【作者】 徐佳；

【导师】 关凤英；

【作者基本信息】 吉林大学 ， 药理学， 2020， 硕士

【摘要】 目的:线粒体动力学即线粒体融合和分裂保持动态平衡的过程,该过程由融合/分裂相关蛋白精确调控完成,对于线粒体代谢、质量和功能有着重要的生理意义,而这些蛋白发生异常可引发线粒体动力学失衡,进而引起线粒体功能障碍并引发各种疾病状态。最新研究发现肝胰岛素抵抗与线粒体动力学失衡密切相关。目前研究集中在线粒体外膜融合蛋白mitofusin 1/2(Mfn1/2)和线粒体分裂蛋白dynamin-like protein 1(Drp1)在肝胰岛素抵抗中的作用。然而,线粒体内膜融合蛋白Optic Atrophy 1(Opa1)异常是否直接引起肝胰岛素抵抗仍是未知的。Sirtuin 1(SIRT1)对糖尿病及线粒体功能的调节具有重要影响,但其是否可以通过调控Opa1改善线粒体功能尚无定论。黄连素(Berberine,BBR)是我国传统中药,其具有降血糖、改善线粒体功能及肝胰岛素抵抗的作用。本课题重在探究BBR是否可以通过激活SIRT1调节线粒体融合蛋白Opa1来改善肝胰岛素抵抗,并探讨其潜在机制。方法:在体内研究中,我们建立db/db糖尿病小鼠模型,设立正常组,模型组,并给予BBR给药治疗4周,首先检测小鼠空腹血糖、血脂、口服葡萄糖耐量、胰岛素耐量,同时对肝脏进行组织形态学及超微结构检测,随后提取肝脏蛋白,测定其SIRT1、Opa1、Mfn1/2、Drp1的表达情况及糖异生关键酶PEPCK、G6pase mRNA表达情况,研究BBR对2型糖尿病小鼠肝胰岛素抵抗的改善作用及与SIRT1调控线粒体动力学相关蛋白的关联。在体外研究中,我们首先对HepG2细胞进行Opa1沉默处理,观察其沉默对线粒体融合/分裂关键蛋白、线粒体复合物I/V蛋白表达、ATP含量、线粒体膜电位及肝胰岛素抵抗关键指标的影响,探讨Opa1沉默是否导致线粒体功能障碍及肝胰岛素抵抗;其次我们建立了棕榈酸(palmitate,PA)诱导的HepG2细胞胰岛素抵抗模型,在模型基础上使用SIRT1过表达及Opa1沉默技术,观察SIRT1/Opa1蛋白表达、ATP含量、线粒体形态及膜电位、肝胰岛素抵抗关键指标;最后,为了探究黄连素是否可以通过调节SIRT1改善Opa1的表达来治疗肝胰岛素抵抗,我们在PA刺激的基础上给予BBR治疗,同时建立SIRT1沉默细胞,观察SIRT1沉默后BBR对Opa1蛋白表达、ATP含量、线粒体膜电位及肝胰岛素抵抗关键指标的影响。结果:在体内研究中,与正常组相比,糖尿病小鼠空腹血糖、低密度脂蛋白、总胆固醇、甘油三酯表达明显升高;HE染色显示肝细胞高度肿胀、排列紊乱且出现空泡;电镜结果显示线粒体体积变小,线粒体嵴较稀疏;PEPCK/G6pase mRNA表达水平明显升高。Western结果显示,模型组肝脏中线粒体融合蛋白Opa1/Mfn1及SIRT1表达明显下调,而分裂蛋白Drp1表达则明显升高。在给予BBR治疗4周后,空腹血糖、低密度脂蛋白、总胆固醇、甘油三酯明显下降;HE染色显示药物可改善肝脏的形态学表现,超微结构检查结果显示线粒体之间出现了融合,嵴稀疏的状态也得到明显改善;PEPCK/G6pase mRNA表达水平明显降低。Western结果显示,在肝脏中给药组线粒体融合蛋白Opa1/Mfn1及SIRT1表达明显上调,而分裂蛋白Drp1表达则明显下调,说明SIRT1/Opa1通路与BBR改善糖尿病小鼠肝胰岛素抵抗状态有关联。在体外研究中,Opa1沉默对线粒体融合蛋白表达无影响,却增加了线粒体分裂蛋白的表达;使线粒体复合物I的蛋白表达、ATP含量及线粒体膜电位明显下降,Opa1沉默还使PEPCK/G6pase表达明显上调且损害了pAKT对胰岛素的敏感性,说明Opa1沉默可以损伤线粒体功能并造成肝胰岛素抵抗。在PA模型基础上使用SIRT1过表达技术,发现SIRT1/Opa1蛋白表达、ATP含量、线粒体膜电位明显上调;线粒体形态得到很大改善;pAKT对胰岛素的敏感性上升且葡萄糖消耗增加;在此基础上进行Opa1沉默干预发现这些作用被逆转。最后,在PA刺激的基础上给予BBR治疗,发现Opa1蛋白表达、ATP含量、线粒体膜电位上调,pAKT对胰岛素的敏感性上升且葡萄糖消耗增加,这些作用在SIRT1沉默后被削弱。结论:Opa1沉默介导的线粒体融合/分裂失衡可导致肝细胞胰岛素抵抗,而SIRT1通过激活Opa1改善肝细胞胰岛素抵抗。黄连素作为一个有效的降糖药,可能通过调节SIRT1/Opa1通路改善肝胰岛素抵抗,进而治疗2型糖尿病。更多还原

【Abstract】 Objective: Mitochondrial dynamics,the processes of mitochondrial fusion and fission maintain homeostasis,are precisely regulated by fusion/fission-related proteins,and play an important physiological role in mitochondrial metabolism,quality and function.The aberrant changes of these proteins can trigger mitochondrial dynamics imbalance,which cause mitochondrial dysfunctions and result various disease states.Recent studies have found that hepatic insulin resistance is closely related to mitochondrial dynamics imbalance.Extensive researches have focused on the role of outer mitochondrial membrane fusion proteines mitofusin 1/2(Mfn1/2)and mitochondrial fission protein Dynamin-like protein 1(Drp1)in hepatic insulin resistance.However,whether mitochondrial inner membrane fusion protein Optic Atrophy 1(Opa1)abnormality directly causes hepatic insulin resistance is still unknown.Sirtuin 1(SIRT1)has important effects on the regulation of diabetes and mitochondrial function,but whether it can improve mitochondrial function by regulating Opa1 is inconclusive.Berberine(BBR)is a traditional Chinese medicine in China,which has the effects of lowering blood glucose,improving mitochondrial function and hepatic insulin resistance.This article focuses on exploring whether berberine can prevent hepatic insulin resistance by activating SIRT1 to regulate mitochondrial fusion protein Opa1,and exploring its potential mechanism.Methods:In the in vivo study,8-weeks old wild type mice and db/db mice were purchased.Wild type mice defined as control group and db/db mice were divided into 2 group which established as model group and BBR treatment group.First,the mice were tested for fasting blood glucose,blood lipid,oral glucose tolerance,and insulin tolerance.At the same time,the liver was morphologically tested,and then liver proteins were extracted,and the expressions of SIRT1,Opa1,Mfn1 / 2,Drp1 and PEPCK/G6 pase mRNA expression were measured to investigate the effect of BBR on improving hepatic insulin resistance in type 2 diabetic mice and its relationship with SIRT1 regulating mitochondrial dynamics.In the in vitro study,firstly,we performed Opal silencing HepG2 cells,and observed the effects of silencing on key proteins of mitochondrial fusion/fission,mitochondrial complex I/V protein expression,ATP content,mitochondrial membrane potential,and key indicators of hepatic insulin resistance to investigate whether Opa1 silencing caused mitochondrial dysfunction and hepatic insulin resistance.Secondly,we established a palmitate(PA)-induced model of insulin resistance in HepG2 cells.Based on the model,we used SIRT1 overexpression and Opa1 silencing technology to observe SIRT1/Opa1 protein expression,ATP content,mitochondrial morphology and membrane potential,key indicators of hepatic insulin resistance;Finally,in order to explore whether BBR can treat hepatic insulin resistance by regulating SIRT1 to improve Opa1 expression,we gave BBR treatment on the basis of PA stimulation,while establishing SIRT1 silenced cells,and observed the expression of Opa1 protein,ATP content,mitochondrial membrane potential and key indicators of hepatic insulin resistance.Results:Compared with the normal group,the expression of fasting blood glucose,low-density lipoprotein,total cholesterol,and triglyceride in diabetic mice was significantly increased in in vivo studies.HE staining showed that hepatocytes were highly swollen,disorderly arranged,and appeared vacuoles;electron microscopy results showed that mitochondrial ridges were thinner and smaller in morphology;the expression of PEPCK/G6 pase mRNA was significantly increased.Western blotting results showed that the expression of mitochondrial fusion proteins Opa1/Mfn1 and SIRT1 in the model group was significantly down-regulated,while the expression of fission protein Drp1 was significantly increased in the model group.After 4 weeks of BBR treatment,fasting blood glucose,low-density lipoprotein,total cholesterol,and triglycerides were significantly reduced;HE staining showed that the drug could improve the morphological performance of the liver,and the sparse state of mitochondria was also significantly improved;PEPCK/G6 pase mRNA expression was significantly reduced.Western blotting results showed that the expression of mitochondrial fusion proteins Opa1/Mfn1 and SIRT1 in the liver of the model group was significantly down-regulated,while the expression of the mitotic protein Drp1 was significantly increased,indicating that SIRT1/Opa1 pathway is associated with BBR improving hepatic insulin resistance in diabetic mice.In in vitro studies,Opa1 silencing had no effect on mitochondrial fusion protein expression,but increased the expression of mitochondrial fission protein and the expression of mitochondrial complex I,ATP content,and mitochondrial membrane potential were significantly reduced.Opa1 silencing also caused the expression of PEPCK/G6 pase significantly up-regulated and impaired the sensitivity of pAKT to insulin,indicating that Opa1 silencing can impair mitochondrial function and cause hepatic insulin resistance.Using SIRT1 overexpression technology based on the PA model,it was found that the protein expression of SIRT1/Opa1,ATP content,and mitochondrial membrane potential were significantly up-regulated;mitochondrial morphology was greatly improved;the sensitivity of pAKT to insulin and glucose consumption were increased;and these effects were reversed when Opal was silenced.Finally,BBR treatment was given on the basis of PA stimulation.It was found that Opa1 protein expression,ATP content,and mitochondrial membrane potential were up-regulated.The sensitivity of pAKT to insulin and glucose consumption were also increased.But these effects were reversed after SIRT1 silencing.Conclusion: Opa1 silencing mediated mitochondrial fusion/fission imbalance could lead to hepatocyte insulin resistance,while SIRT1 improves hepatocyte insulin resistance by activating Opa1.As an effective hypoglycemic drug,Berberine may improve hepatic insulin resistance by regulating SIRT1/Opa1 pathway to treat type 2 diabetes.更多还原