【作者】 王伟伟；

【导师】 孙连坤；

【作者基本信息】 吉林大学 ， 病理学与病理生理学， 2014， 博士

【摘要】 能量代谢障碍是脑缺血再灌注损伤的重要原因。脑缺血再灌注时线粒体氧化磷酸化障碍在导致ATP生成减少的同时，还产生大量的活性氧(reactive oxygenspecies，ROS)，引发氧化应激。脑缺血再灌注时，除了ATP含量减少、自由基生成增加等引起细胞损伤之外，一些适应性调控机制在脑缺血再灌注过程中的作用也引起了人们的关注。内质网应激与自噬是细胞对损伤刺激的适应性反应。内质网应激-自噬可以维持细胞内环境的稳态平衡，具有一定的保护性作用。内质网应激时未折叠蛋白反应可以激活自噬，自噬又可以通过降解错误折叠或未折叠蛋白减轻内质网的负荷，抑制内质网应激的过度激活，同时产生的降解产物也为机体细胞新蛋白质的合成、细胞结构的重建以及ATP的生成提供原料。但过度激活的内质网应激-自噬又能够加重细胞损伤，甚至导致细胞死亡。最近的研究结果提示，内质网应激-自噬在脑缺血再灌注引起的能量代谢障碍和氧化应激过程发挥重要的作用，但其作用机制尚不十分明了。Keap1-Nrf2-ARE信号转导途径是机体氧化应激条件下主要的防御机制。自噬相关蛋白p62作为一种多功能蛋白，不仅仅具有清除损伤的细胞器、降解异常聚集的蛋白等作用，而且，具有多种蛋白相互作用区的p62在细胞应激、细胞生存等多种信号转导途径中发挥衔接分子的作用。已有研究表明在氧化应激条件下，p62可与Nrf2之间形成一个正反馈环路，通过Keap1-Nrf2-ARE信号途径，促进抗氧化基因的表达。因此，探讨多功能蛋白p62在脑缺血再灌注损伤过程中的作用，可能进一步阐明细胞自噬与氧化应激之间的调控机制。自噬晚期自噬体和溶酶体的融合是一个典型的囊泡融合事件。NSF，作为一种ATP酶，通过利用自身水解ATP产生的能量，介导囊泡融合完成后SNARE复合物的解离，释放SNARE进入下一个囊泡融合进程，是囊泡融合顺利进行必需的一个关键蛋白。体外研究表明，只有细胞质内可溶性的NSF在ATP存在条件下才能介导囊泡的融合，如果可溶性的NSF发生聚集或ATP缺失将会丧失该功能。可见，ATP产生与囊泡融合关键蛋白NSF的功能存在着关联，通过复制体外细胞实验性脑缺血再灌注模型，观察能量代谢障碍时囊泡融合关键蛋白NSF的变化，进而探讨脑缺血损伤过程中能量代谢障碍与细胞自噬之间可能的调控机制。目的：本研究基于能量代谢障碍/氧化应激在引起细胞损伤的同时，可能会激活细胞内质网应激-自噬等适应性反应，探讨内质网应激-自噬在细胞能量代谢障碍与氧化应激中的作用，为阐明脑缺血再灌注损伤的机制提供新的线索。方法：（1）体内实验：1）线栓法阻塞大脑中动脉复制大鼠局灶性脑缺血再灌注模型。2）TTC染色和HE染色判断脑缺血再灌注后脑皮质损伤情况。3）免疫组织化学染色检测Keap1和Nrf2的表达；免疫印迹检测内质网应激相关蛋白、自噬相关蛋白和凋亡相关蛋白的表达、RT-PCR法检测Keap1-Nrf2-ARE下游基因的表达。（2）体外实验：1）建立体外培养PC12细胞ATP缺失再恢复模型；MTT法检测细胞生存率的变化；Western Blot检测自噬和内质网应激相关蛋白的表达。2）建立体外培养CHO细胞ATP缺失再恢复模型；LDH法检测细胞的损伤改变；差速离心和线性甘油梯度离心获取不同蛋白组分；Western Blot检测不同组分NSF蛋白表达。结果：（1）体内实验：1）神经缺陷评分、TTC及HE染色结果表明，随着缺血时间的延长，大鼠脑缺血再灌注引发的大脑皮质损伤逐渐加重。2）缺血1H再灌注24h，大脑皮质泛素化蛋白表达增加，Grp78表达升高，Bcl-2的表达升高，Bax的表达下降；随着缺血时间的延长，至缺血3H再灌注24h，大脑皮质泛素化蛋白表达明显增加， CHOP/GADD153表达显著升高，Bax表达升高，Bcl-2表达下降。3）缺血1H再灌注24h及缺血1.5H再灌注24h时，自噬共轭蛋白Atg12-Atg5和LC3-PE表达增加；在缺血3H再灌注24h，自噬共轭蛋白Atg12-Atg5和LC3-PE表达显著下降。4）脑缺血1H再灌注24h时，Nrf2核定位增加，上调其下游抗氧化基因NQO1、GCLM和HO1mRNA的表达。随着缺血时间的延长，缺血3H再灌注24h时，Nrf2核定位减少，激活下游抗氧化基因NQO1和HO1的能力下降。5）随着缺血时间的延长，p62mRNA表达水平先增加后降低，而p62蛋白水平仅仅在缺血3H再灌注24h显著增加。（2）体外实验：1）无糖条件下轻度ATP缺失再恢复，PC12细胞生存率略有升高，此时LC3-II和p62蛋白变化不明显；有糖条件下轻度ATP缺失再恢复，PC12细胞生存率相对于重度ATP缺失再恢复明显升高，此时Grp78蛋白表达显著升高，p62蛋白表达显著下降；重度ATP缺失再恢复，PC12细胞生存率明显下降，此时LC3-II和p62蛋白表达显著升高；3-MA抑制自噬降低PC12细胞在ATP缺失条件下的生存率。2）ATP缺失引起CHO细胞细胞质内可溶性NSF蛋白聚集和细胞损伤。NSF高表达可减轻ATP缺失再恢复诱导的CHO细胞损伤。结论：1.内质网应激-自噬途径参与调控脑缺血再灌注损伤。短时间脑缺血后再灌注，内质网应激-自噬的激活起一定的保护性作用；长时间脑缺血后再灌注，内质网应激反应过度激活，同时机体自噬的降解能力下降，加重脑损伤。2.脑缺血再灌注时，自噬通过p62与Keap1-Nrf2-ARE信号途径相关联，共同作用减轻氧化应激、抑制内质网应激的过度激活。3.体外实验抑制自噬能够加重细胞损伤，提示内质网应激-自噬途径在体外培养细胞能量代谢障碍过程中具有一定的细胞保护作用。4.体外实验发现，能量代谢障碍导致细胞质内可溶性的自噬相关蛋白NSF聚集失活，丧失介导囊泡融合的功能，进而可能抑制自噬降解途径，引发细胞损伤。综上，我们认为脑缺血导致的脑细胞损伤与能量代谢密切相关，而适度的内质网应激-自噬在能量代谢障碍引起的细胞损伤中具有一定的保护性作用。表明脑细胞内质网应激-自噬的相关研究可能为脑缺血再灌注损伤的预防与治疗提供了新的线索。更多还原

【Abstract】 Energy metabolism disorder and oxidative stress are the major causes of cerebralischemia-reperfusion injury. Mitochondrial oxidative phosphorylation disorders aftercerebral ischemia-reperfusion can lead to decreased production of ATP andsimultaneously generate a large amount of reactive oxygen species (ROS), whichresults in oxidative stress. During cerebral ischemia-reperfusion, the decrease of ATPand the increase of free radicals can cause cell damaged, in addition, some adaptiveregulation mechanisms attract researchers’ attention.Endoplasmic reticulum stress and autophagy are two independent adaptiveresponses to cell stress injury. Endoplasmic reticulum stress-autophagy can maintaincell homeostasis, playing a protective role. The unfolded protein response uponendoplasmic reticulum stress can activate autophagy. Autophagy can alleviate theoverload of endoplasmic reticulum by degradation of misfolded or unfolded proteins,inhibiting overactivation of endoplasmic reticulum stress. In addition, autophagy canprovide raw materials for the synthesis of new proteins, reconstruction of cellstructure and ATP generation with the release of degradation products. However,excessive activation of endoplasmic reticulum stress-autophagy can increase celldamage or even cause cell death. Recent studies suggest that endoplasmic reticulumstress-autophagy plays a key role in energy metabolism disorder and oxidative stressinduced by cerebral ischemia-reperfusion, but the mechanism is not yet very clear.Keap1-Nrf2-ARE signaling pathway is a key cell defense mechanism underoxidative stress condition. As a multifunctional protein, autophagy-related protein p62not only can remove damaged cellular organelles and abnormal protein aggregation,but also can work as an adapter molecule in a variety of signal transduction pathways,such as cell stress and cell survival. Previous studies have shown that under oxidativestress condition, p62can form a positive feedback loop with Nrf2, promotes the xpression of antioxidant genes through the Keap1-Nrf2-ARE signaling pathway.Therefore, to explore the role of multifunctional protein p62in cerebral ischemia-reperfusion injury, may further clarify the regulation mechanism between autophagyand oxidative stress.The membrane fusion of autophagosome and lysosomes at the late stage ofautophagy pathway is one of the typical vesicle fusion events. Autophagy-relatedprotein NSF, as an ATPase, upon hydrolysis of ATP energy by itself, mediates therelease of SNARE complexes after the completion of vesicle fusion into the nextvesicle fusion process. NSF is a key protein indispensable for vesicle fusion process.In vitro studies show that only the the cytoplasmic soluble NSF can mediate theprocess of vesicle fusion through hydrolysis of ATP, NSF will lose its function ifaggregation and inactivation occurs or under ATP depletion condition. Thus, theremay exist some association between ATP generation and vesicle fusion. We observedchange of the vesicle fusion key protein NSF upon energy metabolism disorder by invitro experimental cerebral ischemia-reperfusion model, in order to further investigatethe possible regulation mechanisms between energy metabolism and autophagyduring cerebral-ischemic reperfusion injury.Objective:In this study, based on energy metabolism disorder/oxidative stress can inducecell injury，which may also activate some adaptive responses such as endoplasmicreticulum stress-autophagy, we investigated the role of endoplasmic reticulumstress-autophagy during energy metabolism disorder and oxidative stress in order toprovide new clues for the mechanism of cerebral ischemia-reperfusion injury.Methods:(1) In vivo:1) Rat transient middle cerebral artery occlusion (tMCAO) was induced by the suturemethod.2) TTC staining and HE staining were used to determine cortical injury after cerebralischemia-reperfusion.3) Immunohistochemistry, RT-PCR and western blot were used to detect theexpression of endoplasmic reticulum stress associated proteins, autophagy associattedproteins, proteins of Keap1-Nrf2-ARE pathway and expression of downstream genesin the cerebral cortex after ischemia-reperfusion. (2) In vitro:1) CHO cell culture ATP depletion and recovery model was induced by exposure to amixture of2-deoxy-D-glucose (2-DG,5mM) and oligomycin (2.5uM). CHO cellmedium LDH activities were determined to assess cell injury. Differentialcentrifugation and linear glycerol gradient centrifugation were used to get thedifferent protein fraction. Western blot was used to detect the expression of NSFprotein.1) PC12cell culture ATP depletion and recovery model was induced by exposure to amixture of2-deoxy-D-glucose (2-DG,5mM) and oligomycin (2.5uM). MTT methodwas used to detect cell viability. Western blot was used to detect the expression ofautophagy and endoplasmic reticulum stress associated proteins.Results:(1) In vivo:1) The results of neurological deficit scores, TTC and H&E staining revealed thatcerebral cortex injury was aggravated with prolongation of ischaemia afterischemia-reperfusion.2) After1hour of tMCAO and24hours of reperfusion, the expression ofubiquitinated proteins in the cortex increased, the expression of Grp78and Bcl-2alsoincreased, but the expression of Bax decreased. With prolongation of ischaemia, at3hours of ischaemia and24hours of reperfusion, the formation of ubiquitinated proteinaggregates and CHOP/GADD153expression notablely increased. Meanwhile, theexpression of Bax increased and expression of Bcl-2decreased.3) The expression of ubiquitinated proteins in the cortex increases after1hour and1.5hours of ischaemia and24hours of reperfusion, indicative of the accumulation ofprotein aggregates.Meanwhile, the expression of the autophagy conjugate proteinsAtg12-Atg5and LC3-PE, two factors essential for autophagy, also increased.However, at3hours of ischaemia and24hours of reperfusion, the formation ofubiquitinated protein aggregates notablely increased, while the expression ofAtg12-Atg5and LC3-PE decreased.3) After1hour and1.5hours of ischaemia and24hours of reperfusion, the expressionof the autophagy conjugate proteins Atg12-Atg5and LC3-PE, two factors essentialfor autophagy, increased. However, at3hours of ischaemia and24hours ofreperfusion, the expression of Atg12-Atg5and LC3-PE decreased. 4）The nuclear localization of Nrf2increases after1hour or1.5hours of ischaemiaand24hours of reperfusion. This RT-PCR analysis confirmed a marked increase inthe expression of the Nrf2down-stream target antioxidant genes, NQO1, GCLM andHO1. Prolongation to3hours of ischaemia followed by24hours of reperfusion,resulted in a decrease in the nucleic localization of Nrf2and inactivation of the Nrf2down-stream antioxidant target genes.5) The expression of p62increased dramatically after3hours of tMCAO and24hoursof reperfusion, while p62mRNA expression firstly increased and then decreased withprolongation of ischaemic time.(2) In vitro:1) Mild ATP depletion following by24hour recovery under glucose-free condition,the viability of PC12cell slightly elevated, while the protein levels of LC3-II and p62did not have significant changes; Mild ATP depletion following by24hour recoverywith the existence of glucose, the viability of PC12cell was higher than that in severeATP depletion group, Grp78protein significantly increased and p62proteinsignificantly decreased; Severe ATP depletion following by24hour recovery, theviability of PC12cell significant decreased, the protein levels of LC3-II and p62dramatically increased. Inhibition of autophagy by3-MA reduced the viability ofPC12cells upon ATP depletion.2) ATP depletion causes the aggregation of cytoplasmic soluble protein NSF in CHOcell culture and cell injury. Overexpression of NSF mitigates ATP depletion/recovery-induced cell injury.Conclusion:1. Endoplasmic reticulum stress-autophagy pathway is involved in cerebral ischemia-reperfusion injury. Upon short-time cerebral ischemia-reperfusion, the activation ofendoplasmic reticulum stress-autophagy plays a protective role; Upon long-timecerebral ischemia-reperfusion, the excessive activation of endoplasmic reticulumstress response and the decrease of autophagic degradation can aggravate braindamage.2. During cerebral ischaemia-reperfusion, autophagy may be involved in theKeap1-Nrf2-ARE signalling pathway through p62, which collectively induces theexpression of ARE downstream antioxidant proteins, playing a role in alleviating cellinjury induced by oxidative stress and overactivation of endoplasmic reticulum stress. 3. The inhibition of autophagy aggravates cell death, which suggest that endoplasmicreticulum stress-autophagy pathway play a protective role on energy metabolismdisorder.4. Energy metabolism disorder leads to aggregation and inactivation of cytoplasmicsoluble NSF protein, losing its function in mediating vesicle fusion, which results inthe malfunction of autophagic degradation and causes cell damage. Overexpression ofNSF can reduce cell damage induced by ATP depletion by supplying somecytoplasmic soluble NSF.Overall, we think that the cell death induced by brain ischemia-reperfusion isclosely related with energy metabolism disorder/oxidative stress, while mildendoplasmic reticulum stress-autophagy have some protective effect in this process.The study about endoplasmic reticulum stress-autophagy in brain cells probablyprovide new clue for the prevention and therapy of brain ischemia-reperfusion.更多还原