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
罗杰 1图拉妮萨·喀迪尔 1廖师师 1谢景远 1潘锐 1陈榕 1孟庆涛1
作者信息
- 1. 武汉大学人民医院麻醉科 湖北 武汉 430060
- 折叠
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).结论:IIR和HR均可导致Sestrin2蛋白表达上调并激活内质网应激(ERS).抑制Sestrin2基因,加重了缺氧复氧诱导的Caco-2细胞损伤,它可能是通过削弱Caco-2的抗氧化功能,上调CHOP表达,加剧Caco-2细胞凋亡从而加重Caco-2细胞损伤.
Abstract
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,ATF4 and CHOP were increased in the IIR group(P<0.05).The apoptotic protein Bax was increased,the anti-apoptotic protein Bcl2 was decreased,and the expression level of Sestrin2 protein was up-regulat-ed(P<0.05).Compared with those in the NC group,the protein expression levels of BIP,ATF4,CHOP and Sestrin2 in the HR group were significantly increased(P<0.05),and the apoptosis rate was increased(P<0.05).However,compared with those in the HR-sicont group,only CHOP pro-tein expression level was significantly increased in the HR-siSESN2 group(P<0.05),and apoptosis level was significantly increased after SESN2 inhibition(P<0.05).Conclusion:Both IIR and HR lead to up-regulation of Sestrin2 protein expression and activation of ERS.Silencing Sestrin2 gene ex-pression aggravates the damage of Caco-2 cells induced by hypoxia and reoxygenation,which may be through weakening the antioxidant function of Caco-2,upregulating CHOP expression,intensifying the apoptosis of Caco-2 cells and thus aggravating the damage of Caco-2 cells.
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Key words
Sestrin2/Intestinal Ischemia-Reperfusion Injury/Hypoxia/Reoxygenation Injury/Endoplasmic Reticulum Stress Response/Apoptosis引用本文复制引用
基金项目
国家自然科学基金面上项目(82172155)
湖北省重点实验室开放项目(2021KEY032)
出版年
2024
NETL
NSTL
万方数据