帕立骨化醇抑制氧化应激减轻小鼠肝细胞紧密连接受损

Paricalcitol inhibition of oxidative stress alleviates the damage of hepatocyte tight junction in mice

目的探讨帕立骨化醇(Pal)对氧化应激诱导的肝细胞紧密连接受损的影响及其机制研究。方法体内实验:采用胆总管结扎构建胆汁淤积性肝损伤模型。小鼠随机分为对照组(control)、模型组(BDL)、治疗组(BDL+Pal)。通过HE染色,探究各组小鼠肝组织病理形态变化。体外培养人肝癌细胞系HepG2,分为空白组、模型组(400μmol/L H_(2)O_(2))、治疗组(400μmol/L H_(2)O_(2)+20 nmol/L Pal)。Western blot检测各组紧密连接蛋白1(ZO-1)、闭合蛋白(occludin)、磷酸化p65(p-p65)、磷酸化ERK(p-ERK)、磷酸化肌球蛋白II调节轻链(p-MLC)蛋白水平。结果与对照组相比,模型组p-p65、p-ERK、p-MLC蛋白水平显著升高(P<0.0001或P<0.01或P<0.001);ZO-1、occludin蛋白表达水平显著降低(P<0.01);HE染色显示肝细胞坏死及炎性细胞浸润增多。而治疗组相对于模型组,上述水平呈相反趋势。结论Pal通过抑制胆汁淤积性小鼠及HepG2细胞中的氧化应激,从而减轻肝细胞紧密连接受损,其机制可能与抑制活性氧及NF-κB/p65、ERK信号通路有关。

Objective To explore the impact of paricalcitol(Pal)on the oxidative stress-induced tight junction damage of mouse hepatocytes and its mechanism.Methods A model of cholestatic liver injury was created by routine bile duct ligation.The mice were randomly divided into control group(control),model group(BDL)and treatment group(BDL+Pal).HE staining microscopy was used to observe the morphological changes of liver tissues.The human hepatoma cell line HepG2 was cultured and divided into blank group,model group(400μmol/L H_(2)O_(2))and treatment group(400μmol/L H_(2)O_(2)+20 nmol/L Pal).Western blot was used to examine the level of tight junction protein 1(ZO-1),occludin,phosphorylated p65(p-p65),phosphorylated ERK(p-ERK)and phosphorylated myosin II regulated light chain(p-MLC)protein were checked in each group.Results Compared with the control group,the level of p-p65,p-ERK and p-MLC in the model group was significantly increased(P<0.0001 or P<0.01 or P<0.001).The protein expression of ZO-1 and occludin was significantly decreased(P<0.01).HE staining microscopy showed an increased hepatocyte necrosis and inflammatory cell infiltration.In contrast,the above levels in the treatment group showed an opposite trend relative to the model group.Conclusions Pal is able to alleviate the damage of hepatocyte tight junctions by inhibiting oxidative stress in cholestatic mice and HepG2 cells.Its mechanism is potentially related to the inhibition of reactive oxygen species and NF-κB/p65 and ERK signaling pathways.