mTOR介导糖尿病加重脑缺血再灌注损伤的机制研究

The mechanism of diabetes enhanced ischemic brain damage is poorly understood. Mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase,which integrates a variety of cellular signals and regulates protein syntheses, cell proliferation and death. Our preliminary data showed that cerebral ischemia activated mTOR pathway and inhibited sirtuin-3(SIRT3). Inhibition of mTOR by rapamycin significantly reduced ischemic brain damage and prevented the fall of SIRT3. Furthermore,diabetic ischemia further reduced the levels of SIRT3 post-ischemically compared with non-diabetic ischemic rats,and rapamycin prevented diabetes-induced postischemic seizures. We hypothesize that diabetes aggravates ischemic brain damage through activation of mTOR and inhibition of SIRT3 that subsequently leads to acetylation of cyclophilin D (Cyp-D), a critical step in formation of mitochondrial permeability transition pore (MPTP). In the proposed study, we plan to 1) Delineate the role of mTOR in diabetes-enhanced ischemic brain damage by treating diabetic and cerebral ischemic animals with rapamycin, using PCR array, Western blotting, histology and immunohistochemistry to examine the ischemic outcomes and phosphorylation of mTOR pathway related proteins, and measuring mitochondrial function in rapamycin treated and non-treated animals. 2) Identify the upstream pathways (including Ras-MEK-ERK1/2, PI3K-Akt, AMPK) through which diabetic ischemia activates mTOR using specific inhibitors and SiRNA knockdown of a specific gene based on the findings from the inhibitor study in cell culture models that mimic the in vivo diabetic ischemic conditions. 3) Determine whether rapamycin exerts its neuroprotection through mTOR-SIRT3 pathway using gene transfection to overexpress SIRT3, measurements of SIRT3 activity, acetylation of Cyp-D, co-immunoprecipitation of Cyp-D and adenine nucleotide translocase (ANT). Successful completion of the proposed project will delineate the role of mTOR-SIRT pathway in diabetic ischemia damage, as well as therapeutic effects and action mechanism of rapamycin.

糖尿病加重脑缺血损伤，其机制不明。雷帕霉素靶蛋白mTOR是一种丝氨酸/苏氨酸蛋白激酶。它整合多种细胞外信号，调节蛋白合成，细胞增殖及死亡。本课题组前期研究发现，mTOR在糖尿病脑缺血再灌后激活，而抑制mTOR则可以显著减轻脑缺血损伤及高糖诱发的缺血后癫痫。同时mTOR激活伴随SIRT3减低，抑制mTOR则使SIRT3升高。研究发现，SIRT3通过环素D脱乙酰化来阻滞MPTP开放进而减少促凋亡因子释放及细胞死亡。我们将通过糖尿病脑缺血再灌注动物及细胞培养模型，结合雷帕霉素干预、病理、蛋白印迹、SiRNA、基因过表达等方法阐明mTOR分子通路在高糖加重脑缺血损伤中的作用机制及高糖激活mTOR的上游分子通路，同时验证 mTOR通过抑制SIRT3，导致MPTP开放、线粒体功能异常而加重高糖脑缺血损伤。课题研究结果将有助于加深对高糖加重脑缺血损伤机制的理解，并为其治疗提供理论依据和实验基础。

糖尿病加重脑缺血再灌注损伤，其机制研究尚不清晰。本研究采用动物和细胞模型模拟糖尿病模型后制作脑缺血再灌注模型，探讨糖尿病脑缺血再灌对 mTOR 分子通路的影响以及抑制 mTOR 信号通路对糖尿病脑缺血损伤的机制。通过形态学（HE染色，TUNEL 染色）观察神经元损伤情况，判断雷帕霉素干预是否能够减轻脑组织损伤；应用蛋白印记、PCR以及免疫组化染色技术等确定缺血再灌注后 mTOR 通路及其上下游蛋白的表达水平，在此基础上采用抑制剂判断高血糖是通过哪条通路激活 mTOR。 结果表明，HE染色可见脑缺血再灌注损伤后神经元坏死明显，糖尿病组损伤尤为显著（P<0.05,vs 正常糖组），TUNEL染色观察凋亡明显，给予雷帕霉素后神经元损伤显著降低（P<0.05）。免疫印迹检测，mTOR通路（mTOR，P70S6K,P-S6）被激活（P<0.05,vs 假手术组），糖尿病组升高尤其显著（P<0.05,vs 正常糖组），AMPK和Ras/ERK通路在此过程中也被激活（P<0.05,vs 正常糖组），而给予雷帕霉素后均显著降低（P<0.05,vs 糖尿病组）。凋亡启动因子Cytocrome c、自噬标志性蛋白LC3和Beclin1和线粒体裂解蛋白Drp1均显著升高（P<0.05,vs 正常糖组），给予雷帕霉素后其表达均显著降低（P<0.05,vs 糖尿病组）。而Sirt3蛋白在糖尿病组显著降低（P<0.05,vs 正常糖组），给予雷帕霉素后显著升高（P<0.05,vs 糖尿病组）。综合上述结果，糖尿病脑缺血再灌注损伤过程中，坏死、凋亡和自噬三种细胞死亡形式均存在。高糖可通过能量代谢障碍而激活AMPK通路，进而激活细胞自噬，线粒体动态平衡被打破，线粒体裂解蛋白表达升高，造成线粒体内Cytocrome c释放，启动细胞坏死和凋亡。Ras/ERK通路也被激活，可能参与了细胞坏死和凋亡过程。而mTOR通路被激活可能是细胞自我修复的一种体现。雷帕霉素的神经保护效果可能是雷帕霉素增加了Sirt3的表达，而高水平 Sirt3 增加了 Cyp-D脱乙酰化从而阻止 MPTP 开放，稳定了线粒体动态平衡。