STAT3在对乙酰氨基酚所致小鼠肝损伤后肝细胞再生中的作用  

Role of STAT3 in hepatocyte regeneration after acetaminophen-induced hepatocellular injury in mice

作　　者：余旺 章礼久[1] 路燕 宋莎莎[1] YU Wang;ZHANG Lijiu;LU Yan;SONG Shasha(Department of Gastroenterology, The Second Affiliated Hospital of Anhui Medical University, Hefei 230601, China)

机构地区：[1]安徽医科大学第二附属医院消化内科,合肥230601

出　　处：《临床肝胆病杂志》2021年第4期857-862,共6页Journal of Clinical Hepatology

基　　金：国家自然科学基金青年科学基金项目(81800524)。

摘　　要：目的探讨STAT3在对乙酰氨基酚(APAP)致小鼠肝细胞损伤后肝细胞增殖中的作用。方法体外培养小鼠正常肝细胞AML12,APAP(1、2.5、5、10、20 mmol/L)刺激12、24或48 h,等体积PBS作为对照组。筛选出最佳刺激浓度和作用时间后,AG490(10、50、100μmol/L)作用AML12。CCK8法检测AML12细胞活力。RT-PCR检测AML12中PCNA、CyclinD1、Ki67的mRNA表达水平。Western Blot法检测STAT3、p-STAT3及PCNA、CyclinD1表达水平。计量资料多组间比较采用单因素方差分析,进一步两两比较采用LSD-t检验。结果APAP作用24 h及48 h后,与对照组比较,各浓度组AML12细胞活力均降低(P值均<0.05);APAP浓度为2.5 mmol/L时,细胞活力分别为0.717±0.027、0.752±0.014,与对照组比较差异均有统计学意义(P值均<0.05),能够满足后续实验条件。与对照组比较,24 h APAP(2.5 mmol/L)组PCNA、CyclinD1及Ki67 mRNA的表达均降低(P值均<0.01);与24 h APAP组比较,48 h APAP(2.5 mmol/L)组PCNA、CyclinD1及Ki67 mRNA的表达均升高(P<0.01),因此选择APAP 2.5 mmol/L、刺激时间48 h来模拟体外损伤AML12细胞后肝细胞再生的模型。加入AG490,与对照组比较,10、50μmol/L AG490组细胞活力变化无统计学意义,余各组细胞活力均降低(P值均<0.01);与APAP组比较,AG490(50μmol/L)+APAP组和AG490(100μmol/L)+APAP组细胞活力降低(P值均<0.01),因此选择50μmol/L AG490作为后续实验处理浓度。与对照组比较,APAP组p-STAT3的蛋白水平升高(P<0.01),而AG490组、APAP+AG490组降低(P值均<0.05);与APAP组比较,APAP+AG490组PCNA、CyclinD1蛋白水平及PCNA、CyclinD1、Ki67 mRNA表达均降低(P值均<0.05)。结论STAT3参与APAP诱导小鼠肝细胞损伤后的细胞增殖,而AG490作为STAT3抑制剂通过抑制STAT3磷酸化从而抑制APAP肝损伤后肝细胞增殖。Objective To investigate the role of STAT3 in hepatocyte proliferation after acetaminophen(APAP)-induced hepatocellular injury in mice.Methods Normal mouse AML12 hepatocytes were cultured in vitro and were stimulated by APAP(1,2.5,5,10,and 20 mmol/L)for 12,24 or 48 hours,and the hepatocytes treated with an equal volume of phosphate buffered saline were established as control group.After the optimal stimulation concentration and duration of action were screened out,AML12 hepatocytes were treated with AG490(10,50,and 100μmol/L).The CCK-8 assay was used to measure the viability of AML12 hepatocytes;RT-PCR was used to measure the mRNA expression levels of PCNA,CyclinD1,and Ki67 in AML12 hepatocytes,and Western blot was used to measure the protein expression levels of STAT3,p-STAT3,PCNA,and CyclinD1.A one-way analysis of variance was used for comparison of continuous data between multiple groups,and the least significant difference t-test was used for further comparison between two groups.Results After 24 and 48 hours of APAP treatment,compared with the control group,all concentration groups had a significant reduction in the viability of AML12 hepatocytes(all P<0.05),with a viability of 0.717±0.0271 and 0.752±0.0141,respectively,when the concentration of APAP was 2.5 mmol/L,which was significantly different from that in the control group(all P<0.05)and met the conditions of subsequent experiment.Compared with the control group,the 24-hour APAP(2.5 mmol/L)group had significant reductions in the mRNA expression of PCNA,CyclinD1,and Ki67(all P<0.01);compared with the 24-hour APAP group,the 48-hour APAP(2.5 mmol/L)group had significant increases in the mRNA expression of PCNA,CyclinD1,and Ki67(all P<0.01);therefore,a model of hepatocyte regeneration after in vitro AML12 hepatocyte injury was established by stimulation with APAP 2.5 mmol/L for 48 hours.After the addition of AG490,there was no significant difference in viability between the control group and the 10 and 50μmol/L AG490 groups,and the other groups had a s

关 键 词：化学性与药物性肝损伤 STAT3转录因子 醋氨酚 肝再生 

分 类 号：R575[医药卫生—消化系统]