IRF-1通过JNK通路调节细胞自噬在肝缺血再灌注损伤中的作用

发布时间：2018-06-09 05:30

本文选题：IRF-1 + JNK　；参考：《天津医科大学》2017年硕士论文

【摘要】：目的:肝脏缺血再灌注(ischemia reperfusion,IR)损伤是临床常见的病理过程,常发生在休克、创伤、肝切除和肝移植后,可导致术后肝功能恢复延迟甚至移植肝无功能,严重影响肝脏手术的临床效果与患者生存预后。延长肝缺血耐受时间、减少缺血再灌注损伤、保护移植肝功能是临床亟待解决的重要问题。本研究旨在探讨小鼠肝脏缺血再灌注过程中,IRF-1表达对细胞自噬的影响,IRF-1调节JNK通路对肝缺血再灌注损伤自噬与损伤的作用,研究肝脏缺血再灌注损伤的可能机制,为肝脏缺血再灌注损伤的防治提供可能的方法。方法:雄性成年C57BL/6小鼠,随机分为假手术组(Sham组),缺血再灌注0h、2h、6h、12h、24h组。采取夹闭肝左叶和中叶肝门制备70%肝缺血再灌注模型,检测各组小鼠血清丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)水平。HE染色观察肝脏组织病理学改变,TUNEL法观察肝细胞凋亡情况。免疫组织化学检测各组肝组织PCNA、Bcl-2和p-JNK的阳性表达,透射电镜检测小鼠肝组织细胞内自噬体的数量,Western blot检测小鼠肝组织IRF-1、JNK、p-JNK、Beclin-1、p62、LC3、Bcl-2、Caspase-3与Cleave Caspase-3蛋白表达变化。采用肝细胞系AML12细胞建立缺氧/复氧(H/R)细胞模型以模拟肝缺血再灌注,进一步验证体外实验IRF-1调控JNK诱导自噬性死亡在缺氧/复氧中的作用。通过Ad IRF-1、JNK si RNA预处理转染AML12细胞,Western blot检测各组AML12细胞IRF-1、JNK、p-JNK、Beclin-1、p62、LC3、Bcl-2、Caspase-3与Cleave Caspase-3蛋白表达变化,免疫细胞荧光检测AML12细胞p-JNK的表达变化,共聚焦显微镜和透射电镜观察各组AML12细胞内自噬体变化。使用雷帕霉素(Rap)与3-MA预处理AML12细胞,MTT检测各组AML12细胞生存率,Western blot检测各组AML12细胞内Beclin-1、p62、LC3、Bcl-2、Caspase-3与Cleave Caspase-3蛋白表达变化,以明确自噬对H/R处理诱导AML12细胞凋亡的影响。结果:IR组小鼠血清ALT、AST水平随着再灌注时间增加明显上升(P0.05);IR组小鼠肝组织随着再灌注时间增加损伤逐渐加重,肝组织细胞水肿、气球样变,肝窦区狭窄、中央静脉淤血和肝细胞坏死等病理变化。与Sham组相比,IR组小鼠肝组织凋亡细胞随着再灌注时间的增长而明显增多(P0.05)。与Sham组相比,IR组小鼠肝组织PCNA阳性表达的细胞数随着再灌注时间增加而升高,在6h达到高峰。IR组小鼠肝组织Bcl-2阳性表达的细胞数随着再灌注时间增加而减少,在12h表达最少。IR组小鼠肝组织p-JNK阳性表达的细胞数在再灌注2h达峰值,然后随再灌注时间增加而减少。与Sham组相比,再灌注6h组小鼠肝组织的自噬体的数量明显升高。IR组小鼠肝组织IRF-1、JNK与p-JNK蛋白水平随再灌注时间增加而升高,且均在2h达高峰,凋亡蛋白Cleave Caspase-3蛋白水平随着再灌注时间增加而升高,在6h达高峰,Caspase-3蛋白水平没有明显变化,而抗凋亡蛋白Bcl-2蛋白水平减少。自噬相关蛋白Beclin-1、p62、LC3表达水平增加,在6h达高峰。Ad IRF-1组AML12细胞IRF-1、JNK与p-JNK蛋白水平较Ad GFP组升高。免疫荧光结果Ad IRF-1组AML12细胞p-JNK表达水平较Ad GFP组升高。相比Ad GFP组,Ad IRF-1组AML12细胞自噬相关蛋白Beclin-1、p62、LC3表达水平增加,Caspase-3与Cleave Caspase-3蛋白水平升高,而Bcl-2蛋白水平降低。相比si RNA-NC组,JNK si RNA组AML12细胞JNK和p-JNK表达减少,Beclin-1、p62、LC3、Caspase-3与Cleave Caspase-3水平降低,而Bcl-2蛋白水平升高。共聚焦和透射电镜结果说明Ad IRF-1组AML12细胞自噬体增加,而JNK si RNA组自噬体减少。使用自噬激动剂和抑制剂预处理AML12细胞后,Rap组AML12细胞生存率较IR组明显降低(P0.001),而3-MA组AML12细胞生存率较IR组明显升高(P0.01)。Rap组Beclin-1、p62、LC3与Caspase-3与Cleave Caspase-3蛋白表达水平均较IR组显著升高,而3-MA组较IR组明显减少。结论:小鼠肝脏缺血再灌注诱导肝组织IRF-1与JNK信号通路表达增加,自噬水平升高,自噬体数量增加,自噬相关蛋白Beclin-1、p62、LC3的表达增高。同时,凋亡相关蛋白Cleave Caspase-3蛋白表达增加。肝缺血再灌注过程中,IRF-1通过调控JNK信号通路增加肝细胞自噬水平,促进肝细胞凋亡,加重缺血再灌注损伤。
[Abstract]:Objective: ischemia reperfusion (IR) injury is a common clinical pathological process, which often occurs after shock, trauma, hepatectomy and liver transplantation, which can lead to delayed recovery of liver function and no function of liver transplantation, which seriously affects the clinical effect of liver surgery and the survival prognosis of the patients. The purpose of this study is to explore the effect of IRF-1 expression on autophagy in the process of liver ischemia reperfusion in mice. IRF-1 regulates the role of JNK pathway in the autophagy and injury of liver ischemia-reperfusion injury, and studies the possible mechanism of liver ischemia reperfusion injury. Methods: the prevention and control of hepatic ischemia reperfusion injury was provided. Methods: male adult C57BL/6 mice were randomly divided into sham operation group (group Sham), ischemia reperfusion 0h, 2h, 6h, 12h, 24h group. A model of 70% liver ischemia reperfusion was made by clamping the left lobe of the liver and the middle lobe of the liver. The serum alanine transaminase (ALT) and aspartate aminotransferase (aspartate aminotransferase) were measured. (AST) the pathological changes of liver tissue were observed by the level of.HE staining, and the apoptosis of liver cells was observed by TUNEL method. The positive expression of PCNA, Bcl-2 and p-JNK in liver tissues of each group was detected by immunohistochemistry. The number of autophagic bodies in the liver tissues of mice was detected by transmission electron microscopy. Western blot detected the IRF-1, JNK, p-JNK, Beclin-1, Beclin-1. The changes in the expression of pase-3 and Cleave Caspase-3 protein. The hypoxia / reoxygenation (H/R) cell model was established by using hepatocyte line AML12 cells to simulate hepatic ischemia reperfusion. The effect of IRF-1 on the regulation of JNK induced autophagic death in hypoxia / reoxygenation in vitro was further verified. AML12 cells IRF-1, JNK, p-JNK, Beclin-1, p62, LC3, Bcl-2, Caspase-3 and Cleave Caspase-3 protein expression changes, immunofluorescence detection of the expression of AML12 cells, confocal microscopy and transmission electron microscopy to observe the changes in the autophagic cells in each group. Group AML12 cell survival rate and Western blot detection of Beclin-1, p62, LC3, Bcl-2, Caspase-3 and Cleave Caspase-3 protein expression in each group of AML12 cells to determine the effect of autophagy on the apoptosis induced by H/R treatment. With the time of reperfusion, the injury gradually increased, the hepatic tissue cell edema, the balloon like change, the stenosis of the hepatic sinus, the central venous congestion and the necrosis of the liver cells. Compared with the Sham group, the apoptotic cells of the liver tissue in the IR group increased significantly (P0.05). Compared with the group Sham, the PCNA positive form of the liver tissue in the group IR mice was compared with that of the group IR. The number of cells increased with the increase of reperfusion time. The number of Bcl-2 positive cells expressed in the liver tissue of the.IR group at the peak of 6h decreased with the increase of reperfusion time. The number of p-JNK positive cells in the liver tissue of the least.IR group in the.IR group was at the peak of the reperfusion 2h, and then decreased with the increase of the reperfusion time. And Sham group. In contrast, the number of autophagic bodies in the liver tissue of the 6h group was significantly increased in the liver tissue of.IR mice, and the level of JNK and p-JNK protein increased with the increase of reperfusion time, and at the peak of 2h, the level of the apoptotic protein Cleave Caspase-3 protein increased with the increase of reperfusion time, and the level of Caspase-3 protein was not obvious at the peak of 6h. The level of anti apoptotic protein Bcl-2 protein decreased. The expression level of autophagy related protein Beclin-1, p62 and LC3 increased, and the IRF-1, JNK and p-JNK protein levels of AML12 cells in the peak.Ad IRF-1 group were higher than those of the Ad group. The level of cellular autophagy related protein Beclin-1, p62, LC3 increased, and the level of Caspase-3 and Cleave Caspase-3 increased, while the level of Bcl-2 protein decreased. The results of electron microscopy showed that the autophagic body of AML12 cells in the Ad IRF-1 group was increased, but the autophagic body of the JNK Si RNA group decreased. The survival rate of AML12 cell in Rap group was significantly lower than that of the IR group after the pretreatment of AML12 cells with autophagic agonists and inhibitors, while the survival rate of 3-MA group was more than that of Xian Shenggao. The expression level of leave Caspase-3 protein was significantly higher than that in the IR group, while the 3-MA group was significantly lower than the IR group. Conclusion: the expression of IRF-1 and JNK signaling in liver tissues of mice was increased, the level of autophagy increased, the number of autophagic increased, the expression of autophagic related protein Beclin-1, p62 and LC3 increased. Meanwhile, the apoptosis related protein was Cleave Cas The expression of pase-3 protein is increased. In the course of liver ischemia and reperfusion, IRF-1 increases the autophagy level of hepatocyte by regulating the JNK signaling pathway, promotes the apoptosis of liver cells and aggravates the ischemia reperfusion injury.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R657.3

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