胰高血糖素样肽-1对晚期蛋白氧化产物诱导的胰岛微血管内皮细胞损伤的保护作用及机制
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摘要
研究背景
糖尿病(diabetes mellitus, DM)的患病率在全球逐年增加,已成为继心血管疾病和肿瘤之后威胁人类健康的第三大慢性非传染性疾病。随着我国人们生活水平的提高及生活方式的改变,2型糖尿病(T2DM)的发病率也呈现出高速发展的趋势。根据2013年最新流行病学调查研究结果,我国18岁及以上成人中,按照国际最新临床诊断标准进行诊断的糖尿病估测患病率为11.6%,约1.139亿人,而更令人震惊的是中国成年人群中糖尿病前期(IGT)患病率已高达50.1%,且糖尿病发病呈现年轻化趋势。糖尿病所引起的各种急慢性并发症,特别是血管并发症已成为其致残致死的主要原因,严重影响人类的生活质量和寿命。
胰岛素抵抗(insulin resistance, IR)和胰岛β细胞功能减退是2型糖尿病发病的两大主要原因,现有的糖尿病治疗手段通过促进胰岛素分泌、改善胰岛素抵抗、抑制葡萄糖的吸收及抑制肝糖输出为主,这些手段虽能起到改善血糖的作用,但随着病程的进展,仍然无法阻止胰岛β细胞功能的进行性衰竭。近年来更多的研究支持胰岛微循环障碍与β细胞的损伤及功能衰竭密切相关。胰岛是一个高度血管化的微器官,胰岛微血管既是胰岛细胞与机体的关键连接,又是其赖以生存的微环境。胰岛具有独特的微血管系统,含有密集的毛细血管网络,毛细血管网的密度大约是周围外分泌组织的5倍,丰富的血供不仅能够满足胰岛自身对氧气及营养物质的需要,更重要的是使胰岛能够迅速感知体内代谢环境的变化,通过血液循环将分泌的各种激素快速分布到相应的靶组织,从而调节机体代谢平衡。因此,胰岛微循环系统是胰岛内分泌功能重要的调节器。胰岛微循环结构和功能的完整性有赖于另一群目前开始被关注的细胞即胰岛微血管内皮细胞(islet microvascular endothelial cells, IMECs)。IMECs是构成胰岛微循环组织屏障的最主要成分,与胰岛内分泌功能关系密切,直接参与糖尿病胰岛功能衰竭的发生发展过程。随着胰岛微血管在2型糖尿病发病中的作用被逐渐揭示,Tal MG2009年提出：β细胞功能衰竭很可能是胰岛微血管病变的表现,而各种原因导致的胰岛微血管内皮细胞的功能紊乱及损伤是引起胰岛微血管病变的重要因素,继而引起胰岛微循环的缺血缺氧,最终导致胰岛β细胞功能的进行性衰竭。因此,深入研究胰岛微血管内皮细胞功能紊乱及损伤的病理生理机制,探讨有效改善的方法和手段,对于保护胰岛细胞功能寻找防治2型糖尿病发病的新靶点具有重大意义。
血管内皮细胞功能损伤及紊乱是糖尿病循环系统并发症发生发展的前提及基础。糖毒性、脂毒性、血液流变学及血流动力学改变、组织抗氧化能力减弱等因素与血管内皮细胞功能紊乱息息相关,主要表现为舒张血管因子一氧化氮合成减少,缩血管因子内皮素1产生增加,直接刺激动脉内膜下平滑肌细胞增生及纤维连接蛋白和胶原IV的表达。高血糖时血管内皮细胞合成和释放细胞间黏附因子1(ICAM-1)及血管细胞黏附因子1(VCAM-1)增多,增加白细胞和血小板的粘附能力并导致炎症细胞的活化及内膜炎症损伤,这种内皮细胞功能改变使中层平滑肌细胞向内膜下迁移,细胞内脂质沉积,从而加速血管动脉粥样硬化的形成。而胰岛微血管内皮细胞作为全身血管内皮细胞的一种也必然遭受到糖尿病状态下各种致病因素的损伤导致内皮细胞功能障碍,并进而影响到胰岛微循环的功能状态而最终导致胰岛细胞功能损伤。
高级氧化蛋白产物(advanced oxidative protein products, AOPPs)是次氯酸对蛋白质氧化修饰生成的含双酪氨酸的蛋白交联物,是一种新发现的蛋白类大分子氧化应激标志物。最初在透析患者中发现AOPPs的积聚,随后在糖尿病和肥胖患者中也发现了AOPPs的积聚,AOPPs是一种氧化应激的蛋白标志物,它的结构和生物学作用类似于晚期糖基化终末产物(AGEs),也可通过受体RAGE活化血管内皮细胞NADPH氧化酶,激活氧化还原反应敏感的细胞内信号传导途径如ERK1/2、p38MAPK途径以及NF-kB的核转位,从而诱发血管内皮细胞的炎症和氧化应激损伤。慢性AOPPs负荷可以导致动脉壁氧化低密度脂蛋白的沉积增加,加剧动脉内膜及内皮细胞的氧化应激及炎症损伤导致高脂血症家兔动脉粥样硬化斑块的形成明显加速。在CKD患者,循环AOPPs水平和颈动脉粥样硬化病变程度密切相关,提示AOPPs潴留可能参与了CKD的加速性动脉粥样硬化。Gradinaru D等探讨了糖尿病前期及糖尿病阶段患者中AOPPs水平与内皮源性NO合成酶、氧化应激、代谢谱及其他导致动脉硬化的因素之间的关系,发现AOPPs与正常对照人群相比显著身高,并显著抑制内皮源性一氧化氮合成酶的活性,导致内皮源性NO合成减少,在老年糖尿病患者中AOPPs水平的高低与糖尿病动脉粥样硬化紊乱的程度密切相关。Bansal S等对印度265例2型糖尿病患者进行了RAGE的基因多态性分析及其与氧化应激产物、对氧磷酶及糖尿病大血管并发症的相关性分析,发现RAGE基因多态性中-429T/C基因型与血清氧化应激产物的水平及糖尿病大血管并发症的发生具有正相关性。由此可见,AOPPs不仅是氧化应激的标志物,其本身也作为一种强效促炎性因子加速了机体动脉粥样硬化进程,损伤全身的大血管及微血管系统。然而,有关AOPPs在胰岛微循环中的作用尤其是对胰岛微血管内皮细胞功能的影响仍少见报道。
GLP-1是主要由肠道内分泌L细胞分泌的多肽,作为一种安全、有效的促胰岛素分泌剂越来越受到关注,是治疗2型糖尿病的一种新途径。越来越多的研究表明,GLP-1除了具有促进胰岛p细胞增殖、促进胰岛素分泌、抑制食欲、减缓胃肠道平滑肌的蠕动、抑制胰高血糖素释放等作用之外,还有胰外的心血管保护作用。GLP-1受体(GLP-1R)在血管平滑肌、心肌、心内膜、冠脉内皮均有表达,GLP-1R激动剂对心肌的缺血一再灌注损伤具有保护作用,并能改善心脏功能。exendin-4能显著缩小体外离体心脏心肌梗塞面积,exendin-4和GLP-1均能使左室收缩功能增强。Timmers等同样发现exendin-4持续治疗可显著减少冠状动脉结扎后实验猪的心肌梗塞面积,并促进心肌收缩和舒张功能的恢复。GLP-1可抑制单核细胞粘附于人主动脉血管内皮细胞,减轻血管性炎症损伤,阻止动脉粥样硬化的进展。在胰岛素抵抗和高血压的大鼠体内补充GLP-1,可以改善血管内皮功能、改善血压以及心功能。在临床病例观察研究中GLP-1可使2型糖尿病合并稳定冠心病患者的血管内皮功能得到改善。近年有研究者证实,在体外用糖基化终末产物诱导内皮细胞损伤,发现GLP-1可通过其受体显著下调RAGE受体的表达和细胞间粘附分子-1的水平,从而拮抗AGEs-RAGE轴导致的内皮细胞氧化应激损伤,减少血管损伤后内膜的增厚和平滑肌的增殖,具有潜在的抗炎和抗动脉粥样硬化作用。Exendin-4可经由PI3K/Akt-eNOS信号传导通路而促进内皮细胞增殖。在内皮细胞功能障碍和动脉粥样硬化的小鼠模型研究中,GLP-1受体激动剂利拉鲁肽可改善血管内皮细胞功能障碍,并减轻小鼠的动脉粥样硬化程度。2012年国内研究者发现GLP-1可通过激活PI3K/Akt信号转导通路减轻AGEs诱导的人脐静脉内皮细胞的凋亡,这种抗凋亡作用是通过对AGEs诱导的内皮细胞Bc1-2、Bax、cyto-c表达来实现的。2013年Erdogdu.O等进行了GLP-1对脂毒性导致的人冠状动脉内皮细胞损伤的研究,结果发现GLP-1的干预可通过PKA/PI3K/Akt/eNOS、p38MAPK及JNK激酶依赖的途径改善动脉内皮细胞体外的增殖、迁移及血管形成能力,并减少其凋亡。
由此可见,GLP-1除了其优秀的降糖作用之外,其对保护内皮细胞、改善内皮细胞功能同样具有重要优势,而PKA/PI3K/Akt是其发挥多器官保护作用的主要上游通路。那么,在胰岛微血管内皮细胞,GLP-1对糖尿病状态下AOPPs蓄积导致的胰岛微血管内皮细胞的损伤是否也具有同样的保护作用呢?是否可通过改善胰岛微血管内皮细胞的功能进而改善胰岛微循环并最终延缓胰岛β细胞的进行性衰退呢?这些都是值得我们深入研究的重要课题。
因此,探求GLP-1独立于降糖作用之外的胰岛微血管保护作用及机制,挖掘新的保护胰岛细胞功能的治疗靶标,对广大的T2DM患者来说具有十分重要的意义。本研究建立AOPP修饰的大鼠血清白蛋白(AOPP-modified RSAAOPP-RSA)诱导大鼠微血管内皮细胞(islet microvascular endothelial cells, IMECs)损伤研究模型,探讨GLP-1干预对AOPPs诱导的胰岛微血管内皮细胞凋亡、增殖、迁移以及再血管化能力损伤的保护作用及其相关机制,以期为GLP-1治疗延缓胰岛细胞功能的进行性衰竭提供新视点。
目的
本课题拟在建立AOPPs诱导大鼠IMECs损伤为研究模型的基础上,通过观测GLP-1干预对AOPPs诱导内皮细胞凋亡、增殖、迁移及再血管化能力的作用,并检测其对凋亡相关蛋白及PI3K/Akt/eNOS通路的影响,初步探讨GLP-1对AOPPs诱导IMECs功能紊乱的保护作用及分子机制。
内容
课题分以下三大部分：
第一部分AOPPs对大鼠胰岛微血管内皮细胞凋亡的影响及机制
目的
探讨AOPPs对大鼠IMECs细胞凋亡的影响及其机制。
方法
1.体外制备、鉴定晚期蛋白氧化产物：
将20mg/ml大鼠血清白蛋白(RSA)与等体积的40mmol/1次氯酸混合,放置30min,制备出RSA与次氯酸摩尔比为1：140的AOPP。经由无内毒素PBS透析24h,除去过多游离的次氯酸。0.22μm的微孔滤膜过滤除菌。
2.大鼠胰岛细胞的分离、纯化：
利用胶原酶P胰管灌注结合Ficol-400密度梯度消化、分离及纯化大鼠胰岛,体外行DTZ染色、台酚蓝染色,将细胞团浓度调整为500IEQ/ml,以吸管移入细胞培养瓶内,置于34℃、5%CO2二氧化碳恒温培养箱中培养；
3.磁珠纯化胰岛微血管内皮细胞(IMECs)及培养鉴定：
纯化后的胰岛细胞经体外培养3-5d时可见有梭形细胞从胰岛内长出,7-9d细胞逐渐融合成片。将UEA-1包被的M-450免疫磁珠纯化后的胰岛微血管内皮细胞接种在2%明胶包被的细胞培养瓶中培养。免疫荧光检测培养皿中内皮细胞主要标志物第Ⅷ因子相关抗原(vWF)和吞噬Dil标记的乙酰化低密度脂蛋白(Dil-Ac-LDL)能力。
4.实验分组：
不同浓度AOPPs对细胞凋亡的影响：分5组,依次为正常对照组(不加任何干预剂),阴性对照组(200μg/mLRSA),100μg/mL、200μg/mL、300μg/mLAOPPs组,分别作用细胞48h。AOPPs作用不同时间对细胞凋亡的影响：分5组,依次为200μg/mL AOPPs分别作用内皮细胞0、12、24、48、72h。
5. Hoechst33258及Annexin V-FITC/PI定量分析细胞凋亡率：
细胞经各组不同处理后,4%多聚甲醛于4℃处理细胞5min, Hoechst33258染色5min,封片后荧光显微镜观察细胞形态。将各组处理后细胞经胰酶消化,离心后弃上清,PBS洗涤后加入Annexin V和碘化丙啶(P1)避光放置15min,流式细胞仪检测,Cell Quest软件分析细胞早期凋亡率。
6.DHE荧光探针检测细胞内活性氧(ROS)：
直接采用AOPPs200ug/ml干预48小时为干预条件探讨AOPPs导致IMECs细胞凋亡的机制。实验分组：空白对照组,阴性对照组(RSA200ug/ml), AOPPs200ug/ml组,AOPPs200ug/ml+apocynin (NADPH氧化酶阻断剂)组。收集各组细胞后无血清DMEM培养基洗3次,每管加入101μM的DHE工作液2.5m1重悬细胞,37℃5%CO2细胞培养箱内避光孵育40min,洗涤细胞2次。激光共聚焦显微镜观察细胞内DHE荧光分布,各组细胞随机选取3个视野对其扫描并分析相对荧光强度。
7. Western blotting检测细胞Bcl-2、Bax蛋白的表达
收集各组细胞,提取各组细胞总蛋白,采用BCA法测定蛋白浓度。每组蛋白质样本50μg,以12%SDS-PAGE进行电泳,转膜,5%脱脂奶粉封闭2h,洗膜,加入RSA-TBS稀释的抗Bcl-2单克隆抗体(1：1000),抗Bax(1:2000)单克隆抗体,4℃孵育过夜,加入辣根过氧化物酶标记的二抗(1：2000)杂交1h,ECL超敏发光液进行显色,结果用ImageJ分析软件对目的条带进行灰度值分析。
8.细胞凋亡蛋白酶caspase-9、-3活性的检测
实验分组及干预同上。胰酶消化各组细胞,各组细胞孵育48h,消化、离心后、用细胞裂解液重悬,再加入对应的反应底物。37℃水浴2h后,加样至96孔培养板,最后用酶联免疫检测仪测OD值,检测波长为405nm。
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。相关分析采用Spearman相关分析法。P<0.050具有统计学意义。
结果
1.不同浓度AOPPs干预48h对IMECs细胞凋亡的影响
100、200、300μg/mL AOPPs作用IMECs48h后,凋亡率分别为14.57±4.7,24.20±±4.30,35.53±±6.47,均显著高于空白对照组(5.87±1.51)和阴性对照组RSA(6.90±1.42)(均P<0.05)。非参数Spearman相关分析表明：AOPPs浓度(100、200、300μg/mL)与大鼠IMECs的凋亡率间存在显著正相关关系(r=0.949,P=0.000)。
2.比较AOPPs不同作用时间对IMECs细胞凋亡的影响
200μg/mL AOPPs作用时间(12h、24h、48h、72h)后的细胞凋亡率分别为11.61±1.94,16.69±3.17,23.48±4.69,32.80±7.13,均显著高于control组0h的凋亡率(6.10±2.07)(F=17.854,P=0.000)。非参数Spearman相关分析表明：AOPPs作用时间与细胞凋亡率值与存在显著正相关关系(r=0.950,P=0.000)。
3. AOPPs (200μg/mL)干预48h对IMECs细胞内活性氧的影响
IMECs阴性对照组(RSA对照)荧光值为27.49±±4.75,给予AOPPs200ug/ml作用48h后荧光值为58.43±2.71,显著高于对照组(P=0.000),提示AOPPs刺激内皮细胞ROS生成增加。在给予NADPH氧化酶抑制剂apocynin后,细胞内ROS荧光度值显著降低(30.45±4.35),与AOPPs200ug/ml组相比具有统计学差异(P=0.000)
4. AOPPs(200μg/mL)干预48h对IMECs细胞凋亡蛋白Bc1-2、Bax表达的影响
各组细胞经干预48h后,细胞Bcl-2蛋白的表达具有统计学差异(F=4.675,P=0.036)。其中AOPPs200ug/ml组可显著降低抗凋亡蛋白Bcl-2表达量(0.60±0.09),明显低于RSA对照组(P=0.021)。给予NADPH氧化酶抑制剂apocynin后Bcl-2表达量则显著升高(0.87±0.14),与单独AOPPs干预组相比具有统计学差异(P=0.033)。AOPPs200ug/ml干预组显著升高了Bax蛋白表达量(1.35±0.19),与RSA对照相比具有统计学差异(P=0.008)。给予apocynin干预后Bax表达量则显著降低(0.97±0.15),与单独AOPPs干预组相比具有统计学差异(P=0.021)。
5. AOPPs(200μg/mL)干预48h对细胞凋亡蛋白酶caspase-9、-3活性的影响
各组IMECs在干预48h后,细胞中caspase-9、-3活性各组细胞之间具有统计学差异(F分别为144.224和311.617,均P=0.000)。AOPPs作用IMECs细胞48h后,细胞中caspase-9、-3活性(分别为1.244±0.08,1.01±0.04)显著高于其空白对照及RSA阴性对照组(均P<0.05),apocynin提前预处理组细胞中caspase-9、-3活性则显著降低(分别为0.45±0.06,0.40±±0.04)与单独AOPPs干预组相比差异具有统计学意义(均P<0.05)。
结论
AOPPs可呈剂量、时间依赖性诱导胰岛微血管内皮细胞凋亡。AOPPs作用于IMECs细胞48h后,可显著增加细胞内ROS水平、促凋亡蛋白Bax表达,降低抗凋亡蛋白Bcl-2表达,同时增强凋亡蛋白酶caspase-9、-3的活性。而NADPH氧化酶阻断剂Apocynin可阻断AOPPs这一系列效应。因此推测AOPPs可能主要通过NADPH氧化酶途径增加细胞内活性氧水平加剧细胞内氧化应激损伤,进而通过对凋亡相关蛋白Bcl-2/Bax表达、caspase-9、-3活性的调控诱导IMECs细胞的凋亡。
第二部分AOPPs对大鼠胰岛微血管内皮细胞增殖、迁移及再血管化功能的影响及机制
目的
研究AOPPs对IMECs细胞增殖、迁移及再血管化功能的的影响,进而探明AOPPs在胰岛微血管内皮细胞损伤及功能紊乱中的作用及机制。
方法
1.实验分组：
不同浓度AOPPs对细胞增殖、迁移及血管化功能的影响：分6组,依次为空白对照组、阴性对照组(200μg/mLRSA)、 AOPPs100μg/mL、300μg/mL组以及AOPPs200μg/ml+NADPH氧化酶抑制剂组(Apocynin)。观察细胞迁移能力时为排除细胞凋亡影响我们选取干预时间为24h,而观察细胞增殖能力及血管化能力时则选取干预时间为48h。
AOPPs200μg/mL不同作用时间对细胞增殖活性的影响：分5组,依次为200μg/mL AOPPs分别作用内皮细胞0、12、24、48、72h。
2.CCK-8检测细胞增殖活性
96孔板铺板细胞,分组如上所示,在各组干预不同时间,每孔加入cck-8(cell counting kit-8细胞计数试剂)溶液10ul,37℃,5%C02培养箱避光孵育1小时,酶标仪450nm波长处读取吸光度值,反映细胞的增殖活性。
3. Transwell小室检测AOPPs对IMECs细胞体外迁移能力的影响
各组细胞撤血清饥饿12-24h,调整细胞密度至1×105,上室分别加入胰岛微血管内皮细胞悬液200μ1、RSA、不同浓度的AOPPs(100μg/mL、200μg/mL300μg/mL)以及apocynin,下室加人600μl含10%胎牛血清的DMEM培养液,置于37℃培养24小时,固定染色并计数,用棉签轻轻擦掉上层未迁移细胞。
4. Matrigel基质胶中检测AOPPs对IMECs再血管化能力的影响
将缺乏生长因子的高浓度Matrigel基质胶在冰上过夜融化,均匀铺于无菌的96孔板上(70μ L/孔),放于温箱中30min成胶。将5000大鼠胰岛微血管内皮细胞用无血清培养液种植于每孔中,分别加入RSA、不同浓度的AOPPs(100μg/mL,200μg/mL,300μg/mL)以及apocynin。置于37℃、C02培养箱中作用48小时,低倍镜下倒置显微镜照相,每孔随机取3个视野。
5. AOPPs对胰岛微血管内皮细胞NADPH氧化酶活性的影响
各组细胞干预48h后制备细胞匀浆,测定细胞匀浆蛋白含量,将各组100μg细胞蛋白加至96孔测量板中,各孔加入暗适应的光泽精5μ M及底物NADPH100μM。用VICTOR1420多标记分析系统动态检测各组NADPH依赖的02-产生的荧光量,间接反映02-的产量及NADPH氧化酶活性。所测得的数据用“CPS”表示,即每秒通过光子数：counts per second.
6. Western blotting法检测AOPPs对IMECs细胞p-Akt、p-eNOS的表达
取同步生长的胰岛微血管内皮细胞,依次给予阴性对照组(200μg/mLRSA)、AOPPs100μg/mL、AOPPs200μg/mL、AOPPs300μg/mL以及Apocynin干预,作用时间为48小时。具体实验步骤和方法同第一部分。
7. AOPPs对胰岛微血管内皮细胞NO水平的影响
按照碧云天生物科技公司一氧化氮检测试剂盒的标准操作流程测定细胞内NO的水平,DMEM10%FBS稀释标准品,在96孔板中加入50μl/孔的标准品及细胞培养液上清,依次在各孔中加入室温Griess Reagent I, Griess Reagent II,540nm测定吸光度。
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AOPPs对IMECs细胞增殖活力的影响
100、200、300μg/mLAOPPs作用IMECs48h后,OD值分别为1.02±0.06,0.75±0.07,0.55±0.08,均显著低于RSA阴性对照组(1.29±0.08),P=0.000。200μg/mLAOPPs在给予NADPH氧化酶阻断剂apocynin预处理后细胞活力显著增加为(1.22±0.12),与单独AOPPs处理组相比差异具有统计学意义(均P<0.05)。 AOPPs的浓度(100,200,300μg/mL)与IMECs的OD值存在显著负相关关系(r=-0.949,P=0.000)。
2.比较AOPPs不同作用时间对细胞增殖活力的影响
200μg/mL AOPPs作用时间(12h、24h、48h、72h)后的细胞OD值分别为1.11±0.08,0.96±0.07,0.75±0.07,0.53±0.11,均显著低于control组的OD值(1.29±0.08)(vs. control, P=0.000). AOPPs作用时间与细胞活力间存在显著负相关关系(r=-0.972,P=0.000)。
3. AOPPs对胰岛微血管内皮细胞迁移能力的影响
AOPPs100、200、300μg/mLAOPPs作用IMECs24h后,其穿透至下室的细胞数量分别为266.00±11.14,184.00±9.17,135.00±10.44,均显著低于空白对照、RSA阴性对照组(P=0.000)。Apocynin预处理组穿透至下室的细胞数量明显增加(305.67±12.22),与单独AOPPs处理组相比差异具有统计学意义(均P<0.05)。AOPPs的浓度(100、200、300μg/mL)与IMECs迁移能力存在显著负相关关系(r=-0.972,P=0.000)。
4. AOPPs对IMECs细胞Matrigel基质中形成三维结构能力的影响
AOPPs100、200、300μg/mLAOPPs作用IMECs48h后,细胞在Matrigel基质胶中形成三维结构的数量分别为7.33±0.58,5.67±0.57,3.33±0.57,均显著低于空白对照、RSA阴性对照组(均P<0.05)。 Apocynin预处理组形成三维结构的数量明显增加(9.33±1.53),与单独AOPPs处理组相比差异具有统计学意义(均P<0.05)。AOPPs的浓度(100、200、300μg/mL)与IMECs的迁移能力存在显著负相关关系(r=-0.978,P7=0.000)。
5. AOPPs对胰岛微血管内皮细胞NADPH氧化酶活性的影响
随着AOPPs浓度的增加,NADPH依赖性的02-的产量逐渐增强,300μg/ml时达高峰。AOPPs (100、200、300μg/mL)干预组与空白对照、RSA阴性对照及apocynin阻断剂组相比具有统计学差异(F=59.389,P=0.000)=AOPPs的浓度(100、200、300μg/mL)与NADPH氧化酶活性存在显著负相关关系(r=-0.949,P7=0.000)。
6. AOPPs对IMECs中p-Akt、p-eNOS表达的影响
各组间p-Akt、p-eNOS的表达具有统计学差异(F=32.631,P=0.000)和(F=21.791,P=0.000). p-Akt、p-eNOS的表达随AOPPs干预浓度的增加而显著降低,与RSA对照组相比具有统计学差异(均P<0.05)。而在给予NADPH氧化酶阻断剂apocynin后二者表达量显著增加,与无apocynin干预组相比具有统计学差异。AOPPs的浓度(100,200,300μg/mL)与p-Akt、p-eNOS表达存在显著负相关关系(r=-0.949,P=0.000)和(r=-0.926,P=0.000)。
7. AOPPs对胰岛微血管内皮细胞内NO水平的影响
各处理组细胞内NO水平具有统计学差异(F=27.649,P=0.000),其中AOPPs (100,200,300μg/mL)干预后细胞内NO水平显著降低,分别为15.13±1.48,12.14±1.44,8.87±1.74,与空白对照(19.56±0.81)及RSA对照(18.64±1.36)相比具有统计学差异(均P<0.05)。给予Apocynin预处理的细胞内NO水平则显著增加为(17.89±1.25),与单独AOPPs干预相比差异具有统计学意义。AOPPs的浓度(100,200,300μg/mL)与IMECs的细胞内NO水平存在显著负相关关系(r=-0.896,P=0.000)。
结论
AOPPs干预可显著降低胰岛微血管内皮细胞增殖能力、迁移能力及再血管化能力,增强了细胞内NADPH氧化酶的活性,显著降低了细胞内磷酸化Akt及eNOS的表达水平以及细胞内NO的水平。我们推测：一方面AOPPs可通过下调PI3K/Akt通路中磷酸化Akt及eNOS的表达水平,减少细胞NO的水平；另一方面可通过增强NADPH氧化酶活性加强了NADPH氧化酶介导的eNOS脱偶联导致细胞内NO水平的下降。细胞内NO水平的降低最终导致胰岛微血管内皮细胞的损伤及功能紊乱。
第三部分GLP-1对AOPPs诱导的IMECs细胞损伤及功能紊乱的保护作用及机制
目的
研究GLP-1对AOPPs诱导的IMECs细胞凋亡、增殖、迁移及再血管化能力的保护作用及其对PI3K/Akt及其下游分子信号通路的影响。
方法
1.实验分组
分4组,依次为阴性对照组(200μg/mLRSA)、AOPPs200μg/mL、AOPPs200μg/mL+100nmol/LGLP-1组、AOPPs200μg/mL+100nmol/L GLP-1+10μmol/LLY294002组(P13K特异性抑制剂LY294002预处理1h后方加入AOPP-RSA和GLP-1)。用无血清培养基稀释各种药物储存液,作用一定时间后收集各组细胞。其中观察细胞迁移能力时为排除细胞凋亡影响我们选取干预时间为24h,而观察细胞凋亡、增殖能力及血管化能力时则选取干预时间为48h。
2.Annexin V-FITC/PI双染色法检测细胞凋亡(方法同第一部分)
3.CCK-8检测细胞增殖活性(方法同第二部分)
4. Transwell小室检测GLP-1对AOPPs诱导的IMECs体外迁移能力
方法同第二部分
5. Matrigel基质胶中检测GLP-1对AOPPs诱导的IMECs再血管化能力的影响(方法同第二部分)
6.DHE荧光探针检测细胞内活性氧(ROS)(方法同第一部分)
7. Western blotting法检测IMECs细胞RAGE、Bc1-2、Bax、p-Akt, p-eNOS蛋白的表达(方法同第一、二部分)
8.GLP-1对胰岛微血管内皮细胞内NO水平的影响(方法同第二部分)统计学处理
采用SPSS13.0进行统计分析,计量实验数据以均数±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.GLP-1对AOPPs诱导的IMECs细胞凋亡的影响
各处理组间IMECs细胞的凋亡率有显著差异(F=43.433,P=0.000)。200μg/mL AOPPs作用IMECs48h后,凋亡率为25.05±3.46,显著高于RSA对照组8.57±0.86(P<0.05)。在给予GLP-1(100nmol/L)后细胞凋亡率显著降低(P<0.05)。而提前给予PI3K抑制剂LY294002预处理后再给予AOPPs及GLP-1干预的细胞组其细胞凋亡率显著增加(P<0.05)。
2.GLP-1对AOPPs诱导的IMECs细胞增值活性的影响
AOPPs200μg/mL诱导胰岛微血管内皮细胞48h后的OD值为0.65±0.08,与RSA对照组相比,有显著差异(P=0.000)。给予GLP-1联合处理组的细胞OD值为1.13±0.14,显著高于AOPPs单独干预组(P=0.001)。LY294002预处理组的OD值为0.72±0.10,与AOPPs+GLP-1组相比显著降低(P=0.001),LY294002预处理可部分阻断GLP-1恢复细胞活力的效应。
3.GLP-1对AOPPs诱导的IMECs细胞迁移能力的影响
在给予AOPPs200μg/mL刺激24小时后,由Transwell上室穿透至下室的细胞数量显著减少196.33±15.50,与RSA对照组(306.00±13.11)相比具有统计学差异(P<0.05)。在给予GLP-1(100nmol/L)后发生迁移的细胞数量显著增加(296.67±17.62)(P<0.05)。而提前给予PI3K抑制剂LY294002预处理后再给予AOPPs及GLP-1干预的细胞组其迁移细胞率则显著减少(216.00±21.07)(P<0.05)。
4.GLP-1对AOPPs诱导的IMECs在Matrigel基质中形成三维结构的影响
AOPPs200μg/mL干预48小时组细胞形成三维结构的数量(4.67±0.58)与RSA阴性对照(9.33±0.58)相比显著减少,差异具有统计学意义(P=0.000)。在给予GLP-1(100nmol/L)后形成三维结构的数量显著增加(8.00±1.00)(P<0.05)。而提前给予PI3K抑制剂LY294002预处理后再给予AOPPs及GLP-1干预的细胞形成三维结构的数量则未见显著增加(6.00±1.00)(P<0.05)。
5.GLP-1对AOPPs诱导的IMECs细胞NADPH氧化酶活性的影响
AOPPs200μg/mL显著增加胰岛微血管内皮细胞NADPH依赖性的O2-的产量(367.67±21.94),与RSA对照组相比,有显著差异(P=0.000)。给予GLP-1联合处理后NADPH依赖性的O2-的产量显著减少(158.00±13.00),差异具有统计学意义。LY294002预处理组其细胞NADPH依赖性的O2-的产量与单独给予GLP-1干预组相比又显著增加(348.33±18.23),P<0.05。
6.GLP-1对AOPPs诱导的对IMECs内活性氧(ROS)的影响
AOPPs显著增加了细胞内ROS的水平,其荧光强度为(53.85±4.15)与RSA对照相比(24.37±2.84)具有统计学差异(P=0.000)。GLP-1(100nmol/L)干预组细胞内ROS水平显著下降为(30.02±5.12),而提前给予LY294002预处理组细胞内ROS水平又显著升高(48.65±6.31),均P<0.05。
7.GLP-1对AOPPs诱导的IMECs细胞RAGE表达的影响
RSA对照组其蛋白水平为(0.33±0.05), AOPPs干预后显著增加了细胞RAGE表达的水平(0.60±0.09)与RSA对照组相比具有统计学差异(P<0.05)。GLP-1(100nmol/L)干预可显著下调细胞RAGE表达水平(0.37±0.06)(P=0.004),而提前给予LY294002预处理并不能阻断GLP-1下调RAGE表达的作用(0.39±0.08),均P>0.05。
8.GLP-1对AOPPs诱导的IMECs f中bcl-2、Bax、p-Akt、p-eNOS的表达AOPPs200μg/mL干预显著降低了抗凋亡蛋白bcl-2、p-Akt、p-eNOS的表达,而增加了促凋亡蛋白Bax蛋白的表达,与RSA对照组相比差异具有统计学意义(均P<0.05)。 GLP-1干预后可显著增加bcl-2、p-Akt、p-eNOS表达而降低Bax表达(均P<0.05)。LY294002预处理可显著抑制GLP-1介导的bcl-2、p-Akt、 p-eNOS的表达增加以及Bax表达降低(均P<0.05)。
9.GLP-1对AOPPs诱导的IMECs细胞内NO水平的影响
AOPPs200μg/mL干预组显著降低了细胞内NO的水平(10.05±1.67),与RSA对照组(18.85±1.87)相比具有统计学差异,P<0.05。GLP-1(100nmol/L)干预后显著升高了细胞内NO的水平(17.35±1.39),与单纯AOPPs干预组相比差异具有统计学意义(P<0.05)。而PI3K抑制剂LY294002的预处理则显著削弱了GLP-1的这一保护性调节作用,而使细胞内NO的水平显著降低(10.93±1.45)。
结论
本部分实验首次证明GLP-1可降低AOPPs导致的胰岛微血管内皮细胞的凋亡,显著改善被AOPPs损伤的胰岛微血管内皮细胞的增殖、迁移及再血管化能力。GLP-1改善胰岛微血管内皮细胞功能损伤及紊乱的作用主要通过以下两方面途径来完成：一是通过下调RAGE受体表达、抑制NADPH氧化酶活性、减少细胞内活性氧而改善了氧化应激损伤及NADPH氧化酶介导的eNOS脱偶联,升高了细胞内NO的水平；二是激活PI3K/Akt信号转导通路,上调抗凋亡蛋白Bcl-2表达,减低促凋亡蛋白Bax表达,增加磷酸化内皮型一氧化氮合成酶(p-eNOS)表达升高细胞内NO水平,最终减少内皮细胞凋亡并改善内皮细胞功能紊乱。
Background
The prevalence rate of diabetes mellitus (DM) is rising year and year, and has become the third chronic non-communicable diseases like cardiovascular disease and tumor which were the global threat of human health. With the improvement of people's living and the changes of life style, the morbidity of type2diabetes (T2DM) presents a tendency of rapid increasing in our country. According to the latest result from epidemiological survey in2013, the estimated prevalence of DM was11.6%which meant there were about1.139hundred million patients in adults of18years old and above diagnosed with the newest international clinical criteria. The more amazed result was that the prevalence of impaired glucose tolerance (IGT) was up to50.1%in Chinese adult and the onset of DM became younger in age. Acute and chronic complications caused by DM, specially the diabetic macro-and micro-vascular complications, become the main cause of deaths and disabilities and severely impact the quality of life and lifetime.
Insulin resistance (IR) and β cells hypofunction are two major reasons for T2DM. Current treatment includes promoting insulin secretion, improving insulin resistance, inhibiting the absorption of glucose and inhibiting hepatic glucose output. These treatments still cannot stop progressive failure of β cells function with the course of disease proceeding, even they can improve the blood glucose. In recent years, more studies supposed that micro-circulation disorder of β cells was closely related to the injury and hypofunction of (3cells. Islet is a organ of high vascularity. Islet capillaries are not only the crucial link between islet cells and the body, but also the microenvironment for cells to live by. Islet has unique microvascular system which contains intensive capillary network with the density of5times as peripheric exocrine organization. The abundant blood supply could not only satisfy its need for oxygen and nutrition, but also could regulate the metabolic balance in the body when it rapidly percept the changes of metabolic environment and distribute various hormones to relevant target tissue via blood circulation. Therefore the microcirculatory system is a important regulator for islet endocrine function. The intact structure and function of islet microcirculation depend on the islet microvascular endothelial cells (IMECs) which have caused researcher's concern for these years. IMECs, the most elements of islet microcirculation-tissue barriers, have a close relationship with islet endocrine function and directly participate in the development of islet hypofunction. With the effect of islet capillaries in the pathogenesis of T2DM being revealed, Tal MG proposed that β cells hypofunction was probably the representation of diseased islet capillaries in2009. He said that the dysfunction and injury of IMECs was the important factor resulting in the disease of islet capillaries, which could cause anoxia and ischemic injury in islet microcirculation and eventually cause progressive dysfunction of β cells. Therefore, to deeply study the physiopathologic mechanism in dysfunction and injury of IMECs and the effective treatment has exerting great impact on protecting the function of β cells and looking for new targets to prevent T2DM.
The dysfunction and injury of vascular endothelial cells (VEC) is the precondition and basis of the development in diabetic microvascular complications. Glucotoxicity, lipotoxicity, the change of hemarheology and haemodynamics and reduced antioxidant capacity of tissue are closely linked to dysfunction of vascular endothelial cells. The vasodilator factor NO reduce synthesis and the vasoconstrictor factor endothelin-1increase production, which can directly stimulate the hyperplasia of intimal arterial smooth muscle and the expression of fibronectin and collagen IV. In the state of hyperglycemia, the synthesis and release of intercellular adhersion molecule1(ICAM-1) and vascular cell adhesion molecule1(VCAM-1) increased by VECs, which could enhance the adherence of leucocyte and platelet resulting in activation of inflammatory cells and intimal inflammation. The change could induce medial smooth muscle cells migrating under intima and lipid depositing in cells, which accelerated formation of arterial atherosclerosis. As a kind of VECs, IMECs should be damaged by different pathogenic factors In the state of hyperglycemia, which could result in dysfunction of endothelial cells and islet microcirculation, specially the dysfunction of islet cells.
Advanced oxidative protein products (AOPPs), considered as a novel marker of oxidant-mediated protein damage, are dityrosine-containing and cross-linking protein products mainly formed during oxidative stress by reaction of plasma albumin with chlorinated oxidants. AOPPs were firstly found in the plasma of patients undergoing dialysis and were subsequently found in subjects with diabetes. The structure and biological function of AOPPs was similar to advanced glycation end products (AGEs) which could combine with RAGE (receptor of AGEs) to activate NADPH oxidase, intra-cellular signaling of redox reaction such as ERK1/2, p38MAPK and translocation of nuclear factor-KB resulting in inflammation and oxidative stress in VECs. Chronic accumulation of AOPPs aggravated accumulation of oxidized low density lipoprotein (LDL) in arterial wall and worsen inflammation and oxidative stress in artery to accelerate atherosclerosis in a hyperlipidemic model. The level of AOPPs closely correlated with the degree of carotid atherosclerosis in patients with CKD, which meant that accumulation of AOPPs could play a role in atherosclerosis of patients with CKD. Gradinaru D and his team discussed the relation between level of AOPPs and the factors leading to atherosclerosis such as endothelium NO synthetase, oxidative stress and metabolic profiling in patients with diabetes or prediabetes. They found that AOPPs significantly increased compared with normal control group and inhibited activity of endothelium NO synthetase to reduce synthesis of NO, which suggested that the level of AOPPs closely correlated with the degree of diabetic atherosclerosis in old patients with DM. Bansal S and team conducted an analysis of gene polymorphism of RAGE and its relation with oxidative stress products, paraoxonase and diabetic macro-vascular complications in265Indian patients with T2DM. They found that there existed a positive correlation between the genotype of429T/C and level of oxidative stress products, development of diabetic macro-vascular complications. Thus, apart from being regarded as oxidative stress makers, AOPPs has also been shown to be a pro-inflammatory factor in accelerating the progress of atherosclerosis and damaging vascular system in our body. However, few studies aimed at the effect of AOPPs in islet microcirculation, especially in the function of IMECs.
GLP-1is a polypeptide secreted by the intestinal L cell. GLP-1is best known as a safe and effective insulinotropic hormone and has been proposed as prospective approach to clinical treatment of T2DM. More and more studies indicated that GLP-1not only stimulated survival and proliferation, increased insulin secretion, inhibited appetite, retarded peristalsis of gastrointestinal smooth muscle and inhibited release of glucagon, but also plays an important role in diabetic cardiovascular complications. The receptor of GLP-1(GLP-1R) expressed in vascular smooth muscle, cardiac muscle, endocardium and coronary endothelium. The agonist of GLP-1R could protect cardiac muscle from ischemic-reperfusion and improved the function of heart. On one hand, exendin-4significantly reduced the area of myocardial infarction in vitro, on the other hand, exendin-4and GLP-1could enhance left ventricular systolic function. Timmers and his team also found that exendin-4sustaining treatment could significantly reduce the area of myocardial infarction and promote the recovery of myocardial contractive and systolic function in a pig model after ligation of coronary artery. GLP-1could inhibit monocyte from adhering to aortic vascular endothelial cells, alleviate vascular inflammatory injury and stop the progress of AS. Supplementing GLP-1in rats with IR and hypertension could improve the function of vascular endothelium, blood pressure and cardiac function. In the clinical study, GLP-1could improve the vascular endothelial function of patients with T2DM and stable CAD. In recent years, researchers suggested that GLP-1significantly blocked the oxidative stress of endothelial cell induced by AGEs-RAGE through the down-regulation of RAGE and ICAM-1and reduced intimal hyperplasia and smooth muscle hyperplasia after the injury of endothelial cells induced by AGEs in vitro, which meant that GLP-1had potential effect of anti-inflammatory and anti-atherosclerosis. Exendin-4promoted the proliferation of endothelial cells through PI3K/Akt-eNOS signal pathway. In the study of rats model with endothelial cell dysfunction and AS, liraglutide, GLP-1receptor agonist, could improve dysfunction of VECs and alleviate the degree of AS. In2012, chinese researchers found that GLP-1could alleviate the apoptosis of human umbilical vein endothelial cells (HUVECs) induced by AGEs through activation of PI3K/Akt signal pathway by the means of regulating the expression of Bcl-2, Bax and cyto-c in the endothelial cells. In2013, Erdogadu.O and his team conducted a study about the effect of GLP-1in human coronary artery endothelial cells (HCAECs) induced by lipotoxicity, and found that GLP-1could improve the proliferation, metastasis and vasculogenesis of aortic endothelial cells and reduced the apoptosis through PKA/PI3K/Akt/eNOS, p38MAPK and JNK kinase-dependent pathway.
Thus, apart from the hypoglycemic effect, GLP-1also play a important role in protecting endothelial cells and improving the function of them, which through PKA/PI3K/Akt pathway. However, did GLP-1have the same protective effect in the injury of IMECs induced by AOPPs under the state of DM? Did it can improve the function of IMECs and islet microcirculation so that delay the progressive dysfunction of Pcells? All of these are important issues for our discussion.
It is very important to investigate the effects of GLP-1on islet microcirculation and its possible mechanisms for discovering a new therapeutic target to protect the function of islet cells in T2DM patients. In this study, we established the damaged model by treating IMECs with AOPP-modified RSA (AOPP-RSA), in order to explore the effects and related mechanisms of GLP-1in the apoptosis, proliferation, migration and revascularization of IMECs induced by AOPPs, and try to provide a new viewpoint for GLP-1treatment which could delay the progressive dysfunction of β-cells.
Objectives
In this project we aim to on the base of damaged model induced by AOPPs, to observe the effects of GLP-1on the apoptosis, proliferation, migration and revascularization of IMECs induced by AOPPs, and to investigate the effect of GLP-1on apoptosis-relative proteins expression and PI3K/Akt/eNOS pathway, also to discuss the protective role of GLP-1and its molecular mechanisms in the dysfunction of IMECs induced by AOPPs.
Content
The whole project includes three parts:
Part I The effect of AOPPs on the apoptosis of islet microvascular endothelial cells (IMECs) and its possible mechanism
Objectives
To explore the effect and mechanism of AOPPs on the apoptosis of IMECs
Methods
1. Preparation of AOPP-RSA
20mg/ml rat serum albumin (RSA) was mixed with the same volume of hypochlorous (40mmol/1), and then was placed for30min. The molar ratio of RSA and hypochlorous in AOPP was1:140. Excessive dissociative hypochlorous was removed by dialysis with non-endotoxin PBS for24h. The solution was sterilized by passing it through a0.22micron bacteria-retentive filter.
2. Isolation and digestion of rat islet cells
We used a modified method of collagenase digestion and Ficoll density gradient separation for isolation and digestion of islet from the rat. The cells were stained with DTZ and typan blue, and the concentration of cells was adjusted to500IEQ/ml. These cells were cultured at34℃in a humidified atmosphere of5%CO2.
3. Purification and identification of IMECs
The growth of spindle cells in the islet was observed after culturing3-5days in vitro and the proliferation of cells was observed in7-9days. IMECs which were carried by the2%gelatin were cultured after immunomagnetic beads. These cells were identified for von Willebrand factor (vWF) antigen and Dil-acetylated low density lipoprotein (Dil-Ac-LDL) by immunofluorescence.
4. Groups:
The effect of different concentrations of AOPPs on the apoptosis of IMECs: AOPPs-RSA treated the cells at concentrations of100,200,300μg/mL for48h. The effect of AOPPs on the apoptosis of IMECs for different time:AOPPs-RSA alone (final concentration200μg/mL) incubated with cells for0,12,24,48,72h respectively.
5. The apoptosis of IMECs with Hoechst33258dye staining and AnnexinV-FITC/PI
Cells were treated with4%paraformaldehyde for5min in4℃after indicated treatment. The morphology of cells apoptosis were observed by fluorescence microscope after Hoechst33258dye staining. Cells were added AnnexinV and PI after the digestion with trypsin, and then placed for15min protecting from light. The early apoptosis rate was detected with flow cytometry and analyzed by Cell Quest software.
6. The ROS level in IMECs detected by DHE fluorescent probe
The cells were divided into4groups:normal control group, RSA control group,200μg/ml AOPPs group and200μg/ml AOPPs+apocynin group, and all the groups were accepted identified treatment for48h. Cells were collected and cultured in the solution of DHE at37℃for30min. After the removal of too much solution, the distributions of DCF in cells were observed by laser scanning confocal microscope. All the groups were selected3horizons to be scanned and analyzed the relative intensity of fluorescence.
7. The expression of BcI-2and Bax in IMECs with Western blot
The total protein of all groups were extracted and detected by BCA method. After electrophoresis in12%SDS-PAGE, transfer, blocking and washing, all the groups were incubated with anti-Bcl-2monoclonal antibody(1:1000) and anti-Bax monoclonal antibody(1:2000) at4℃over night. Cells were conducted hybridization with secondary antibodies labeled with horseradish peroxidase for1h and coloration, and the results were analyzed by ImageJ software.
8. The activities of caspase-9and-3were detected by ELISA
Cells of all the groups were accepted identified treatment for48h and then were collected. Cells were then added to the reaction substrate correspondingly. Incubated in37℃water bath for2h, finally using ELISA meter measured OD values at the detection wavelength of405nm.
Statistical Analysis
All analyses were carried out with SPSS13.0software. Data were expressed as mean±standard deviation (SD). Differences between groups were tested by one-way ANOVA followed by a LSD test. We used Spearman correlation analysis for correlation analysis. Statistical significance was defined as two-sided P<0.05.
Results
1. The effect of different concentration of AOPPs on the apoptosis of IMECs for48h
After treatment with100,200,300μg/ml AOPPs for48h, the apoptosis rate were14.57±4.7,24.20±4.30,35.53±6.47, respectively, and significantly higher than those of normal control group (5.87±1.51) and RSA control group (6.90±1.42)(P<0.05). Spearman correlation analysis showed that a significant positive correlation existed between the apoptosis rate of IMECs and the AOPPs concentrations (100,200,300ug/ml)(r=0.949, P=0.000).
2. The effect of AOPPs on the apoptosis of IMECs for different time
The apoptosis rate of200μg/ml AOPPs incubated for different time (12,24,48,72h) were11.61±1.94,16.69±3.17,23.48±4.69,32.80±7.13, respectively, and significantly higher than control group (0h)(6.10±2.07)(F=17.854, P=0.000). ntrol group (5.87±1.51) and RSA control group (6.90±1.42)(P<0.05). Spearman correlation analysis showed that a significant positive correlation existed between the apoptosis rate of IMECs and time (r=0.950, P=0.000).
3. The effect of AOPPs on ROS level of IMECs
The fluorescence value of ROS in200μg/ml AOPPs group was58.43±2.71, and significantly higher than that of RSA control group (27.49±4.75)(P=0.000), which suggested that AOPPs stimulated the prodiction of ROS in IMECs. Treated IMECs with apocynin (inhibitor of NADPH oxidase) for48h, the fluorescence value of ROS was significantly down to30.45±4.35compared with the200μg/ml AOPPs group (P=0.000).
4. The effect of AOPPs on the expression of Bcl-2, Bax of IMECs
After identified treatment for48h, the expressions of Bcl-2were significantly different among the groups (F=4.675, P=0.036). The expression of Bcl-2in200μg/ml AOPPs group was0.60±0.09, which significantly lower than that of RSA control group (P=0.021). Treated IMECs with apocynin (inhibitor of NADPH oxidase) for48h, the expression of Bcl-2was significantly up to0.87±0.14compared with the200μg/ml AOPPs group (P=0.033). The expression of Bax in200μg/ml AOPPs group was1.35±0.19, which significantly higher than that of RSA control group (P=0.008). Treated IMECs with apocynin, the expression of Bax was significantly down to0.97±0.15compared with the200μg/ml AOPPs group (P=0.021).
5. The effect of AOPPs on the activities of caspase-9,-3in IMECs
After identified treatment for48h, the activities of caspase-9,-3were significantly different among the groups (F=144.224and311.617, P=0.000and P=0.000). The activities of caspase-9,-3were1.24±0.08and1.01±0.04, which significantly higher than those of normal control group (P<0.05) and RSA control group (P<0.05). Pretreatment IMECs with apocynin, the activities of caspase-9,-3were down to 0.45±0.06and0.40±0.04compared with the200μg/ml AOPPs group (P<0.05and P<0.05).
Conclusions
Apoptosis was induced by AOPPs in a dose, time-dependent manner. Exposure to AOPPs for48h caused a significant increase in ROS generation, expression of Bax and activities of caspase-9and-3, but a decrease in expression of Bcl-2. However, co-incubation with apocynin (inhibitor of NADPH oxidase) attenuated these AOPPs-induced effects in IMECs. Therefore, we speculated that AOPPs aggravated the oxidative stress injury through activity of NADPH oxidase and increasing the level of ROS, and then induced the apoptosis of IMECs by the regulation of Bcl-2/Bax, caspase-9and-3.
Part Ⅱ The effect and mechanism of AOPPs on the proliferation, migration activity and revascularization of IMECs
Objectives
To explore the effect of AOPPs on the proliferation, migration activity and revascularization of IMECs, and then to investigate the mechanism of AOPPs on the injury and dysfunction of IMECs
Method
1. Group:
The effect of different concentrations of AOPPs on the proliferation, migration activity and revascularization of IMECs:the cells were divided into6groups:normal control group, RSA control group,100μg/ml AOPPs group,200μg/ml AOPPs group,300μg/ml AOPPs group and200μg/ml AOPPs+apocynin group. We chose24h as intervention time to observe the migration activity of IMECs excluding the effect of apoptosis and48h to observe the the proliferation and revascularization of IMECs.
The effect of AOPPs on the proliferation of IMECs for different time:200μg/ml AOPPs alone incubated with cells forO,12,24,48,72h respectively.
2. The proliferation activity of IMECs with CCK-8method
The cells were planted in96-well plates. After each experimental treatment, we added10μl cell counting kit-8solution into each hole, and incubated at37℃in a humidified atmosphere of5%CO2for1h. The OD value was detected by ELISA with wavelengths of450nm to reflect the proliferation activity.
3. The effect of AOPPs on the migration activity of IMECs with Transwell assay
The cells were cultured in serum-free medium for12-24h and the density of cells was adjusted to1×105. The upper-well was added in200μl cell suspension of IMECs, RSA, different concentration of AOPPs (100μg/ml,200μg/ml,300μg/ml) and apocynin, and the lower-well was added in600μl DMEM with10%fetal bovine serum. The cells were cultured at37℃for24h. After the fixation and staining, we counted the cells and gently erased the non-migrated cells in the upper-well by swab.
4. The effect of AOPPs on the revascularization of IMECs with matrigel
The high concentration of matrigel was placed in96-well plates (70μl/hole) and formed into gel in incubator for30min after melting over night. The IMECs were planted in each hole with serum-free medium and added in RSA, different concentration of AOPPs (100μg/ml,200μg/ml,300μg/ml) and apocynin. The cells were incubated at37℃in a humidified atmosphere of5%CO2for48h and detected by invert microscope with3views for each hole.
5. The effect of AOPPs on the activity of NADPH oxidase in IMECs
After each experimental treatment for48h, we prepared the cell homogenate and detected the protein content in them.100μg proteins of each group were planted in96-well plates, and added in5μm lucigenin and100μm substrate of NADPH. The fluorescence quantum produced by NADPH-dependent O2-was detected with VICTOR1420multi-label analyzer, which indirectly reflected the production of O2-and activity of NAPDH oxidase. The data were expressed as counts per second (CPS).
6. The effect of AOPPs on the expression of p-Akt and p-eNOS in IMECs with Western blot
The cells were treated with200μg/ml RSA and different concentration of AOPPs (100,200,300μg/ml) and apocynin for48h. The detailed experimental procedure and methods referred to part1.
7. The effect of AOPPs on the level of NO in IMECs
According to standard operating procedure of NO detection kit produced from Beyotime, we detected the level of NO in the cells. The standard substance was diluted with DMEM and10%FBS, and placed50μl of each hole in96-well plates with the supernatant. The absorbance was detected by the wavelength of540nm after adding Griess Reagent I and Griess Reagent Ⅱ at room temperature.
Statistical Analysis
All analyses were carried out with SPSS13.0software. Data were 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 AOPPs on the proliferation of IMECs
After treatment with100,200,300μg/ml AOPPs for48h, the OD value were1.02±0.06,0.75±0.07,0.55±0.08, respectively, and significantly lower than that of RSA control group (1.29±0.08)(P=0.000). Pretreated IMECs with apocynin, the OD value was up to1.22±0.12compared with the200μg/ml AOPPs group (P<0.05). A significant negative correlation existed between the OD value of IMECs and the AOPPs concentrations (100,200,300μg/ml)(r=-0.949, P=0.000).
2. The effect of AOPPs on the proliferation of IMECs for different time
The OD values of200μg/ml AOPPs incubated for different time (12,24,48,72h) were1.11±0.08,0.96±0.07,0.75±0.07,0.53±0.11, respectively, and significantly lower than that of RSA control group (1.29±0.08)(P=0.000). A significant negative correlation existed between the proliferation of IMECs and time (r=-0.972, P=0.000).
3. The effect of AOPPs on the migration activity of IMECs
After treatment with100,200,300μg/ml AOPPs for48h, the cell counts of penetrating to lower-well were266.00±11.14,184.00±9.17,135.00±10.44, respectively, and significantly lower than those of nomal control group and RSA control group (P=0.000and P=0.000). Pretreated IMECs with apocynin, the cell count of penetrating to lower-well was up to305.67±12.22compared with the200μg/ml AOPPs group (P<0.05). A significant negative correlation existed between the migration activity of IMECs and the AOPPs concentrations (100,200,300μg/ml)(r=-0.972, P=0.000).
4. The effect of AOPPs on the capacity of forming3d structure of IMECs in matrigel
After treatment with100,200,300μg/ml AOPPs for48h, the counts of forming3d structure of IMECs in matrigel were7.33±0.58,5.67±0.57,3.33±0.57, respectively, and significantly lower than those of nomal control group (P<0.05) and RSA control group (P<0.05). Pretreated IMECs with apocynin, the count of forming3d structure in matrigel was up to9.33±1.53compared with the200μg/ml AOPPs group (P<0.05). A significant negative correlation existed between the migration activity of IMECs and the AOPPs concentrations (100,200,300μg/ml)(r=-0.978, P=0.000).
5. The effect of AOPPs on the activity of NADPH oxidase of IMECs
The production of NADPH-dependent O32-increased with the increase of AOPPs concentration, and the300μg/ml AOPPs group reached the peak. The productions of NADPH-dependent O2-were significantly different among the groups (F=59.389, P=0.000). A significant negative correlation existed between the activity of NADPH oxidase of IMECs and the AOPPs concentrations (100,200,300μg/ml)(r=-0.949, P=0.000).
6. The effect of AOPPs on the expression of p-Akt and p-eNOS of IMECs
The expressions of p-Akt and p-eNOS of IMECs were significantly different among the groups (F=32.631, P=0.000) and (F=21.791, P=0.000). With the AOPPs concentration increasing, the expression of p-Akt and p-eNOS significantly decreased, compared with those of RSA control group (P<0.05) and (P<0.05). Treated IMECs with apocynin, the expressions of p-Akt and p-eNOS significantly increased compared with other groups. A significant negative correlation existed between the expression of p-Akt, p-eNOS and the AOPPs concentrations (100,200,300μg/ml)(r=-0.949, P=0.000) and (r=-0.926, P=0.000).
7. The effect of AOPPs on the level of NO in IMECs
The levels of NO of IMECs were significantly different among the groups (F=27.649, P=0.000). After treatment with100,200,300μg/ml AOPPs for48h, the levels of NO were15.13±1.48,12.14±1.44,8.87±1.47, respectively, and significantly lower than those of nomal control group (19.56±0.81)(P<0.05) and RSA control group (18.64±1.36)(P<0.05). Pretreated IMECs with apocynin, the level of NO was up to17.89±1.25compared with the200μg/ml AOPPs group. A significant negative correlation existed between the levels of NO in IMECs and the AOPPs concentrations (100,200,300μg/ml)(r=-0.896, P=0.000).
Conclusions
AOPPs could significantly lower the proliferation, migration activity and revascularization of IMECs, enhanced the activity of NADPH oxidase, and decreased the expressions of p-Akt, p-eNOS and the level of NO. Therefore, we speculated that AOPPS decreased the level of NO not only through the down-regulation of p-Akt and p-eNOS in PI3K/Akt pathway but also via increasing the activity of NADPH oxidase to enhance eNOS uncoupling. More importantly, the decreased NO level induced the injury and dysfunction of IMECs.
Part III The protection of GLP-1and its mechanism against the damage and dysfunction of IMECs cells induced by AOPPs
Objective:
Our study investigated the protective effect of GLP-1on the apoptosis, proliferation, migration, the capacity of revascularization and the influence on PI3K/Akt and its downstream molecule signal pathway in IMECs cells induced by AOPPs
Method:
1. Group:
The whole experiment divided into negative control group (200μg/mLRSA), AOPPs200μg/mL group, AOPPs200μg/mL±100nmol/LGLP-1group, AOPPs200μg/mL+100nmol/L GLP-1±10μmol/L LY294002group (added APOO-RSA and GLP-1after preprocessing1hour by LY294002, the specific inhibitor of PI3K). Serum-free medium was used to attenuaate all kinds of stock solution, and then collected cells after effect for a certain time. We chose24hours as the intervention time in case to exclude the influence of apoptosis when we observed the capacity of migration, while we chose48hours as the intervention time when we observed the apoptosis, proliferation, and the capacity of revascularization.
2. Apoptosis detected by Annexin V-FITC/PI staining technique
The method was the same as we mentioned in the first part.
3. Cell proliferation activity detected by using CCK-8
The method was the same as we mentioned in the second part.
4. The effect of GLP-1on the migration in vitro of IMECs cells induced by AOPPs was measured by Transwell chamber
The method was the same as we mentioned in the second part.
5. The capacity of revascularization of IMECs cells detected by using Matrigel
The method was the same as we mentioned in the second part.
6. The ROS level in IMECs cells was measured by DHE fluorescence probe
The method was the same as we mentioned in the first part.
7. The expressions of RAGE, Bcl-2, Bax, p-Akt, p-eNOS were detected by Western blotting
The method was the same as we mentioned in the first and second part.
8. The NO level in islet microvascular endothelial cell
The method was the same as we mentioned in the first and second part.
Statistical analyses
Statistical analyse was performed using SPSS version13.0software. Quantitative data normality was assessed with the One-Way ANOVA test and is expressed as mean±SD. Comparisons between quantitative data were conducted using LSD tests. A value of p<0.05was considered statistically significant.
Results
1. The influence of GLP-1on the apoptosis in AOPPs induced IMECs cells
The apoptosis rate of IMECs cells have significant difference between treatment groups (F=43.433, P=43.433). Apoptosis rate was25.05±3.46%about48hours after200μg/mL AOPPs effect on IMECs cells, and was significantly higher than the RSA control group8.57±0.86%(P<0.05). The apoptosis rate was significantly lower (P<0.05) after giving the treatment of GLP-1(100nmol/L). The apoptosis rate increased significantly (P<0.05) in the group which was first pre-processed by PI3K inhibitors LY294002and then intervened by AOPPs and GLP-1.
2. The influence of GLP-1on the proliferation vitality in IMECs cells induced by AOPPs
The OD value of AOPPs200mu g/mL inducing pancreatic microvascular endothelial cells after48h was0.65±0.08, and was significant differences (P=0.000) compared with RSA control group. The OD value of the combined treatment group giving GLP-1was1.13±0.14, significantly higher than that of AOPPs individual intervention group (P=0.001). The OD value of LY294002pre-processing group was0.72±0.10, decreased significantly (P=0.001) compared with AOPPs±glp-1group, and suggested that LY294002pre-processing could partially blocks the effect of GLP-1to restore cell vitality.
3. The influence of GLP-1to the ability of migration in IMECs cells induced by AOPPs.
After stimulation by giving AOPPs200μg/mL for24hours, the quantity of cells which migrated from Transwell upper chamber to the lower chamber was196.33±15.50, and significantly reduced compared with RSA control group (306.00±13.11)(P<0.05). The quantity of cells which migrated after giving GLP-1(100nmol/L) was296.67±17.62, and was significantly increased (P<0.05). And the cell migration rate of the group first pre-processing by PI3K inhibitors LY294002and then intervened by AOPPs and GLP-1was significantly reduced (216.00±21.07)(P<0.05).
4. The influence of GLP-1on the three-dimensional structure which formated in the Matrigel in IMECs cells induced by AOPPs
The quantity of the three-dimensional structure was4.67±0.58in the group of AOPPs200μg/mL intervented48hours, and was significantly reduced compared with RSA negative control (9.33±0.58)the number of cells form a three-dimensional structure (4.67±0.58)(P=0.000). The quantity of the three-dimensional structure was significantly increased (8.00±1.00)(P<0.05) by giving GLP-1(100nmol/L). And the quantity of the three-dimensional structure of the group first pre-processing by PI3K inhibitors LY294002and then intervened by AOPPs and GLP-1did not see a significant increased (6.00±1.00)(P<0.05).
5. The influence of GLP-1on the activity of NADPH oxidase in IMECs cells induced by AOPPs
The NADPH dependent02-production in islet microvascular endothelial cells was (367.67±21.94) by giving AOPPs200μg/mL, and significantly increased compared with RSA control group (P=0.000). The NADPH dependent O2production of the combined treatment group giving GLP-1was significant
糖尿病(diabetes mellitus, DM)的患病率在全球逐年增加,已成为继心血管疾病和肿瘤之后威胁人类健康的第三大慢性非传染性疾病。随着我国人们生活水平的提高及生活方式的改变,2型糖尿病(T2DM)的发病率也呈现出高速发展的趋势。根据2013年最新流行病学调查研究结果,我国18岁及以上成人中,按照国际最新临床诊断标准进行诊断的糖尿病估测患病率为11.6%,约1.139亿人,而更令人震惊的是中国成年人群中糖尿病前期(IGT)患病率已高达50.1%,且糖尿病发病呈现年轻化趋势。糖尿病所引起的各种急慢性并发症,特别是血管并发症已成为其致残致死的主要原因,严重影响人类的生活质量和寿命。
胰岛素抵抗(insulin resistance, IR)和胰岛β细胞功能减退是2型糖尿病发病的两大主要原因,现有的糖尿病治疗手段通过促进胰岛素分泌、改善胰岛素抵抗、抑制葡萄糖的吸收及抑制肝糖输出为主,这些手段虽能起到改善血糖的作用,但随着病程的进展,仍然无法阻止胰岛β细胞功能的进行性衰竭。近年来更多的研究支持胰岛微循环障碍与β细胞的损伤及功能衰竭密切相关。胰岛是一个高度血管化的微器官,胰岛微血管既是胰岛细胞与机体的关键连接,又是其赖以生存的微环境。胰岛具有独特的微血管系统,含有密集的毛细血管网络,毛细血管网的密度大约是周围外分泌组织的5倍,丰富的血供不仅能够满足胰岛自身对氧气及营养物质的需要,更重要的是使胰岛能够迅速感知体内代谢环境的变化,通过血液循环将分泌的各种激素快速分布到相应的靶组织,从而调节机体代谢平衡。因此,胰岛微循环系统是胰岛内分泌功能重要的调节器。胰岛微循环结构和功能的完整性有赖于另一群目前开始被关注的细胞即胰岛微血管内皮细胞(islet microvascular endothelial cells, IMECs)。IMECs是构成胰岛微循环组织屏障的最主要成分,与胰岛内分泌功能关系密切,直接参与糖尿病胰岛功能衰竭的发生发展过程。随着胰岛微血管在2型糖尿病发病中的作用被逐渐揭示,Tal MG2009年提出：β细胞功能衰竭很可能是胰岛微血管病变的表现,而各种原因导致的胰岛微血管内皮细胞的功能紊乱及损伤是引起胰岛微血管病变的重要因素,继而引起胰岛微循环的缺血缺氧,最终导致胰岛β细胞功能的进行性衰竭。因此,深入研究胰岛微血管内皮细胞功能紊乱及损伤的病理生理机制,探讨有效改善的方法和手段,对于保护胰岛细胞功能寻找防治2型糖尿病发病的新靶点具有重大意义。
血管内皮细胞功能损伤及紊乱是糖尿病循环系统并发症发生发展的前提及基础。糖毒性、脂毒性、血液流变学及血流动力学改变、组织抗氧化能力减弱等因素与血管内皮细胞功能紊乱息息相关,主要表现为舒张血管因子一氧化氮合成减少,缩血管因子内皮素1产生增加,直接刺激动脉内膜下平滑肌细胞增生及纤维连接蛋白和胶原IV的表达。高血糖时血管内皮细胞合成和释放细胞间黏附因子1(ICAM-1)及血管细胞黏附因子1(VCAM-1)增多,增加白细胞和血小板的粘附能力并导致炎症细胞的活化及内膜炎症损伤,这种内皮细胞功能改变使中层平滑肌细胞向内膜下迁移,细胞内脂质沉积,从而加速血管动脉粥样硬化的形成。而胰岛微血管内皮细胞作为全身血管内皮细胞的一种也必然遭受到糖尿病状态下各种致病因素的损伤导致内皮细胞功能障碍,并进而影响到胰岛微循环的功能状态而最终导致胰岛细胞功能损伤。
高级氧化蛋白产物(advanced oxidative protein products, AOPPs)是次氯酸对蛋白质氧化修饰生成的含双酪氨酸的蛋白交联物,是一种新发现的蛋白类大分子氧化应激标志物。最初在透析患者中发现AOPPs的积聚,随后在糖尿病和肥胖患者中也发现了AOPPs的积聚,AOPPs是一种氧化应激的蛋白标志物,它的结构和生物学作用类似于晚期糖基化终末产物(AGEs),也可通过受体RAGE活化血管内皮细胞NADPH氧化酶,激活氧化还原反应敏感的细胞内信号传导途径如ERK1/2、p38MAPK途径以及NF-kB的核转位,从而诱发血管内皮细胞的炎症和氧化应激损伤。慢性AOPPs负荷可以导致动脉壁氧化低密度脂蛋白的沉积增加,加剧动脉内膜及内皮细胞的氧化应激及炎症损伤导致高脂血症家兔动脉粥样硬化斑块的形成明显加速。在CKD患者,循环AOPPs水平和颈动脉粥样硬化病变程度密切相关,提示AOPPs潴留可能参与了CKD的加速性动脉粥样硬化。Gradinaru D等探讨了糖尿病前期及糖尿病阶段患者中AOPPs水平与内皮源性NO合成酶、氧化应激、代谢谱及其他导致动脉硬化的因素之间的关系,发现AOPPs与正常对照人群相比显著身高,并显著抑制内皮源性一氧化氮合成酶的活性,导致内皮源性NO合成减少,在老年糖尿病患者中AOPPs水平的高低与糖尿病动脉粥样硬化紊乱的程度密切相关。Bansal S等对印度265例2型糖尿病患者进行了RAGE的基因多态性分析及其与氧化应激产物、对氧磷酶及糖尿病大血管并发症的相关性分析,发现RAGE基因多态性中-429T/C基因型与血清氧化应激产物的水平及糖尿病大血管并发症的发生具有正相关性。由此可见,AOPPs不仅是氧化应激的标志物,其本身也作为一种强效促炎性因子加速了机体动脉粥样硬化进程,损伤全身的大血管及微血管系统。然而,有关AOPPs在胰岛微循环中的作用尤其是对胰岛微血管内皮细胞功能的影响仍少见报道。
GLP-1是主要由肠道内分泌L细胞分泌的多肽,作为一种安全、有效的促胰岛素分泌剂越来越受到关注,是治疗2型糖尿病的一种新途径。越来越多的研究表明,GLP-1除了具有促进胰岛p细胞增殖、促进胰岛素分泌、抑制食欲、减缓胃肠道平滑肌的蠕动、抑制胰高血糖素释放等作用之外,还有胰外的心血管保护作用。GLP-1受体(GLP-1R)在血管平滑肌、心肌、心内膜、冠脉内皮均有表达,GLP-1R激动剂对心肌的缺血一再灌注损伤具有保护作用,并能改善心脏功能。exendin-4能显著缩小体外离体心脏心肌梗塞面积,exendin-4和GLP-1均能使左室收缩功能增强。Timmers等同样发现exendin-4持续治疗可显著减少冠状动脉结扎后实验猪的心肌梗塞面积,并促进心肌收缩和舒张功能的恢复。GLP-1可抑制单核细胞粘附于人主动脉血管内皮细胞,减轻血管性炎症损伤,阻止动脉粥样硬化的进展。在胰岛素抵抗和高血压的大鼠体内补充GLP-1,可以改善血管内皮功能、改善血压以及心功能。在临床病例观察研究中GLP-1可使2型糖尿病合并稳定冠心病患者的血管内皮功能得到改善。近年有研究者证实,在体外用糖基化终末产物诱导内皮细胞损伤,发现GLP-1可通过其受体显著下调RAGE受体的表达和细胞间粘附分子-1的水平,从而拮抗AGEs-RAGE轴导致的内皮细胞氧化应激损伤,减少血管损伤后内膜的增厚和平滑肌的增殖,具有潜在的抗炎和抗动脉粥样硬化作用。Exendin-4可经由PI3K/Akt-eNOS信号传导通路而促进内皮细胞增殖。在内皮细胞功能障碍和动脉粥样硬化的小鼠模型研究中,GLP-1受体激动剂利拉鲁肽可改善血管内皮细胞功能障碍,并减轻小鼠的动脉粥样硬化程度。2012年国内研究者发现GLP-1可通过激活PI3K/Akt信号转导通路减轻AGEs诱导的人脐静脉内皮细胞的凋亡,这种抗凋亡作用是通过对AGEs诱导的内皮细胞Bc1-2、Bax、cyto-c表达来实现的。2013年Erdogdu.O等进行了GLP-1对脂毒性导致的人冠状动脉内皮细胞损伤的研究,结果发现GLP-1的干预可通过PKA/PI3K/Akt/eNOS、p38MAPK及JNK激酶依赖的途径改善动脉内皮细胞体外的增殖、迁移及血管形成能力,并减少其凋亡。
由此可见,GLP-1除了其优秀的降糖作用之外,其对保护内皮细胞、改善内皮细胞功能同样具有重要优势,而PKA/PI3K/Akt是其发挥多器官保护作用的主要上游通路。那么,在胰岛微血管内皮细胞,GLP-1对糖尿病状态下AOPPs蓄积导致的胰岛微血管内皮细胞的损伤是否也具有同样的保护作用呢?是否可通过改善胰岛微血管内皮细胞的功能进而改善胰岛微循环并最终延缓胰岛β细胞的进行性衰退呢?这些都是值得我们深入研究的重要课题。
因此,探求GLP-1独立于降糖作用之外的胰岛微血管保护作用及机制,挖掘新的保护胰岛细胞功能的治疗靶标,对广大的T2DM患者来说具有十分重要的意义。本研究建立AOPP修饰的大鼠血清白蛋白(AOPP-modified RSAAOPP-RSA)诱导大鼠微血管内皮细胞(islet microvascular endothelial cells, IMECs)损伤研究模型,探讨GLP-1干预对AOPPs诱导的胰岛微血管内皮细胞凋亡、增殖、迁移以及再血管化能力损伤的保护作用及其相关机制,以期为GLP-1治疗延缓胰岛细胞功能的进行性衰竭提供新视点。
目的
本课题拟在建立AOPPs诱导大鼠IMECs损伤为研究模型的基础上,通过观测GLP-1干预对AOPPs诱导内皮细胞凋亡、增殖、迁移及再血管化能力的作用,并检测其对凋亡相关蛋白及PI3K/Akt/eNOS通路的影响,初步探讨GLP-1对AOPPs诱导IMECs功能紊乱的保护作用及分子机制。
内容
课题分以下三大部分：
第一部分AOPPs对大鼠胰岛微血管内皮细胞凋亡的影响及机制
目的
探讨AOPPs对大鼠IMECs细胞凋亡的影响及其机制。
方法
1.体外制备、鉴定晚期蛋白氧化产物：
将20mg/ml大鼠血清白蛋白(RSA)与等体积的40mmol/1次氯酸混合,放置30min,制备出RSA与次氯酸摩尔比为1：140的AOPP。经由无内毒素PBS透析24h,除去过多游离的次氯酸。0.22μm的微孔滤膜过滤除菌。
2.大鼠胰岛细胞的分离、纯化：
利用胶原酶P胰管灌注结合Ficol-400密度梯度消化、分离及纯化大鼠胰岛,体外行DTZ染色、台酚蓝染色,将细胞团浓度调整为500IEQ/ml,以吸管移入细胞培养瓶内,置于34℃、5%CO2二氧化碳恒温培养箱中培养；
3.磁珠纯化胰岛微血管内皮细胞(IMECs)及培养鉴定：
纯化后的胰岛细胞经体外培养3-5d时可见有梭形细胞从胰岛内长出,7-9d细胞逐渐融合成片。将UEA-1包被的M-450免疫磁珠纯化后的胰岛微血管内皮细胞接种在2%明胶包被的细胞培养瓶中培养。免疫荧光检测培养皿中内皮细胞主要标志物第Ⅷ因子相关抗原(vWF)和吞噬Dil标记的乙酰化低密度脂蛋白(Dil-Ac-LDL)能力。
4.实验分组：
不同浓度AOPPs对细胞凋亡的影响：分5组,依次为正常对照组(不加任何干预剂),阴性对照组(200μg/mLRSA),100μg/mL、200μg/mL、300μg/mLAOPPs组,分别作用细胞48h。AOPPs作用不同时间对细胞凋亡的影响：分5组,依次为200μg/mL AOPPs分别作用内皮细胞0、12、24、48、72h。
5. Hoechst33258及Annexin V-FITC/PI定量分析细胞凋亡率：
细胞经各组不同处理后,4%多聚甲醛于4℃处理细胞5min, Hoechst33258染色5min,封片后荧光显微镜观察细胞形态。将各组处理后细胞经胰酶消化,离心后弃上清,PBS洗涤后加入Annexin V和碘化丙啶(P1)避光放置15min,流式细胞仪检测,Cell Quest软件分析细胞早期凋亡率。
6.DHE荧光探针检测细胞内活性氧(ROS)：
直接采用AOPPs200ug/ml干预48小时为干预条件探讨AOPPs导致IMECs细胞凋亡的机制。实验分组：空白对照组,阴性对照组(RSA200ug/ml), AOPPs200ug/ml组,AOPPs200ug/ml+apocynin (NADPH氧化酶阻断剂)组。收集各组细胞后无血清DMEM培养基洗3次,每管加入101μM的DHE工作液2.5m1重悬细胞,37℃5%CO2细胞培养箱内避光孵育40min,洗涤细胞2次。激光共聚焦显微镜观察细胞内DHE荧光分布,各组细胞随机选取3个视野对其扫描并分析相对荧光强度。
7. Western blotting检测细胞Bcl-2、Bax蛋白的表达
收集各组细胞,提取各组细胞总蛋白,采用BCA法测定蛋白浓度。每组蛋白质样本50μg,以12%SDS-PAGE进行电泳,转膜,5%脱脂奶粉封闭2h,洗膜,加入RSA-TBS稀释的抗Bcl-2单克隆抗体(1：1000),抗Bax(1:2000)单克隆抗体,4℃孵育过夜,加入辣根过氧化物酶标记的二抗(1：2000)杂交1h,ECL超敏发光液进行显色,结果用ImageJ分析软件对目的条带进行灰度值分析。
8.细胞凋亡蛋白酶caspase-9、-3活性的检测
实验分组及干预同上。胰酶消化各组细胞,各组细胞孵育48h,消化、离心后、用细胞裂解液重悬,再加入对应的反应底物。37℃水浴2h后,加样至96孔培养板,最后用酶联免疫检测仪测OD值,检测波长为405nm。
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。相关分析采用Spearman相关分析法。P<0.050具有统计学意义。
结果
1.不同浓度AOPPs干预48h对IMECs细胞凋亡的影响
100、200、300μg/mL AOPPs作用IMECs48h后,凋亡率分别为14.57±4.7,24.20±±4.30,35.53±±6.47,均显著高于空白对照组(5.87±1.51)和阴性对照组RSA(6.90±1.42)(均P<0.05)。非参数Spearman相关分析表明：AOPPs浓度(100、200、300μg/mL)与大鼠IMECs的凋亡率间存在显著正相关关系(r=0.949,P=0.000)。
2.比较AOPPs不同作用时间对IMECs细胞凋亡的影响
200μg/mL AOPPs作用时间(12h、24h、48h、72h)后的细胞凋亡率分别为11.61±1.94,16.69±3.17,23.48±4.69,32.80±7.13,均显著高于control组0h的凋亡率(6.10±2.07)(F=17.854,P=0.000)。非参数Spearman相关分析表明：AOPPs作用时间与细胞凋亡率值与存在显著正相关关系(r=0.950,P=0.000)。
3. AOPPs (200μg/mL)干预48h对IMECs细胞内活性氧的影响
IMECs阴性对照组(RSA对照)荧光值为27.49±±4.75,给予AOPPs200ug/ml作用48h后荧光值为58.43±2.71,显著高于对照组(P=0.000),提示AOPPs刺激内皮细胞ROS生成增加。在给予NADPH氧化酶抑制剂apocynin后,细胞内ROS荧光度值显著降低(30.45±4.35),与AOPPs200ug/ml组相比具有统计学差异(P=0.000)
4. AOPPs(200μg/mL)干预48h对IMECs细胞凋亡蛋白Bc1-2、Bax表达的影响
各组细胞经干预48h后,细胞Bcl-2蛋白的表达具有统计学差异(F=4.675,P=0.036)。其中AOPPs200ug/ml组可显著降低抗凋亡蛋白Bcl-2表达量(0.60±0.09),明显低于RSA对照组(P=0.021)。给予NADPH氧化酶抑制剂apocynin后Bcl-2表达量则显著升高(0.87±0.14),与单独AOPPs干预组相比具有统计学差异(P=0.033)。AOPPs200ug/ml干预组显著升高了Bax蛋白表达量(1.35±0.19),与RSA对照相比具有统计学差异(P=0.008)。给予apocynin干预后Bax表达量则显著降低(0.97±0.15),与单独AOPPs干预组相比具有统计学差异(P=0.021)。
5. AOPPs(200μg/mL)干预48h对细胞凋亡蛋白酶caspase-9、-3活性的影响
各组IMECs在干预48h后,细胞中caspase-9、-3活性各组细胞之间具有统计学差异(F分别为144.224和311.617,均P=0.000)。AOPPs作用IMECs细胞48h后,细胞中caspase-9、-3活性(分别为1.244±0.08,1.01±0.04)显著高于其空白对照及RSA阴性对照组(均P<0.05),apocynin提前预处理组细胞中caspase-9、-3活性则显著降低(分别为0.45±0.06,0.40±±0.04)与单独AOPPs干预组相比差异具有统计学意义(均P<0.05)。
结论
AOPPs可呈剂量、时间依赖性诱导胰岛微血管内皮细胞凋亡。AOPPs作用于IMECs细胞48h后,可显著增加细胞内ROS水平、促凋亡蛋白Bax表达,降低抗凋亡蛋白Bcl-2表达,同时增强凋亡蛋白酶caspase-9、-3的活性。而NADPH氧化酶阻断剂Apocynin可阻断AOPPs这一系列效应。因此推测AOPPs可能主要通过NADPH氧化酶途径增加细胞内活性氧水平加剧细胞内氧化应激损伤,进而通过对凋亡相关蛋白Bcl-2/Bax表达、caspase-9、-3活性的调控诱导IMECs细胞的凋亡。
第二部分AOPPs对大鼠胰岛微血管内皮细胞增殖、迁移及再血管化功能的影响及机制
目的
研究AOPPs对IMECs细胞增殖、迁移及再血管化功能的的影响,进而探明AOPPs在胰岛微血管内皮细胞损伤及功能紊乱中的作用及机制。
方法
1.实验分组：
不同浓度AOPPs对细胞增殖、迁移及血管化功能的影响：分6组,依次为空白对照组、阴性对照组(200μg/mLRSA)、 AOPPs100μg/mL、300μg/mL组以及AOPPs200μg/ml+NADPH氧化酶抑制剂组(Apocynin)。观察细胞迁移能力时为排除细胞凋亡影响我们选取干预时间为24h,而观察细胞增殖能力及血管化能力时则选取干预时间为48h。
AOPPs200μg/mL不同作用时间对细胞增殖活性的影响：分5组,依次为200μg/mL AOPPs分别作用内皮细胞0、12、24、48、72h。
2.CCK-8检测细胞增殖活性
96孔板铺板细胞,分组如上所示,在各组干预不同时间,每孔加入cck-8(cell counting kit-8细胞计数试剂)溶液10ul,37℃,5%C02培养箱避光孵育1小时,酶标仪450nm波长处读取吸光度值,反映细胞的增殖活性。
3. Transwell小室检测AOPPs对IMECs细胞体外迁移能力的影响
各组细胞撤血清饥饿12-24h,调整细胞密度至1×105,上室分别加入胰岛微血管内皮细胞悬液200μ1、RSA、不同浓度的AOPPs(100μg/mL、200μg/mL300μg/mL)以及apocynin,下室加人600μl含10%胎牛血清的DMEM培养液,置于37℃培养24小时,固定染色并计数,用棉签轻轻擦掉上层未迁移细胞。
4. Matrigel基质胶中检测AOPPs对IMECs再血管化能力的影响
将缺乏生长因子的高浓度Matrigel基质胶在冰上过夜融化,均匀铺于无菌的96孔板上(70μ L/孔),放于温箱中30min成胶。将5000大鼠胰岛微血管内皮细胞用无血清培养液种植于每孔中,分别加入RSA、不同浓度的AOPPs(100μg/mL,200μg/mL,300μg/mL)以及apocynin。置于37℃、C02培养箱中作用48小时,低倍镜下倒置显微镜照相,每孔随机取3个视野。
5. AOPPs对胰岛微血管内皮细胞NADPH氧化酶活性的影响
各组细胞干预48h后制备细胞匀浆,测定细胞匀浆蛋白含量,将各组100μg细胞蛋白加至96孔测量板中,各孔加入暗适应的光泽精5μ M及底物NADPH100μM。用VICTOR1420多标记分析系统动态检测各组NADPH依赖的02-产生的荧光量,间接反映02-的产量及NADPH氧化酶活性。所测得的数据用“CPS”表示,即每秒通过光子数：counts per second.
6. Western blotting法检测AOPPs对IMECs细胞p-Akt、p-eNOS的表达
取同步生长的胰岛微血管内皮细胞,依次给予阴性对照组(200μg/mLRSA)、AOPPs100μg/mL、AOPPs200μg/mL、AOPPs300μg/mL以及Apocynin干预,作用时间为48小时。具体实验步骤和方法同第一部分。
7. AOPPs对胰岛微血管内皮细胞NO水平的影响
按照碧云天生物科技公司一氧化氮检测试剂盒的标准操作流程测定细胞内NO的水平,DMEM10%FBS稀释标准品,在96孔板中加入50μl/孔的标准品及细胞培养液上清,依次在各孔中加入室温Griess Reagent I, Griess Reagent II,540nm测定吸光度。
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AOPPs对IMECs细胞增殖活力的影响
100、200、300μg/mLAOPPs作用IMECs48h后,OD值分别为1.02±0.06,0.75±0.07,0.55±0.08,均显著低于RSA阴性对照组(1.29±0.08),P=0.000。200μg/mLAOPPs在给予NADPH氧化酶阻断剂apocynin预处理后细胞活力显著增加为(1.22±0.12),与单独AOPPs处理组相比差异具有统计学意义(均P<0.05)。 AOPPs的浓度(100,200,300μg/mL)与IMECs的OD值存在显著负相关关系(r=-0.949,P=0.000)。
2.比较AOPPs不同作用时间对细胞增殖活力的影响
200μg/mL AOPPs作用时间(12h、24h、48h、72h)后的细胞OD值分别为1.11±0.08,0.96±0.07,0.75±0.07,0.53±0.11,均显著低于control组的OD值(1.29±0.08)(vs. control, P=0.000). AOPPs作用时间与细胞活力间存在显著负相关关系(r=-0.972,P=0.000)。
3. AOPPs对胰岛微血管内皮细胞迁移能力的影响
AOPPs100、200、300μg/mLAOPPs作用IMECs24h后,其穿透至下室的细胞数量分别为266.00±11.14,184.00±9.17,135.00±10.44,均显著低于空白对照、RSA阴性对照组(P=0.000)。Apocynin预处理组穿透至下室的细胞数量明显增加(305.67±12.22),与单独AOPPs处理组相比差异具有统计学意义(均P<0.05)。AOPPs的浓度(100、200、300μg/mL)与IMECs迁移能力存在显著负相关关系(r=-0.972,P=0.000)。
4. AOPPs对IMECs细胞Matrigel基质中形成三维结构能力的影响
AOPPs100、200、300μg/mLAOPPs作用IMECs48h后,细胞在Matrigel基质胶中形成三维结构的数量分别为7.33±0.58,5.67±0.57,3.33±0.57,均显著低于空白对照、RSA阴性对照组(均P<0.05)。 Apocynin预处理组形成三维结构的数量明显增加(9.33±1.53),与单独AOPPs处理组相比差异具有统计学意义(均P<0.05)。AOPPs的浓度(100、200、300μg/mL)与IMECs的迁移能力存在显著负相关关系(r=-0.978,P7=0.000)。
5. AOPPs对胰岛微血管内皮细胞NADPH氧化酶活性的影响
随着AOPPs浓度的增加,NADPH依赖性的02-的产量逐渐增强,300μg/ml时达高峰。AOPPs (100、200、300μg/mL)干预组与空白对照、RSA阴性对照及apocynin阻断剂组相比具有统计学差异(F=59.389,P=0.000)=AOPPs的浓度(100、200、300μg/mL)与NADPH氧化酶活性存在显著负相关关系(r=-0.949,P7=0.000)。
6. AOPPs对IMECs中p-Akt、p-eNOS表达的影响
各组间p-Akt、p-eNOS的表达具有统计学差异(F=32.631,P=0.000)和(F=21.791,P=0.000). p-Akt、p-eNOS的表达随AOPPs干预浓度的增加而显著降低,与RSA对照组相比具有统计学差异(均P<0.05)。而在给予NADPH氧化酶阻断剂apocynin后二者表达量显著增加,与无apocynin干预组相比具有统计学差异。AOPPs的浓度(100,200,300μg/mL)与p-Akt、p-eNOS表达存在显著负相关关系(r=-0.949,P=0.000)和(r=-0.926,P=0.000)。
7. AOPPs对胰岛微血管内皮细胞内NO水平的影响
各处理组细胞内NO水平具有统计学差异(F=27.649,P=0.000),其中AOPPs (100,200,300μg/mL)干预后细胞内NO水平显著降低,分别为15.13±1.48,12.14±1.44,8.87±1.74,与空白对照(19.56±0.81)及RSA对照(18.64±1.36)相比具有统计学差异(均P<0.05)。给予Apocynin预处理的细胞内NO水平则显著增加为(17.89±1.25),与单独AOPPs干预相比差异具有统计学意义。AOPPs的浓度(100,200,300μg/mL)与IMECs的细胞内NO水平存在显著负相关关系(r=-0.896,P=0.000)。
结论
AOPPs干预可显著降低胰岛微血管内皮细胞增殖能力、迁移能力及再血管化能力,增强了细胞内NADPH氧化酶的活性,显著降低了细胞内磷酸化Akt及eNOS的表达水平以及细胞内NO的水平。我们推测：一方面AOPPs可通过下调PI3K/Akt通路中磷酸化Akt及eNOS的表达水平,减少细胞NO的水平；另一方面可通过增强NADPH氧化酶活性加强了NADPH氧化酶介导的eNOS脱偶联导致细胞内NO水平的下降。细胞内NO水平的降低最终导致胰岛微血管内皮细胞的损伤及功能紊乱。
第三部分GLP-1对AOPPs诱导的IMECs细胞损伤及功能紊乱的保护作用及机制
目的
研究GLP-1对AOPPs诱导的IMECs细胞凋亡、增殖、迁移及再血管化能力的保护作用及其对PI3K/Akt及其下游分子信号通路的影响。
方法
1.实验分组
分4组,依次为阴性对照组(200μg/mLRSA)、AOPPs200μg/mL、AOPPs200μg/mL+100nmol/LGLP-1组、AOPPs200μg/mL+100nmol/L GLP-1+10μmol/LLY294002组(P13K特异性抑制剂LY294002预处理1h后方加入AOPP-RSA和GLP-1)。用无血清培养基稀释各种药物储存液,作用一定时间后收集各组细胞。其中观察细胞迁移能力时为排除细胞凋亡影响我们选取干预时间为24h,而观察细胞凋亡、增殖能力及血管化能力时则选取干预时间为48h。
2.Annexin V-FITC/PI双染色法检测细胞凋亡(方法同第一部分)
3.CCK-8检测细胞增殖活性(方法同第二部分)
4. Transwell小室检测GLP-1对AOPPs诱导的IMECs体外迁移能力
方法同第二部分
5. Matrigel基质胶中检测GLP-1对AOPPs诱导的IMECs再血管化能力的影响(方法同第二部分)
6.DHE荧光探针检测细胞内活性氧(ROS)(方法同第一部分)
7. Western blotting法检测IMECs细胞RAGE、Bc1-2、Bax、p-Akt, p-eNOS蛋白的表达(方法同第一、二部分)
8.GLP-1对胰岛微血管内皮细胞内NO水平的影响(方法同第二部分)统计学处理
采用SPSS13.0进行统计分析,计量实验数据以均数±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.GLP-1对AOPPs诱导的IMECs细胞凋亡的影响
各处理组间IMECs细胞的凋亡率有显著差异(F=43.433,P=0.000)。200μg/mL AOPPs作用IMECs48h后,凋亡率为25.05±3.46,显著高于RSA对照组8.57±0.86(P<0.05)。在给予GLP-1(100nmol/L)后细胞凋亡率显著降低(P<0.05)。而提前给予PI3K抑制剂LY294002预处理后再给予AOPPs及GLP-1干预的细胞组其细胞凋亡率显著增加(P<0.05)。
2.GLP-1对AOPPs诱导的IMECs细胞增值活性的影响
AOPPs200μg/mL诱导胰岛微血管内皮细胞48h后的OD值为0.65±0.08,与RSA对照组相比,有显著差异(P=0.000)。给予GLP-1联合处理组的细胞OD值为1.13±0.14,显著高于AOPPs单独干预组(P=0.001)。LY294002预处理组的OD值为0.72±0.10,与AOPPs+GLP-1组相比显著降低(P=0.001),LY294002预处理可部分阻断GLP-1恢复细胞活力的效应。
3.GLP-1对AOPPs诱导的IMECs细胞迁移能力的影响
在给予AOPPs200μg/mL刺激24小时后,由Transwell上室穿透至下室的细胞数量显著减少196.33±15.50,与RSA对照组(306.00±13.11)相比具有统计学差异(P<0.05)。在给予GLP-1(100nmol/L)后发生迁移的细胞数量显著增加(296.67±17.62)(P<0.05)。而提前给予PI3K抑制剂LY294002预处理后再给予AOPPs及GLP-1干预的细胞组其迁移细胞率则显著减少(216.00±21.07)(P<0.05)。
4.GLP-1对AOPPs诱导的IMECs在Matrigel基质中形成三维结构的影响
AOPPs200μg/mL干预48小时组细胞形成三维结构的数量(4.67±0.58)与RSA阴性对照(9.33±0.58)相比显著减少,差异具有统计学意义(P=0.000)。在给予GLP-1(100nmol/L)后形成三维结构的数量显著增加(8.00±1.00)(P<0.05)。而提前给予PI3K抑制剂LY294002预处理后再给予AOPPs及GLP-1干预的细胞形成三维结构的数量则未见显著增加(6.00±1.00)(P<0.05)。
5.GLP-1对AOPPs诱导的IMECs细胞NADPH氧化酶活性的影响
AOPPs200μg/mL显著增加胰岛微血管内皮细胞NADPH依赖性的O2-的产量(367.67±21.94),与RSA对照组相比,有显著差异(P=0.000)。给予GLP-1联合处理后NADPH依赖性的O2-的产量显著减少(158.00±13.00),差异具有统计学意义。LY294002预处理组其细胞NADPH依赖性的O2-的产量与单独给予GLP-1干预组相比又显著增加(348.33±18.23),P<0.05。
6.GLP-1对AOPPs诱导的对IMECs内活性氧(ROS)的影响
AOPPs显著增加了细胞内ROS的水平,其荧光强度为(53.85±4.15)与RSA对照相比(24.37±2.84)具有统计学差异(P=0.000)。GLP-1(100nmol/L)干预组细胞内ROS水平显著下降为(30.02±5.12),而提前给予LY294002预处理组细胞内ROS水平又显著升高(48.65±6.31),均P<0.05。
7.GLP-1对AOPPs诱导的IMECs细胞RAGE表达的影响
RSA对照组其蛋白水平为(0.33±0.05), AOPPs干预后显著增加了细胞RAGE表达的水平(0.60±0.09)与RSA对照组相比具有统计学差异(P<0.05)。GLP-1(100nmol/L)干预可显著下调细胞RAGE表达水平(0.37±0.06)(P=0.004),而提前给予LY294002预处理并不能阻断GLP-1下调RAGE表达的作用(0.39±0.08),均P>0.05。
8.GLP-1对AOPPs诱导的IMECs f中bcl-2、Bax、p-Akt、p-eNOS的表达AOPPs200μg/mL干预显著降低了抗凋亡蛋白bcl-2、p-Akt、p-eNOS的表达,而增加了促凋亡蛋白Bax蛋白的表达,与RSA对照组相比差异具有统计学意义(均P<0.05)。 GLP-1干预后可显著增加bcl-2、p-Akt、p-eNOS表达而降低Bax表达(均P<0.05)。LY294002预处理可显著抑制GLP-1介导的bcl-2、p-Akt、 p-eNOS的表达增加以及Bax表达降低(均P<0.05)。
9.GLP-1对AOPPs诱导的IMECs细胞内NO水平的影响
AOPPs200μg/mL干预组显著降低了细胞内NO的水平(10.05±1.67),与RSA对照组(18.85±1.87)相比具有统计学差异,P<0.05。GLP-1(100nmol/L)干预后显著升高了细胞内NO的水平(17.35±1.39),与单纯AOPPs干预组相比差异具有统计学意义(P<0.05)。而PI3K抑制剂LY294002的预处理则显著削弱了GLP-1的这一保护性调节作用,而使细胞内NO的水平显著降低(10.93±1.45)。
结论
本部分实验首次证明GLP-1可降低AOPPs导致的胰岛微血管内皮细胞的凋亡,显著改善被AOPPs损伤的胰岛微血管内皮细胞的增殖、迁移及再血管化能力。GLP-1改善胰岛微血管内皮细胞功能损伤及紊乱的作用主要通过以下两方面途径来完成：一是通过下调RAGE受体表达、抑制NADPH氧化酶活性、减少细胞内活性氧而改善了氧化应激损伤及NADPH氧化酶介导的eNOS脱偶联,升高了细胞内NO的水平；二是激活PI3K/Akt信号转导通路,上调抗凋亡蛋白Bcl-2表达,减低促凋亡蛋白Bax表达,增加磷酸化内皮型一氧化氮合成酶(p-eNOS)表达升高细胞内NO水平,最终减少内皮细胞凋亡并改善内皮细胞功能紊乱。
Background
The prevalence rate of diabetes mellitus (DM) is rising year and year, and has become the third chronic non-communicable diseases like cardiovascular disease and tumor which were the global threat of human health. With the improvement of people's living and the changes of life style, the morbidity of type2diabetes (T2DM) presents a tendency of rapid increasing in our country. According to the latest result from epidemiological survey in2013, the estimated prevalence of DM was11.6%which meant there were about1.139hundred million patients in adults of18years old and above diagnosed with the newest international clinical criteria. The more amazed result was that the prevalence of impaired glucose tolerance (IGT) was up to50.1%in Chinese adult and the onset of DM became younger in age. Acute and chronic complications caused by DM, specially the diabetic macro-and micro-vascular complications, become the main cause of deaths and disabilities and severely impact the quality of life and lifetime.
Insulin resistance (IR) and β cells hypofunction are two major reasons for T2DM. Current treatment includes promoting insulin secretion, improving insulin resistance, inhibiting the absorption of glucose and inhibiting hepatic glucose output. These treatments still cannot stop progressive failure of β cells function with the course of disease proceeding, even they can improve the blood glucose. In recent years, more studies supposed that micro-circulation disorder of β cells was closely related to the injury and hypofunction of (3cells. Islet is a organ of high vascularity. Islet capillaries are not only the crucial link between islet cells and the body, but also the microenvironment for cells to live by. Islet has unique microvascular system which contains intensive capillary network with the density of5times as peripheric exocrine organization. The abundant blood supply could not only satisfy its need for oxygen and nutrition, but also could regulate the metabolic balance in the body when it rapidly percept the changes of metabolic environment and distribute various hormones to relevant target tissue via blood circulation. Therefore the microcirculatory system is a important regulator for islet endocrine function. The intact structure and function of islet microcirculation depend on the islet microvascular endothelial cells (IMECs) which have caused researcher's concern for these years. IMECs, the most elements of islet microcirculation-tissue barriers, have a close relationship with islet endocrine function and directly participate in the development of islet hypofunction. With the effect of islet capillaries in the pathogenesis of T2DM being revealed, Tal MG proposed that β cells hypofunction was probably the representation of diseased islet capillaries in2009. He said that the dysfunction and injury of IMECs was the important factor resulting in the disease of islet capillaries, which could cause anoxia and ischemic injury in islet microcirculation and eventually cause progressive dysfunction of β cells. Therefore, to deeply study the physiopathologic mechanism in dysfunction and injury of IMECs and the effective treatment has exerting great impact on protecting the function of β cells and looking for new targets to prevent T2DM.
The dysfunction and injury of vascular endothelial cells (VEC) is the precondition and basis of the development in diabetic microvascular complications. Glucotoxicity, lipotoxicity, the change of hemarheology and haemodynamics and reduced antioxidant capacity of tissue are closely linked to dysfunction of vascular endothelial cells. The vasodilator factor NO reduce synthesis and the vasoconstrictor factor endothelin-1increase production, which can directly stimulate the hyperplasia of intimal arterial smooth muscle and the expression of fibronectin and collagen IV. In the state of hyperglycemia, the synthesis and release of intercellular adhersion molecule1(ICAM-1) and vascular cell adhesion molecule1(VCAM-1) increased by VECs, which could enhance the adherence of leucocyte and platelet resulting in activation of inflammatory cells and intimal inflammation. The change could induce medial smooth muscle cells migrating under intima and lipid depositing in cells, which accelerated formation of arterial atherosclerosis. As a kind of VECs, IMECs should be damaged by different pathogenic factors In the state of hyperglycemia, which could result in dysfunction of endothelial cells and islet microcirculation, specially the dysfunction of islet cells.
Advanced oxidative protein products (AOPPs), considered as a novel marker of oxidant-mediated protein damage, are dityrosine-containing and cross-linking protein products mainly formed during oxidative stress by reaction of plasma albumin with chlorinated oxidants. AOPPs were firstly found in the plasma of patients undergoing dialysis and were subsequently found in subjects with diabetes. The structure and biological function of AOPPs was similar to advanced glycation end products (AGEs) which could combine with RAGE (receptor of AGEs) to activate NADPH oxidase, intra-cellular signaling of redox reaction such as ERK1/2, p38MAPK and translocation of nuclear factor-KB resulting in inflammation and oxidative stress in VECs. Chronic accumulation of AOPPs aggravated accumulation of oxidized low density lipoprotein (LDL) in arterial wall and worsen inflammation and oxidative stress in artery to accelerate atherosclerosis in a hyperlipidemic model. The level of AOPPs closely correlated with the degree of carotid atherosclerosis in patients with CKD, which meant that accumulation of AOPPs could play a role in atherosclerosis of patients with CKD. Gradinaru D and his team discussed the relation between level of AOPPs and the factors leading to atherosclerosis such as endothelium NO synthetase, oxidative stress and metabolic profiling in patients with diabetes or prediabetes. They found that AOPPs significantly increased compared with normal control group and inhibited activity of endothelium NO synthetase to reduce synthesis of NO, which suggested that the level of AOPPs closely correlated with the degree of diabetic atherosclerosis in old patients with DM. Bansal S and team conducted an analysis of gene polymorphism of RAGE and its relation with oxidative stress products, paraoxonase and diabetic macro-vascular complications in265Indian patients with T2DM. They found that there existed a positive correlation between the genotype of429T/C and level of oxidative stress products, development of diabetic macro-vascular complications. Thus, apart from being regarded as oxidative stress makers, AOPPs has also been shown to be a pro-inflammatory factor in accelerating the progress of atherosclerosis and damaging vascular system in our body. However, few studies aimed at the effect of AOPPs in islet microcirculation, especially in the function of IMECs.
GLP-1is a polypeptide secreted by the intestinal L cell. GLP-1is best known as a safe and effective insulinotropic hormone and has been proposed as prospective approach to clinical treatment of T2DM. More and more studies indicated that GLP-1not only stimulated survival and proliferation, increased insulin secretion, inhibited appetite, retarded peristalsis of gastrointestinal smooth muscle and inhibited release of glucagon, but also plays an important role in diabetic cardiovascular complications. The receptor of GLP-1(GLP-1R) expressed in vascular smooth muscle, cardiac muscle, endocardium and coronary endothelium. The agonist of GLP-1R could protect cardiac muscle from ischemic-reperfusion and improved the function of heart. On one hand, exendin-4significantly reduced the area of myocardial infarction in vitro, on the other hand, exendin-4and GLP-1could enhance left ventricular systolic function. Timmers and his team also found that exendin-4sustaining treatment could significantly reduce the area of myocardial infarction and promote the recovery of myocardial contractive and systolic function in a pig model after ligation of coronary artery. GLP-1could inhibit monocyte from adhering to aortic vascular endothelial cells, alleviate vascular inflammatory injury and stop the progress of AS. Supplementing GLP-1in rats with IR and hypertension could improve the function of vascular endothelium, blood pressure and cardiac function. In the clinical study, GLP-1could improve the vascular endothelial function of patients with T2DM and stable CAD. In recent years, researchers suggested that GLP-1significantly blocked the oxidative stress of endothelial cell induced by AGEs-RAGE through the down-regulation of RAGE and ICAM-1and reduced intimal hyperplasia and smooth muscle hyperplasia after the injury of endothelial cells induced by AGEs in vitro, which meant that GLP-1had potential effect of anti-inflammatory and anti-atherosclerosis. Exendin-4promoted the proliferation of endothelial cells through PI3K/Akt-eNOS signal pathway. In the study of rats model with endothelial cell dysfunction and AS, liraglutide, GLP-1receptor agonist, could improve dysfunction of VECs and alleviate the degree of AS. In2012, chinese researchers found that GLP-1could alleviate the apoptosis of human umbilical vein endothelial cells (HUVECs) induced by AGEs through activation of PI3K/Akt signal pathway by the means of regulating the expression of Bcl-2, Bax and cyto-c in the endothelial cells. In2013, Erdogadu.O and his team conducted a study about the effect of GLP-1in human coronary artery endothelial cells (HCAECs) induced by lipotoxicity, and found that GLP-1could improve the proliferation, metastasis and vasculogenesis of aortic endothelial cells and reduced the apoptosis through PKA/PI3K/Akt/eNOS, p38MAPK and JNK kinase-dependent pathway.
Thus, apart from the hypoglycemic effect, GLP-1also play a important role in protecting endothelial cells and improving the function of them, which through PKA/PI3K/Akt pathway. However, did GLP-1have the same protective effect in the injury of IMECs induced by AOPPs under the state of DM? Did it can improve the function of IMECs and islet microcirculation so that delay the progressive dysfunction of Pcells? All of these are important issues for our discussion.
It is very important to investigate the effects of GLP-1on islet microcirculation and its possible mechanisms for discovering a new therapeutic target to protect the function of islet cells in T2DM patients. In this study, we established the damaged model by treating IMECs with AOPP-modified RSA (AOPP-RSA), in order to explore the effects and related mechanisms of GLP-1in the apoptosis, proliferation, migration and revascularization of IMECs induced by AOPPs, and try to provide a new viewpoint for GLP-1treatment which could delay the progressive dysfunction of β-cells.
Objectives
In this project we aim to on the base of damaged model induced by AOPPs, to observe the effects of GLP-1on the apoptosis, proliferation, migration and revascularization of IMECs induced by AOPPs, and to investigate the effect of GLP-1on apoptosis-relative proteins expression and PI3K/Akt/eNOS pathway, also to discuss the protective role of GLP-1and its molecular mechanisms in the dysfunction of IMECs induced by AOPPs.
Content
The whole project includes three parts:
Part I The effect of AOPPs on the apoptosis of islet microvascular endothelial cells (IMECs) and its possible mechanism
Objectives
To explore the effect and mechanism of AOPPs on the apoptosis of IMECs
Methods
1. Preparation of AOPP-RSA
20mg/ml rat serum albumin (RSA) was mixed with the same volume of hypochlorous (40mmol/1), and then was placed for30min. The molar ratio of RSA and hypochlorous in AOPP was1:140. Excessive dissociative hypochlorous was removed by dialysis with non-endotoxin PBS for24h. The solution was sterilized by passing it through a0.22micron bacteria-retentive filter.
2. Isolation and digestion of rat islet cells
We used a modified method of collagenase digestion and Ficoll density gradient separation for isolation and digestion of islet from the rat. The cells were stained with DTZ and typan blue, and the concentration of cells was adjusted to500IEQ/ml. These cells were cultured at34℃in a humidified atmosphere of5%CO2.
3. Purification and identification of IMECs
The growth of spindle cells in the islet was observed after culturing3-5days in vitro and the proliferation of cells was observed in7-9days. IMECs which were carried by the2%gelatin were cultured after immunomagnetic beads. These cells were identified for von Willebrand factor (vWF) antigen and Dil-acetylated low density lipoprotein (Dil-Ac-LDL) by immunofluorescence.
4. Groups:
The effect of different concentrations of AOPPs on the apoptosis of IMECs: AOPPs-RSA treated the cells at concentrations of100,200,300μg/mL for48h. The effect of AOPPs on the apoptosis of IMECs for different time:AOPPs-RSA alone (final concentration200μg/mL) incubated with cells for0,12,24,48,72h respectively.
5. The apoptosis of IMECs with Hoechst33258dye staining and AnnexinV-FITC/PI
Cells were treated with4%paraformaldehyde for5min in4℃after indicated treatment. The morphology of cells apoptosis were observed by fluorescence microscope after Hoechst33258dye staining. Cells were added AnnexinV and PI after the digestion with trypsin, and then placed for15min protecting from light. The early apoptosis rate was detected with flow cytometry and analyzed by Cell Quest software.
6. The ROS level in IMECs detected by DHE fluorescent probe
The cells were divided into4groups:normal control group, RSA control group,200μg/ml AOPPs group and200μg/ml AOPPs+apocynin group, and all the groups were accepted identified treatment for48h. Cells were collected and cultured in the solution of DHE at37℃for30min. After the removal of too much solution, the distributions of DCF in cells were observed by laser scanning confocal microscope. All the groups were selected3horizons to be scanned and analyzed the relative intensity of fluorescence.
7. The expression of BcI-2and Bax in IMECs with Western blot
The total protein of all groups were extracted and detected by BCA method. After electrophoresis in12%SDS-PAGE, transfer, blocking and washing, all the groups were incubated with anti-Bcl-2monoclonal antibody(1:1000) and anti-Bax monoclonal antibody(1:2000) at4℃over night. Cells were conducted hybridization with secondary antibodies labeled with horseradish peroxidase for1h and coloration, and the results were analyzed by ImageJ software.
8. The activities of caspase-9and-3were detected by ELISA
Cells of all the groups were accepted identified treatment for48h and then were collected. Cells were then added to the reaction substrate correspondingly. Incubated in37℃water bath for2h, finally using ELISA meter measured OD values at the detection wavelength of405nm.
Statistical Analysis
All analyses were carried out with SPSS13.0software. Data were expressed as mean±standard deviation (SD). Differences between groups were tested by one-way ANOVA followed by a LSD test. We used Spearman correlation analysis for correlation analysis. Statistical significance was defined as two-sided P<0.05.
Results
1. The effect of different concentration of AOPPs on the apoptosis of IMECs for48h
After treatment with100,200,300μg/ml AOPPs for48h, the apoptosis rate were14.57±4.7,24.20±4.30,35.53±6.47, respectively, and significantly higher than those of normal control group (5.87±1.51) and RSA control group (6.90±1.42)(P<0.05). Spearman correlation analysis showed that a significant positive correlation existed between the apoptosis rate of IMECs and the AOPPs concentrations (100,200,300ug/ml)(r=0.949, P=0.000).
2. The effect of AOPPs on the apoptosis of IMECs for different time
The apoptosis rate of200μg/ml AOPPs incubated for different time (12,24,48,72h) were11.61±1.94,16.69±3.17,23.48±4.69,32.80±7.13, respectively, and significantly higher than control group (0h)(6.10±2.07)(F=17.854, P=0.000). ntrol group (5.87±1.51) and RSA control group (6.90±1.42)(P<0.05). Spearman correlation analysis showed that a significant positive correlation existed between the apoptosis rate of IMECs and time (r=0.950, P=0.000).
3. The effect of AOPPs on ROS level of IMECs
The fluorescence value of ROS in200μg/ml AOPPs group was58.43±2.71, and significantly higher than that of RSA control group (27.49±4.75)(P=0.000), which suggested that AOPPs stimulated the prodiction of ROS in IMECs. Treated IMECs with apocynin (inhibitor of NADPH oxidase) for48h, the fluorescence value of ROS was significantly down to30.45±4.35compared with the200μg/ml AOPPs group (P=0.000).
4. The effect of AOPPs on the expression of Bcl-2, Bax of IMECs
After identified treatment for48h, the expressions of Bcl-2were significantly different among the groups (F=4.675, P=0.036). The expression of Bcl-2in200μg/ml AOPPs group was0.60±0.09, which significantly lower than that of RSA control group (P=0.021). Treated IMECs with apocynin (inhibitor of NADPH oxidase) for48h, the expression of Bcl-2was significantly up to0.87±0.14compared with the200μg/ml AOPPs group (P=0.033). The expression of Bax in200μg/ml AOPPs group was1.35±0.19, which significantly higher than that of RSA control group (P=0.008). Treated IMECs with apocynin, the expression of Bax was significantly down to0.97±0.15compared with the200μg/ml AOPPs group (P=0.021).
5. The effect of AOPPs on the activities of caspase-9,-3in IMECs
After identified treatment for48h, the activities of caspase-9,-3were significantly different among the groups (F=144.224and311.617, P=0.000and P=0.000). The activities of caspase-9,-3were1.24±0.08and1.01±0.04, which significantly higher than those of normal control group (P<0.05) and RSA control group (P<0.05). Pretreatment IMECs with apocynin, the activities of caspase-9,-3were down to 0.45±0.06and0.40±0.04compared with the200μg/ml AOPPs group (P<0.05and P<0.05).
Conclusions
Apoptosis was induced by AOPPs in a dose, time-dependent manner. Exposure to AOPPs for48h caused a significant increase in ROS generation, expression of Bax and activities of caspase-9and-3, but a decrease in expression of Bcl-2. However, co-incubation with apocynin (inhibitor of NADPH oxidase) attenuated these AOPPs-induced effects in IMECs. Therefore, we speculated that AOPPs aggravated the oxidative stress injury through activity of NADPH oxidase and increasing the level of ROS, and then induced the apoptosis of IMECs by the regulation of Bcl-2/Bax, caspase-9and-3.
Part Ⅱ The effect and mechanism of AOPPs on the proliferation, migration activity and revascularization of IMECs
Objectives
To explore the effect of AOPPs on the proliferation, migration activity and revascularization of IMECs, and then to investigate the mechanism of AOPPs on the injury and dysfunction of IMECs
Method
1. Group:
The effect of different concentrations of AOPPs on the proliferation, migration activity and revascularization of IMECs:the cells were divided into6groups:normal control group, RSA control group,100μg/ml AOPPs group,200μg/ml AOPPs group,300μg/ml AOPPs group and200μg/ml AOPPs+apocynin group. We chose24h as intervention time to observe the migration activity of IMECs excluding the effect of apoptosis and48h to observe the the proliferation and revascularization of IMECs.
The effect of AOPPs on the proliferation of IMECs for different time:200μg/ml AOPPs alone incubated with cells forO,12,24,48,72h respectively.
2. The proliferation activity of IMECs with CCK-8method
The cells were planted in96-well plates. After each experimental treatment, we added10μl cell counting kit-8solution into each hole, and incubated at37℃in a humidified atmosphere of5%CO2for1h. The OD value was detected by ELISA with wavelengths of450nm to reflect the proliferation activity.
3. The effect of AOPPs on the migration activity of IMECs with Transwell assay
The cells were cultured in serum-free medium for12-24h and the density of cells was adjusted to1×105. The upper-well was added in200μl cell suspension of IMECs, RSA, different concentration of AOPPs (100μg/ml,200μg/ml,300μg/ml) and apocynin, and the lower-well was added in600μl DMEM with10%fetal bovine serum. The cells were cultured at37℃for24h. After the fixation and staining, we counted the cells and gently erased the non-migrated cells in the upper-well by swab.
4. The effect of AOPPs on the revascularization of IMECs with matrigel
The high concentration of matrigel was placed in96-well plates (70μl/hole) and formed into gel in incubator for30min after melting over night. The IMECs were planted in each hole with serum-free medium and added in RSA, different concentration of AOPPs (100μg/ml,200μg/ml,300μg/ml) and apocynin. The cells were incubated at37℃in a humidified atmosphere of5%CO2for48h and detected by invert microscope with3views for each hole.
5. The effect of AOPPs on the activity of NADPH oxidase in IMECs
After each experimental treatment for48h, we prepared the cell homogenate and detected the protein content in them.100μg proteins of each group were planted in96-well plates, and added in5μm lucigenin and100μm substrate of NADPH. The fluorescence quantum produced by NADPH-dependent O2-was detected with VICTOR1420multi-label analyzer, which indirectly reflected the production of O2-and activity of NAPDH oxidase. The data were expressed as counts per second (CPS).
6. The effect of AOPPs on the expression of p-Akt and p-eNOS in IMECs with Western blot
The cells were treated with200μg/ml RSA and different concentration of AOPPs (100,200,300μg/ml) and apocynin for48h. The detailed experimental procedure and methods referred to part1.
7. The effect of AOPPs on the level of NO in IMECs
According to standard operating procedure of NO detection kit produced from Beyotime, we detected the level of NO in the cells. The standard substance was diluted with DMEM and10%FBS, and placed50μl of each hole in96-well plates with the supernatant. The absorbance was detected by the wavelength of540nm after adding Griess Reagent I and Griess Reagent Ⅱ at room temperature.
Statistical Analysis
All analyses were carried out with SPSS13.0software. Data were 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 AOPPs on the proliferation of IMECs
After treatment with100,200,300μg/ml AOPPs for48h, the OD value were1.02±0.06,0.75±0.07,0.55±0.08, respectively, and significantly lower than that of RSA control group (1.29±0.08)(P=0.000). Pretreated IMECs with apocynin, the OD value was up to1.22±0.12compared with the200μg/ml AOPPs group (P<0.05). A significant negative correlation existed between the OD value of IMECs and the AOPPs concentrations (100,200,300μg/ml)(r=-0.949, P=0.000).
2. The effect of AOPPs on the proliferation of IMECs for different time
The OD values of200μg/ml AOPPs incubated for different time (12,24,48,72h) were1.11±0.08,0.96±0.07,0.75±0.07,0.53±0.11, respectively, and significantly lower than that of RSA control group (1.29±0.08)(P=0.000). A significant negative correlation existed between the proliferation of IMECs and time (r=-0.972, P=0.000).
3. The effect of AOPPs on the migration activity of IMECs
After treatment with100,200,300μg/ml AOPPs for48h, the cell counts of penetrating to lower-well were266.00±11.14,184.00±9.17,135.00±10.44, respectively, and significantly lower than those of nomal control group and RSA control group (P=0.000and P=0.000). Pretreated IMECs with apocynin, the cell count of penetrating to lower-well was up to305.67±12.22compared with the200μg/ml AOPPs group (P<0.05). A significant negative correlation existed between the migration activity of IMECs and the AOPPs concentrations (100,200,300μg/ml)(r=-0.972, P=0.000).
4. The effect of AOPPs on the capacity of forming3d structure of IMECs in matrigel
After treatment with100,200,300μg/ml AOPPs for48h, the counts of forming3d structure of IMECs in matrigel were7.33±0.58,5.67±0.57,3.33±0.57, respectively, and significantly lower than those of nomal control group (P<0.05) and RSA control group (P<0.05). Pretreated IMECs with apocynin, the count of forming3d structure in matrigel was up to9.33±1.53compared with the200μg/ml AOPPs group (P<0.05). A significant negative correlation existed between the migration activity of IMECs and the AOPPs concentrations (100,200,300μg/ml)(r=-0.978, P=0.000).
5. The effect of AOPPs on the activity of NADPH oxidase of IMECs
The production of NADPH-dependent O32-increased with the increase of AOPPs concentration, and the300μg/ml AOPPs group reached the peak. The productions of NADPH-dependent O2-were significantly different among the groups (F=59.389, P=0.000). A significant negative correlation existed between the activity of NADPH oxidase of IMECs and the AOPPs concentrations (100,200,300μg/ml)(r=-0.949, P=0.000).
6. The effect of AOPPs on the expression of p-Akt and p-eNOS of IMECs
The expressions of p-Akt and p-eNOS of IMECs were significantly different among the groups (F=32.631, P=0.000) and (F=21.791, P=0.000). With the AOPPs concentration increasing, the expression of p-Akt and p-eNOS significantly decreased, compared with those of RSA control group (P<0.05) and (P<0.05). Treated IMECs with apocynin, the expressions of p-Akt and p-eNOS significantly increased compared with other groups. A significant negative correlation existed between the expression of p-Akt, p-eNOS and the AOPPs concentrations (100,200,300μg/ml)(r=-0.949, P=0.000) and (r=-0.926, P=0.000).
7. The effect of AOPPs on the level of NO in IMECs
The levels of NO of IMECs were significantly different among the groups (F=27.649, P=0.000). After treatment with100,200,300μg/ml AOPPs for48h, the levels of NO were15.13±1.48,12.14±1.44,8.87±1.47, respectively, and significantly lower than those of nomal control group (19.56±0.81)(P<0.05) and RSA control group (18.64±1.36)(P<0.05). Pretreated IMECs with apocynin, the level of NO was up to17.89±1.25compared with the200μg/ml AOPPs group. A significant negative correlation existed between the levels of NO in IMECs and the AOPPs concentrations (100,200,300μg/ml)(r=-0.896, P=0.000).
Conclusions
AOPPs could significantly lower the proliferation, migration activity and revascularization of IMECs, enhanced the activity of NADPH oxidase, and decreased the expressions of p-Akt, p-eNOS and the level of NO. Therefore, we speculated that AOPPS decreased the level of NO not only through the down-regulation of p-Akt and p-eNOS in PI3K/Akt pathway but also via increasing the activity of NADPH oxidase to enhance eNOS uncoupling. More importantly, the decreased NO level induced the injury and dysfunction of IMECs.
Part III The protection of GLP-1and its mechanism against the damage and dysfunction of IMECs cells induced by AOPPs
Objective:
Our study investigated the protective effect of GLP-1on the apoptosis, proliferation, migration, the capacity of revascularization and the influence on PI3K/Akt and its downstream molecule signal pathway in IMECs cells induced by AOPPs
Method:
1. Group:
The whole experiment divided into negative control group (200μg/mLRSA), AOPPs200μg/mL group, AOPPs200μg/mL±100nmol/LGLP-1group, AOPPs200μg/mL+100nmol/L GLP-1±10μmol/L LY294002group (added APOO-RSA and GLP-1after preprocessing1hour by LY294002, the specific inhibitor of PI3K). Serum-free medium was used to attenuaate all kinds of stock solution, and then collected cells after effect for a certain time. We chose24hours as the intervention time in case to exclude the influence of apoptosis when we observed the capacity of migration, while we chose48hours as the intervention time when we observed the apoptosis, proliferation, and the capacity of revascularization.
2. Apoptosis detected by Annexin V-FITC/PI staining technique
The method was the same as we mentioned in the first part.
3. Cell proliferation activity detected by using CCK-8
The method was the same as we mentioned in the second part.
4. The effect of GLP-1on the migration in vitro of IMECs cells induced by AOPPs was measured by Transwell chamber
The method was the same as we mentioned in the second part.
5. The capacity of revascularization of IMECs cells detected by using Matrigel
The method was the same as we mentioned in the second part.
6. The ROS level in IMECs cells was measured by DHE fluorescence probe
The method was the same as we mentioned in the first part.
7. The expressions of RAGE, Bcl-2, Bax, p-Akt, p-eNOS were detected by Western blotting
The method was the same as we mentioned in the first and second part.
8. The NO level in islet microvascular endothelial cell
The method was the same as we mentioned in the first and second part.
Statistical analyses
Statistical analyse was performed using SPSS version13.0software. Quantitative data normality was assessed with the One-Way ANOVA test and is expressed as mean±SD. Comparisons between quantitative data were conducted using LSD tests. A value of p<0.05was considered statistically significant.
Results
1. The influence of GLP-1on the apoptosis in AOPPs induced IMECs cells
The apoptosis rate of IMECs cells have significant difference between treatment groups (F=43.433, P=43.433). Apoptosis rate was25.05±3.46%about48hours after200μg/mL AOPPs effect on IMECs cells, and was significantly higher than the RSA control group8.57±0.86%(P<0.05). The apoptosis rate was significantly lower (P<0.05) after giving the treatment of GLP-1(100nmol/L). The apoptosis rate increased significantly (P<0.05) in the group which was first pre-processed by PI3K inhibitors LY294002and then intervened by AOPPs and GLP-1.
2. The influence of GLP-1on the proliferation vitality in IMECs cells induced by AOPPs
The OD value of AOPPs200mu g/mL inducing pancreatic microvascular endothelial cells after48h was0.65±0.08, and was significant differences (P=0.000) compared with RSA control group. The OD value of the combined treatment group giving GLP-1was1.13±0.14, significantly higher than that of AOPPs individual intervention group (P=0.001). The OD value of LY294002pre-processing group was0.72±0.10, decreased significantly (P=0.001) compared with AOPPs±glp-1group, and suggested that LY294002pre-processing could partially blocks the effect of GLP-1to restore cell vitality.
3. The influence of GLP-1to the ability of migration in IMECs cells induced by AOPPs.
After stimulation by giving AOPPs200μg/mL for24hours, the quantity of cells which migrated from Transwell upper chamber to the lower chamber was196.33±15.50, and significantly reduced compared with RSA control group (306.00±13.11)(P<0.05). The quantity of cells which migrated after giving GLP-1(100nmol/L) was296.67±17.62, and was significantly increased (P<0.05). And the cell migration rate of the group first pre-processing by PI3K inhibitors LY294002and then intervened by AOPPs and GLP-1was significantly reduced (216.00±21.07)(P<0.05).
4. The influence of GLP-1on the three-dimensional structure which formated in the Matrigel in IMECs cells induced by AOPPs
The quantity of the three-dimensional structure was4.67±0.58in the group of AOPPs200μg/mL intervented48hours, and was significantly reduced compared with RSA negative control (9.33±0.58)the number of cells form a three-dimensional structure (4.67±0.58)(P=0.000). The quantity of the three-dimensional structure was significantly increased (8.00±1.00)(P<0.05) by giving GLP-1(100nmol/L). And the quantity of the three-dimensional structure of the group first pre-processing by PI3K inhibitors LY294002and then intervened by AOPPs and GLP-1did not see a significant increased (6.00±1.00)(P<0.05).
5. The influence of GLP-1on the activity of NADPH oxidase in IMECs cells induced by AOPPs
The NADPH dependent02-production in islet microvascular endothelial cells was (367.67±21.94) by giving AOPPs200μg/mL, and significantly increased compared with RSA control group (P=0.000). The NADPH dependent O2production of the combined treatment group giving GLP-1was significant
引文
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