蛋白磷酸酶4显性负性突变体介导的活性抑制改善棕榈酸诱导的肝细胞脂性凋亡

Inhibition of Protein Phosphatase 4 Activity by Dominant Negative Mutant Ameliorates Palmitate-induced Lipoapoptosis in Hepatocytes

目的探讨抑制蛋白磷酸酶4(protein phosphatase 4,PP4)活性对肝细胞脂性凋亡的影响。方法以400μmol/L的棕榈酸(palmitic acid,PA)处理HepG2细胞24 h建立肝细胞脂性凋亡模型;以西方饮食喂养C57BL/6J小鼠16w建立非酒精性脂肪肝炎(non-alcoholic steatohepatitis,NASH)模型;构建磷酸酶活性缺失的显性负性突变体AD-PP4RL腺病毒载体;转染AD-PP4RL至HepG2细胞腺病毒以抑制肝细胞中PP4的活性;尾静脉注射AD-PP4RL以抑制肝脏中PP4的活性;转染RAC1慢病毒至HepG2细胞中抑制RAC1的表达;BoDIPY染色检测细胞及组织中脂质蓄积情况;TUNEL染色检测细胞凋亡水平;免疫沉淀结合丝苏氨基酸磷酸酶活性检测法测定PP4活力;GTP酶活性检测试剂盒检测RAC1活性;Western印迹检测脂质代谢及脂性凋亡相关蛋白的表达水平。结果①在PA处理的HepG2细胞及西方饮食喂养诱导的NASH小鼠肝脏中PP4活性升高;②过表达磷酸酶活性缺失突变的腺病毒载体AD-PP4RL能有效抑制PP4活性;③抑制PP4活性降低PA诱导的HepG2细胞及NASH小鼠肝脏中脂性凋亡水平,下调脂性凋亡相关基因PUMA、Bim及c-caspase3的水平;④RAC1介导了在PP4在肝细胞脂性凋亡中的作用。结论抑制PP4活性可以通过抑制RAC1介导的JNK信号通路的激活,改善肝细胞脂性凋亡。

Objective To investigate the effect of protein phosphate 4(PP4)activation on hepatocyte lipoapoptosis.Methods HepG2 cells were treated with 400μmol/L palmitic acid(PA)for 24 hours to establish hepatocyte lipoapoptosis model.C57BL/6J mice were fed with Western diet for 16 weeks to establish non-alcoholic steatohepatitis(NASH)model.The PP4 dominant negative mutant adenovirus vector AD-PP4RL was constructed.HepG2 cells were transfected with AD-PP4RL adenovirus to inhibit the activity of PP4 in hepatocytes.Mice were intravenously injected through the tail vein with AD-PP4RL adenovirus to inhibit the activity of PP4 in the liver.HepG2 cells were transfected with RAC1 lentivirus to inhibit the expression of RAC1.The lipid accumulation was detected by BoDIPY staining in cells and tissues.The levels of apoptosis were detected by TUNEL staining.The activity of PP4 was determined by immunoprecipitation combined with serine-threonine phosphatase activity assay kit.The activity of RAC1 was detected by GTPase activity assay kit.The expression levels of lipid metabolism and lipid apoptosis related proteins were detected by Western blotting.Results①The activity of PP4 was increased in HepG2 cells treated with PA and in the livers of NASH mice induced by Western diet.②The overexpression of dominant negative mutant adenovirus vector AD-PP4RL inhibited the activity of PP4 effectively.③Inhibition of PP4 activity decreased the level of lipoapoptosis in PA-treated HepG2 cells and livers of NASH mice,as well as the expression levels of lipoapoptosis related genes such as PUMA,Bim and c-caspase3.④RAC1 mediates the role of PP4 in hepatocyte lipoapoptosis.Conclusion Inhibition of PP4 activity alleviates hepatocyte lipoapoptosis by inhibiting the RAC1-mediated JNK activation and lipoapoptosis signaling pathway.