乙型肝炎病毒全x蛋白体外诱导肝星状细胞炎症反应并促进肝细胞凋亡*

目的 探究乙型肝炎病毒(HBV)全x蛋白(HBwx)诱导肝星状细胞(HSCs)炎症反应和促进肝细胞凋亡的作用及其机制。方法 取LX2细胞,分为对照组、脂多糖(LPS)处理组、转染HBwx处理组和LPS联合转染HBwx处理组。采用Western blot法检测自噬相关蛋白p62和LC3及炎症相关蛋白NLRP3和pro-IL表达,采用ELISA法检测培养上清IL-1β水平;收集上述各组LX2细胞培养上清刺激HL7702细胞培养,使用流式细胞仪检测HL7702细胞凋亡。结果 经HBwx质粒转染的LX2细胞在细胞质表达HBwx蛋白;与正常组比,LPS组、HBwx组和LPS联合HBwx组LC3表达显著减低,而p62表达显著增强(P<0.05);与正常组比,LPS组、HBwx组和LPS联合HBwx组NLRP3和pro-IL1表达显著增强(P<0.05);与正常组比,LPS组、HBwx组和LPS联合HBwx组IL-1β分泌显著增多(P<0.05);与正常组比,LPS、HBwx和LPS联合HBwx处理HL-7702细胞凋亡水平显著增强(P<0.05);与LPS组或HBwx组比,LPS联合HBwx处理HL-7702细胞凋亡水平进一步增强(P<0.05)。结论 HBwx作用于HSCs抑制自噬并促进细胞炎症反应,HSCs分泌IL-1β,诱导肝细胞凋亡。

Hepatitis B whole x gene promotes inflammation reaction of hepatic stellate cells and triggers hepatocyte apoptosis in vitro

Objective The purpose of this experiment was to investigate hepatitis B whole x (HBwx) gene promotes inflammation reaction of hepatic stellate cells (HSCs) and triggers hepatocyte apoptosis in vitro. Methods The LX2 cells were divided into control, lipopolysaccharide (LPS)-, HBwx transfected and LPS and HBwx transfection combination-intervened groups. The cell p62 and LC3 as well as NOD-like receptor protein 3 (NLRP3) and pro-IL expression were detected by Western blot, and supernatant IL-1β levels were detected by ELISA. The HL7702 hepatocytes were co-cultured by the supernatants of LX2 cells intervened mentioned above. The cell apoptosis was detected by FCM. Results The HBwx protein was clearly expressed in the cytoplasm of transfected LX2 cells; the LC3 expression decreased, while p62 expression increased in LPS-, HBwx- and LPS and HBwx combination-intervened groups as compared to in the control (P<0.05); the NLRP3 and pro-IL expression increased in LPS-, HBwx- and LPS and HBwx combination-intervened groups as compared to in the control (P<0.05); the supernatant IL-1β levels increased in LPS-, HBwx- and LPS and HBwx combination-intervened groups as compared to in the control (P<0.05); the apoptosis of HL-7702 cells significantly increased in LPS-, HBwx- and especially in LPS and HBwx combination-intervened groups as compared to in the control (P<0.05). Conclusions HBwx promotes inflammatory reaction in HSCs by inhibition of cell autophagy and induces hepatocyte apoptosis.