【作者】 王静；

【导师】 张丽晖；

【作者基本信息】 山西医科大学 ， 内科学（专业学位）， 2021， 硕士

【摘要】 目的:1、通过对自发性高血压大鼠进行干预,观察GLP-1对大鼠血压的影响;2、探讨GLP-1对高血压诱导的心肌肥厚的作用;3、探究GLP-1对心肌肥厚影响的分子机制。方法:选用8只体重为200-250g的8周龄雄性Wistar–Kyoto(WKY)大鼠和24只同周龄同体重的雄性自发性高血压大鼠(SHRs),SHRs随机分为三组:SHR组(n=8),利拉鲁肽组(n=8),阿格列汀组(n=8);WKY组作为对照组,利拉鲁肽组皮下注射利拉鲁肽(300μg/kg,2次/日),阿格列汀组通过灌胃给药(20 mg/kg,1次/日),干预7周。实验结束后,注射戊巴比妥(100 mg/kg)处理大鼠,采用BL-420数据采集和分析系统监测大鼠的收缩压及舒张压,取出心脏并计算心脏重量(HW)/体重(BW)的比值,将组织固定、包埋、切片、染色,显微镜下观察组织学的改变;提取组织蛋白后,通过Western-blot检测心肌肥厚标志基因及通路蛋白表达情况;为了进一步明确GLP-1发挥作用的分子机制,在心肌细胞中加入AMPK抑制剂compound C或mTOR激活剂MHY1485进行孵育,检测心肌肥厚标志基因及通路蛋白的表达变化;最后分析数据。结果:1.GLP-1可以降低自发性高血压大鼠血压。为了观察GLP-1对大鼠血压的影响,我们监测了GLP-1治疗后血压的变化,结果显示:与SHR组相比,利拉鲁肽或阿格列汀干预7周后,收缩压、舒张压、平均动脉压均明显降低(P<0.05),且收缩压下降比较显著,两种药物的降压作用无统计学差异。2.GLP-1可以改善高血压诱导的心肌肥厚。在GLP-1干预高血压大鼠7周后,我们监测大鼠心脏的重量及体重的变化,并计算心重/体重的比值,发现SHR组HW/BW比值显著增加(P<0.05),同时心肌肥厚标志基因(ANP,BNP,β-MHC)表达明显升高(P<0.05),而GLP-1干预后这些指标有所下降(P<0.05)。同时镜下观察心肌组织学的改变,结果显示:通过HE染色发现,与对照组相比,SHR组心肌细胞的横截面积增大,而利拉鲁肽或阿格列汀干预后,这种现象可以得到改善;通过Masson染色评估胶原的沉积及心肌纤维化程度,发现SHR组心室壁广泛间质纤维化,心肌间质及血管周围可见大量胶原蛋白沉积,利拉鲁肽或阿格列汀干预后胶原蛋白的沉积减少;电镜下观察心肌的超微结构,发现正常心肌细胞线粒体紧密,排列整齐,肌原纤维完整。而长期压力负荷导致线粒体减少、肿胀、肌原纤维断裂,导致能量供应不足,最终引起心肌肥厚,然而,利拉鲁肽或阿格列汀干预后可逆转这些现象。组织学的改变与心肌肥厚标志物的表达结果一致。以上这些结果表明,GLP-1可以改善高血压诱导的心肌肥厚。3.GLP-1通过调节RAAS相关蛋白的表达抑制心肌肥厚。为了研究GLP-1对RAAS系统的影响,采用ELISA方法检测血清中血管紧张素II(Ang Ⅱ)的水平,Western blot方法检测Ang Ⅱ受体及血管紧张素转换酶2(ACE2)的表达,结果显示,与对照组相比,SHR组Ang Ⅱ水平及Ang Ⅱ 1型受体(AT1R)表达增强,而2型受体(AT2R)及ACE2的表达降低,导致AT1R/AT2R比值增加(P<0.05)。利拉鲁肽或阿格列汀干预后这些改变得到了逆转(P<0.05)。这些结果表明GLP-1可以调节RAAS蛋白的表达。4.GLP-1通过AMPK/mTOR/p70S6K通路逆转心肌肥厚。为了验证AMPK/mTOR/p70S6K信号通路在GLP-1抗心肌肥厚中的作用,我们通过Western blot方法检测了通路中相关蛋白的表达,结果显示:与对照组相比,SHR组AMPK的磷酸化水平降低,mTOR、p70S6K、4EBP1的磷酸化水平升高(P<0.05),利拉鲁肽或阿格列汀干预后,AMPK的表达显著升高(P<0.05),而下游分子的蛋白表达有所下降(P<0.05)。而AMPK、mTOR、p70S6K、4EBP1的总蛋白水平在干预前后无明显变化。同时,也发现随着AMPK的激活,GLP-1R的表达升高,因此,我们认为GLP-1R可能参与调节AMPK/mTOR通路。为了进一步明确AMPK/mTOR途径在GLP-1介导心肌肥厚中的作用,我们在细胞实验中加入AMPK抑制剂compound C或mTOR激活剂MHY1485,发现相比于Ang Ⅱ+GLP-1组,心肌肥厚标志物表达增加,表明compound C和MHY1485消除了GLP-1的抗肥厚作用,并提高了mTOR,p70S6K和4-EBP1的磷酸化水平。以上结果显示GLP-1通过AMPK/mTOR/p70S6K通路改善心肌肥厚。结论:GLP-1可以通过抑制Ang Ⅱ/AT1R/ACE2以及激活AMPK/mTOR/p70S6K信号通路发挥抗心肌肥厚作用。更多还原

【Abstract】 Objective:1.Observe the effect of GLP-1 on the blood pressure by intervening in spontaneously hypertensive rats;2.Explore the effect of GLP-1 on myocardial hypertrophy induced by hypertension;3.Explore the molecular mechanism of the effect of GLP-1 on myocardial hypertrophy.Methods:8 male Wistar–Kyoto(WKY)rats and 24 male SHRs: weight,200–250 g;age,8weeks.SHRs were divided randomly into three groups: the SHR group(n = 8),liraglutide group(n = 8),and alogliptin group(n = 8).WKY rats were used as the control group.The liraglutide group was subcutaneously injected with liraglutide(300 μg/kg,bid),and the alogliptin group was administered alogliptin(20 mg/kg,Qd.)by gastric gavage.All treatments lasted 7 weeks.At the end of the experimental period,all rats were euthanized with a dose of pentobarbital(100 mg/kg),and the BL-420 data acquisition and analysis system was used to monitor the systolic and diastolic blood pressure of the rats.The heart was harvested and calculate the heart weight(HW)/body weight(BW)ratio.The tissue was fixed,embedded,sliced,stained,and observed histological changes under the microscope;after the tissue protein was extracted,the expression of cardiac hypertrophy marker and pathway proteins was detected by Western-blot.To further assess the role of GLP-1 mediated protection from cardiac hypertrophy,cardiomyocytes were incubated with the AMPK inhibitor compound C or m TOR activator MHY1485,the expression of cardiac hypertrophy marker and pathway proteins was detected;Finally,the data was analyzed.Results:1.GLP-1 reduced blood pressure in SHRsTo observe the effect of GLP-1 on blood pressure in rats,we monitored blood pressure changes after GLP-1 treatment.SBP,DBP,MAP were sigznificantly decreased at 7 weeks after liraglutide or alogliptin treatment compared with the SHR group(P <0.05),and the systolic blood pressure decreased more significantly,and there was no statistical difference in the blood pressure lowering effects of the two drugs.2.GLP-1 attenuated cardiac hypertrophy induced by hypertensionTo investigate the effect of GLP-1 in hypertrophic hearts in vivo,we established a model of cardiac hypertrophy by pressure overload.SHRs were treated with liraglutide or alogliptin for 7 weeks,during which we measured HW and BW and calculated the HW/BW ratio.The HW/BW ratio was obviously increased in SHRs(P < 0.05).Western blot showed that the levels of hypertrophic markers(ANP,BNP,β-MHC)in rat hearts were increased in the SHR group compared with the WKY group(P < 0.05).GLP-1treatment significantly inhibited ANP,BNP,and β-MHC levels and the HW/BW ratio(P< 0.05).Hematoxylin and eosin staining indicated that hypertension remarkably increased the cell cross-sectional area of myocardial tissue,compared with WKY group,cell crosssectional area decreased after liraglutide or alogliptin treatment.Collagen deposition was evaluated using Masson’s trichrome staining.The SHR group showed extensive interstitial fibrosis in the ventricular wall and a significant increase in collagen deposition in both the perivascular region and intermyocardium.Liraglutide or alogliptin administration significantly reversed these changes.TEM showed abundant tight,neatly arranged mitochondria and intact myofibrils in the cytoplasm of cardiomyocytes in the WKY group.In contrast,reduced and swollen mitochondria and ruptured myofibrils were seen in the SHR group.However,these phenomena were reversed by liraglutide or alogliptin treatment.Alterations in morphology were consistent with the gene expression of hypertrophic markers.These results suggest that GLP-1 ameliorates the cardiac hypertrophy induced by hypertension.3.Effects of GLP-1 on the RAAS in SHRsTo evaluate the effect of GLP-1 on the RAAS,we examined components of the RAAS.In this experiment,the concentration of Ang II in serum was measured by ELISA,and the protein expression of Ang II receptors and ACE2 were detected using Western blot.Hypertension caused a significant increase in the levels of Ang II and AT1 R in the heart tissue compared with the WKY group.However,the protein levels of AT2 R and ACE2 were decreased in the SHR group compared with the WKY group(P < 0.05).However,administration of liraglutide or alogliptin reduced the Ang II and AT1 R levels and upregulated the AT2 R and ACE2 levels,as evidenced by a reduced AT1R/AT2 R ratio(P < 0.05).These results indicate that GLP-1 reduces blood pressure and exerts cardiovascular protection by regulating RAAS proteins expression.4.GLP-1 reverses cardiac hypertrophy via the AMPK/m TOR/p70S6 K pathwayTo elucidate the underlying molecular mechanism of the anti-hypertrophic effect of GLP-1,we extracted protein from myocardium in each group and examined the effect of GLP-1 treatment on the expression of proteins involved in the AMPK/m TOR/p70S6 K signaling pathway by Western blot.The results show that decreased p-AMPK levels and increased p-m TOR,p-p70S6 K,and p-4EBP1 levels were observed in the SHR group compared with the WKY group(P < 0.05).However,treatment with liraglutide or alogliptin remarkably increased the p-AMPK level,which prevented activation of pm TOR,p-p70S6 K,and p-4EBP1(P < 0.05).Nevertheless,the levels of total AMPK,m TOR,and p70S6 K proteins showed no obvious differences between the SHR and WKY groups.Meanwhile,along with AMPK activation,the expression level of GLP-1R was increased,so we considered that GLP-1R may participate in the regulation of the AMPK/m TOR pathway.To further assess the role of AMPK/m TOR signaling in GLP-1mediated protection from cardiac hypertrophy,cardiomyocytes were incubated with the AMPK inhibitor compound C or m TOR activator MHY1485 increased expression of hypertrophic markers relative to the Ang II + GLP-1 group,suggesting that the antihypertrophic effect of GLP-1 is abolished by compound C and MHY1485,and increased phosphorylation levels of m TOR,p70S6 K and 4-EBP1.Our results showed that GLP-1ameliorates cardiac remodeling via the AMPK/m TOR/p70S6 K signaling pathway.Conclusion:GLP-1 plays a protective role in cardiac hypertrophy by suppressing the Ang II/AT1R/ACE2 pathway and activating the AMPK/m TOR/p70S6 K pathway.更多还原