Salubrinal对铜负荷诱导的PERK/eIF2α内质网应激信号通路介导肝细胞凋亡的干预作用  

Copper Loading Induces PERK/eIF2α Endoplasmic Reticulum Stress Signaling Pathway-Mediated Hepatocyte Apoptosis and Its Intervention by Salubrinal

作　　者：陈永华 杨文明[2] 江海林[1] 唐露露 CHEN Yong-Hua;YANG Wen-Ming;JIANG Hai-Lin;TANG Lu-Lu(Graduate Institute,Anhui University of Chinese Medicine,Heifei 230031,China;Encephalopathy Center,First Affiliated Hospital of Anhui University of Chinese Medicine,Hefei 230038,China)

机构地区：[1]安徽中医药大学研究生院,合肥230031 [2]安徽中医药大学第一附属医院脑病中心,合肥230038

出　　处：《中国生物化学与分子生物学报》2018年第9期990-996,共7页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金项目资助(No.81373599)~~

摘　　要：Wilson病是一种以肝损害为常见临床表现的常染色体隐性遗传铜代谢障碍性疾病,但铜蓄积导致的肝细胞损害的具体分子机制尚不明确。本研究拟采用硫酸铜模拟肝细胞铜负荷进行体外实验,选用Western印迹法测试铜负荷肝细胞内蛋白激酶R样内质网激酶(protein kinase R-like endoplasmic reticulum kinase,PERK)及p-eIF2α蛋白质的表达;并探究特异性eIF2α磷酸化抑制剂Salubrinal对铜负荷诱导的肝细胞凋亡、胱天蛋白酶-3活性、C/EBP同源蛋白(C/EBP homologous protein,CHOP)mRNA转录的影响。选用流式细胞仪测试肝细胞凋亡率;比色法测试肝细胞内的胱天蛋白酶-3活性;Real-time PCR法检测肝细胞内CHOP mRNA转录水平。本研究结果显示:(1)铜负荷显示出时间依赖性地增加肝细胞内PERK及p-eIF2α蛋白质表达(P<0.05,P<0.01)。(2)相比较对照组,铜负荷培养肝细胞24 h明显增加了肝细胞的凋亡率(P<0.01),增强了肝细胞内的胱天蛋白酶-3活性,促进肝细胞内CHOP mRNA的转录,加入特异性eIF2α磷酸化抑制剂Salubrinal后可抑制上述过程。(3)相比较对照组,铜负荷促进肝细胞PERK及p-eIF2α蛋白质表达,加入特异性eIF2α磷酸化抑制剂Salubrinal后,对铜负荷诱导的肝细胞PERK蛋白质表达无影响(P>0.05),但可显著抑制铜负荷诱导的肝细胞p-eIF2α蛋白质表达(P<0.01)。本研究结果提示,铜负荷可以诱发肝细胞内质网应激,铜负荷引起的肝细胞凋亡机制与激活内质网应激PERK/eIF2α信号通路密切相关,Salubrinal具有干预该信号通路作用,能够抑制铜负荷后肝细胞的凋亡。The Wilson ＇s disease is an autosomal recessive copper metabolism disorder with liver damage as the most common clinical manifestations. However,the molecular mechanism of hepatocyte damages caused by copper accumulation is unclear. In this study,copper sulfate was used to simulate thecopper overload of hepatocytes in vitro. The abundance of protein kinase R-like endoplasmic reticulum kinase（ PERK） and p-eIF2α proteins in hepatocytes was measured by Western blotting,and the effect of specific eIF2α phosphorylation inhibitor Salubrinal on hepatocytes apoptosis,caspase-3 activity and C/EBP homologous protein（ CHOP） mRNA transcription induced by copper overload were investigated,respectively. The rate of hepatocytes apoptosis was tested by flow cytometry,the caspase-3 activity in hepatocytes was measured by colorimetric assay,and the CHOP mRNA level in hepatocytes was measured by real-time PCR. The results of this study are as follows：（ 1） The copper overload hepatocytes showed a time dependent increase in the expression of PERK and p-eIF2α protein（ P〈 0. 05,P 〈0. 01）.（ 2）Compared with the control group,copper overload-cultured hepatocytes in 24 h significantly increased the hepatocytes apoptosis rate（ P〈 0. 01）,enhanced caspase-3 activity,increased transcription CHOP mRNA. And adding specific eIF2α phosphorylation inhibitor Salubrinal could inhibit the process.（ 3）Compared with the control group,copper overload promoted the expression of PERK and p-eIF2α proteins in hepatocytes. After adding the specific eIF2α phosphorylation inhibitor Salubrinal,it could not inhibit the expression of PERK proteins in hepatocytes（ P 〉0. 05）,but it could significantly inhibit the expression of p-eIF2α proteins in hepatocytes（ P〈 0. 01）. This study showed that copper overloaded hepatocytes can induce the endoplasmic reticulum stress of hepatocytes. Moreover,the mechanism of hepatocytes apoptosis is closely related to the activation of endoplasmic reticulum stress PERK/eIF2αsig

关 键 词：铜负荷 凋亡 内质网应激 蛋白激酶R样内质网激酶 OUMS29人肝细胞系 Salubrinal 

分 类 号：R-331[医药卫生]