【作者】 周密；

【导师】 夏中元；

【作者基本信息】 武汉大学 ， 麻醉学， 2016， 博士

【摘要】 缺血性脑血管病占脑血管病绝大部分,其有效的治疗方法是及时的恢复缺血区域的血液灌注。然而,在脑缺血一段时间恢复再灌注后会导致再灌注损伤(Reperfusion Injury),因此需要采取有效的脑保护措施,尽可能的降低缺血再灌注损伤。研究表明,缺血预处理(ischemic preconditioning, IPR)和缺血后处理(ischemic postconditioning, IPO)均能有效减轻脑缺血再灌注损伤,但由于自身局限性使这两种方式在临床实践中的应用受到极大的限制。而在器官缺血后,在更能耐受缺血的远隔器官如肢体施行缺血后处理即远端缺血后处理(remote ischemic postconditioning, RIPoC),可控性强、操作方便,在临床实践中具有广泛的应用价值。虽然RIPoC对脑缺血再灌注损伤能够产生良好的保护作用且作用靶点多样,但其作用机制并未完全阐明。自噬(autophagy)在缺血再灌注损伤中发挥重要作用,但对于自噬在脑缺血再灌注损伤中起保护作用还是促进神经细胞死亡,目前的研究尚存在争议。现普遍认为：生理状态下,自噬在细胞内持续低水平进行以维持细胞正常功能；当细胞处于适度应激状态时,如氧化应激、缺血、饥饿等,自噬可以显著增强,以维持细胞的结构与功能；当细胞处于强烈应激状态下,过度自噬将会发生,从而使细胞失去活力。在机体处于健康状况下时,经氧化磷酸化反应线粒体能够为细胞正常活动提供能量和生物合成的底物,同时,线粒体代谢过程中ROS的异常积累会引起线粒体损伤；受损的线粒体通过自噬被选择性清除而维持细胞内环境的稳定,即线粒体自噬(mitophagy)。调控机体适度的自噬水平,特别是选择性调控线粒体自噬水平对维持细胞正常结构功能并防治一些疾病发生发展十分重要。研究表明,不论是IPR还是IPO均可通过调节细胞自噬水平来诱导对缺血神经元的保护作用。然而到目前为止,在脑缺血再灌注损伤过程中神经元的生存和死亡是如何通过自噬水平的高低来决定还不太清楚。远端缺血后处理对脑缺血再灌注损伤的保护作用是否与调节线粒体自噬有关,尚无研究报道。Parkin与DJ-1为帕金森病相关蛋白,均可通过调控氧化应激水平而维持线粒体功能,并在调控的线粒体自噬中具有重要作用。本研究将在SD成年大鼠上,建立短暂性局灶性脑缺血再灌注模型及肢体远端缺血后处理模型,首先明确远端缺血后处理对脑缺血再灌注损伤具有保护作用,然后应用线粒体自噬抑制剂研究Parkin/DJ-1调控的线粒体自噬在远端缺血后处理减轻脑缺血再灌注中的作用,探讨其可能的作用机制,为其今后应用于临床提供理论指导和实验依据。本实验研究分为两个部分：1.远端缺血后处理对大鼠局灶性脑缺血再灌注损伤的保护作用。2. Parkin/DJ-1介导的线粒体自噬在远端缺血后处理减轻局灶性脑缺血再灌注损伤中的作用。第一部分远端缺血后处理对大鼠局灶性脑缺血再灌注损伤的保护作用目的：探讨RIPoC对大鼠局灶性脑缺血再灌注损伤的保护作用与氧化应激、神经元凋亡相关性方法：成年雄性SD大鼠39只,体重280-320g,采用随机数字表法,将其分为3组(n=13)：假手术组(S组)、缺血再灌注组(I/R组)、I/R+远端缺血后处理组(I/R+RIPo组)。采用大脑中动脉阻断法制备大鼠局灶性脑缺血再灌注损伤模型。于再灌注开始时,I/R+RIPoC组立即夹闭双侧股动脉实行10min缺血/10min再灌注,共计3个循环。于再灌注24h,行神经功能缺陷评分(NDS)；采用TTC染色测定脑梗死体积,计算脑梗死体积百分比；TUNEL法检测缺血半暗区神经元凋亡情况；测定缺血半暗区SOD的活性及MDA、15-F2t-Isoprostane的含量。结果：1、RIPoC减轻缺血再灌注后脑梗死体积,改善大鼠的神经功能：S组大鼠脑组织TTC染色未见梗死灶,无神经功能障碍；与S组比较,I/R组、]I/R+RIPoC组脑梗死体积百分比、NDS评分显著升高(P<0.05)；与I/R组比较,I/R+RIPoC组脑梗死体积百分比、NDS评分显著降低(P<0.05),表明RIPoC减轻了缺血再灌注后脑梗死体积,改善了大鼠的神经功能。2、RIPoC减轻大鼠缺血半暗区神经元凋亡,抑制氧化应激反应：与S组比较,I/R组、I/R+RIPoC组缺血半暗区神经元凋亡指数、MDA和15-F2t-Isoprostane含量显著升高,I/R组缺血半暗区SOD活性降低(P<0.05)；与I/R组比较,I/R+RIPoC组缺血半暗区神经元凋亡指数、MDA和15-F2t-Isoprostane含量显著降低、SOD活性升高(P<0.05),表明RIPoC减轻了大鼠缺血半暗区神经元凋亡,抑制了氧化应激反应。结论：RIPoC可有效的减轻大鼠局灶性脑缺血再灌注损伤,其与抑制氧化应激反应,减少神经元凋亡有关。第二部分Parkin/DJ-1介导的线粒体自噬在远端缺血后处理减轻局灶性脑缺血再灌注损伤中的作用目的：探讨Parkin/DJ-1介导线粒体自噬在RIPoC减轻大鼠局灶性脑缺血再灌注损伤中的作用。方法：成年雄性SD大鼠96只,体重280-320g,采用随机数字表法,将其分为5组：假手术组(S组)、缺血再灌注组(I/R组)、I/R+远端缺血后处理组(I/R+RIPo组)、I/R+RIPoC+线粒体自噬抑制剂组(I/R+RIPoC+M组)和I/R+RIPoC+生理盐水组(I/R+RIPoC+NS组)。采用大脑中动脉阻断法制备大鼠局灶性脑缺血再灌注损伤模型。于再灌注开始时,I/R+RIPoC组、I/R+RIPoC+M组和I/R+RIPoC+NS组立即夹闭双侧股动脉实行10min缺血/10min再灌注,共计3个循环；I/R+RIPoC+M组、I/R+RIPoC+NS组在缺血前5 min分别腹腔注射线粒体分裂抑制剂(mitochondrial-division inhibitor-1, Mdivi-1) 3mg/kg和等容量的生理盐水。于再灌注24h,行神经功能缺陷评分(NDS)；采用TTC染色测定脑梗死体积,计算脑梗死体积百分比；透射电镜观察缺血半暗区线粒体自噬体；流式细胞术检测缺血半暗区神经元线粒体膜电位和线粒体质量；Western blot法检测缺血半暗区LC3-Ⅰ、LC3-Ⅱ、Parkin和DJ-1蛋白表达水平,计算LC3-Ⅱ/Ⅰ比值；免疫荧光染色观察Parkin和DJ-1蛋白的亚细胞定位；LC3-Ⅱ/Ⅰ比值、脑梗死体积百分比分别与Parkin和DJ-1蛋白表达水平行直线相关分析。结果：1、RIPoC减轻缺血再灌注后脑梗死体积,改善大鼠的神经功能,mdivi-1阻抑RIPoC的作用：S组大鼠脑组织TTC染色未见梗死灶,无神经功能障碍；与S组比较,其余4组脑梗死体积百分比、NDS评分显著升高(P<0.05)；与I/R组比较,I/R+RIPoC组和I/R+RIPoC+NS脑梗死体积百分比、NDS评分显著降低(P<0.05),表明RIPoC减轻了缺血再灌注后脑梗死体积,改善了大鼠的神经功能；与I/R+RIPoC组和I/R+RIPoC+NS组比较,I/R+RIPoC+M组脑梗死体积百分比、NDS评分显著升高(P<0.05),而I/R组和I/R+RIPoC+M组间脑梗死体积百分比、NDS评分比较差异无统计学意义(P>0.05),表明抑制线粒体自噬会阻抑RIPoC的脑保护作用。2、脑缺血再灌注诱导缺血半暗区神经元发生线粒体自噬,而RIPoC增加了缺血半暗区线粒体自噬的水平,mdivi-1阻抑RIPoC的这种作用：1)透射电镜显示：S组神经元细胞结构正常,线粒体膜完整,未见线粒体自噬体；I/R组可见典型双层膜包裹的线粒体自噬体,还可观察到线粒体自噬溶酶体以及自噬溶酶体内不能降解的线粒体残体。流式细胞术检测显示：与S组比较,I/R组缺血半暗区神经元线粒体膜电位和线粒体质量分别减低到56.5%和64.8%(P<0.05)。以上结果表明脑缺血再灌注显著诱导缺血半暗区线粒体自噬的发生。2)LC3-II/I比值的变化来评价自噬水平,与S组比较,其余4组缺血半暗区LC3-II/I比值升高(P<0.05)；与I/R组比较,I/R+RIPoC组和I/R+RIPoC+NS缺血半暗区LC3-II/I比值升高(P<0.05),表明RIPoC增加了缺血半暗区神经元自噬的水平；与I/R+RIPoC组和I/R+RIPoC+NS组比较,]I/R+RIPoC+M组缺血半暗区LC3-II/I比值降低(P<0.05),而I/R组和I/R+RIPoC+M组间LC3-Ⅱ/Ⅰ比较差异无统计学意义(P>0.05),表明]mdivi-1阻抑RIPoC引起的缺血半暗区神经元自噬水平的增加。3、RIPoC上调大鼠脑缺血半暗区Parkin、DJ-1蛋白表达水平,mdivi-1未能抑制RIPoC的作用：与S组比较,其余4组缺血半暗区Parkin、DJ-1蛋白表达上调(P<0.05)；与I/R组比较,I/R+RIPoC组和I/R+RIPoC+NS缺血半暗区Parkin、DJ-1蛋白表达上调(P<0.05),表明RIPoC上调大鼠脑缺血半暗区Parkin, DJ-1蛋白表达水平；I/R+RIPoC组、I/R+RIPoC+NS组、I/R+RIPoC+M组之间两两比较,Parkin、DJ-1蛋白表达差异无统计学意义(P>0.05),表明mdivi-1未能抑制IRIPoC对大鼠缺血半暗区Parkin、DJ-1蛋白表达水平的上调。4、免疫荧光染色观察Parkin和DJ-1蛋白的亚细胞定位,结果显示：与S组比较,I/R组黄色荧光在脑缺血半暗区神经元线粒体上显著增强,表明脑缺血再灌注诱导Parkin和DJ-1蛋白向线粒体转位；与I/R组比较,I/R+RIPoC组脑缺血半暗区神经元线粒体上黄色荧光也显著增强,表明RIPoC进一步促进Parkin和DJ-1蛋白向线粒体转位。5、大鼠脑缺血半暗区Parkin和DJ-1蛋白表达水平分别与LC3-Ⅱ/Ⅰ比值、脑梗死体积百分比行直线相关分析,结果显示：大鼠脑缺血半暗区LC3-Ⅱ/Ⅰ比值与Parkin、DJ-1蛋白表达水平呈正相关,脑梗死体积百分比与Parkin、DJ-1蛋白表达水平呈负相关。结论：RIPoC减轻大鼠局灶性脑缺血再灌注损伤,与其上调Parkin、DJ-1蛋白表达水平,促进Parkin、DJ-1蛋白向线粒体转位,从而增强线粒体自噬有关,即Parkin/DJ-1介导线粒体自噬参与了远端缺血后处理的脑保护作用。更多还原

【Abstract】 BackgroundThe ischemic cerebrovascular disease accounts for the vast majority of all cerebrovascular diseases and the effective treatment method for such a disease is the timely restoration of blood perfusion in the ischemic area. However, restoration of reperfusion after cerebral ischemia for a period of time will lead to reperfusion injury, and therefore, effective measures for cerebral protection are needed to reduce ischemia/reperfusion injury as long as possible. Both ischemic preconditioning (IPR) and ischemic postconditioning (IPO) can reduce the cerebral ischemia/reperfusion injury, while their applications in clinical practices are restricted because of their own limitations. Remote ischemic postconditioning (RIPoC) is performed after ischemia on distant organs that can better tolerate ischemia, such as limbs. It is controllable and convenient in operations, with a wide range of application values in clinical practices. Although RIPoC can yield good protective effects on cerebral ischemia/reperfusion injuries and demonstrates diverse targets, its mechanism of action has not been fully elucidated.Autophagy plays an important role in the ischemia/reperfusion injury, while current studies are still controversial over whether autophagy plays a protective role or promotes the death of nerve cells in the cerebral ischemia/reperfusion injury. It is now generally agreed that under physiological conditions, autophagy is ongoing at a low level in cells so as to maintain their normal function. When cells are under moderate stress, such as oxidative stress, ischemia and hunger, etc, autophagy can be significantly enhanced to maintain the structure and function of cells and when cells are under intense stress, excessive autophagy will occur, thereby causing cell death. Under physiological conditions, mitochondria provide energy and substrates for biosynthesis for cell survivals via oxidative phosphorylation, and meanwhile, the abnormal accumulation of ROS during the process of mitochondrial metabolism can cause mitochondrial damages. The damaged mitochondria are selectively cleared via autophagy so as to maintain the homeostasis of cells, namely mitophagy. Moderate regulation of the autophagy level of organisms, especially the selective regulation of mitophagy level, is very important in maintaining the normal structures and function of cells and in preventing the development and progression of some diseases.Neuroprotective effects induced by IPR and IPO are associated with the level of autophagy regulation, but it remains elusive how the autophagy level in cerebral ischemia/reperfusion decides the survival and death of cells. To date, there have been no reports on whether the protective effects of RIPoC on the ischemia/reperfusion injury are associated with the regulation of mitophagy. As Parkinson’s disease-related proteins, Parkin and DJ-1 can maintain mitochondrial function by regulating the level of oxidative stress and play important roles in the regulation of mitophagy.In this study, transient focal cerebral ischemia/reperfusion and limb remote IPO models will be established on SD rats.First, clarifying the neuroprotective effects of RIPoC against cerebral ischemia/reperfusion injury, and then mitophagy inhibitors will be utilized to study the effects of mitophagy regulated by Parkin/Dj-1 on alleviating the cerebral ischemia/reperfusion through RIPoC, provide necessary theoretical guidance and experimental basis for future clinical applications by exploring its possible mechanism of protection.Our study include the following two parts:1. Protective effects of RIPoC against focal cerebral ischemia/reperfusion injury in rats 2. Role of mitophagy controlled by Parkin/DJ-1 in RIPoC-induced mitigation of focal cerebral ischemia-reperfusion injury in rats.Part one:Protective effects of RIPoC against focal cerebral ischemia/reperfusion injury in ratsObjective:To explore whether the protective effects of RIPoC against focal cerebral ischemia/reperfusion injury in rats is related to oxidative stress and neuronal apoptosis.Methods:39 adult male SD rats weighing 280 to 320g were randomized into three groups (n =13), namely the sham operation group (S group), the ischemia/reperfusion group (I/R group), the I/R+RIPoC group. The focal cerebral ischemia/reperfusion injury models of rats were established using middle cerebral artery occlusion. The lOmin ischemia/lOmin reperfusion in 3 circles was performed on rats whose bilateral femoral arteries were clamped instantly at the commencement of reperfusion in the I/R+RIPoC group. At 24h of reperfusion, the neurological deficit score (NDS) was conducted, and the volume of cerebral infarction was measured using TTC staining and the percentage of volume of cerebral infarct was calculated. The neuronal apoptosis in the ischemic penumbra was detected using TUNEL assay, the activities of SOD and the contents of MDA and 15-F2t-Isoprostane in the ischemic penumbra were determined.Results:1、RIPoC alleviated the volume of cerebral infaction after ischemia/reperfusion, and improved the neurological functions of rats:No infarction was observed in the cerebral tissues of rats using TTC staining and no neurological disorders in the S group. Compared with the S group, the percentage of cerebral infarct volume and NDS in the I/R and I/R+RIPoC groups were significantly increased (P<0.05). Compared with the I/R group, the percentage of cerebral infarct volume and NDS in the I/R+RIPoC group was significantly decreased (P<0.05), indicating that RIPoC reduces the cerebral infarct volume after ischema/reperfusion and improves the neurological functions of rats.2、RIPoC alleviated neuronal apoptosis in the ischemic penumbra of rats and inhibited the oxidative stress:Compared with the S group, the cell apoptosis index and the contents of MDA and 15-F2t-Isoprostane in the ischemic penumbra of in the I/R and I/R+RIPoC groups were increased significantly, and the activities of SOD in the ischemic penumbra in the I/R group were decreased (P<0.05). Compared with the I/R group, the cell apoptosis index and the contents of MDA and 15-F2t-Isoprostane in the ischemic penumbra in the I/R+RIPoC group were significantly decreased and the activities of SOD were increased (P<0.05), indicating that IPoC alleviates the neuronal apoptosis in the ischemic penumbra of rats and inhibits the oxidative stress.Conclusions:RIPoC can effective alleviate focal cerebral ischemia/reperfusion injury in rats by inhibiting oxidative stress and reducing neuronal apoptosis.Part two:Role of mitophagy controlled by Parkin/DJ-1 in RIPoC-induced mitigation of focal cerebral ischemia-reperfusion injury in rats.Objective:To evaluate the role of mitophagy controlled by Parkin/DJ-1 in RIPoC-induced mitigation of focal cerebral ischemia/reperfusion injury in rats.Methods:96 adult male SD rats weighing 280 to 320g were randomized into five groups, namely the sham operation group (S group), the ischemia/reperfusion group (I/R group), the I/R+RIPo group, the I/R+RIPoC+mitophagy inhibitor group (I/R+RIPoC+M group) and the I/R+RIPoC+normal saline group (I/R+RIPoC+NS group). The focal cerebral ischemia/reperfusion injury models of rats were established using middle cerebral artery occlusion. The 10min ischemia/10min reperfusion in 3 circles was performed on rats whose bilateral femoral arteries were clamped instantly at the commencement of reperfusion in the I/R+RIPoC, I/R+RIPoC+M and I/R+RIPoC+NS groups, while rats in the I/R+RIPoC+M and I/R+RIPoC+NS groups were injected intraperitoneally with mitochondrial-division inhibitor-1 (Mdivi-1) at 3mg/kg and normal saline of an equal volume 5min before ischemia, respectively. At 24h of reperfusion, the neurological deficit score (NDS) was conducted, and the volume of cerebral infarction was measured using TTC staining and the percentage of volume of cerebral infarct was calculated. Mitophagosomes in the ischemic penumbra were observed using the transmission election microscopy. Mitochondrial membrane potential (MMP) and mitochondrial mass of neurons in the ischemic penumbra were detected using flow cytometry. The expression levels of LC3-I, LC3-Ⅱ, Parkin and DJ-1 in the ischemic penumbra were detected using Western blot assay and the LC3-Ⅱ/Ⅰ ratio was calculated, and the subcellular localizations of Parkin and DJ-1 proteins were observed using immunofluorescence staining. The linear correlation analysis was performed between the LC3-Ⅱ/Ⅰ ratio and the expression levels of Parkin and DJ-1 and between the percentage of volume of cerebral infarct and the protein expression levels of Parkin and DJ-1, respectively.Results:1、RIPoC alleviated the volume of cerebral infaction after ischemia/reperfusion and improved the neurological functions of rats, while mdivi-1 suppressed the effects of limb remote IPO:No infarction was observed in the cerebral tissues of rats using TTC staining and no neurological disorders in the S group. Compared with the S group, the percentage of cerebral infarct volume and NDS in the other four groups were significantly increased (P<0.05). Compared with the I/R group, the percentage of cerebral infarct volume and NDS in the I/R+RIPoC and I/R+RIPoC+NS group were significantly decreased (P<0.05), indicating that RIPoC reduces the cerebral infarct volume after ischema/reperfusion and improves the neurological functions of rats. Compared with the I/R+RIPoC and I/R+RIPoC+NS group, The percentage of cerebral infarct volume and NDS in the I/R+RIPoC+M group were significantly increased (P<0.05). The percentage of cerebral infarct volume and NDS between the I/R group and the I/R+RIPoC+M were not significantly different (P>0.05), showing that inhibition of mitophagy can suppress the cerebral protective effects of RIPoC.2、Cerebral ischemia/reperfusion induced the mitophagy of neurons in the ischemic penumbra, while RIPoC increased the mitophagy level in the ischemic penumbra and mdivi-1 suppressed such an effect of limb remote IPO:1) The transmission electron microscopy revealed that structures of neuronal cells in the S group were normal, the mitochondrial membranes were intact and no mitophagy was observed. Typical Mitophagosomes wrapped in double membranes were seen in the I/R group, together with Mitolysosomes and non-degradable mitochondrial residues therein. Flow cytometry demonstrated that compared with the S group, the MMP and mitochondrial mass of neurons in the ischemic penumbra were decreased to 56.5% and 64.8% (P<0.05) respectively in the I/R group. The above results suggest that cerebral ischemia/reperfusion significantly induces mitophagy the in the ischemic penumbra.2) The changes in the LC3-Ⅱ/Ⅰ ratio were utilized to evaluate the level of autophagy. Compared with the S group, the LC3-Ⅱ/Ⅰ ratio was increased in the other 4 groups (P<0.05). Compared with the I/R group, the LC3-Ⅱ/Ⅰ ratio in the I/R+RIPoC group and I/R+RIPoC+NS groups were increased (P<0.05), indicating that RIPoC increases the level of autophagy of neurons in the ischemic penumbra. Compared with the I/R+RIPoC and I/R+RIPoC+NS group, the LC3-Ⅱ/Ⅰ ratio in the I/R+RIPoC+M group was decreased (P<0.05), while no deference between the I/R group and I/R+RIPoC+M group (P>0.05), showing that mdivi-1 suppresses the increase in the autophagy level of neurons in the ischemic penumbra induced by RIPoC.3、RIPoC upregulated the expression levels of Parkin and DJ-1 in the cerebral ischemic penumbra of rats, while mdivi-1 failed to inhibit the effects of RIPoC. Compared with the S group, the expression levels of Parkin and DJ-1 in the cerebral ischemic penumbra of rats in the other 4 groups were upregulated (P<0.05). Compared with the I/R group, the expression levels of Parkin and DJ-1 in the cerebral ischemic penumbra of rats in the I/R+RIPoC and I/R+RIPoC+NS group were upregulated (P<0.05), indicating that RIPoC upregulates the expression levels of Parkin and DJ-1 in the cerebral ischemic penumbra of rats. Comparison between any two of the I/R+RIPoC group, I/R+RIPoC+NS group and I/R+RIPoC+M group showed that protein expression levels of Parkin and DJ-1 were not significantly different (P> 0.05), indicating that mdivi-1 fails to inhibit the upregulation of the expression levels of Parkin and DJ-1 in the ischemic penumbra of rats by RIPoC.4、The subcellular localizations of Parkin and DJ-1 proteins were observed using immunofluorescence staining and the results showed that compared with the S group, the yellow fluorescence in the neural mitochondria of cerebral ischemic penumbra was significantly enhanced in the I/R group, suggesting that cerebral ischemia reperfusion induces the translocation of Parkin and DJ-1 proteins to mitochondria. Besides, compared with the I/R group, the yellow fluorescence in the neural mitochondria of cerebral ischemic penumbra was also significantly enhanced in the I/R+RIPoC group, indicating that RIPoC further promotes the translocation of Parkin and DJ-1 proteins to mitochondria.5、The linear correlation analysis was conducted between the expression levels of Parkin and DJ-1 in the cerebral ischemic penumbra of rats and the LC3-Ⅱ/Ⅰ ratio and between the expression levels of Parkin and DJ-1 in the cerebral ischemic penumbra of rats and the percentage of volume of cerebral infarct, respectively. The results showed that the LC3-Ⅱ/Ⅰ ratio in the cerebral ischemic penumbra of rats was positively correlated with the expression levels of Parkin and DJ-1 and that the percentage of volume of cerebral infarct was negatively correlated with the expression levels of Parkin and DJ-1.Conclusions:RIPoC alleviates the focal cerebral ischemia/reperfusion injury in rats, which is associated with its upregulation of the expression levels of Parkin and DJ-1 and promotion of translocation of Parkin and DJ-1 proteins to mitochondria, thereby enhancing mitophagy. That is to say, mitophagy controlled by Parkin/DJ-1 is involved in the cerebral protective effects of RIPoC.更多还原