脑缺血再灌注损伤中线粒体缝隙连接蛋白43对神经血管单元的保护作用及丹参多酚酸干预

发布时间：2018-04-10 03:38

本文选题：线粒体缝隙连接蛋白43　切入点：线粒体　出处：《吉林大学》2016年博士论文

【摘要】：背景:神经血管单元由神经元、神经胶质细胞和血管内皮细胞三部分共同组成,此定义的提出强调了三者及其相互联系在神经系统中的重要地位。缝隙连接实现了细胞群信息传递、代谢底物交换和离子平衡上的同步化,并且在神经元兴奋性、血管舒缩及内环境稳态的调节中发挥了重要作用。在神经系统中缝隙连接蛋白43(connexin43,Cx43)为缝隙连接的最主要组成成分。缺血性脑损伤主要是由于局部脑血流中断,组织缺氧,其代谢功能无法正常运转,导致以神经元为主,胶质细胞及血管内皮细胞共同组成的神经血管单元受损,最终机体的功能受到破坏。近来发现,在星形胶质细胞的线粒体上也存在Cx43的表达,但是其在缺血性脑损伤过程中的变化及所扮演的角色尚未探知。鉴于线粒体在能量代谢、信号转导、蛋白磷酸化等方面的重要作用,我们选择以缝隙连接蛋白——特别是分布于线粒体内膜上的Cx43的分布变化及功能为研究对象,拟通过分析其在缺血再灌注(ischemia-reperfusion,IR)损伤中变化规律及作用机制,探索干预线粒体缝隙连接对神经保护的意义,为脑梗塞的治疗提供新的靶点。目的:观察线粒体内Cx43的分布及表达变化,分析其在脑IR损伤中变化规律,进一步探索相关作用机制,同时评价丹参多酚酸的保护作用。方法:体外实验中,以原代培养的星形胶质细胞糖氧剥夺模型(oxygen glucose deprivation,OGD)为基础,实验分为对照组,单纯OGD组(模型组),缝隙连接通道阻断剂—甘珀酸(carbenoxolone,CBX)干预组,线粒体ATP敏感性钾通道激动剂—二氮嗪(diazoxide,DZX)干预组,DZX+线粒体ATP敏感性钾通道抑制剂—5-羟奎酸(5-hydroxydecanoic acid,5-HD)干预组和丹参多酚酸(salvianolic acid,SA)干预组。首先,检测各干预剂下细胞的相对存活率,同时分析各组细胞凋亡情况,电镜下观察各组细胞及细胞内线粒体形态。进而,检测各干预剂刺激下线粒体膜电位的变化,通过免疫荧光定位观察星形胶质细胞中Cx43的分布情况。体内实验中,选用体重约为250~290 g的雄性Wistar大鼠,采用左侧大脑中动脉栓塞(middle cerebral artery occlusion,MCAO)模型。实验分为假手术组(对照组)、单纯IR组(模型组)、CBX干预组、DZX干预组、DZX+5-HD干预组、蛋白激酶C(protein kinase C,PKC)抑制剂—Ro-31-8425+DZX干预组、PKC激动剂—佛波酯(phorbol-12-myristate-13-acetate,PMA)+5-HD干预组和SA干预组。首先,染色观察比较各干预药物对大鼠脑梗死面积、神经功能学评分的作用,评价各干预药物对大鼠脑梗塞后凋亡情况的影响,继而观察脑皮层梗死区线粒体形态,同时通过超氧化物歧化酶(superoxide dismutase,SOD)活力和丙二醛(malondialdehyde,MDA)含量的检测评价各干预剂下线粒体功能的变化,最后对脑皮层梗死区Cx43、p-Cx43、PKC、p-PKC蛋白的表达含量进行定量,探讨不同干预剂的不同作用,分析其机制。结果:1、组织、细胞形态学:与单纯模型组比,CBX和DZX干预组脑梗死面积明显减小,细胞的凋亡程度降低(P0.01),且5-HD降低了DZX对梗死面积的缩小作用(P0.01);同样与模型组相比,SA组明显减少了梗死面积,降低了细胞凋亡程度(P0.01)。2、神经功能学评分:与对照组比较,其余各组评分均增高(P0.01),表现为梗塞区对侧的肢体肌力降低,但除对照组外,其余各组之间评分无显著差异。3、线粒体形态及功能:线粒体形态方面,与对照组相比,其余各组脑皮层梗死区线粒体形态损伤明显,主要表现为线粒体肿胀,棘断裂,基质透明化,其中单纯模型组和DZX+5-HD组线粒体损伤较明显(P0.05);线粒体功能方面,与假手术组比较,单纯模型组可见明显的SOD活力减低、MDA含量增高(P0.01),而CBX、DZX、PMA、SA干预剂皆能一定程度上提高SOD活力、降低MDA含量(P0.05),对线粒体功能起到保护作用。4、Cx43定位与定量:与对照组比,单纯OGD组星形胶质细胞整体Cx43含量减低,特别是细胞膜上分布含量减低,而细胞内Cx43的含量趋近增多,并且可见线粒体与Cx43共定位;在线粒体蛋白定量中,与假手术组比,模型组脑皮层梗死区线粒体Cx43、p-Cx43含量明显减低(P0.01),而p-Cx43降低更为明显(P0.01),在CBX、DZX、PMA、SA干预调节下,与IR组比,线粒体Cx43、p-Cx43明显升高(P0.05),且p-Cx43增高更为明显(P0.05)。结论:线粒体Cx43可以在脑IR损伤中起到保护神经血管单元的作用,这种保护作用是通过线粒体ATP敏感性钾通道调节PKC活性实现的,并且线粒体Cx43也参与了丹参多酚酸对脑IR损伤的保护过程。
[Abstract]:Background: the neurovascular unit consists of neurons, glial cells and vascular endothelial cells composed of three parts together, the proposed definition emphasizes the importance of the three and their relationship in the nervous system. The gap junction cell group information transmission, synchronization of metabolic substrate exchange and ion balance, and neuronal excitability homeostasis, vasomotor and play an important role in regulating the gap. In the nervous system of connexin 43 (connexin43, Cx43) is the main composition of gap junctions. Ischemic brain damage is mainly due to local cerebral blood flow interruption, tissue hypoxia, metabolic dysfunctional, leading to neuronal. The neurovascular unit glial cells and vascular endothelial cells composed of damaged body, eventually destroyed function. Recently, in astrocyte mitochondria also The expression of Cx43, but in the process of ischemic brain injury and the change of the role has not been penetrated. In view of the mitochondria in energy metabolism, signal transduction, protein phosphorylation plays an important role, we choose the distribution and change of function in connexin -- especially in the distribution of mitochondria membrane Cx43 as the research object, through the analysis in the ischemia reperfusion (ischemia-reperfusion, IR) changes and mechanism of injury, explore the involvement of mitochondrial gap junction on the neuroprotective significance, provide a new target for the treatment of cerebral infarction. Objective: To observe the distribution and expression of mitochondrial Cx43, analyze its changes in the IR of brain injury, to further explore the mechanism, and to evaluate the protective effect of salvianolic acid. Methods: in vitro, the astroglia cells cultured in oxygen glucose deprivation. Type (oxygen glucose deprivation, OGD) as the basis, the experiment was divided into control group, OGD group (model group), a gap junction blocker - carbenoxolone (carbenoxolone, CBX) intervention group, mitochondrial ATP sensitive potassium channel agonist - two diazoxide (diazoxide, DZX) intervention group, DZX+ of mitochondrial ATP sensitive potassium channel inhibitor 5- hydroxyl acid (5-hydroxydecanoic acid, qui 5-HD) intervention group and salvianolate (salvianolic acid SA) intervention group. First of all, to detect the intervention agents cell survival rate, cell apoptosis was also analysis, observe the mitochondrial morphology of each cell and cell in electron microscope. Then, change detection the intervention agent under the stimulation of mitochondrial membrane potential, by immunofluorescence to observe the distribution of Cx43 in astrocytes. In vivo, the weight is about 250~290 g in male Wistar rats by left middle cerebral artery (middle cerebral artery occlusion, embolization MCAO). Model rats were divided into sham operation group (control group), IR group (model group), CBX group, DZX group, DZX+5-HD group, protein kinase C (protein kinase C, PKC) - Ro-31-8425+DZX inhibitor intervention group, PKC - agonist phorbol ester (phorbol-12-myristate-13-acetate, PMA) +5-HD intervention group and SA intervention group. First of all, the drug intervention was observed on cerebral infarction area in rats, the neurological scores of the evaluation of the impact of the intervention effect of drugs on apoptosis after cerebral infarction in rats, and then observe the cerebral cortex infarction region of mitochondrial morphology, the superoxide dismutase enzyme (superoxide dismutase, SOD) activity and malondialdehyde (malondialdehyde, MDA) were detected to evaluate the intervention changes of mitochondrial function agent, at the end of the cerebral infarct zone in Cx43, p-Cx43, PKC, p-PKC protein expression containing The amount of quantitative, to explore the different effects of different intervention agents, analysis of its mechanism. Results: 1, tissue, cell morphology and simple model group, CBX intervention group and DZX cerebral infarction area was decreased, the degree of apoptosis cells decreased (P0.01), and the decrease of 5-HD to reduce the effect of DZX on infarct size (P0.01) the same; compared with the model group, SA group significantly reduced infarct size, reduced the degree of apoptosis (P0.01).2, neurological score: compared with the control group, the other groups were increased (P0.01), decreased the contralateral limb muscle strength in infarction area, but in addition to the control group, the other groups between there were no significant differences in.3, mitochondrial morphology and mitochondrial morphology and function: compared with the control group, the infarct area of each cerebral cortex mitochondria injury obviously, mainly for mitochondrial swelling, spine fracture, matrix transparent, simple model group and DZX+5 -HD group of mitochondrial damage is obvious (P0.05); mitochondrial function, compared with the sham group, pure model group showed obvious SOD activity decreased, MDA content increased (P0.01), CBX, DZX, PMA, SA all intervention agents can improve the activity of SOD, decrease the content of MDA (P0.05), to the protective effect of.4 on mitochondrial function, Cx43 positioning and quantitative: compared with control group, OGD group decreased astrocyte overall Cx43 content, especially the content distribution to reduce the cell membrane, and intracellular Cx43 content reaching increased, and the co localization of Cx43 and mitochondria; mitochondrial proteins in quantitative, and sham operation group than in model group, cerebral cortex infarction area of mitochondrial Cx43, p-Cx43 were significantly decreased (P0.01), and p-Cx43 was significantly lower (P0.01), CBX, DZX, PMA, SA intervention, compared with group IR, mitochondrial Cx43, p-Cx43 increased significantly (P0.05), and higher p-Cx43 (obviously P0.05). Conclusion: mitochondrial Cx43 can play a protective role in the IR damage of brain, which is mediated by mitochondrial ATP sensitive potassium channel to regulate PKC activity. Mitochondrial Cx43 is also involved in the protective process of salvianolic acid on brain IR injury.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R743

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