白芍总苷调节RhoA/ROCK1信号通路对大鼠肝缺血再灌注损伤的影响

Effect of total glucosides of paeony on hepatic ischemia-reperfusion injury in rats by regulating RhoA/ROCK1 signaling pathway

目的 探讨白芍总苷通过调节Ras同源基因家族成员A/Rho相关卷曲螺旋蛋白激酶1(RhoA/ROCK1)信号通路对大鼠肝缺血再灌注损伤的影响。方法 通过阻断肝动脉和门静脉1 h建立肝缺血再灌注损伤大鼠模型,设假手术组、模型组、白芍总苷(100 mg/kg)组、白芍总苷+Rhosin(100 mg/kg+40 mg/kg)组和白芍总苷+LPA(100 mg/kg+1 mg/kg)组,每组8只。连续治疗6周后,检测各组大鼠血清肝功能指标[丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)、总胆红素(TBiL)];HE、TUNEL染色法观察肝组织病理改变和细胞凋亡,并计算肝损伤评分和凋亡指数(AI);分光光度法检测肝组织中氧化应激指标[超氧化物歧化酶(SOD)、过氧化氢酶(CAT)活性和丙二醛(MDA)含量],ELISA检测炎症因子[白细胞介素(IL)-1β、IL-6、肿瘤坏死因子-α(TNF-α)]含量;通过反转录聚合酶链反应(RT-PCR)、蛋白免疫印迹(Western blotting)法检测肝组织中RhoA、ROCK1、核因子-κB p65(NF-κB p65)、B细胞淋巴瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、活化型半胱氨酸蛋白酶-3(C-Caspase-3)m RNA和蛋白表达。结果 与模型组比较,白芍总苷组和白芍总苷+Rhosin组血清ALT、AST、TBiL水平显著降低(P<0.05);肝组织病理改变和细胞凋亡状况均明显改善,肝损伤评分和细胞AI均显著降低(P<0.05);SOD、CAT活性显著升高,MDA、IL-1β、IL-6、TNF-α含量显著降低(P<0.05);RhoA、ROCK1、NF-κB p65、Bax、C-Caspase-3m RNA和蛋白表达均显著降低,Bcl-2m RNA和蛋白表达均显著升高(P<0.05)。与白芍总苷组相比,Rhosin可显著增强白芍总苷对模型大鼠肝功能、肝组织病变、细胞凋亡、氧化应激、炎症及RhoA/ROCK1信号通路相关m RNA和蛋白表达的作用;LPA则显著逆转了上述调控作用。结论 白芍总苷可能通过抑制RhoA/ROCK1信号通路,减轻氧化应激、炎症和细胞凋亡,从而对大鼠肝缺血再灌注损伤起到一定的保护作用。

Objective To investigate the effect of total glucosides of paeony on hepatic ischemia-reperfusion injury in rats by regulating RhoA/Rock1 signaling pathway.Methods The rat model with hepatic ischemia-reperfusion injury was established by blocking hepatic artery and portal vein for 1 h.And the sham operation group,model group,total glucosides of paeony(100 mg/kg)group,total glucosides of paeony+TGP(100 mg/kg+40 mg/kg)group,and total glucosides of paeony+LPA(100 mg/kg+1 mg/kg)group were set up,with 8 rats in each group.After 6 weeks of continuous treatment,the level of ALT,AST,TBiL in serum were measured,the hepatic tissue pathological changes and cell apoptosis were observed through HE or TUNEL staining,the hepatic injury score and apoptosis index(AI)were calculated.The oxidative stress indexes(SOD,CAT,and the content of MDA)in hepatic tissue were detected by spectrophotometer.The level of inflammatory factors(IL-1β,IL-6,TNF-α)were detected by ELISA method.The mRNA and protein expressions of RhoA,ROCK1,NF-κB p65,Bcl-2,Bax,C-Caspase-3 were in hepatic tissue were detected by reverse transcription-polymerase chain reaction(RT-PCR)or Western blotting.Results Compared with model group,the level of ALT,AST,TBiL in serum of total glucosides of paeony group and total glucosides of paeony+Rhosin group were significantly decreased(P<0.05).The pathological changes and cell apoptosis of hepatic tissue were significantly improved,the hepatic injury score and AI were significantly decreased(P<0.05).The activity of SOD,CAT were significantly increased,and the content of MDA,IL-1β,IL-6,TNF-αwere significantly decreased(P<0.05).The mRNA and protein expression of RhoA,ROCK1,NF-κB p65,Bax,C-Caspase-3 were significantly decreased,while the mRNA and protein expression of Bcl-2 were significantly increased(P<0.05).Compared with total glucosides of paeony group,Rhosin could significantly enhance the effect of total glucosides of paeony on hepatic function,hepatic lesion,apoptosis,oxidative stress,inflammation,the expression of RhoA/ROCK1 signaling pathway-related mRNA and protein in HIR rats,while LPA could reversed the regulatory effect.Conclusion Total glucosides of paeony can alleviate oxidative stress,inflammation,cell apoptosis by inhibiting the RhoA/ROCK1 signaling pathway,thus playing a certain protective role against hepatic ischemia-reperfusion injury in rats.