桂枝通过调控FXR通路改善ANIT诱导的肝内胆汁淤积大鼠模型的机制研究

Mechanism of Cinnamomi Ramulus improving rat model of intrahepatic cholestasis induced by ANIT by regulating FXR pathway

研究桂枝通过调控法尼醇X受体(farnesoid X receptor,FXR)通路改善α-萘异硫氰酸酯(ANIT)诱导大鼠肝内胆汁淤积的作用及机制。将40只雄性SD大鼠随机分为正常组、模型组、熊去氧胆酸(UDCA)组(60 mg·kg-1)、桂枝60 mg·kg-1治疗组、桂枝20 mg·kg-1治疗组5组,每组8只。除正常组和模型组外,其余各组按对应浓度连续水溶液0. 005 m L·g-1体质量灌胃,每天1次,灌胃7 d;除正常对照组外,其余各组于灌胃第5天予100 mg·kg-1ANIT灌胃造模,每天1次,灌胃3 d。末次给药24 h后腹主动脉取血,检测血清谷丙转氨酶(ALT)、谷草转氨酶(AST)、总胆红素(TBi L)、总胆汁酸(TBA)水平;取大鼠肝脏组织1. 5~2 cm,经10%甲醛固定,进行石蜡包埋切片,并行HE染色,免疫组化(IHC)分析FXR的表达;取大鼠肝脏组织提取RNA及蛋白,检测FXR mRNA表达及FXR下游分别与胆汁酸合成、解毒、转运相关的SHP,UGT2B4,BSEP蛋白的表达情况。结果表明,与正常组比较,模型组血清ALT,AST,TBi L,TBA的水平升高(P<0. 01),肝脏损伤严重,FXR蛋白和mRNA表达降低,SHP,UGT2B4,BSEP蛋白表达均降低(P<0. 05,P<0. 01);与模型组比较,药物组能在不同程度上降低血清ALT,AST,TBi L,TBA水平(P<0. 05,P<0. 01),改善肝脏组织损伤,增加FXR蛋白灰度值,促进FXR mRNA和FXR,SHP,BSEP和UGT2B4蛋白的表达(P <0. 05,P <0. 01)。桂枝可以缓解ANIT诱导的肝内胆汁淤积,减轻肝细胞损伤,降低血清ALT,AST,TBi L,TBA水平。其机制可能是通过FXR-SHP,FXR-UGT2B4,FXR-BSEP信号通路来进行干预。故而,在肝内胆汁淤积的发病过程中,可以尝试在桂枝改善肝内胆汁淤积方面做更进一步的探索和尝试,为临床治疗肝内胆汁淤积提供有效药物。

To study the mechanism and action of Cinnamomi Ramulus in ameliorating intrahepatic cholestasis induced byα-isothiocyanate(ANIT)in rats by regulating FXR pathway.Forty SD rats were randomly divided into normal group,model group,positive control(ursodeoxycholic acid)group(60 mg·kg-1),Cinnamomi Ramulus treatment(60 mg·kg-1·d-1)group,and Cinnamomi Ramulus treatment(20 mg·kg-1·d-1)group,with 8 rats in each group.Except for the normal control group,the other groups were intragastrically administered with the corresponding concentrations of continuous aqueous solution(0.005 m L·g-1),once a day,for 7 days.Except for the normal group,the other groups were treated with ANIT(100 mg·kg-1),once a day,for 3 days.Blood was taken from the abdominal aorta 24 hours after the last administration,and serum alanine aminotransferase(ALT),aspartate aminotransferase(AST),total bilirubin(TBi L),and total bile acid(TBA)were measured.1.5-2 cm of rat liver tissue was taken.After fixation with10%formaldehyde,paraffin-embedded sections were taken,HE staining was performed,and immunohistochemistry(IHC)was used to analyze the expression of FXR.RNA and protein were extracted from rat liver tissue to detect FXR mRNA expression,as well as bile acid synthesis and detoxification,transport related SHP,UGT2 B4,BSEP protein expressions at downstream of FXR.Compared with the normal group,serum ALT,AST,TBi L,and TBA levels were elevated in the model group(P<0.01),liver damage was severe,FXR protein’s optical density decreased,FXR mRNA expression decreased,and SHP,UGT2 B4,BSEP protein expressions were decreased(P<0.05,P<0.01).Compared with the model group,the drug group could reduce serum ALT,AST,TB,TBA levels to different degrees(P<0.05,P<0.01),alleviate liver tissue damage,increase the optical density of FXR protein,and promote the expressions of FXR mRNA and FXR,SHP,BSEP and UGT2 B4 proteins(P<0.05,P<0.01).Cinnamomi Ramulus can alleviate ANIT-induced intrahepatic cholestasis,and reduce hepatocyte injury and serum ALT,AST,TBi L and TBA levels.The mechanism may be through FXR-SHP,FXR-UGT2 B4,FXR-BSEP signaling pathways.Therefore,in the pathogenesis of intrahepatic cholestasis,we can try to further explore in alleviating intrahepatic cholestasis with Cinnamomi Ramulus,so as to provide effective drugs for clinical treatment of intrahepatic cholestasis.