Calpain-2 activity promotes aberrant endoplasmic reticulum stress-related apoptosis in hepatocytes  

作　　者：Ru-Jia Xie Xiao-Xia Hu Lu Zheng Shuang Cai Yu-Si Chen Yi Yang Ting Yang Bing Han Qin Yang 

机构地区：[1]Guizhou Provincial Key Laboratory of Pathogenesis and Drug Research on Common Chronic Diseases,College of Basic Medical Sciences,Guizhou Medical University,Guiyang 550025,Guizhou Province,China [2]Department of Pathophysiology,College of Basic Medical Sciences,Guizhou Medical University,Guiyang 550025,Guizhou Province,China [3]Department of Physiology,College of Basic Medical Sciences,Guizhou Medical University,Guiyang 550025,Guizhou Province,China

出　　处：《World Journal of Gastroenterology》2020年第13期1450-1462,共13页世界胃肠病学杂志（英文版）

基　　金：the National Natural Science Foundation of China,No.81560105;the Department of Science and Technology of Guizhou Province,No.LH(2014)7074。

摘　　要：BACKGROUND Calpain-2 is a Ca^2+-dependent cysteine protease,and high calpain-2 activity can enhance apoptosis mediated by multiple triggers.AIM To investigate whether calpain-2 can modulate aberrant endoplasmic reticulum(ER)stress-related apoptosis in rat hepatocyte BRL-3A cells.METHODS BRL-3A cells were treated with varying doses of dithiothreitol(DTT),and their viability and apoptosis were quantified by 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2-H-tetrazolium bromide and flow cytometry.The expression of ER stress-and apoptosis-related proteins was detected by Western blot analysis.The protease activity of calpain-2 was determined using a fluorescent substrate,Nsuccinyl-Leu-Leu-Val-Tyr-AMC.Intracellular Ca^2+content,and ER and calpain-2 co-localization were characterized by fluorescent microscopy.The impact of calpain-2 silencing by specific small interfering RNA on caspase-12 activation and apoptosis of BRL-3A cells was quantified.RESULTS DTT exhibited dose-dependent cytotoxicity against BRL-3A cells and treatment with 2 mmol/L DTT triggered BRL-3A cell apoptosis.DTT treatment significantly upregulated 78 kDa glucose-regulated protein,activating transcription factor 4,C/EBP-homologous protein expression by>2-fold,and enhanced PRKR-like ER kinase phosphorylation,caspase-12 and caspase-3 cleavage in BRL-3A cells in a trend of time-dependence.DTT treatment also significantly increased intracellular Ca^2+content,calpain-2 expression,and activity by>2-fold in BRL-3A cells.Furthermore,immunofluorescence revealed that DTT treatment promoted the ER accumulation of calpain-2.Moreover,calpain-2 silencing to decrease calpain-2 expression by 85%significantly mitigated DTT-enhanced calpain-2 expression,caspase-12 cleavage,and apoptosis in BRL-3A cells.CONCLUSION The data indicated that Ca^2+-dependent calpain-2 activity promoted the aberrant ER stress-related apoptosis of rat hepatocytes by activating caspase-12 in the ER.

关 键 词：Calcium Calpain-2 CASPASE-12 Endoplasmic reticulum stress APOPTOSIS Hepatocyte 

分 类 号：R575[医药卫生—消化系统]