AMPK-mTOR介导DCM心肌细胞自噬机制及温阳活血利水法的干预效应

Diabetic cardiomyopathy (DCM) is the leading mainly deadly cause of diabetes mellitus (DM) Complications . but the mechanism is not fully understood. The latest study found that,Under hyperglycemic conditions DM cardiac cells perform a series of changes, starting autophagy, causing DCM, AMPK-mTOR pathway plays a key role in the regulation of DCM. Preliminary clinical studies have confirmed the treatment of Wenyanghuoxuelishui formula-Qiangxinheji effect on Diabetic cardiomyopathy. we hypothesis Qiangxinheji possibly through AMPK-mTOR- mediated autophagy and block its progress in DCM.This project by copying the DCM rat model,detect blood glucose and lipid levels , automatic biochemical analyzer to measure heart function, differential centrifugation to extract mitochondria, using electron microscope to observe mitochondrial autophagy and ultrastructure, using Immunohistochemistry, Western Blot, ELISA, RT-PCR to detect AMPK-mTOR signaling pathway and its downstream mitochondrial autophagy-specific protein TSC2, RHEB and autophagy-related protein Beclin1, LC3-I/Ⅱ,etc. using immunoprecipitation,to detect the protein interactions of AMPK and mTOR , in order to explore the mechanism of autophagy in DCM and the effect of the intervention of Qiangxinheji and provide objective basis of Wenyanghuoxuelishui treating DCM.

糖尿病心肌病(DCM)是糖尿病(DM)并发症致死的首要原因，但其机制尚不完全清楚。最新研究发现，DM心肌细胞在高血糖下发生一系列的变化，启动细胞自噬，引发DCM，AMPK-mTOR通路在DCM调控中起着关键作用。前期临床证实了温阳活血利水方—强心合剂治疗DCM临床疗效肯定。我们假说强心合剂可能通过AMPK-mTOR介导DCM自噬机制而阻断其进程。本项目通过复制DCM大鼠模型，检测大鼠血糖血脂变化，自动生化仪测量心功能，差速离心法提取线粒体，电镜观察心肌细胞线粒体自噬体及超微结构，免疫组化、Western Blot、ELISA、RT-PCR检测AMPK-mTOR信号通路及其下游线粒体自噬特异蛋白TSC2、RHEB及自噬相关蛋白Beclin1、LC3-I/Ⅱ等，免疫共沉淀检测AMPK、mTOR蛋白相互作用，探讨DCM自噬的机制及强心合剂的干预效应，为益气活血方防治DCM提供客观依据。

糖尿病心肌病是最严重的糖尿病并发症且机制不明，预后能力很差，病死率极高。最新研究发现，DM心肌细胞在高血糖下发生一系列的变化，启动细胞自噬，引发DCM，AMPK-mTOR通路在DCM调控中起着关键作用，前期临床证实了温阳活血利水方—强心合剂治疗DCM临床疗效肯定。我们假说强心合剂可能通过AMPK-mTOR介导DCM心肌细胞自噬机制而阻断DCM进程。.本项目通过复制DCM大鼠模型，检测大鼠血糖血脂变化，电镜观察心肌细胞线粒体自噬体及超微结构，免疫组化、Western Blot、ELISA、RT-PCR检测AMPK-mTOR信号通路及其下游线粒体自噬特异蛋白TSC2、RHEB及自噬相关蛋白Beclin1、LC3-I/Ⅱ等表达。探讨DCM心肌细胞自噬的机制及强心合剂的干预效应。.实验结果显示：DCM模型组大鼠心肌组织AMPK、TSC2、Beclin1、LC3-Ⅱ表达水平较空白组升高，LC3-I，mTOR，RHEB表达水平下调。给与强心合剂（QXM）干预后，DCM+HQXM、DCM+MQXM、DCM+LQXM各组大鼠心肌组织AMPK、TSC2、Beclin1、LC3-Ⅱ表达均有不同程度提高，LC3-I，mTOR，RHEB有不同程度下调，DCM+MQXM对上述指标干预效果与DCM+Captopril组比较，无统计学意义（P>0.05）；与DCM组比较，DCM+HQXM干预效果具有显著性差异（P<0.05）。透射电镜观察显示正常组大鼠心肌细胞超微结构正常，肌纤维排列整齐规则紧密，线粒体较丰富。DCM组大鼠心肌纤维排列较紊乱，部分肌纤维破坏，肌小节破坏，线粒体较少，胞浆内有空洞。DCM+HQXM组心肌纤维排列整齐、线粒体丰富，可见大量自噬溶酶体。DCM+MQXM组大鼠心肌纤维排列较整齐，偶见肌纤维排列松散，可见少量自噬溶酶体。DCM+LQXM组和DCM+Captopril组大鼠心肌纤维排列紊乱，线粒体较少，部分线粒体破坏，未见自噬溶酶体。提示强心合剂高剂量干预DCM大鼠，可以协调心肌细胞的自噬水平，从而阻断DCM进程，本探究为温阳活血利水法组方之强心合剂防治DCM提供客观依据。