EP4受体介导的肝缺血再灌注时线粒体保护效应及机制研究

发布时间：2018-03-04 09:28

本文选题：缺血再灌注　切入点：环氧化酶-2　出处：《第二军医大学》2016年硕士论文　论文类型：学位论文

【摘要】：缺血再灌注(I/R)损伤是临床常见的病理现象,创伤与失血性休克、冠脉缺血和体外循环手术以及器官移植手术时I/R损伤不可避免,重者可影响患者预后。I/R损伤机制复杂,其发生机理仍不完全清楚,目前认为主要涉及氧自由基和活性氧(Reactive oxygen species,ROS)的大量产生、炎性损伤以及微循环障碍等过程,而启动这些环节的关键是细胞能量代谢障碍。线粒体作为细胞氧化磷酸化的重要细胞器,其功能改变与I/R损伤密切相关。前列腺素E2(PGE2)是生物体内重要的内源性调节因子。I/R损伤时,PGE2对肝脏组织的炎性反应和微循环灌注起到重要的调节作用。环氧化酶-2(COX-2)是调控体内PGE2生成的重要限速酶,已有研究证明COX-2在肝I/R损伤中发挥重要作用。课题组前期发现抑制COX-2能激活一种细胞内保护机制,即抑制线粒体膜通透性转换孔(MPTP)开放,减轻I/R损伤,但其具体分子机制并不完全清楚。PGE2在细胞膜上存在4种G蛋白偶联受体,即前列腺素E2受体1、2、3和4(PGE2 receptor 1,2,3和4,EP 1,2,3,4)。作为COX-2下游重要的G蛋白偶联受体,已有研究证实EP4在I/R时直接介导了PGE2的肝脏保护效应。但是,EP4信号与对再灌注时线粒体功能的影响并不清楚。本课题通过70%大鼠肝脏热I/R损伤模型,研究了EP4信号在肝脏I/R损伤中的作用和机制。采用可逆性的肝血管阻断法建立稳定、可靠的Sprague Dawley(SD)大鼠70%肝热缺血再灌注模型,通过血清酶学检测、组织病理学观察、荧光分子探针测定线粒体钙容纳力(mitochondria calcium capacity,CRC)以及免疫印迹检测线粒体相关信号通路来研究EP4激动剂对肝I/R时的线粒体保护效应及分子机制。方法:将雄性Sprague Dawley(SD)大鼠(200g-230g)随机化分组:1.假手术组(Sham组):只打开其腹腔,分离血管,不给予夹闭缺血;2.缺血再灌注组(I/R组):采用经典的、可逆性的肝血管阻断法建立稳定、可靠的SD大鼠70%肝热缺血再灌注模型,夹闭肝左叶和肝中叶血管系统,形成肝脏中叶和左叶缺血,松开血管夹行再灌注;3.I/R+缺血预处理(IPC)组:于缺血前给予一次10min的短暂缺血,然后再灌注10min+70%肝脏热缺血60min再灌注;4.I/R+COX-2抑制剂NS-398(NS-398)预处理组:缺血前10min腹腔给予COX-2抑制剂NS-398(30mg/kg)+70%肝脏热缺血60min再灌注;5.I/R+EP4激动剂CAY10598(CAY)处理组:70%肝脏热缺血60min再灌注+于缺血前1.5h、缺血30min和再灌注即刻分三次腹腔内注射总剂量1mg/kg EP4激动剂CAY10598;6.I/R+CAY+Carboxy Atractyloside(CATR)组:70%肝脏热缺血60min再灌注+于缺血前1.5h、缺血30min和再灌注即刻分三次腹腔内注射总剂量1mg/kg EP4激动剂CAY10598+缺血30min前腹腔注射5mg/kg线粒体膜通透性转换孔开放剂Carboxyatractyloside。于再灌注2h和6h,取肝组织和血清。采用聚合酶链式反应(Polymerase Chain Reaction,PCR)及Western Blot检测肝脏I/R损伤后不同时间点EP4受体的表达情况;行血清酶学、组织病理学监测肝损伤、荧光免疫法测定线粒体钙容纳力(CRC);信号机制研究方面采用ELISA法检测肝组织环磷酸腺苷(c AMP)和组织活性氧(ROS)水平;运用Western blot技术检测各组肝组织GSK-3β、p-GSK-3β、ERK1/2、p-ERK1/2、JAK2、p-JAK2、STAT3、p-STAT3蛋白表达情况。结果:1.PCR和Real Time-PCR结果发现,正常肝组织EP1、EP2、EP3和EP4基因均有不同程度的表达,而与sham组比较,I/R后2h肝组织EP1、EP2和EP4受体的m RNA表达均明显升高。IPC能明显下调EP2和EP4受体的基因表达,而使用COX-2抑制剂NS-398预处理能下调EP4受体的基因表达。Western blot结果显示,与Sham组比较,I/R能显著促进再灌注2 h和6h肝组织EP4蛋白表达(P0.05),而NS-398则能明显抑制该作用(P0.05)。2.再灌注时检测血清AST和ALT水平,结果显示:I/R时血清AST和ALT水平显著升高,CAY预处理能明显降低再灌注2h和6h血清AST和ALT水平(P0.05)。再灌注6 h肝组织H-E染色和TUNEL染色结果显示,再灌注6h I/R组肝细胞损伤明显,CAY能明显减轻肝细胞坏死,减少凋亡指数(P0.05),提供肝保护。而MPTP开放剂CATR能显著逆转CAY的保护作用。透射电镜下观察细胞超微结构发现:CAY能减少再灌注2h肝细胞核固缩溶解,线粒体等细胞器形态明显改善。差速离心法制备再灌注2 h和6 h线粒体,使用Flex Station II荧光分析仪测定各组线粒体钙容纳力(CRC)显示:与I/R组比较,再灌注2 h和6 h CAY预处理能显著增加线粒体CRC,抑制MPTP开放,而CATR则能显著逆转CAY的作用(P0.05)。3.再灌注6 h肝组织c AMP和ROS水平检测显示:I/R能诱导肝组织c AMP和ROS水平明显升高(P0.01),与I/R组比较,CAY预处理能进一步促进肝组织c AMP浓度升高(P0.01),降低ROS水平(P0.05)。Western blot结果显示:再灌注2h和6h I/R组肝组织p-ERK1/2表达明显升高,CAY处理能进一步增加肝组织p-ERK1/2表达(P0.05);再灌注2h I/R、I/R+CAY和I/R+CAY+CATR组肝组织p-GSK-3β表达均有不同程度升高,至再灌注6h各组p-GSK-3β表达呈下降趋势,与I/R组和I/R+CAY+CATR组比较,CAY能增加再灌注6h肝组织p-GSK-3β表达(P0.05);再灌注2h和6h,I/R、I/R+CAY和I/R+CAY+CATR组p-JAK2表达均有不同程度升高,其中2h升高最为显著,与I/R组比较,CAY能抑制再灌注时肝组织p-JAK2的表达(P0.05);再灌注2h和6h,I/R和I/R+CAY+CATR组p-STAT3表达均显著升高,而CAY组再灌注2h p-STAT3表达与sham和I/R组比较呈显著下降,至6h时逐渐升高(P0.05),提示CAY能延缓再灌注时肝组织p-STAT3的表达升高。结论:1.作为COX-2信号下游重要位点,EP4直接参与了肝脏I/R损伤的病理过程。2.使用EP4激动剂CAY10598预处理激活EP4信号,能抑制再灌注时MPTP开放,减轻线粒体损伤,减少肝细胞坏死和凋亡。3.EP4可通过下游c AMP-ERK1/2信号进一步影响MPTP调控相关的GSK-3β和JAK2-STAT3信号改变,从而调控MPTP通透性,影响ROS生成,调控肝I/R损伤。
[Abstract]:Ischemia reperfusion (I/R) injury is common in clinic, trauma and hemorrhagic shock, ischemia and coronary bypass surgery and organ transplantation when I/R damage is unavoidable, the weight can affect the prognosis of patients with.I/R injury mechanism is complex, its pathogenesis is still not completely clear, think at present mainly involves oxygen free radicals and reactive oxygen species (Reactive oxygen species, ROS) of the emergence, process of inflammatory injury and microcirculation, and these links are the key to start cell energy metabolism. Mitochondrial oxidative phosphorylation in cells as important organelles, its function is closely related to the I/R injury. Prostaglandin E2 (PGE2) is an important endogenous regulation factor.I/R injury, PGE2 of liver tissue inflammation and microcirculation play an important role of cyclooxygenase -2 (COX-2) is regulated in vivo production of PGE2 heavy The speed limit enzyme, studies have demonstrated that COX-2 play an important role in liver injury in I/R. Our research group found that inhibition of COX-2 can activate a protective mechanism of cells, inhibit the mitochondrial permeability transition pore (MPTP) opening, reduce I/R damage, but its molecular mechanism is not completely clear.PGE2 there are 4 G protein coupled the receptor on the cell membrane, the prostaglandin E2 receptor (PGE2 1,2,3 and 4 receptor 1,2,3 and 4 EP, 1,2,3,4). As COX-2 downstream G protein coupled receptor is important, it has been confirmed that EP4 in I/R mediated liver protective effect of PGE2. However, the EP4 signal and the effects on mitochondrial function during reperfusion. This topic is not clear. By 70% I/R of rat liver heat injury model, and study the mechanism of EP4 signaling in liver I/R injury. Occlusion of hepatic vascular stability by reversible, reliable Sprague Dawley (SD) 7 rats 0% liver ischemia reperfusion model by serum enzyme detection, histopathological observation, fluorescence molecular probe for the determination of mitochondrial calcium capacity (mitochondria calcium capacity, CRC) and Western blot detection of mitochondrial related signal pathway of EP4 agonist on mitochondrial protective effect and molecular mechanism of liver I/R. Methods: male Sprague Dawley (SD) rat (200g-230g) 1. randomization: sham operation group (group Sham): open the abdominal cavity, the separation of blood vessels, not given the occlusion ischemia; 2. ischemia reperfusion group (I/R group): the classical, reversible hepatic vascular occlusion, 70% SD rats liver reliable thermal ischemia reperfusion model, occlusion of left hepatic lobe and middle lobe of liver vascular system, the formation of the middle and the left lobe of liver ischemia reperfusion, loosen the vascular clamp; 3.I/R+ ischemic preconditioning (IPC) group: transient ischemic ischemia in given before a 10min, then Perfusion of liver heat 10min+70% 60min ischemia reperfusion; 4.I/R+COX-2 inhibitor NS-398 (NS-398) 10min before ischemia preconditioning group: intraperitoneal administration of COX-2 inhibitor NS-398 (30mg/kg) +70% 60min liver ischemia reperfusion; 5.I/R+EP4 CAY10598 agonist (CAY) treatment group: 70% hepatic ischemia reperfusion + 60min at 1.5h before ischemia, ischemia and 30min immediately after reperfusion three intraperitoneal injection of 1mg/kg total dose of EP4 agonist CAY10598; 6.I/R+CAY+Carboxy Atractyloside (CATR) group: 70% hepatic ischemia reperfusion + 60min at 1.5h before ischemia, ischemia 30min and reperfusion three intraperitoneal injection of 1mg/kg total dose of EP4 agonist CAY10598+ 30min ischemia before intraperitoneal injection of 5mg/kg mitochondrial membrane the permeability transition pore opening agent Carboxyatractyloside. on reperfusion 2H and 6H, the liver tissue and serum. Using polymerase chain reaction (Polymerase Chain Reaction, PCR) and Western Blo T detection of liver I/R injury at different time points after EP4 receptor expression; serum enzymology, tissue pathology monitoring of liver injury, determination of the capacity of mitochondrial calcium fluorescence immunoassay (CRC); signal mechanism of liver tissue was detected by ELISA method of cyclic adenosine monophosphate (C AMP) and reactive oxygen species (ROS) level; the detection of GSK-3 beta, the liver Western blot p-GSK-3 ERK1/2, p-ERK1/2, beta, JAK2, p-JAK2, STAT3, p-STAT3 protein expression. Results: 1.PCR and Real Time-PCR were found in normal liver tissue EP1, EP2, EP3 and EP4 gene expression in varying degrees, and compared with group sham, liver 2H EP1 I/R, m RNA and EP2 EP4 expression were significantly increased in.IPC could significantly reduce the EP2 and EP4 receptor gene expression, and the use of COX-2 inhibitors of NS-398 pretreatment can down regulate the gene expression of EP4 receptor.Western blot results showed that, compared with Sham group, I/R Reperfusion can significantly promote the EP4 protein expression in liver tissue of 2 h and 6h (P0.05), while NS-398 could inhibit the effect of.2. (P0.05) showed reperfusion serum AST and ALT levels, serum AST and ALT levels were significantly higher in I/R, CAY pretreatment can significantly reduce reperfusion 2H and 6h the serum AST and ALT levels (P0.05). 6 h reperfusion of liver tissue H-E staining and TUNEL staining showed that 6h reperfusion group I/R hepatocellular injury obviously, CAY can significantly reduce the necrosis of liver cells, decrease the apoptosis index (P0.05), to provide protection. The protective effect of liver and MPTP open agent CATR could reverse CAY under transmission electron microscope. Cell ultrastructure showed that CAY can reduce the reperfusion of 2H liver cell nuclear pyknosis dissolved, mitochondria morphology improved significantly. The differential centrifugation was prepared after reperfusion for 2 h and 6 h mitochondria, determination of mitochondrial calcium content using Flex Station II fluorescence analyzer Nali (CRC) showed that: compared with group I/R, reperfusion 2 h and 6 h CAY pretreatment could significantly increase mitochondrial CRC, inhibiting the opening of MPTP, while CATR can significantly reverse the effects of CAY (P0.05).3. showed 6 h reperfusion liver C AMP and ROS I/R can induce the liver tissue C AMP and ROS levels were significantly increased (P0.01), compared with I/R group, CAY pretreatment can promote the C AMP concentration in liver tissue increased (P0.01), reduce the level of ROS (P0.05).Western blot showed that 2h of reperfusion and liver tissue 6h I/R group significantly increased the expression of p-ERK1/2, CAY treatment can further increase the liver the expression of p-ERK1/2 (P0.05); 2H reperfusion I/R, increased the expression of p-GSK-3 in I/R+CAY and I/R+CAY+CATR group, the expression of 6h p-GSK-3 in each group to reperfusion beta decreased, compared with group I/R and group I/R+CAY+CATR, CAY and 6h in liver tissue perfusion can increase the expression of p-GSK-3 (P0.05); reperfusion Note: 2h and 6h, I/R, I/R+CAY and I/R+CAY+CATR p-JAK2 expression were increased to varying degrees, of which 2H was the most significant, compared with I/R, CAY can inhibit the expression of p-JAK2 in liver tissue during reperfusion (P0.05); reperfusion 2H and 6h, I/R and p-STAT3 expression were significantly increased in group I/R+CAY+CATR, and group CAY reperfusion 2H p-STAT3 expression compared with sham and I/R group were significantly decreased, and 6h increased gradually (P0.05), suggesting that CAY can delay the reperfusion of hepatic p-STAT3 expression increased. Conclusion: the 1. COX-2 signal as important downstream sites, EP4 is directly involved in the pathological process of liver injury in.2. I/R using the EP4 agonist CAY10598 the pretreatment can inhibit the activation of EP4 signal, the reperfusion of the opening of MPTP, alleviate the injury of mitochondria,.3.EP4 reduced liver cell necrosis and apoptosis by AMP-ERK1/2 signal downstream of the C further affect the regulation of MPTP related GSK-3 beta and JAK2-STAT3 signal changes, and Regulating the permeability of MPTP, affecting the formation of ROS and regulating the I/R damage of liver.

【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R657.3

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