Sestrin2通过调节CHOP表达对肠缺血再灌注损伤的影响  

Effect of Sestrin2 on intestinal ischemia-reperfusion injury by modulating CHOP expression

作　　者：罗杰 图拉妮萨·喀迪尔 廖师师 谢景远 潘锐 陈榕[1] 孟庆涛[1] LUO Jie;TULANISA Kadier;LIAO Shishi;XIE Jingyuan;PAN Rui;CHEN Rong;MENG Qingtao(Dept.of Anesthesiology,Renmin Hospital of Wuhan University,Wuhan 430060,Hubei,China)

机构地区：[1]武汉大学人民医院麻醉科,湖北武汉430060

出　　处：《武汉大学学报（医学版）》2024年第9期1038-1043,共6页Medical Journal of Wuhan University

基　　金：国家自然科学基金面上项目(编号:82172155);湖北省重点实验室开放项目(编号:2021KEY032)。

摘　　要：目的:探讨Sestrin2(SESN2)在肠缺血再灌注损伤中的作用及内在调控机制,并探究SESN2与内质网应激之间的关联作用。方法:(1)动物实验:将12只健康雄性成年C57BL/6J小鼠随机分为假手术组(Sham组,n=6)和肠缺血再灌注组(IIR组,n=6)。采用无创动脉夹夹闭肠系膜上动脉根部45 min、恢复供血2 h的方法构建肠缺血再灌注小鼠模型。采用HE染色法和Chiu's病理学损伤评分观察各组小鼠小肠损伤情况。检测各组小鼠小肠组织中氧化应激指标超氧化物歧化酶(SOD)和丙二醛(MDA)水平。免疫印迹法测定各组小鼠小肠组织中葡萄糖调节蛋白78(GRP78/BIP)、活性转录因子4(ATF4)、CCAAT-增强子结合蛋白(CHOP)、Sestrin2、Bcl2和Bax蛋白表达水平。(2)细胞实验:所有细胞实验选择人结直肠腺癌细胞(Caco-2细胞),通过缺氧12 h后复氧2 h建立缺氧复氧(HR)模型,随机分组为对照组(NC组,n=6)、缺氧复氧组(HR组,n=6);在此基础上,对Caco-2细胞转染Sestrin2-siRNA(siSESN2)和对照组siRNA(sicont),随机分为对照+对照组siRNA(NC-sicont组,n=6)、对照+siSESN2(NC-siSESN2组,n=6)、HR+对照组siRNA(HR-sicont组,n=6)和HR+siSESN2(HR-siSESN2组,n=6);免疫印迹法测定各组细胞中SENS2、BIP、ATF4和CHOP蛋白的表达水平,流式细胞学检测各组细胞凋亡水平。结果:与Sham相比,IIR组小鼠小肠组织损伤显著加重,其Chiu's评分和MDA含量显著升高,SOD含量显著降低(P<0.05)。IIR组小鼠肠组织中BIP、ATF4及CHOP蛋白表达水平显著升高(P<0.05),肠细胞凋亡蛋白Bax显著升高、抗凋亡蛋白Bcl2显著降低,与此同时,IIR组小鼠小肠组织中Sestrin2蛋白表达水平较之于Sham组显著升高(P<0.05)。与NC组相比,HR组细胞BIP、ATF4、CHOP及Sestrin2蛋白表达水平显著升高(P<0.05),细胞凋亡率显著增高(P<0.05)。然而,较之于HR-sicont组,HR-siSESN2组中仅发现CHOP蛋白表达水平显著升高(P<0.05),并且抑制SESN2表达后细胞凋亡水平显著升高(P<0.05)�Objective:To investigate the role of Sestrin2(SESN2)in intestinal ischemia-reperfusion injury(IIR)and its underlying regulatory mechanism,and to explore the synergistic effect between SESN2 and endoplasmic reticulum stress.Methods:In vivo,12 healthy male adult C57BL/6J mice were ran-domly divided into the Sham group(n=6)and IIR group(n=6).IIR was established by clamping the superior mesenteric artery for 45 min followed by 120 min reperfusion.The mice were sacrificed 2 hours later and the intestinal tissue was collected,fixed with 10%paraformaldehyde and frozen with liquid nitrogen.HE staining and Chiu's pathological injury scoring were used to observe the small in-testine injury in each group.The levels of oxidative stress indexes of superoxide dismutase(SOD)and malondialdehyde(MDA)in the small intestine of mice in each group were detected.The expression levels of glucose-regulatory protein 78(GRP78/BIP),active transcription factor 4(ATF4),CCAAT-enhancer binding protein(CHOP),Sestrin2,Bcl2 and Bax in the small intestine of each group were determined by Western Blot.In vitro,human colorectal adenocarcinoma cells(Caco-2 cells)were se-lected for all cell experiments,and the hypoxia/reoxygenation(HR)model was established through re-oxygenation for 2 hours after 12 hours of hypoxia.They were randomly divided into control group(NC group,n=6)and hypoxia and reoxygenation group(HR group,n=6).On this basis,Caco-2 cells transfected with Sestrin2-siRNA(siSESN2)and control group siRNA(sicont)were randomly di-vided into:Control+control group siRNA(NC-sicont group,n=6),control+siSESN2(NC-siS-ESN2 group,n=6),HR+control group siRNA(HR-Sicont group,n=6)and HR+siSESN2(HR-SisesN2 group,n=6).The expression levels of SENS2,BIP,ATF4 and CHOP were determined by Western Blot,and apoptosis was detected by flow cytometry.Results:Compared with those in the Sham group,Chiu's score and MDA content in the IIR group were significantly increased,SOD con-tent expression was significantly decreased(P<0.05),and protein expression levels of BIP,AT

关 键 词：Sestrin2 肠缺血再灌注损伤 缺氧复氧损伤 内质网应激反应 细胞凋亡 

分 类 号：R364.4[医药卫生—病理学]