Cystatin C在高压氧预处理诱导大鼠脑缺血耐受中的作用机制研究

发布时间：2018-04-24 11:24

本文选题：脑保护 + 高压氧预处理　；参考：《第四军医大学》2014年硕士论文

【摘要】：背景：目前脑血管疾病已成为成年人死亡致残的第二大原因，但其治疗方法还很有限且疗效不尽人意。我们前期实验已证实，高压氧（hyperbaric oxygen，HBO）预处理可显著缩小实验动物缺血再灌注损伤后梗死容积，改善神经功能表现[1,2]。在对HBO预处理的机制研究中我们证实，HBO预处理可通过上调脑缺血再灌注损伤后大鼠脑缺血半暗带组织内自噬水平，，减少细胞凋亡坏死，发挥脑保护作用[3]。但HBO预处理是如何促进自噬的机制仍然不清楚，对HBO预处理脑保护机制的深入研究有助于探索新的临床干预靶点。胱抑素C（cystatin C）作为一种半胱氨酸蛋白酶抑制剂，可作用于溶酶体系统，调节多种蛋白水解酶的活性。在氧化应激状态下，Cystatin C可显著地促进细胞存活：强烈抑制组织蛋白酶活性，保护细胞内膜结构的稳定性，并通过调节分子信号通路促进自噬，延长细胞寿命。有研究表明外源性补充Cystatin C可提高神经细胞对氧糖剥夺损伤的耐受性[4,85]。我科通过同位素标记相对与绝对定量（i-TRAQ）蛋白筛选技术发现HBO预处理后血清及脑组织中Cystatin C表达均明显提高。但Cystatin C的表达增加是否与HBO预处理脑保护作用有关，尚不清楚。基于上述研究我们推测，HBO预处理通过促进少量氧自由基产生，上调CystatinC的表达，作用于自噬关键调节蛋白mTOR，促进自噬信号通路，从而发挥脑保护效应。本实验运用SD大鼠大脑中动脉阻塞（MCAO）及原代神经元细胞氧糖剥夺（OGD）模型，利用siRNA干扰及外源性补充方法调节Cystatin C蛋白水平，对HBO预处理的脑保护机制进行了深入探索。实验一Cystatin C在HBO预处理诱导大鼠脑缺血耐受中的作用研究目的：探讨Cystatin C在HBO预处理脑保护中的作用及可能机制方法：1)观察HBO预处理后脑缺血半暗带区Cystatin C的表达。成年雄性SD大鼠，HBO预处理（2.5ATA，100%O2，1h/d×5d）后24h，行右侧大脑中动脉阻闭，缺血维持2h后给予再通。检测缺血前、再灌注后2、4、6、12、24h共6个时间点CystatinC蛋白表达。2) Cystatin C对HBO预处理脑保护作用的影响。应用侧脑室注射技术，通过补充外源性Cystatin C蛋白及siRNA干扰方法上调及下调Cystatin C，检测再灌注后24h后大鼠神经功能及脑梗死容积。3)观察HBO预处理后脑缺血半暗带区mTOR磷酸化及自噬水平。检测缺血前、再灌注后2、4、6、12、24h共6个时间点p-mTOR、mTOR及自噬相关蛋白LC3II/I、Beclin-1蛋白表达。免疫荧光双标定位。结果：HBO预处理可有效减小大鼠脑缺血再灌注损伤后脑梗死容积，改善神经功能状况。再灌注后4h开始Cystatin C蛋白表达增加，至少可持续至24h；缺血再灌注损伤后mTOR磷酸化升高，HBO预处理可显著下调mTOR磷酸化，自再灌注后4h开始至少可持续至24h；再灌注后6h开始自噬相关蛋白LC3II及Beclin-1表达增加，至少可持续至24h，mTOR磷酸化改变主要体现在神经元细胞质中，Beclin-1主要在脑缺血半暗带区神经元核中表达。Cystatin C-siRNA抑制HBO预处理诱导的CystatinC的增加，减弱HBO预处理的脑保护作用。外源性补充Cystatin C可模拟HBO预处理发挥脑保护作用。结论：HBO预处理通过上调Cystatin C而诱导大鼠脑缺血耐受，且HBO预处理可抑制再灌注损伤后mTOR磷酸化，促进自噬。实验二Cystatin C在HBO预处理诱导原代神经元氧糖剥夺耐受中的作用及机制研究目的：观察Cystatin C是否参与HBO预处理对原代神经元的保护作用，并探讨HBO预处理是否是通过Cystatin C调节mTOR及自噬而发挥作用。方法：1) HBO预处理对原代神经元氧糖剥夺损伤后Cystatin C表达的影响。孕16-18周SD大鼠，取胚胎鼠脑皮层进行原代神经元培养。第6天行HBO预处理（3.5ATA，90%O2，3h），24h后进行氧糖剥夺（OGD），维持2h后给予复氧复糖。复氧后24h行MTT（检测细胞存活率）、LDH漏出率（检测细胞存活状态）检测及Western Blot（检测Cystatin C蛋白的表达）。2) Cystatin C对HBO预处理脑保护作用的影响。向细胞培养液中外源性补充Cystatin C及siRNA干扰来上调及下调CystatinC，RT-PCR（检测Cystatin C-mRNA，分别于干扰后1、2、3d进行）及Western Blot(检测Cystatin C蛋白，干扰后24h进行)检测不同时间点干预措施效率，复氧24h后检测MTT及LDH漏出率。3) Cystatin C在HBO预处理调节原代神经元细胞内mTOR磷酸化及自噬水平中的作用。复氧24h检测不同处理组细胞内p-mTOR、mTOR及自噬相关蛋白LC3II/I、Beclin-1蛋白表达。结果：免疫荧光染色显示，皮层原代神经元纯度95%。HBO预处理可提高原代神经元氧糖剥夺/复氧损伤24h后细胞存活率、降低细胞毒性，提高Cystatin C的表达。外源性补充Cystatin C可有效上调原代神经元细胞内Cystatin C水平，CystatinC-siRNA可显著抑制Cystatin C表达，最佳转染浓度为10μM。Cystatin C-siRNA抑制了HBO预处理诱导的Cystatin C的增加，减弱HBO预处理的神经元保护作用；外源性补充Cystatin C可模拟HBO预处理的细胞保护作用。上调原代神经元细胞内Cystatin C后，抑制mTOR磷酸化、提高自噬水平（LC3II及Beclin-1表达增加）；下调HBO预处理后Cystatin C表达，mTOR磷酸化升高，自噬水平降低。结论：Cystatin C参与HBO预处理的神经细胞保护作用，且HBO预处理是通过上调Cystatin C、抑制mTOR磷酸化、促进自噬而减轻体外培养大鼠皮层原代神经元的氧糖剥夺/复氧损伤。小结：在体实验与离体实验结果共同提示，Cystatin C在HBO预处理诱导的缺血耐受中发挥重要作用；HBO预处理通过上调Cystatin C蛋白表达并抑制其下游mTOR的磷酸化而促进自噬，从而诱导缺血耐受产生。
[Abstract]:Background: at present, cerebrovascular disease has become the second major cause of death and disability in adults, but the treatment method is still limited and the curative effect is not satisfactory. Our previous experiments have proved that hyperbaric oxygen (HBO) preconditioning can significantly reduce the infarct volume after ischemia reperfusion injury in experimental animals and improve the neurological function of [1,2]. in the experimental animals. In the study of the mechanism of HBO preconditioning, we have confirmed that HBO preconditioning can increase the level of autophagy within the ischemic penumbra of the rat brain after the brain ischemic reperfusion injury, reduce apoptosis and necrosis, and play the role of [3]., but the mechanism of promoting autophagy by HBO preconditioning is still unclear. Further research on the brain protection mechanism of HBO preconditioning It is helpful to explore new targets for clinical intervention.
Cystatin C (cystatin C), as a cysteine protease inhibitor, acts in the lysosome system and regulates the activity of a variety of protein hydrolases. Under oxidative stress, Cystatin C can significantly promote cell survival: strongly inhibit cathepsin activity, protect the stability of the microstructure of the fine cell, and regulate the molecular signaling through the regulation of the activity of cystatin. The road promotes autophagy and prolongs cell life. Studies have shown that exogenous supplementation of Cystatin C can increase the tolerance of nerve cells to oxygen glucose deprivation injury. [4,85]. in our family by isotope labeling relative and absolute quantitative (i-TRAQ) protein screening technique, the expression of Cystatin C in serum and brain tissues is significantly increased after HBO pretreatment, but Cystatin C Whether the increase of expression is related to the protective effect of HBO on brain is unclear.
Based on the above study, we speculate that HBO preconditioning can increase the expression of CystatinC by promoting the production of a small amount of oxygen free radicals, acting on the key regulatory protein mTOR of autophagy, promoting autophagic signaling pathway, and thus exerting the effect of brain protection. In this experiment, the middle cerebral artery occlusion (MCAO) and the primary neuron cell oxygen glucose deprivation (OGD) model in SD rats were used in this experiment. SiRNA interference and exogenous supplementation were used to regulate the level of Cystatin C protein, and the mechanism of HBO pretreatment for brain protection was further explored.
Effect of Experiment 1 Cystatin C on ischemic tolerance induced by HBO pretreatment in rats
Objective: To explore the role and possible mechanism of Cystatin C in HBO preconditioning brain protection: 1) to observe the expression of Cystatin C in cerebral ischemic penumbra after HBO preconditioning. Adult male SD rats, HBO preconditioning (2.5ATA, 100%O2,1h/d x 5d) 24h, right middle cerebral artery occlusion and reperfusion after ischemia, and reperfusion before ischemia and reperfusion. The effect of Cystatin C on the protective effect of HBO pretreatment on the brain protection by.2 Cystatin C at 6 time points in a total of 6 time points. The application of lateral ventricle injection technique, up regulation and downregulation of Cystatin C by supplementing the exogenous Cystatin C protein and siRNA interference method, and detecting the neural function and the volume of cerebral infarction after reperfusion after reperfusion The level of mTOR phosphorylation and autophagy in the ischemic penumbra region of the posterior brain. Before and after reperfusion, 2,4,6,12,24h was detected at 6 time points: p-mTOR, mTOR and autophagy related protein LC3II/I, Beclin-1 protein expression, and immunofluorescence double labeling.
Results: HBO pretreatment could effectively reduce the volume of cerebral infarction after cerebral ischemia-reperfusion injury and improve the state of nerve function. After reperfusion, the expression of Cystatin C protein increased at least at 4h, at least to 24h; mTOR phosphorylation was increased after ischemia reperfusion injury, and mTOR phosphorylation could be reduced significantly by HBO preconditioning, and 4H began to begin at least after reperfusion. After reperfusion, the expression of autophagy related protein LC3II and Beclin-1 increased at least after reperfusion, at least to 24h, and the change of mTOR phosphorylation was mainly reflected in the cytoplasm of neurons. Beclin-1 mainly expressed the CystatinC increase of.Cystatin C-siRNA inhibitory HBO preconditioning induced in the neuronal nuclei of the ischemic penumbra region, and weakened HBO preconditioning. Exogenous Cystatin C can simulate the protective effect of HBO on brain protection.
Conclusion: HBO pretreatment can induce cerebral ischemic tolerance in rats by up regulating Cystatin C, and HBO pretreatment can inhibit mTOR phosphorylation and promote autophagy after reperfusion injury.
Experiment two the role and mechanism of Cystatin C in HBO preconditioning induced oxygen glucose deprivation tolerance in primary neurons
Objective: To observe whether Cystatin C participates in the protective effect of HBO preconditioning on primary neurons and the effect of HBO preconditioning on the regulation of mTOR and autophagy through Cystatin C.
Methods: 1) the effect of HBO preconditioning on the expression of Cystatin C after primary neuron oxygen deprivation injury. 16-18 weeks pregnant SD rats were taken to take the embryonic rat cerebral cortex for primary neuron culture. HBO preconditioning (3.5ATA, 90%O2,3h) was performed on sixth days, oxygen glucose deprivation (OGD) after 24h, and 2h after 2h, and 24h line MTT (detection of cell survival) after reoxygenation. LDH leakage rate (detection of cell survival state) detection and Western Blot (detection of the expression of Cystatin C protein).2) the effect of Cystatin C on the brain protection of HBO preconditioning. Estern Blot (detection of Cystatin C protein, 24h after interference) detection of the efficiency of intervention measures at different time points, the detection of MTT and LDH leakage after reoxygenation 24h, Cystatin C is the role of Cystatin C in the regulation of phosphorylation and autophagy in the primary neurons of the HBO preconditioning. White LC3II/I, Beclin-1 protein expression.
Results: immunofluorescence staining showed that the preconditioning of primary cortical neuron purity 95%.HBO could increase the survival rate of 24h cells after oxygen deprivation / reoxygenation injury in primary neurons, reduce cytotoxicity and increase the expression of Cystatin C. Exogenous supplementation of Cystatin C can effectively increase the level of Cystatin C in the cells of primary neurons, and CystatinC-siRNA can be significantly increased. Inhibition of Cystatin C expression, the optimal transfection concentration of 10 u M.Cystatin C-siRNA inhibited the increase of Cystatin C induced by HBO pretreatment and weakened the neuronal protection of HBO preconditioning. Exogenous Cystatin C could simulate the cell protection of HBO pretreatment. Autophagy level (increased expression of LC3II and Beclin-1), down regulated Cystatin C expression after pretreatment of HBO, increased mTOR phosphorylation and decreased autophagy level.
Conclusion: Cystatin C participates in the neuroprotective effect of HBO preconditioning, and HBO preconditioning is by up regulation of Cystatin C, inhibiting the phosphorylation of mTOR, promoting autophagy and alleviating the oxygen deprivation / reoxygenation injury in the primary neurons of the rat cortex in vitro.
Summary: both in vivo and in vitro results suggest that Cystatin C plays an important role in the ischemic tolerance induced by HBO preconditioning; HBO preconditioning promotes autophagy by up regulating the expression of Cystatin C protein and inhibiting the phosphorylation of the downstream mTOR, thus inducing ischemic tolerance.

【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R743

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