硫氧还蛋白-1小干扰RNA加剧脂多糖诱导的大鼠肝细胞内质网应激  

Thioredoxin-1 siRNA aggravates lipopolysaccharide-induced endoplasmic reticulum stress of hepatocytes in rat

作　　者：王涛[1] 费成平 王宇[1] 刘晓珍[1] 刘夷嫦[1] 刘国庆[1] 闫骏[2] 谷振勇[1] 

机构地区：[1]南通大学医学院法医学系,江苏南通226001 [2]天津市第一中心医院病理科,300100

出　　处：《中华危重病急救医学》2016年第4期298-302,共5页Chinese Critical Care Medicine

基　　金：国家自然科学基金（81273341,81300346）;江苏省高校优势学科建设工程资助项目（PAPD-2011-03-16）

摘　　要：目的探讨脂多糖（LPS）诱导大鼠肝细胞内质网应激（ERS）过程中硫氧还蛋白-1（Trx-1）的变化及其作用机制。方法①体外培养大鼠肝细胞株BRL，取部分细胞，分别用0．1、1．0、5．0、10．0、20．0mg／LLPS处理细胞12h；另取部分细胞，用10．0mg／LLPS分别处理细胞1、3、6、12、24h；均以LPS溶剂磷酸盐缓冲液（PBS）为对照，从量效和时效的角度观察LPS对Trx-1蛋白表达和ERS的影响。②体外培养大鼠肝细胞株BRL，分为溶剂对照组、阴性小干扰RNA（siRNA）对照组、Trx-1 siRNA对照组、LPS刺激组、阴性siRNA＋LPS组、Trx-1 siRNA＋LPS组，Trx-1 siRNA或阴性siRNA转染24h后用10．0mg／LLPS作用12h，观察Trx-1是否参与LPS诱导大鼠肝细胞ERS。用蛋白质免疫印迹试验（Western Blot）检测Trx-1、硫氧还蛋白还原酶（TrxR）和硫氧还蛋白结合蛋白（TXNIP）的蛋白表达，ERS标志性蛋白葡萄糖调节蛋白78（GRP78）和天冬氨酸特异性半胱氨酸蛋白酶12（caspase-12）以及细胞凋亡执行蛋白caspase-3及其底物多聚二磷酸腺苷聚合酶-1（PARP-1）裂解片段的表达；采用细胞计数试剂盒检测细胞活性。结果①与溶剂对照组比较，LPS可诱导大鼠肝细胞Trx-1和TrxR蛋白表达上调、TXNIP蛋白表达下调，引起ERS相关蛋白GRP78、caspase-12上调，抑制细胞活性，且具有一定的剂量和时间依赖效应。②与溶剂对照组比较，除Trx-1及ERS相关蛋白外，LPS还可诱导大鼠肝细胞caspase-3及PARP-1裂解片段明显上调，同时抑制细胞活性；Trx-1 siRNA转染预处理24h能明显引起大鼠肝细胞Trx-1蛋白表达沉默（A值：0．249±0．017比0．531±0．030，P〈0．01），使LPS诱导的ERS及凋亡相关蛋白表达进一步上调[GRP78（A值）：1.247±0.124比0．786±0．048，caspase-12（A值）：0．810±0．017比0．578±0．013，caspase-3（A值）：0．508±0．008比0．308±0．009，PARP-1裂解片段（A值）：0Objective To investigate the change in thioredoxin-1 （Trx-1） in lipopolysaccharide （LPS）-induced endoplasmic reticulum stress （ERS） of hepatocytes in rat and its role. Methods ① The rat hepatocyte line BRL cells were cultured in vitro. Some cells were collected and treated with 0.1, 1.0, 5.0, 10.0, and 20.0 mg/L LPS for 12 hours, while other cells were collected and treated with 10.0 mg/L LPS for 1, 3, 6, 12, and 24 hours. Both of them were compared with LPS solvent phosphate buffer saline （PBS） in order to investigate the effects of LPS on Trx-1 expression and ERS in a time- and dose-dependent manner. ② The rat hepatocyte line BRL cells cultured in vitro were collected, and they were divided into vehicle control, negative siRNA control, Trx-1 siRNA control, LPS stimulation, negative siRNA ＋ LPS, and Trx-1 siRNA ＋ LPS groups. The hepatocytes transfected with Trx-1 siRNA or negative siRNA were treated with 10.0 mg/L LPS for 12 hours in order to investigate the roles of Trx-1 in LPS-induced ERS changes. Expressions of Trx-1,thioredoxin reductase （TrxR） and thioredoxin interacting protein （TXNIP）, ERS-related proteins glucose-regulated protein （GRP78） and caspase-12 as well as apoptosis-related proteins caspase-3 and its substrate cleaved poly （ADP-ribose） polymerase-1 （PARP-1） were determined by Western Blot. The cell viability was measured with cell counting kit-8 （CCK-8） test. Results ① Compared with vehicle control group, LPS could induce an increase in Trx-1 and TrxR protein expressions but a decrease in TXNIP protein expression in hepatocytes, with concomitant up-regulation of GRP78 and caspase-12, along with a decrease in cell viability in a dose- and time-dependent manner. ② Compared with vehicle control group, LPS could induce an increase in caspase-3 and cleaved PARP-1 besides Trx-1 and ERS-related proteins, and inhibit the cell viability. Trx-1 siRNA pretreatment with transfection for 24 hours could reverse LPS-induced Trx-1 up-regulation （A value： 0

关 键 词：硫氧还蛋白 脂多糖 小干扰RNA 内质网应激 大鼠 肝细胞 

分 类 号：R575[医药卫生—消化系统]