MCT2激活Nrf2通路调控H/R视网膜神经节细胞凋亡与焦亡  

Activation of the Nrf2 Pathway by MCT2 Regulates Apoptosis and Pyroptosis in H/R Retinal Ganglion Cells

作　　者：张小敏[1] 刘文 蔡傲竹 ZHANG Xiaomin;LIU Wen;CAI Aozhu(Zhongnan Hospital of Wuhan University,Hubei Wuhan 430070,China)

机构地区：[1]武汉大学中南医院眼科,湖北武汉430070

出　　处：《河北医学》2022年第9期1425-1431,共7页Hebei Medicine

基　　金：湖北省卫生健康委科研项目,(编号:WJ2019Q040)。

摘　　要：目的:探究单羧酸转运蛋白2(monocarboxylate transporters 2,MCT2)对缺氧/复氧(hypoxia/reoxygenation,H/R)后视网膜神经节细胞凋亡与焦亡的影响及机制。方法:将视网膜神经节细胞RGC-5随机分为对照组、H/R组、MCT2组、MCT2+ML385组,通过脂质体将pcDNA3.1-MCT2重组质粒转染至RGC-5细胞,建立细胞H/R模型,并用Nrf2抑制剂ML385处理细胞;细胞进行对应处理后,实时荧光定量PCR和蛋白质印迹(Western blot)法检测转染效率,CCK-8法测定细胞增殖活性,流式细胞术检测细胞凋亡水平,Hoechst/PI染色法检测PI凋亡指数,Western blot法检测细胞内凋亡相关蛋白与焦亡相关蛋白的表达水平,免疫荧光染色观察细胞内核因子E2相关因子(Nrf2)与半胱氨酸天冬氨酸蛋白酶1(Caspase-1)表达。结果:将pcDNA3.1-MCT2转染至RGC-5细胞后,细胞内MCT2 mRNA与蛋白相对表达量均显著上调(P<0.05);经H/R处理后,细胞增殖活性下降,细胞凋亡率增加,PI凋亡指数升高,细胞内Bax和Caspase-3蛋白相对表达量均上调而Bcl-2蛋白相对表达量下调,GSDMD、Caspase-1、NLRP3及IL-1β蛋白相对表达量均上调,Nrf2荧光强度减弱,Caspase-1荧光强度增强,差异均有统计学意义(P<0.05);细胞转染pcDNA3.1-MCT2再经H/R处理后,细胞增殖活性升高,细胞凋亡率减少,PI凋亡指数降低,细胞内Bax和Caspase-3蛋白相对表达量均下调,Bcl-2蛋白相对表达量上调,同时,细胞内GSDMD、Caspase-1、NLRP3及IL-1β蛋白相对表达量均下调,Nrf2荧光强度增强,Caspase-1荧光强度则减弱,差异均有统计学意义(P<0.05);而细胞经转染pcDNA3.1-MCT2与H/R处理后再用Nrf2抑制剂ML385作用,细胞增殖活性下降,细胞凋亡率与PI凋亡指数升高,细胞内Bax和Caspase-3蛋白相对表达量均上调、Bcl-2蛋白相对表达量下调,GSDMD、Caspase-1、NLRP3及IL-1β蛋白相对表达量均上调,Nrf2荧光强度减弱,Caspase-1荧光强度增强,差异均有统计学意义(P<0.05)。结论:MCT2能够抑制H/R诱导的�Objective:To explore the effect and mechanism of monocarboxylate transporters 2(MCT2)on apoptosis and pyrolysis of retinal ganglion cells after hypoxia/reoxygenation(H/R).Methods:Retinal ganglion cells RGC-5 were randomly divided into control group,H/R group,MCT2 group,MCT2+ML385 group,and pcDNA3.1-MCT2 recombinant plasmid was transfected into RGC-5 cells via liposome to establish Cell H/R model,and treat cells with Nrf2 inhibitor ML385.After the cells were treated accordingly,transfection efficiency was measured by real-time fluorescence quantitative PCR and protein blotting(Western blot),cell proliferation activity was measured by CCK-8,apoptosis level was measured by flow cytometry,PI apoptosis index was measured by Hoechst/PI staining,expression levels of apoptosis-related and pyrolysis-related proteins were measured by Western blot,and expression of Nrf2 and Caspase-1 were observed by immunofluorescence staining.Results:After transfection of pcDNA3.1-MCT2 into RGC-5 cells,both intracellular MCT2 mRNA and protein relative expression were significantly upregulated(P<0.05);after H/R treatment,cell proliferation activity decreased,apoptosis rate increased,PI apoptosis index increased,intracellular Bax and Caspase-3 protein relative expression were both upregulated while Bcl-2 protein relative The relative expression of Bax and Caspase-3 proteins was upregulated while that of Bcl-2 protein was downregulated,the relative expression of GSDMD,Caspase-1,NLRP3,and IL-1βproteins were upregulated,the fluorescence intensity of Nrf2 was reduced and that of Caspase-1 was enhanced.After transfected with pcDNA3.1-MCT2 and then treated with H/R,cell proliferation activity increased,apoptosis rate decreased,PI apoptosis index decreased,relative expression of intracellular Bax and Caspase-3 proteins were down-regulated,relative expression of Bcl-2 protein was up-regulated.The relative expression of GSDMD,Caspase-1,NLRP3,and IL-1βwere all down-regulated,the fluorescence intensity of Nrf2 was enhanced and that of Caspase-1 was

关 键 词：单羧酸转运蛋白2 视网膜神经节细胞 细胞凋亡 细胞焦亡 核因子E2相关因子 

分 类 号：R774.6[医药卫生—眼科]