-291.
[7]刘倩,石颖,殷惠军p38MAPK信号通路与内皮细胞激活[J].中国分子心脏病学杂 志,2011,11(5)：308-312.
[8]Zhang S, Rahman M, Zhang S, et al. p38 Mitogen-activated protein kinase signaling regulates streptococcal M1 protein-induced neutrophil activation and lung injury[J]. J Leukoc Biol,2012,91(1):137-145.
[9]Wang L, Li Q, Du J, et al. Advanced glycation end products induce moesin phosphorylation in murine retinal endothelium[J]. Acta Diabetol,2012,49(1):47-55.
[10]Li Q, Liu H, Du J, et al. Advanced glycation end products induce moesin phosphorylation in murine brain endothelium[J]. Brain Res,2011,1373:1-10.
[11]Susztak K, Raff A C, Schiffer M, et al. Glucose-induced reactive oxygen species cause apoptosis of podocytes and podocyte depletion at the onset of diabetic nephropathy[J]. Diabetes,2006,55(1):225-233.
[12]Goettsch C, Goettsch W, Muller G, et al. Nox4 overexpression activates reactive oxygen species and p38 MAPK in human endothelial cells[J]. Biochem Biophys Res Commun,2009,380(2):355-360.
[13]Yeh C H, Sturgis L, Haidacher J, et al. Requirement for p38 and p44/p42 mitogen-activated protein kinases in RAGE-mediated nuclear factor-kappaB transcriptional activation and cytokine secretion[J]. Diabetes,2001,50(6):1495-1504.
[14]Sun C, Liang C, Ren Y, et al. Advanced glycation end products depress function of endothelial progenitor cells via p38 and ERK 1/2 mitogen-activated protein kinase pathways[J]. Basic Res Cardiol,2009,104(1):42-49.
[15]Bhat N R, Feinstein D L, Shen Q, et al. p38 MAPK-mediated transcriptional activation of inducible nitric-oxide synthase in glial cells. Roles of nuclear factors, nuclear factor kappa B, cAMP response element-binding protein, CCAAT/enhancer-binding protein-beta, and activating transcription factor-2[J]. J Biol Chem,2002,277(33):29584-29592.
[16]Nick J A, Avdi N J, Young S K, et al. Selective activation and functional significance of p38alpha mitogen-activated protein kinase in lipopolysaccharide-stimulated neutrophils[J]. J Clin Invest,1999,103(6):851-858.
[17]Lane C, Knight D, Burgess S, et al. Epithelial inducible nitric oxide synthase activity is the major determinant of nitric oxide concentration in exhaled breath[J]. Thorax,2004,59(9):757-760.
[18]Lee J C, Laydon J T, Mcdonnell P C, et al. A protein kinase involved in the regulation of inflammatory cytokine biosynthesis [J]. Nature,1994,372(6508):739-746.
[19]Peng J H, Cui T, Sun Z L, et al. Effects of Puerariae Radix Extract on Endotoxin Receptors and TNF-alpha Expression Induced by Gut-Derived Endotoxin in Chronic Alcoholic Liver Injury[J]. Evid Based Complement Alternat Med,2012,2012:234987.
[20]Wang N, Yin D, Zhang S, et al. TNF-alpha rs 1800629 polymorphism is not associated with HPV infection or cervical cancer in the Chinese population[J]. PLoS One,2012,7(9):e45246.
[21]Brotas A M, Cunha J M, Lago E H, et al. Tumor necrosis factor-alpha and the cytokine network in psoriasis[J]. An Bras Dermatol,2012,87(5):673-681,682-683.
[22]Yanagida M, Jung G, Tanaka Y, et al. Serum proteome analysis in patients with rheumatoid arthritis receiving therapy with etanercept, a chimeric tumor necrosis factor-alpha receptor[J]. Int J Rheum Dis,2012,15(5):486-495.
[23]Spirchez M, Samasca G, Iancu M, et al. Relation of interleukin-6, TNF-alpha and interleukin-1 alpha with disease activity and severity in juvenile idiopathic arthritis patients [J]. Clin Lab,2012,58(3-4):253-260.
[24]苏友新,郑良朴,陈智能,等.糖尿病模型大鼠强骨宝方含药血清对成骨细胞培养上清液IL-6、TNF-α及AGEs的影响[J].中华中医药杂志,2007,22(10)：672-676.
[25]游捷,朱明理,彭伟,等AGEs通过RAGE促进Jurkat细胞分泌肿瘤坏死因子-a[J].中国免疫学杂志,2012,28(7)：594-598.
[2]Xu Y, Wang L, He J, et al. Prevalence and control of diabetes in Chinese adults[J]. JAMA,2013,310(9):948-959.
[3]Intensive blood-glucose control with sulphonylureas or insulin compared with conventional treatment and risk of complications in patients with type 2 diabetes (UKPDS 33). UK Prospective Diabetes Study (UKPDS) Group[J]. Lancet,1998,352(9131):837-853.
[4]Effect of intensive blood-glucose control with metformin on complications in overweight patients with type 2 diabetes (UKPDS 34). UK Prospective Diabetes Study (UKPDS) Group[J]. Lancet,1998,352(9131):854-865.
[5]Baggio L L, Drucker D J. Biology of incretins:GLP-1 and GIP[J]. Gastroenterology,2007,132(6):2131-2157.
[6]Nauck M A. Incretin-based therapies for type 2 diabetes mellitus:properties, functions, and clinical implications[J]. Am J Med,2011,124(1 Suppl):S3-S18.
[7]Liu H, Hu Y, Simpson R W, et al. Glucagon-like peptide-1 attenuates tumour necrosis factor-alpha-mediated induction of plasminogen [corrected] activator inhibitor-1 expression[J]. J Endocrinol,2008,196(1):57-65.
[8]Singh D K, Winocour P, Farrington K. Oxidative stress in early diabetic nephropathy:fueling the fire[J]. Nat Rev Endocrinol,2011,7(3):176-184.
[9]Park C W, Kim H W, Ko S H, et al. Long-term treatment of glucagon-like peptide-1 analog exendin-4 ameliorates diabetic nephropathy through improving metabolic anomalies in db/db mice[J]. J Am Soc Nephrol,2007,18(4):1227-1238.
[10]Perfetti R, Merkel P. Glucagon-like peptide-1:a major regulator of pancreatic beta-cell function[J]. Eur J Endocrinol,2000,143(6):717-725.
[11]Freeman J S. The pathophysiologic role of incretins[J]. J Am Osteopath Assoc,2007,107 Suppl:S6-S9.
[12]Ranganath L R. Incretins:pathophysiological and therapeutic implications of glucose-dependent insulinotropic polypeptide and glucagon-like peptide-1 [J]. J Clin Pathol,2008,61 (4):401-409.
[13]Nauck M, Stockmann F, Ebert R, et al. Reduced incretin effect in type 2 (non-insulin-dependent) diabetes[J].Diabetologia,1986,29(1):46-52.
[14]Nauck M A, Heimesaat M M, Orskov C, et al. Preserved incretin activity of glucagon-like peptide 1 [7-36 amide] but not of synthetic human gastric inhibitory polypeptide in patients with type-2 diabetes mellitus[J]. J Clin Invest,1993,91(1):301-307.
[15]Mannucci E, Ognibene A, Cremasco F, et al. Glucagon-like peptide (GLP)-l and leptin concentrations in obese patients with Type 2 diabetes mellitus[J]. Diabet Med,2000,17(10):713-719.
[16]Vilsboll T, Agerso H, Krarup T, et al. Similar elimination rates of glucagon-like peptide-1 in obese type 2 diabetic patients and healthy subjects[J]. J Clin Endocrinol Metab,2003,88(1):220-224.
[17]Aaboe K, Knop F K, Vilsboll T, et al. KATP channel closure ameliorates the impaired insulinotropic effect of glucose-dependent insulinotropic polypeptide in patients with type 2 diabetes[J]. J Clin Endocrinol Metab,2009,94(2):603-608.
[18]Gautier J F, Choukem S P, Girard J. Physiology of incretins (GIP and GLP-1) and abnormalities in type 2 diabetes[J]. Diabetes Metab,2008,34 Suppl 2:S65-S72.
[19]Muscelli E, Mari A, Casolaro A, et al. Separate impact of obesity and glucose tolerance on the incretin effect in normal subjects and type 2 diabetic patients[J]. Diabetes,2008,57(5):1340-1348.
[20]Daoudi M, Hennuyer N, Borland M G, et al. PPARbeta/delta activation induces enteroendocrine L cell GLP-1 production [J]. Gastroenterology,2011,140(5):1564-1574.
[21]Holst J J, Mcgill M A. Potential new approaches to modifying intestinal GLP-1 secretion in patients with type 2 diabetes mellitus:focus on bile acid sequestrants[J]. Clin Drug Investig,2012,32(1):1-14.
[22]Singh R, Barden A, Mori T, et al. Advanced glycation end-products:a review[J]. Diabetologia,2001,44(2):129-146.
[23]Yan S F, D'Agati V, Schmidt A M, et al. Receptor for Advanced Glycation Endproducts (RAGE):a formidable force in the pathogenesis of the cardiovascular complications of diabetes & aging[J]. Curr Mol Med,2007,7(8):699-710.
[24]Bierhaus A, Hofmann M A, Ziegler R, et al. AGEs and their interaction with AGE-receptors in vascular disease and diabetes mellitus. I. The AGE concept[J]. Cardiovasc Res,1998,37(3):586-600.
[25]Schmidt A M, Hori O, Cao R, et al. RAGE:a novel cellular receptor for advanced glycation end products[J]. Diabetes,1996,45 Suppl 3:S77-S80.
[26]Brownlee M. Biochemistry and molecular cell biology of diabetic complications[J]. Nature,2001,414(6865):813-820.
[27]Singh R, Barden A, Mori T, et al. Advanced glycation end-products:a review[J]. Diabetologia,2001,44(2):129-146.
[28]Chen J, Jin J, Song M, et al. C-reactive protein down-regulates endothelial nitric oxide synthase expression and promotes apoptosis in endothelial progenitor cells through receptor for advanced glycation end-products[J]. Gene,2012,496(2):128-135.
[29]Wendt T M, Tanji N, Guo J, et al. RAGE drives the development of glomerulosclerosis and implicates podocyte activation in the pathogenesis of diabetic nephropathy[J]. Am J Pathol,2003,162(4):1123-1137.
[30]Li Z, Xu J, Xu P, et al. Wnt/beta-catenin signalling pathway mediates high glucose induced cell injury through activation of TRPC6 in podocytes[J]. Cell Prolif,2013,46(1):76-85.
[31]Reiniger N, Lau K, Mccalla D, et al. Deletion of the receptor for advanced glycation end products reduces glomerulosclerosis and preserves renal function in the diabetic OVE26 mouse[J]. Diabetes,2010,59(8):2043-2054.
[32]Bondeva T, Wojciech S, Wolf G. Advanced glycation end products inhibit adhesion ability of differentiated podocytes in a neuropilin-1-dependent manner[J]. Am J Physiol Renal Physiol,2011,301 (4):F852-F870.
[33]Waanders F, van den Berg E, Nagai R, et al. Renoprotective effects of the AGE-inhibitor pyridoxamine in experimental chronic allograft nephropathy in rats[J]. Nephrol Dial Transplant,2008,23(2):518-524.
[34]Cheng C L, Tang Y, Zheng Z, et al. Advanced glycation end-products activate the renin-angiotensin system through the RAGE/PI3-K signaling pathway in podocytes[J]. Clin Invest Med,2012,35(5):E282.
[35]Zhou L L, Cao W, Xie C, et al. The receptor of advanced glycation end products plays a central role in advanced oxidation protein products-induced podocyte apoptosis[J]. Kidney Int,2012,82(7):759-770.
[36]Alikhani M, Maclellan C M, Raptis M, et al. Advanced glycation end products induce apoptosis in fibroblasts through activation of ROS, MAP kinases, and the FOXO1 transcription factor[J]. Am J Physiol Cell Physiol,2007,292(2):C850-C856.
[37]Smith W W, Norton D D, Gorospe M, et al. Phosphorylation of p66Shc and forkhead proteins mediates Abeta toxicity[J]. J Cell Biol,2005,169(2):331-339.
[38]Chuang P Y, Yu Q, Fang W, et al. Advanced glycation endproducts induce podocyte apoptosis by activation of the FOXO4 transcription factor [J]. Kidney Int,2007,72(8):965-976.
[39]Hirose A, Tanikawa T, Mori H, et al. Advanced glycation end products increase endothelial permeability through the RAGE/Rho signaling pathway[J]. FEBS Lett,2010,584(1):61-66.
[40]Yan S F, Ramasamy R, Schmidt A M. The RAGE axis:a fundamental mechanism signaling danger to the vulnerable vasculature[J]. Circ Res,2010,106(5):842-853.
[41]Takuma K, Fang F, Zhang W, et al. RAGE-mediated signaling contributes to intraneuronal transport of amyloid-beta and neuronal dysfunction [J]. Proc Natl Acad Sci U S A,2009,106(47):20021-20026.
[42]Lee B W, Chae H Y, Kwon S J, et al. RAGE ligands induce apoptotic cell death of pancreatic beta-cells via oxidative stress[J]. Int J Mol Med,2010,26(6):813-818.
[43]Zhu Y, Shu T, Lin Y, et al. Inhibition of the receptor for advanced glycation endproducts (RAGE) protects pancreatic beta-cells[J]. Biochem Biophys Res Commun,2011,404(1):159-165.
[44]Costal F, Oliveira E, Raposo A, et al. Dual effect of advanced glycation end products in pancreatic islet apoptosis[J]. Diabetes Metab Res Rev,2013,29(4):296-307.
[1]Elrick H, Stimmler L, Hlad C J, et al. PLASMA INSULIN RESPONSE TO ORAL AND INTRAVENOUS GLUCOSE ADMINISTRATION[J]. J Clin Endocrinol Metab,1964,24:1076-1082.
[2]Vilsboll T, Krarup T, Sonne J, et al. Incretin secretion in relation to meal size and body weight in healthy subjects and people with type 1 and type 2 diabetes mellitus[J]. J Clin Endocrinol Metab,2003,88(6):2706-2713.
[3]李强笃,李斌,张倩,等.糖尿病患者42例心理健康状况调查分析[J].中国煤炭工业医学杂志,2008,11(5)：748-749.
[4]李秀钧,董砚虎,程丽霞.糖尿病研究进展——第16届国际糖尿病联盟大会纪要[J].中华内分泌代谢杂志,1998(2)：72.
[5]李玫,许瑞珍,孙涛,等.糖尿病患者医院社区互动护理模式的建立及运行[J].中华护理杂志,2009,44(8)：703-704.
[6]纪立农.糖尿病患病形势严峻,“狼”真的来了[J].糖尿病天地·文摘(中旬),2012(1)：插3.
[7]Meier J J, Nauck M A. Incretins and the development of type 2 diabetes[J]. Curr Diab Rep,2006,6(3):194-201.
[8]Edie E S. Further Observations on the Treatment of Diabetes Mellitus by Acid Extract of Duodenal Mucous Membrane[J]. Biochem J,1906,1(8-9):446-454.
[9]Elrick H, Stimmler L, Hlad C J, et al. PLASMA INSULIN RESPONSE TO ORAL AND INTRAVENOUS GLUCOSE ADMINISTRATION [J]. J Clin Endocrinol Metab,1964,24:1076-1082.
[10]Freeman J S. The pathophysiologic role of incretins[J]. J Am Osteopath Assoc,2007,107 Suppl:S6-S9.
[11]Ranganath L R. Incretins:pathophysiological and therapeutic implications of glucose-dependent insulinotropic polypeptide and glucagon-like peptide-1[J]. J Clin Pathol.2008,61 (4):401-409.
[12]Knop F K, Vilsboll T, Hojberg P V, et al. Reduced incretin effect in type 2 diabetes:cause or consequence of the diabetic state?[J]. Diabetes,2007,56(8):1951-1959.
[13]Toft-Nielsen M B, Damholt M B, Madsbad S, et al. Determinants of the impaired secretion of glucagon-like peptide-1 in type 2 diabetic patients[J]. J Clin Endocrinol Metab,2001,86(8):3717-3723.
[14]Nauck M A, Heimesaat M M, Orskov C, et al. Preserved incretin activity of glucagon-like peptide 1 [7-36 amide] but not of synthetic human gastric inhibitory polypeptide in patients with type-2 diabetes mellitus[J]. J Clin Invest,1993,91(1):301-307.
[15]Willms B, Werner J, Holst J J, et al. Gastric emptying, glucose responses, and insulin secretion after a liquid test meal:effects of exogenous glucagon-like peptide-1 (GLP-1)-(7-36) amide in type 2 (noninsulin-dependent) diabetic patients[J]. J Clin Endocrinol Metab,1996,81(1):327-332.
[16]Lim G E, Huang G J, Flora N, et al. Insulin regulates glucagon-like peptide-1 secretion from the enteroendocrine L cell[J]. Endocrinology,2009,150(2):580-591.
[17]Daoudi M, Hennuyer N, Borland M G, et al. PPARbeta/delta activation induces enteroendocrine L cell GLP-1 production[J]. Gastroenterology,2011,140(5):1564-1574.
[18]Holst J J, Mcgill M A. Potential new approaches to modifying intestinal GLP-1 secretion in patients with type 2 diabetes mellitus:focus on bile acid sequestrants[J]. Clin Drug Investig,2012,32(1):1-14.
[19]Singh R, Barden A, Mori T, et al. Advanced glycation end-products:a review[J]. Diabetologia,2001,44(2):129-146.
[20]G H A, G R J, H V. Diabetes and advanced glyeoxidation end products[J]. 2006,29:1420-1432.
[21]Schmidt A M, Yan S D, Yan S F, et al. The multiligand receptor RAGE as a progression factor amplifying immune and inflammatory responses[J]. J Clin Invest,2001,108(7):949-955.
[22]Hj H, C F, H R. Receptor for advanced glycation end products (RAGE)-mediated neurite outgrowth and activation of NF-kappaB require the cytoplasmic domain of the receptor but different downstream signaling pathways[J].1999,274:19919-19924.
[23]Vlassara H, Bucala R. Recent progress in advanced glycation and diabetic vascular disease: role of advanced glycation end product receptors[J]. Diabetes,1996,45 Suppl 3:S65-S66.
[24]Y C W, H V, Peten E P. Advanced glycation endproducts up-regulate gene expression found in diabetic glomerular disease[J].1994,91:9436-9440.
[25]Sd. Y, J. F, M. C, et al. RAGE AND AMYLOID-BETA PEPTIDE NEUROTOXICITY IN ALZHEIMERS DISEASE[J].1996,382(6593):685-691.
[26]Y S, Y S, S Y, et al. Upregulation of retinal vascular endothelial growth factor mRNAs in spontaneously diabetic rats without ophthalmoscopic retinopathy.A possible participation of advanced glycation end products in the development of the early phase of diabetic retinopathy[J]. 1998,30:333-339.
[27]Zhu Y, Shu T, Lin Y, et al. Inhibition of the receptor for advanced glycation endproducts (RAGE) protects pancreatic beta-cells[J]. Biochem Biophys Res Commun,2011,404(1):159-165.
[28]Rakonczay Z J, Jarmay K, Kaszaki J, et al. NF-kappaB activation is detrimental in arginine-induced acute pancreatitis[J]. Free Radic Biol Med,2003,34(6):696-709.
[29]Costal F, Oliveira E, Raposo A, et al. Dual effect of advanced glycation end products in pancreatic islet apoptosis[J]. Diabetes Metab Res Rev,2013,29(4):296-307.
[30]Brubaker P L, Schloos J, Drucker D J. Regulation of glucagon-like peptide-1 synthesis and secretion in the GLUTag enteroendocrine cell line[J]. Endocrinology,1998,139(10):4108-4114.
[31]Se S, M G, Ab L. Minireview:Development and differentiation of gut endocrine cells[J].2004,145:2639-2644.
[32]孙慧琳,刘珍珍,李琼,等.胰高血糖素样肽-1对糖基化终末产物诱导的血管内皮细胞损伤的保护作用[J].实用医学杂志,2013,29(3)：496-498.
[33]陈瑜,刘翠萍,茅晓东,等.糖基化终产物诱导小鼠足细胞内质网应激和细胞凋亡的研究[J].医学研究生学报,2013,26(11)：1129-1133.
[34]汪峻岭,刘瑶,郑杰.糖基化终产物对体外培养兔视网膜Muller细胞的影响[J].现代医学,2006,34(4)：229-233.
[1]张玉峰,党中勤,李鲜.慢性胃炎细胞凋亡指数与中医证型相关性研究[J].光明中医,2008,23(5)：557-559.
[2]Zhou L L, Cao W, Xie C, et al. The receptor of advanced glycation end products plays a central role in advanced oxidation protein products-induced podocyte apoptosis[J]. Kidney Int,2012,82(7):759-770.
[3]Costal F, Oliveira E, Raposo A, et al. Dual effect of advanced glycation end products in pancreatic islet apoptosis[J]. Diabetes Metab Res Rev,2013,29(4):296-307.
[4]Neeper M, Schmidt A M, Brett J, et al. Cloning and expression of a cell surface receptor for advanced glycosylation end products of proteins[J]. J Biol Chem,1992,267(21):14998-15004.
[5]Schmidt A M, Yan S D, Yan S F, et al. The multiligand receptor RAGE as a progression factor amplifying immune and inflammatory responses[J]. J Clin Invest,2001,108(7):949-955.
[6]Haslbeck K M, Schleicher E, Bierhaus A, et al. The AGE/RAGE/NF-(kappa)B pathway may contribute to the pathogenesis of polyneuropathy in impaired glucose tolerance (IGT)[J]. Exp Clin Endocrinol Diabetes,2005,113(5):288-291.
[7]Li J, Schmidt A M. Characterization and functional analysis of the promoter of RAGE, the receptor for advanced glycation end products[J]. J Biol Chem,1997,272(26):16498-16506.
[8]Lin L. RAGE on the Toll Road?[J]. Cell Mol Immunol,2006,3(5):351-358.
[9]Py C, Q Y, W F. Advanced glycation endproducts induce podocyte apoptosis by activation of the FOXO4 transcription factor[J].2007,72:965-976.
[10]于青,袁伟杰,刘智辉,等.晚期糖基化终末产物与其受体相互作用对足细胞凋亡的影响[J].中华肾脏病杂志,2008,24(11)：804-809.
[11]游捷,朱明理,彭伟,等AGEs通过RAGE促进Jurkat细胞分泌肿瘤坏死因子-a[J].中国免疫学杂志,2012,28(7)：594-598.
[12]Gd F, S E, Ed M. The role of reactive oxygen species in insulin signaling in the vasculature.[J].2005,7(7/8):1053-1061.
[13]Yamada Y, Ishibashi K, Ishibashi K, et al. The expression of advanced glycation endproduct receptors in rpe cells associated with basal deposits in human maculas[J]. Exp Eye Res,2006,82(5):840-848.
[14]Van Houten B, Woshner V, Santos J H. Role of mitochondrial DNA in toxic responses to oxidative stress[J]. DNA Repair (Amst),2006,5(2):145-152.
[15]Dworakowski R, Alom-Ruiz S P, Shah A M. NADPH oxidase-derived reactive oxygen species in the regulation of endothelial phenotype.[J].2008,60(l):21-28.
[16]Nabeebaccus A, Zhang M, Shah A M. NADPH oxidases and cardiac remodelling[J]. Heart Fail Rev,2011,16(1):5-12.
[17]Zhou Y J, Yang H W, Wang X G, et al. Hepatocyte growth factor prevents advanced glycation end products-induced injury and oxidative stress through a PI3K/Akt-dependent pathway in human endothelial cells[J]. Life Sci,2009,85(19-20):670-677.
[18]Ishibashi Y, Matsui T, Takeuchi M, et al. Glucagon-like peptide-1 (GLP-1) inhibits advanced glycation end product (AGE)-induced up-regulation of VCAM-1 mRNA levels in endothelial cells by suppressing AGE receptor (RAGE) expression[J]. Biochem Biophys Res Commun,2010,391(3):1405-1408.
[19]Wautier M P, Chappey O, Corda S, et al. Activation of NADPH oxidase by AGE links oxidant stress to altered gene expression via RAGE[J]. Am J Physiol Endocrinol Metab,2001,280(5):E685-E694.
[20]Du X,Fu X, Hao Q中国肿瘤临床(英文版),2007,4(5)：343-346.
[21]Xanthis A, Hatzitolios A, Fidani S, et al. Receptor of advanced glycation end products (RAGE) positively regulates CD36 expression and reactive oxygen species production in human monocytes in diabetes[J]. Angiology,2009,60(6):772-779.
[22]Zn O, Ci M, Sj K. Bel-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed all death[J].1993,74:609.
[23]朱国萍,娄阳,徐冲.Bcl-2蛋白家族与细胞凋亡[J].细胞生物学杂志,2001,23(1)：20-23.
[24]曹智刚,袁守军.抑制Bax基因表达与细胞保护的研究进展[J].中国临床药理学与治疗学,2003,8(3)：245-248.
[25]Padanilam B J. Cell death induced by acute renal injury:a perspective on the contributions of apoptosis and necrosis[J]. Am J Physiol Renal Physiol,2003,284(4):F608-F627.
[26]Kumar S, Vaux D L. Apoptosis. A cinderella caspase takes center stage[J]. Science,2002,297(5585):1290-1291.
[27]Wang J, Lenardo M J. Roles of caspases in apoptosis, development, and cytokine maturation revealed by homozygous gene deficiencies[J]. J Cell Sci,2000,113 (Pt 5):753-757.
[28]Lo A C, Woo T T, Wong R L, et al. Apoptosis and other cell death mechanisms after retinal detachment:implications for photoreceptor rescue[J]. Ophthalmologica,2011,226 Suppl 1:10-17.
[29]Le Bras M, Rouy I, Brenner C. The modulation of inter-organelle cross-talk to control apoptosis[J]. Med Chem,2006,2(1):1-12.
[30]Kuranaga E. Beyond apoptosis:caspase regulatory mechanisms and functions in vivo[J]. Genes Cells,2012,17(2):83-97.
[31]Choudhuri T, Pal S, Agwarwal M L, et al. Curcumin induces apoptosis in human breast cancer cells through p53-dependent Bax induction[J]. FEBS Lett,2002,512(1-3):334-340.
[32]Hoffmann S, Podlich D, Hahnel B, et al. Angiotensin II type 1 receptor overexpression in podocytes induces glomerulosclerosis in transgenic rats[J]. J Am Soc Nephrol,2004,15(6):1475-1487.
[33]Ding G, Reddy K, Kapasi A A, et al. Angiotensin II induces apoptosis in rat glomerular epithelial cells[J]. Am J Physiol Renal Physiol,2002,283(1):F173-F180.
[34]Zhou Y J, Wang J H, Zhang J. Hepatocyte growth factor protects human endothelial cells against advanced glycation end products-induced apoptosis [J]. Biochem Biophys Res Commun,2006,344(2):658-666.
[1]Vaccaro O, Eberly L E, Neaton J D, et al. Impact of diabetes and previous myocardial infarction on long-term survival:25-year mortality follow-up of primary screenees of the Multiple Risk Factor Intervention Trial[J]. Arch Intern Med,2004,164(13):1438-1443.
[2]Vlassara H, Bucala R, Striker L. Pathogenic effects of advanced glycosylation:biochemical, biologic, and clinical implications for diabetes and aging[J]. Lab Invest,1994,70(2):138-151.
[3]Scivittaro V, Ganz M B, Weiss M F. AGEs induce oxidative stress and activate protein kinase C-beta(II) in neonatal mesangial cells[J]. Am J Physiol Renal Physiol,2000,278(4):F676-F683.
[4]Yang C W, Vlassara H, Peten E P, et al. Advanced glycation end products up-regulate gene expression found in diabetic glomerular disease[J]. Proc Natl Acad Sci U S A,1994,91 (20):9436-9440.
[5]Haddad E B, Birrell M, Mccluskie K, et al. Role of p38 MAP kinase in LPS-induced airway inflammation in the rat[J]. Br J Pharmacol,2001,132(8):1715-1724.
[6]Haslbeck K M, Schleicher E, Bierhaus A, et al. The AGE/RAGE/NF-(kappa)B pathway may contribute to the pathogenesis of polyneuropathy in impaired glucose tolerance (IGT)[J]. Exp Clin Endocrinol Diabetes,2005,113(5):288-291.
[7]刘倩,石颖,殷惠军p38MAPK信号通路与内皮细胞激活[J].中国分子心脏病学杂 志,2011,11(5)：308-312.
[8]Zhang S, Rahman M, Zhang S, et al. p38 Mitogen-activated protein kinase signaling regulates streptococcal M1 protein-induced neutrophil activation and lung injury[J]. J Leukoc Biol,2012,91(1):137-145.
[9]Wang L, Li Q, Du J, et al. Advanced glycation end products induce moesin phosphorylation in murine retinal endothelium[J]. Acta Diabetol,2012,49(1):47-55.
[10]Li Q, Liu H, Du J, et al. Advanced glycation end products induce moesin phosphorylation in murine brain endothelium[J]. Brain Res,2011,1373:1-10.
[11]Susztak K, Raff A C, Schiffer M, et al. Glucose-induced reactive oxygen species cause apoptosis of podocytes and podocyte depletion at the onset of diabetic nephropathy[J]. Diabetes,2006,55(1):225-233.
[12]Goettsch C, Goettsch W, Muller G, et al. Nox4 overexpression activates reactive oxygen species and p38 MAPK in human endothelial cells[J]. Biochem Biophys Res Commun,2009,380(2):355-360.
[13]Yeh C H, Sturgis L, Haidacher J, et al. Requirement for p38 and p44/p42 mitogen-activated protein kinases in RAGE-mediated nuclear factor-kappaB transcriptional activation and cytokine secretion[J]. Diabetes,2001,50(6):1495-1504.
[14]Sun C, Liang C, Ren Y, et al. Advanced glycation end products depress function of endothelial progenitor cells via p38 and ERK 1/2 mitogen-activated protein kinase pathways[J]. Basic Res Cardiol,2009,104(1):42-49.
[15]Bhat N R, Feinstein D L, Shen Q, et al. p38 MAPK-mediated transcriptional activation of inducible nitric-oxide synthase in glial cells. Roles of nuclear factors, nuclear factor kappa B, cAMP response element-binding protein, CCAAT/enhancer-binding protein-beta, and activating transcription factor-2[J]. J Biol Chem,2002,277(33):29584-29592.
[16]Nick J A, Avdi N J, Young S K, et al. Selective activation and functional significance of p38alpha mitogen-activated protein kinase in lipopolysaccharide-stimulated neutrophils[J]. J Clin Invest,1999,103(6):851-858.
[17]Lane C, Knight D, Burgess S, et al. Epithelial inducible nitric oxide synthase activity is the major determinant of nitric oxide concentration in exhaled breath[J]. Thorax,2004,59(9):757-760.
[18]Lee J C, Laydon J T, Mcdonnell P C, et al. A protein kinase involved in the regulation of inflammatory cytokine biosynthesis [J]. Nature,1994,372(6508):739-746.
[19]Peng J H, Cui T, Sun Z L, et al. Effects of Puerariae Radix Extract on Endotoxin Receptors and TNF-alpha Expression Induced by Gut-Derived Endotoxin in Chronic Alcoholic Liver Injury[J]. Evid Based Complement Alternat Med,2012,2012:234987.
[20]Wang N, Yin D, Zhang S, et al. TNF-alpha rs 1800629 polymorphism is not associated with HPV infection or cervical cancer in the Chinese population[J]. PLoS One,2012,7(9):e45246.
[21]Brotas A M, Cunha J M, Lago E H, et al. Tumor necrosis factor-alpha and the cytokine network in psoriasis[J]. An Bras Dermatol,2012,87(5):673-681,682-683.
[22]Yanagida M, Jung G, Tanaka Y, et al. Serum proteome analysis in patients with rheumatoid arthritis receiving therapy with etanercept, a chimeric tumor necrosis factor-alpha receptor[J]. Int J Rheum Dis,2012,15(5):486-495.
[23]Spirchez M, Samasca G, Iancu M, et al. Relation of interleukin-6, TNF-alpha and interleukin-1 alpha with disease activity and severity in juvenile idiopathic arthritis patients [J]. Clin Lab,2012,58(3-4):253-260.
[24]苏友新,郑良朴,陈智能,等.糖尿病模型大鼠强骨宝方含药血清对成骨细胞培养上清液IL-6、TNF-α及AGEs的影响[J].中华中医药杂志,2007,22(10)：672-676.
[25]游捷,朱明理,彭伟,等AGEs通过RAGE促进Jurkat细胞分泌肿瘤坏死因子-a[J].中国免疫学杂志,2012,28(7)：594-598.
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