生长素、肥胖抑素在动脉粥样硬化中的作用及机制研究
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摘要
生长素(Ghrelin)是迄今发现的唯一的生长激素促分泌剂受体(GHS-R)的内源性配体,在增强食欲,促进胃肠功能以及调节能量代谢等方面发挥重要的作用。近年来研究发现Ghrelin除了特异性的促进生长激素释放外,还与多种病理生理过程密切相关,如肥胖、心血管疾病、糖尿病及衰老。近来从大鼠的胃组织中成功分离出一段Ghrein相关肽命名为肥胖抑素(obestatin),研究证实Obestatin具有和Ghrelin相反的作用,Obestatin的发现进一步拓展了Ghrelin的相关领域,有研究报道Obestatin水平在冠心病体内升高,Obstatin可以加重离体心脏的缺血再灌注损伤。但迄今未见有关Ghrelin和Obestatin对动脉粥样斑块消退的影响的相关报道。
为此,本研究拟从以下几个方面探讨Ghrelin和Obestatin对动脉粥样硬化的影响:①离体研究Ghrelin和Obestatin对AngⅡ诱导的脐静脉内皮细胞NO释放,细胞内氧化应激、内皮细胞增殖和迁移、炎症因子产生的影响及信号通路研究;②在体研究Ghrelin对动脉粥样硬化小鼠动脉粥样硬化斑块和炎症因子以及血管壁NFκB的影响
第一章:生长素对人脐静脉内皮细胞功能影响及机制研究
目的:观察生长素对AngⅡ所致的脐静脉内皮细胞活性氧生成、NO释放,内皮细胞增殖、迁移、单核内皮细胞粘附和TNFα、IL-8、MCP-1水平和基因表达的作用以及涉及的信号通路。
方法:①采用不同浓度的AngⅡ(0(?)24小时)孵育HUVEC-12,获得最佳观察点,并观察(10~(-9)(?)10~(-6)mol/L)生长素对AngⅡ损伤的影响。②加入MAPK信号通路抑制剂PD98059(25μmol/L)、GHSRla受体阻断剂([Dys~3]GHRP-625μmol/L)、NFκB通路抑制剂PDTC1μmol/L预处理HUVEC-12细胞观察对生长素影响AngⅡ诱导的内皮细胞损伤的作用。采用硝酸还原酶法测细胞培养上清NO;WST-8法测细胞增殖;Transwell小室测细胞迁移;荧光探针法测细胞内的活性氧生成。RT-PCR法测细胞中TNFα、IL-8、MCP-1基因表达;ELISA法测细胞培养上清TNFα、IL-8、MCP-1浓度;Western blot测细胞中totalNFκBp65,ERK1/2、total Akt、p-Akt、P-NFκBp65,P-ERK1/2、eNOS蛋白表达。
结果:①AngⅡ呈浓度和时间依赖性地抑制HUVEC-12细胞分泌NO,生长素呈浓度依赖性地逆转AngⅡ对NO生成的减少。PD98059预处理对AngⅡ诱导的NO生成无影响,而[Dys~3]GHRP-6(25μmol/L)处理阻断生长素对NO生成的升高作用。②AngⅡ呈浓度和时间依赖性地促进内皮细胞迁移和增殖。生长素抑制AngⅡ引起的内皮细胞迁移和增殖呈浓度的时间依赖性。PD98059(25μmol/L)预处理可以抑制AngⅡ促内皮细胞迁移和增殖作用,[Dys~3]GHRP-6(25μmol/L)预处理阻断生长素对内皮细胞迁移和增殖的抑制作用。③AngⅡ浓度和时间依赖性地促进HUVEC-12细胞ROS生成增加。生长素呈浓度依赖性抑制AngⅡ对ROS生成的促进作用。[Dys~3]GHRP-6预处理可阻断生长素对ROS生成的抑制作用。④生长素可浓度依赖性地减弱AngⅡ诱导的TNFα、IL-8、MCP-1的释放和时间依赖性地减弱这些炎症因子的基因表达。[Dys~3]GHRP-6预处理阻断生长素对TNFα、IL-8、MCP-1释放和基因表达的影响。⑤生长素可增加内皮细胞pAkt,eNOS表达,减少p-NFκBp65和p-ERK1/2的表达,同时减少单核内皮细胞粘附。
结论:生长素可抑制AngⅡ诱导的内皮细胞损伤、炎症反应和氧化应激,其机制涉及PI3K/Akt途径,影响ERK1/2和NFκB通路活化。
第二章肥胖抑素对人脐静脉内皮细胞功能的影响及
机制研究
目的:观察肥胖抑素对脐静脉内皮细胞活性氧形成、内皮细胞NO释放、内皮细胞增殖、迁移,单核内皮细胞粘附和TNFα、IL-8、MCP-1水平和基因表达的作用以及涉及的信号通路。
方法:①采用不同浓度AngⅡ(0(?)24h)孵育HUVEC-12细胞,获得最佳观察点,并观察(10~(10)-10~(-7)mol/L)obestatin对AngⅡ损伤的影响。②加入MAPK信号通路抑制剂PD98059预处理HUVEC-12细胞后再用肥胖抑素和AngⅡ孵育HUVEC-12,采用方法同第一章。
结果:①AngⅡ抑制NO释放同第一章,肥胖抑素抑制HUVEC-12细胞释放NO。肥胖抑素进一步加重AngⅡ引起的HUVEC-12细胞释放NO减少,呈浓度和时间依赖性。肥胖抑素抑制eNOS表达。有可能通过抑制PI3K途径抑制NO分泌。②肥胖抑素呈剂量和时间依赖性地增加内皮细胞的增殖,相同剂量生长素可以拮抗肥胖抑素促内皮细胞增殖作用。③肥胖抑素对AngⅡ诱导的内皮细胞迁移起促进作用,而本身对内皮细胞迁移没作用。PD98059能阻断肥胖抑素+AngⅡ引起的内皮细胞迁移作用。④肥胖抑素浓度依赖和时间依赖性地刺激的HUVEC-12细胞ROS增加。⑤肥胖抑素时间和浓度依赖性地增加AngⅡ诱导的HUVEC-12细胞TNFα、IL-8、MCP-1水平和基因表达。PD98059和PDTC能阻断肥胖抑素增加AngⅡ诱导的HUVEC-12细胞TNFα、IL-8、MCP-1水平和基因表达的作用。⑥肥胖抑素增加p-ERK1/2、p-NFκBp65蛋白表达,减少p-AKt、eNOS蛋白表达和促进单核内皮细胞粘附,及促进AngⅡ诱导的单核内皮细胞粘附。
结论:肥胖抑素促进AngⅡ诱导的HUVEC-12内皮细胞损伤、炎症反应和氧化应激,其机制可能与抑制PI3K/Akt、激活ERK1/2、NFκB通路有关。
第三章生长素对apoE基因敲除小鼠动脉粥样斑块的影响
目的:观察生长素对apoE~(-/-)小鼠血浆IL-8、MCP-1、TNFα水平和血管壁NFκBp65表达及动脉粥样斑块的影响。
方法:8周龄apoE~(-/-)小鼠饲以西方类型膳食12周建立动脉粥样硬化模型。遗传背景相同的8周龄C57BL/6J小鼠饲以同类型膳食作对照。第8周时模型组分为腹腔内注射生长素(100μg/kg)组和注射生理盐水(0.1ml)组,C57BL/6J小鼠亦给予生理盐水(0.1ml)腹腔内注射。第12周时眼眶取血,分离血浆ELISA法测IL-8、MCP-1、TNFα,处死小鼠取主动脉进行大体标本观察和HE及油红O染色、免疫组化分析。
结果:①体视显微镜HE和油红O染色及图像分析仪测量显示:C56BL/6J小鼠无动脉斑块形成,apoE~(-/-)组和ApoE~(-/-)+生长素组均有动脉斑块形成,但apoE~(-/-)+生长素组比ApoE~(-/-)组斑块减少。②与C57BL/6J小鼠相比,apoE~(-/-)小鼠血浆中TNFα、IL-8、MCP-1水平升高。而apoE~(-/-)+生长素组较apoE~(-/-)组TNFα、IL-8、MCP-1显著降低。③与C57BL/6J小鼠相比,apoE~(-/-)小鼠血管壁NFκBp65免疫组化阳性细胞积分光密度值明显升高。生长素可明显降低apoE~(-/-)小鼠主动脉NFκBp65表达。
结论:生长素通过抑制炎症反应减少ApoE~(-/-)小鼠动脉粥样斑块形成。
Ghrelin is the only endogenous ligand of growth hormone secretagogue receptor up to now,it play a important role in the increasing appetide,improving stomach intestine function and adjusting energy metabolism.Recent study suggest that apart from promoting growth hormone releasing,they are related with multiple pathophysiology process,for example:obesity、cardiolvascular disease、Diabetes mellitus and senescence.Recently researchers successfully isolated a length of ghrelin associated peptide named obestatin from the stomach of rat.study demonstrate that obestatin has opposite effect to ghrelin.The found of obestatin was expand the ghrelin associated study field.there some report demonstrate that the level of obestatin in the coronary disease increase,obestatin can accelerate the cardic ischemia reperfusion injury.But there are no report about the effect of Ghrelin and Obestatin in the regression of atherosclerosis plaque.
Therefore,we explore ghrelin and obestatin's effect on the atherosclerosis from below aspect:Ⅰ-In vitro study the effect of ghrelin and Obestatin on the AngⅡinduced human umbilicus endothelial cell NO release and cell oxidative stress、proliferation、migration and inflammatory factor production and the signal pathway involved in;Ⅱ-In vivo study the impact on the atherosclerosis plaque of apoE~(-/-)mice and inflammatory factor level and NFκBp65 expression of vascular wall.
ChapterⅠ:impact and mechanism of Ghrelin on the function of human umbilical endothelial cell
Object:To observe the effect of Ghrelin on human umbilicus endothelial cell ROS production、NO release and proliferation、migration and the adhesion of moncyte to endothelial cell、TNFα、IL-8、MCP-1 level and gene express and signal path way involved in.
Method:I use different concentration of AngⅡ(10~(-9)-10~(-6)mol/L) incubate HUVEC-12 in vitro(0(?)24h),to aquire best observation,and observe(10~(-9)(?)10~(-6)mol/L)Ghrelin's effect on AngⅡinduced injury.Ⅱpretreated with the inhibitor of MAPK signal pathway PD98059 25μmol/L、GHS-Rla receptor block[Dys~3]GHRP-6 25μmol/L,and NFκB inhibitor PDTC1μmol/Land then observe the role to the effect of Ghrelin on AngⅡinduced injury,adopt nitrate reductase method measure the NO of supernatant.WST-8 method measure the proliferation of cell, transwell method measure the cell migration.Measuring endothelilal cell ROS with fluorescent compound 2',7'-dichlorofuorenscein(DCF) method.RT-PCRmethod measure the gene expression of TNFα、IL-8、MCP-1.ELISA method measure the level of TNFα、IL-8、MCP-1 in the supernatant.westernblot measure the expression of total Akt、ERK1/2、totalNFκBp65、p-Akt、p-ERK1/2、p-NFκBp65、eNOS.
Result:I AngⅡinhibit HUVEC-12 releasing NO with dose and time dependent response.Ghrelin reverse AngⅡinduced NO release decreased with dose-dependent response.Pretreated with PD98059 has no effect to AngⅡinduced NO release decreased,but[Dys~3]GHRP-6 pretreated block ghrelin's effect of increasing NO release.ⅡAngⅡpromote endothelial cell proliferation and migration Ghrelin inhibit AngⅡinduced endothelial cell proliferation and migration with dose and time dependent.Pretreated with PD98059 can inhibit AngⅡinduced endothelial cell proliferation and migration,pretreated with [Dys~3]GHRP-6 block the inhibited effect of Ghrelin on endothelial migration and proliferation.ⅢAngⅡincreased endothelial cell ROS production with time and dose response.Ghrelin inhibit AngⅡinduced endothelial proliferation and migration with dose dependent response.pretreated with[Dys~3]GHRP-6 block the inhibited effect of ghrelin on ROS production.ⅣGhrelin attenuate AngⅡinduced TNFα、IL-8、MCP-1 level with dose depent response and gene expression with time dependent response.Pretreated with[Dys~3]GHRP-6 block the effect of ghrelin on TNFα、IL-8、MCP-1release and gene expression.Ⅴghreln can increase the endothelial cell expression of pAkt,eNOS,decrease p-NFκBp65 and p-ERK1/2 expression,and meanwhile decrease monocyte adhesion to endothelial cell
Conclusion:ghrelin inhibit AngⅡinduced endothelial injury、inflammatory response and oxidative stress,the mechanism involved in PI3K/Akt signal pathway,affect ERK1/2 and NFκB signal pathway activation
ChapterⅡ:the effect and mechanism of obestatin on human umbilicus endothelial cell function
Objective:To observe the effect of obestatin on HUVEC-12 ROS production、NO release and proliferation、migration and the adhesion of moncyte to endothelial cell、TNFα、IL-8、MCP-1 level and gene expression and the signal pathway involved in.
Method:Different concentration of AngⅡ(0(?)24h)incubate HUVEC-12 in vitro to observe best point of time and concentration,and observe obestatin interfere's effect to the AngⅡinduced injury.Ⅱpretreated HUVEC-12 with inhibitor of MAPK signal pathway PD98059、NFκB signal pathway PDTC,and then incubate HUVEC-12 with obestatin and AngⅡ。Adopt the same method as chapterⅠ.
Result:ⅠAngⅡinhibit HUVEC-12 release NO the same as chapterⅠ,obestatin further accelerate AngⅡinhibit HUVEC-12 release NO with time and dose dependent response,obestatin inhibit eNOS expression,and may inhibit NO secretion through PI3K pathway.Ⅱobestatin increase HUVEC-12 proliferation,the same dose of ghrelin can opposite obestatin's effect.Ⅲobestatin itself has no effect on HUVEC-12 migration,but promote AngⅡinduced HUVEC-12 migration.PD98059 can block the effect of obestatin+AngⅡinduced HUVEC-12 migration.Ⅳobestatin increase AngⅡinduced HUVEC-12 ROS production with dose and time dependent response.Ⅴobestatin increase AngⅡinduced HUVEC-12's TNFα、IL-8、MCP-1 level and gene expression.PD98059 and PDTC block obestatin's effect.Ⅵobestatin increase p-ERK1/2、p-NFκBp65protein expression,decrease p-AKt、 eNOS,and promote monocyte adhesion to endothelial cell,and accelerate AngⅡ's effect.
Conclusion:obestatin promote AngⅡinduced HUVEC-12 injury、inflammatory response、oxidative stress,the mechanism may be related to inhibit PI3K/Akt、activate ERKl/2、NFκB signal pathway.
ChapterⅢthe effect of ghrelin on the regression of atherosclerosis plaque in ApoE~(-/-)mice aorta
Objective:To observe the effect of ghrelin on reducing the apoE~(-/-) mice plasma IL-8、MCP-1、TNFαlevel and the NFκBp65 expression in vascular wall and the regression of atherosclerotic plaque.
Method:8 week ApoE~(-/-)mice feed with western style meals,and the same age mice C57BL/6J feed with same meals as control.In the eighth week,ApoE~(-/-)mice was assign to ghrelin intraperitoneal injection and saline injection group randomly in the twelfth week.All of the groupswere drawed blood from eye sockets,isolate plasma to measure IL-8、MCP-1、TNFαby ELISA.Mice were killed to be observed undered stereomicroscope and paraffin imbedding for HE and immunohistochemistry,and frozen section for red oil stain.
Result:Ⅰthe result of stereomicroscope、HE、oil red stain and image analysis equipment measurement demonstrate that no plaque at C57BL/6J mice vessels,and both apoE~(-/-)group and ApoE~(-/-)+ghrelin group has atherosclerosis plaque at vessels.Ⅱcontrast to C57BL/6J mice, apoE~(-/-)mice has higher plasma TNFα、IL-8、MCP-1 level,but apoE~(-/)+ghrelin mice has lower TNFα、IL-8、MCP-1 level than ApoE~(-/)mice.ⅢContrast to C57BL/6J mice,apoE~(-/-)mice NFκBp65 immunohistochemistry positive cell integral calculus value are increase obviously,ghrelin can decrease the expression of NFκBp65 in apoE~(-/-)mice aorta.
Conclusion:Ghrelin can inhibit inflammatory response to decrease ApoE~(-/-)mice atherosclerosis plaque formation.
为此,本研究拟从以下几个方面探讨Ghrelin和Obestatin对动脉粥样硬化的影响:①离体研究Ghrelin和Obestatin对AngⅡ诱导的脐静脉内皮细胞NO释放,细胞内氧化应激、内皮细胞增殖和迁移、炎症因子产生的影响及信号通路研究;②在体研究Ghrelin对动脉粥样硬化小鼠动脉粥样硬化斑块和炎症因子以及血管壁NFκB的影响
第一章:生长素对人脐静脉内皮细胞功能影响及机制研究
目的:观察生长素对AngⅡ所致的脐静脉内皮细胞活性氧生成、NO释放,内皮细胞增殖、迁移、单核内皮细胞粘附和TNFα、IL-8、MCP-1水平和基因表达的作用以及涉及的信号通路。
方法:①采用不同浓度的AngⅡ(0(?)24小时)孵育HUVEC-12,获得最佳观察点,并观察(10~(-9)(?)10~(-6)mol/L)生长素对AngⅡ损伤的影响。②加入MAPK信号通路抑制剂PD98059(25μmol/L)、GHSRla受体阻断剂([Dys~3]GHRP-625μmol/L)、NFκB通路抑制剂PDTC1μmol/L预处理HUVEC-12细胞观察对生长素影响AngⅡ诱导的内皮细胞损伤的作用。采用硝酸还原酶法测细胞培养上清NO;WST-8法测细胞增殖;Transwell小室测细胞迁移;荧光探针法测细胞内的活性氧生成。RT-PCR法测细胞中TNFα、IL-8、MCP-1基因表达;ELISA法测细胞培养上清TNFα、IL-8、MCP-1浓度;Western blot测细胞中totalNFκBp65,ERK1/2、total Akt、p-Akt、P-NFκBp65,P-ERK1/2、eNOS蛋白表达。
结果:①AngⅡ呈浓度和时间依赖性地抑制HUVEC-12细胞分泌NO,生长素呈浓度依赖性地逆转AngⅡ对NO生成的减少。PD98059预处理对AngⅡ诱导的NO生成无影响,而[Dys~3]GHRP-6(25μmol/L)处理阻断生长素对NO生成的升高作用。②AngⅡ呈浓度和时间依赖性地促进内皮细胞迁移和增殖。生长素抑制AngⅡ引起的内皮细胞迁移和增殖呈浓度的时间依赖性。PD98059(25μmol/L)预处理可以抑制AngⅡ促内皮细胞迁移和增殖作用,[Dys~3]GHRP-6(25μmol/L)预处理阻断生长素对内皮细胞迁移和增殖的抑制作用。③AngⅡ浓度和时间依赖性地促进HUVEC-12细胞ROS生成增加。生长素呈浓度依赖性抑制AngⅡ对ROS生成的促进作用。[Dys~3]GHRP-6预处理可阻断生长素对ROS生成的抑制作用。④生长素可浓度依赖性地减弱AngⅡ诱导的TNFα、IL-8、MCP-1的释放和时间依赖性地减弱这些炎症因子的基因表达。[Dys~3]GHRP-6预处理阻断生长素对TNFα、IL-8、MCP-1释放和基因表达的影响。⑤生长素可增加内皮细胞pAkt,eNOS表达,减少p-NFκBp65和p-ERK1/2的表达,同时减少单核内皮细胞粘附。
结论:生长素可抑制AngⅡ诱导的内皮细胞损伤、炎症反应和氧化应激,其机制涉及PI3K/Akt途径,影响ERK1/2和NFκB通路活化。
第二章肥胖抑素对人脐静脉内皮细胞功能的影响及
机制研究
目的:观察肥胖抑素对脐静脉内皮细胞活性氧形成、内皮细胞NO释放、内皮细胞增殖、迁移,单核内皮细胞粘附和TNFα、IL-8、MCP-1水平和基因表达的作用以及涉及的信号通路。
方法:①采用不同浓度AngⅡ(0(?)24h)孵育HUVEC-12细胞,获得最佳观察点,并观察(10~(10)-10~(-7)mol/L)obestatin对AngⅡ损伤的影响。②加入MAPK信号通路抑制剂PD98059预处理HUVEC-12细胞后再用肥胖抑素和AngⅡ孵育HUVEC-12,采用方法同第一章。
结果:①AngⅡ抑制NO释放同第一章,肥胖抑素抑制HUVEC-12细胞释放NO。肥胖抑素进一步加重AngⅡ引起的HUVEC-12细胞释放NO减少,呈浓度和时间依赖性。肥胖抑素抑制eNOS表达。有可能通过抑制PI3K途径抑制NO分泌。②肥胖抑素呈剂量和时间依赖性地增加内皮细胞的增殖,相同剂量生长素可以拮抗肥胖抑素促内皮细胞增殖作用。③肥胖抑素对AngⅡ诱导的内皮细胞迁移起促进作用,而本身对内皮细胞迁移没作用。PD98059能阻断肥胖抑素+AngⅡ引起的内皮细胞迁移作用。④肥胖抑素浓度依赖和时间依赖性地刺激的HUVEC-12细胞ROS增加。⑤肥胖抑素时间和浓度依赖性地增加AngⅡ诱导的HUVEC-12细胞TNFα、IL-8、MCP-1水平和基因表达。PD98059和PDTC能阻断肥胖抑素增加AngⅡ诱导的HUVEC-12细胞TNFα、IL-8、MCP-1水平和基因表达的作用。⑥肥胖抑素增加p-ERK1/2、p-NFκBp65蛋白表达,减少p-AKt、eNOS蛋白表达和促进单核内皮细胞粘附,及促进AngⅡ诱导的单核内皮细胞粘附。
结论:肥胖抑素促进AngⅡ诱导的HUVEC-12内皮细胞损伤、炎症反应和氧化应激,其机制可能与抑制PI3K/Akt、激活ERK1/2、NFκB通路有关。
第三章生长素对apoE基因敲除小鼠动脉粥样斑块的影响
目的:观察生长素对apoE~(-/-)小鼠血浆IL-8、MCP-1、TNFα水平和血管壁NFκBp65表达及动脉粥样斑块的影响。
方法:8周龄apoE~(-/-)小鼠饲以西方类型膳食12周建立动脉粥样硬化模型。遗传背景相同的8周龄C57BL/6J小鼠饲以同类型膳食作对照。第8周时模型组分为腹腔内注射生长素(100μg/kg)组和注射生理盐水(0.1ml)组,C57BL/6J小鼠亦给予生理盐水(0.1ml)腹腔内注射。第12周时眼眶取血,分离血浆ELISA法测IL-8、MCP-1、TNFα,处死小鼠取主动脉进行大体标本观察和HE及油红O染色、免疫组化分析。
结果:①体视显微镜HE和油红O染色及图像分析仪测量显示:C56BL/6J小鼠无动脉斑块形成,apoE~(-/-)组和ApoE~(-/-)+生长素组均有动脉斑块形成,但apoE~(-/-)+生长素组比ApoE~(-/-)组斑块减少。②与C57BL/6J小鼠相比,apoE~(-/-)小鼠血浆中TNFα、IL-8、MCP-1水平升高。而apoE~(-/-)+生长素组较apoE~(-/-)组TNFα、IL-8、MCP-1显著降低。③与C57BL/6J小鼠相比,apoE~(-/-)小鼠血管壁NFκBp65免疫组化阳性细胞积分光密度值明显升高。生长素可明显降低apoE~(-/-)小鼠主动脉NFκBp65表达。
结论:生长素通过抑制炎症反应减少ApoE~(-/-)小鼠动脉粥样斑块形成。
Ghrelin is the only endogenous ligand of growth hormone secretagogue receptor up to now,it play a important role in the increasing appetide,improving stomach intestine function and adjusting energy metabolism.Recent study suggest that apart from promoting growth hormone releasing,they are related with multiple pathophysiology process,for example:obesity、cardiolvascular disease、Diabetes mellitus and senescence.Recently researchers successfully isolated a length of ghrelin associated peptide named obestatin from the stomach of rat.study demonstrate that obestatin has opposite effect to ghrelin.The found of obestatin was expand the ghrelin associated study field.there some report demonstrate that the level of obestatin in the coronary disease increase,obestatin can accelerate the cardic ischemia reperfusion injury.But there are no report about the effect of Ghrelin and Obestatin in the regression of atherosclerosis plaque.
Therefore,we explore ghrelin and obestatin's effect on the atherosclerosis from below aspect:Ⅰ-In vitro study the effect of ghrelin and Obestatin on the AngⅡinduced human umbilicus endothelial cell NO release and cell oxidative stress、proliferation、migration and inflammatory factor production and the signal pathway involved in;Ⅱ-In vivo study the impact on the atherosclerosis plaque of apoE~(-/-)mice and inflammatory factor level and NFκBp65 expression of vascular wall.
ChapterⅠ:impact and mechanism of Ghrelin on the function of human umbilical endothelial cell
Object:To observe the effect of Ghrelin on human umbilicus endothelial cell ROS production、NO release and proliferation、migration and the adhesion of moncyte to endothelial cell、TNFα、IL-8、MCP-1 level and gene express and signal path way involved in.
Method:I use different concentration of AngⅡ(10~(-9)-10~(-6)mol/L) incubate HUVEC-12 in vitro(0(?)24h),to aquire best observation,and observe(10~(-9)(?)10~(-6)mol/L)Ghrelin's effect on AngⅡinduced injury.Ⅱpretreated with the inhibitor of MAPK signal pathway PD98059 25μmol/L、GHS-Rla receptor block[Dys~3]GHRP-6 25μmol/L,and NFκB inhibitor PDTC1μmol/Land then observe the role to the effect of Ghrelin on AngⅡinduced injury,adopt nitrate reductase method measure the NO of supernatant.WST-8 method measure the proliferation of cell, transwell method measure the cell migration.Measuring endothelilal cell ROS with fluorescent compound 2',7'-dichlorofuorenscein(DCF) method.RT-PCRmethod measure the gene expression of TNFα、IL-8、MCP-1.ELISA method measure the level of TNFα、IL-8、MCP-1 in the supernatant.westernblot measure the expression of total Akt、ERK1/2、totalNFκBp65、p-Akt、p-ERK1/2、p-NFκBp65、eNOS.
Result:I AngⅡinhibit HUVEC-12 releasing NO with dose and time dependent response.Ghrelin reverse AngⅡinduced NO release decreased with dose-dependent response.Pretreated with PD98059 has no effect to AngⅡinduced NO release decreased,but[Dys~3]GHRP-6 pretreated block ghrelin's effect of increasing NO release.ⅡAngⅡpromote endothelial cell proliferation and migration Ghrelin inhibit AngⅡinduced endothelial cell proliferation and migration with dose and time dependent.Pretreated with PD98059 can inhibit AngⅡinduced endothelial cell proliferation and migration,pretreated with [Dys~3]GHRP-6 block the inhibited effect of Ghrelin on endothelial migration and proliferation.ⅢAngⅡincreased endothelial cell ROS production with time and dose response.Ghrelin inhibit AngⅡinduced endothelial proliferation and migration with dose dependent response.pretreated with[Dys~3]GHRP-6 block the inhibited effect of ghrelin on ROS production.ⅣGhrelin attenuate AngⅡinduced TNFα、IL-8、MCP-1 level with dose depent response and gene expression with time dependent response.Pretreated with[Dys~3]GHRP-6 block the effect of ghrelin on TNFα、IL-8、MCP-1release and gene expression.Ⅴghreln can increase the endothelial cell expression of pAkt,eNOS,decrease p-NFκBp65 and p-ERK1/2 expression,and meanwhile decrease monocyte adhesion to endothelial cell
Conclusion:ghrelin inhibit AngⅡinduced endothelial injury、inflammatory response and oxidative stress,the mechanism involved in PI3K/Akt signal pathway,affect ERK1/2 and NFκB signal pathway activation
ChapterⅡ:the effect and mechanism of obestatin on human umbilicus endothelial cell function
Objective:To observe the effect of obestatin on HUVEC-12 ROS production、NO release and proliferation、migration and the adhesion of moncyte to endothelial cell、TNFα、IL-8、MCP-1 level and gene expression and the signal pathway involved in.
Method:Different concentration of AngⅡ(0(?)24h)incubate HUVEC-12 in vitro to observe best point of time and concentration,and observe obestatin interfere's effect to the AngⅡinduced injury.Ⅱpretreated HUVEC-12 with inhibitor of MAPK signal pathway PD98059、NFκB signal pathway PDTC,and then incubate HUVEC-12 with obestatin and AngⅡ。Adopt the same method as chapterⅠ.
Result:ⅠAngⅡinhibit HUVEC-12 release NO the same as chapterⅠ,obestatin further accelerate AngⅡinhibit HUVEC-12 release NO with time and dose dependent response,obestatin inhibit eNOS expression,and may inhibit NO secretion through PI3K pathway.Ⅱobestatin increase HUVEC-12 proliferation,the same dose of ghrelin can opposite obestatin's effect.Ⅲobestatin itself has no effect on HUVEC-12 migration,but promote AngⅡinduced HUVEC-12 migration.PD98059 can block the effect of obestatin+AngⅡinduced HUVEC-12 migration.Ⅳobestatin increase AngⅡinduced HUVEC-12 ROS production with dose and time dependent response.Ⅴobestatin increase AngⅡinduced HUVEC-12's TNFα、IL-8、MCP-1 level and gene expression.PD98059 and PDTC block obestatin's effect.Ⅵobestatin increase p-ERK1/2、p-NFκBp65protein expression,decrease p-AKt、 eNOS,and promote monocyte adhesion to endothelial cell,and accelerate AngⅡ's effect.
Conclusion:obestatin promote AngⅡinduced HUVEC-12 injury、inflammatory response、oxidative stress,the mechanism may be related to inhibit PI3K/Akt、activate ERKl/2、NFκB signal pathway.
ChapterⅢthe effect of ghrelin on the regression of atherosclerosis plaque in ApoE~(-/-)mice aorta
Objective:To observe the effect of ghrelin on reducing the apoE~(-/-) mice plasma IL-8、MCP-1、TNFαlevel and the NFκBp65 expression in vascular wall and the regression of atherosclerotic plaque.
Method:8 week ApoE~(-/-)mice feed with western style meals,and the same age mice C57BL/6J feed with same meals as control.In the eighth week,ApoE~(-/-)mice was assign to ghrelin intraperitoneal injection and saline injection group randomly in the twelfth week.All of the groupswere drawed blood from eye sockets,isolate plasma to measure IL-8、MCP-1、TNFαby ELISA.Mice were killed to be observed undered stereomicroscope and paraffin imbedding for HE and immunohistochemistry,and frozen section for red oil stain.
Result:Ⅰthe result of stereomicroscope、HE、oil red stain and image analysis equipment measurement demonstrate that no plaque at C57BL/6J mice vessels,and both apoE~(-/-)group and ApoE~(-/-)+ghrelin group has atherosclerosis plaque at vessels.Ⅱcontrast to C57BL/6J mice, apoE~(-/-)mice has higher plasma TNFα、IL-8、MCP-1 level,but apoE~(-/)+ghrelin mice has lower TNFα、IL-8、MCP-1 level than ApoE~(-/)mice.ⅢContrast to C57BL/6J mice,apoE~(-/-)mice NFκBp65 immunohistochemistry positive cell integral calculus value are increase obviously,ghrelin can decrease the expression of NFκBp65 in apoE~(-/-)mice aorta.
Conclusion:Ghrelin can inhibit inflammatory response to decrease ApoE~(-/-)mice atherosclerosis plaque formation.
引文
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