基于PINK1/Parkin调控的线粒体自噬在糖尿病肠缺血再灌注所致脓毒症高易感性中的作用及机制

Our previous studies have shown that mitophagy dysfunction plays a crucial role in the systemic inflammatory response syndrome (SIRS) and sepsis after intestinal ischemia reperfusion (IIR). Most important of all, sepsis can be induced by IIR, glucose metabolism and intestinal flora in diabetic patients can trigger each other, resulting in increased susceptibility of sepsis and worse prognosis. To date, its underlying mechenisms are still unknown..Recent studies indicated that PINK1 and Parkin were the key factors in regulating mitophagy. Our preliminary study also showed that the protein complex of PINK1 and Parkin was found significantly upregulated in diabetic intestinal, but the expression of PINK1 and Parkin decreased siganificantly. Interestingly, IIR injury and the incidence of sepsis have been reduced by the using of autophagy inducers. Therefore, we speculate that PINK1/Parkin regulated mitophagy is the key regulators on increased susceptibility of sepsis induced by IIR in diabetic mice..To address them, the IIR model of PINK1-/-, Parkin-/- mice(on the background of C57BL/6) were applied to observe the incidence of SIRS, sepsis and mitophagy. Furthermore, the anoxia-reoxygenation cell model of intestinal epithelial cells and RAW 264.7 murine macrophages exposed to high glucose were also used to investigate the effects of short interfering RNA (siRNA)-mediated PINK1, Parkin, AMPK, mTOR, Nrf2 knockdown on the effect of inflammation, oxidant, apoptosis, and the role of PINK1/Parkin in the proposed project. .In this study, we aim to elucidate the roles and mechanisms of PINK1/Parkin regulated mitophagy on increased susceptibility of sepsis induced by intestinal ischemia-reperfusion in diabetic mice, and invetigate the effects on the mitophagy sigaling pathway, such as AMPK/mTOR/Nrf2. This investigation will provide the theoretical evidences and new ideas for optimal therapies on sepsis induced by intestinal ischemia-reperfusion in diabetic patients.

肠缺血/再灌注(IIR)可致脓毒症，糖尿病状态下糖代谢紊乱与肠道菌群失调相互促发形成恶性循环，致使糖尿病IIR后脓毒症易感性增高，预后更差。研究表明其与线粒体自噬功能紊乱密切相关，但其具体调控机制尚待明确。新近发现PINK1与Parkin是调控线粒体自噬的关键因子。我们预实验显示糖尿病肠组织PINK1与Parkin复合体形成虽增加，但两者的表达水平显著降低。而应用自噬诱导剂可减轻糖尿病IIR后肠道损伤并降低脓毒症发生率。据此我们推测PINK1/Parkin 调控的线粒体自噬是糖尿病IIR后脓毒症易感性增高的关键途径。本项目拟采用基因敲除小鼠、原代肠粘膜上皮细胞、单核巨噬细胞为研究对象，探讨糖尿病IIR致脓毒症病程中PINK1/Parkin调控的线粒体自噬的具体作用及分子机制, 并观察其对线粒体自噬通路AMPK/mTOR/Nrf2的影响，为糖尿病IIR后脓毒症的防治提供有效的治疗靶点和思路。

肠缺血/再灌注(IIR)可致脓毒症，糖尿病状态下糖代谢紊乱与肠道菌群失调相互促发形成恶性循环，致使糖尿病IIR后脓毒症易感性增高，预后更差。研究表明其与线粒体自噬功能紊乱密切相关，但其具体调控机制尚待明确。新近发现PINK1与Parkin是调控线粒体自噬的关键因子。我们预实验显示糖尿病肠组织PINK1与Parkin复合体形成虽增加，但两者的表达水平显著降低。而应用自噬诱导剂可减轻糖尿病IIR后肠道损伤并降低脓毒症发生率。据此我们推测PINK1/Parkin 调控的线粒体自噬是糖尿病IIR后脓毒症易感性增高的关键途径。本项目应用野生型小鼠制备了糖尿病模型、肠缺血再灌注模型及糖尿病肠缺血再灌注模型，通过自噬激动剂和抑制剂、缺血后处理、Nrf2激活剂及Nrf2拮抗剂干预后，应用形态学、生物化学、分子生物学等技术检测了肠缺血再灌注后肠组织及肾组织的组织学变化及相关分子通路蛋白PINK1/Parkin的变化，评估小肠及肾组织损伤情况，同时检测了小鼠氧化应激相关指标、炎性因子、凋亡及自噬相关水平。结果发现：1.糖尿病状态下肠缺血再灌注（IIR）后肠道易损性增加、氧化应激水平及炎性因子增强、自噬水平增强，引起相关通路蛋白的变化；2.自噬在小鼠肠缺血再灌注（IIR）中的作用-自噬激动剂通过减少细胞凋亡，降低氧化应激水平及炎症因子而对肠道损伤有保护作用；3.缺血后处理(IPO)通过Akt /GSK-3β/ Nrf2途径的氧化应激增强自噬，减轻了肠缺血再灌注损伤；4.缺血后处理通过Nrf2调节自噬、抗氧化和抗炎症作用减轻肠道缺血再灌注诱导的急性肾损伤。