二硫苏糖醇抑制序列相似性家族134成员B(FAM134B)介导的内质网自噬促进大鼠肝细胞凋亡  

Dithiothreitol promotes apoptosis in rat hepatocytes by inhibiting family with sequence similarity 134 member B(FAM134B)-mediated endoplasmic reticulophagy

作　　者：郭怡歆 韩冰[1] 杨婷[1] 陈雨丝 杨毅 梁猜 李佳瑶 杨勤[1] 谢汝佳[1] GUO Yixin;HAN Bing;YANG Ting;CHEN Yusi;YANG Yi;LIANG Cai;LI Jiayao;YANG Qin;XIE Rujia(Guizhou Provincial Key Laboratory of Pathogenesis&Drug Research on Common Chronic Diseases,Department of Pathophysiology,Guizhou Medical University,Guiyang 550025,China)

机构地区：[1]贵州医科大学,贵州省常见慢性疾病发病机制及药物研究重点实验室,基础医学院病理生理学教研室,贵州贵阳550025

出　　处：《细胞与分子免疫学杂志》2022年第12期1104-1110,共7页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(81560105);贵州省科学技术基金(黔科合基础-ZK[2021]一般365);贵州省科技计划项目(黔科合平台人才[2019]5801号);贵州医科大学国家自然科学基金培育项目(20NSP016)。

摘　　要：目的探讨序列相似性家族134成员B(FAM134B)介导的内质网自噬在内质网应激(ERS)诱导的肝细胞凋亡中的作用及分子机制。方法采用(0、0.5、1.0、2.0、4.0、6.0)mmol/L的二硫苏糖醇(DTT)处理BRL-3A大鼠肝细胞48 h,实时无标记细胞动态分析(RTCA)检测肝细胞增殖,流式细胞术检测细胞凋亡,Western blot法检测ERS及内质网自噬相关蛋白、内质网-线粒体接触位点相关蛋白及线粒体凋亡途经相关蛋白表达的影响。内质网及溶酶体的荧光探针检测DTT处理细胞后二者的荧光共定位;线粒体钙离子荧光探针罗丹明2乙酰氧基甲酯(Rhod-2 AM)检测DTT对线粒体中钙离子水平的影响。结果DTT可显著抑制肝细胞的增殖并促进细胞凋亡。DTT处理肝细胞后ERS及内质网自噬相关蛋白、内质网-线粒体接触位点相关蛋白及线粒体凋亡途经相关蛋白的表达均显著上调。DTT处理细胞后内质网和溶酶体的荧光共定位显著减弱,同时,线粒体内钙离子荧光强度明显增强。结论DTT通过抑制FAM134B介导的内质网自噬,进而增加线粒体内Ca^(2+)的水平,促进肝细胞凋亡。Objective To investigate the effect of family with sequence similarity 134 member B(FAM134B)-mediated endoplasmic reticulophagy on apoptosis of hepatocytes induced by endoplasmic reticulum stress(ERS)and identify its potential regulatory mechanism.Methods BRL-3A cells were treated with 0,0.5,1.0,2.0,4.0,6.0 mmol/L dithiothreitol(DTT)for 48 hours.The effect of DTT treatment on the proliferation and apoptosis was analyzed using real time cellular dynamic analysis(RTCA)and flow cytometry.The level of proteins related to ERS,endoplasmic reticulophagy,mitochondria-endoplasmic reticulum contact sites(MERCs),and mitochondrial apoptosis pathway were determined using Western blot analysis.Co-localization of ER and lysosomes were detected using ER and lysosomal fluorescence probes.A Ca^(2+)fluorescence probe was used to detect the level of Ca^(2+)in mitochondria.Results DTT treatment significantly inhibited cell proliferation and promoted apoptosis in hepatocytes.The levels of proteins related to ERS and endoplasmic reticulophagy,MERCs and the mitochondrial apoptosis pathway significantly increased in BRL-3A cells treated with DTT.DTT treatment decreased the ER-lysosome co-localization and enhanced the fluorescence intensity of Ca^(2+)in mitochondria.Conclusion DTT aggravates hepatocyte apoptosis by inhibiting FAM134B-mediated endoplasmic reticulophagy and enhancing the level of mitochondrial Ca^(2+).

关 键 词：肝细胞 内质网自噬 序列相似性家族134成员B(FAM134B) 线粒体凋亡途经 细胞凋亡 

分 类 号：Q255[生物学—细胞生物学] R392.12[医药卫生—免疫学] Q731[医药卫生—基础医学]