水蛭对肝内胆汁淤积大鼠模型法尼醇X受体通路的调节作用

Regulation effect of leech on farnesol X receptor pathway in rat model of intrahepatic cholestasis

目的研究水蛭对肝内胆汁淤积大鼠模型法尼醇X受体(FXR)通路的调节作用。方法选取40只8周龄SPF级雄性SD大鼠,按照随机数字表法分为正常组、模型组、熊去氧胆酸(UDCA)组、水蛭高剂量组、水蛭低剂量组,每组8只。UDCA组按60 mg/kg的剂量灌胃UDCA溶液,水蛭高、低剂量组分别按50 mg/kg和20 mg/kg剂量灌胃水蛭颗粒剂,1次/d,连续7 d,正常组和模型组予以等量生理盐水。除正常组外,其他各组大鼠于灌胃第5天予以100 mg/kgα-异硫氰酸萘酯(ANIT)灌胃造模,灌胃3 d,1次/d,正常组予以等量生理盐水。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(TBil)、总胆汁酸(TBA)水平;取肝脏组织进行HE染色镜检,观察大鼠肝脏组织病理情况;采用实时荧光定量PCR与Western blot检测FXR及下游小异源二聚体伴侣受体(SHP)、尿苷二磷酸葡萄糖苷酸转移2B4(UGT2B4)、胆盐输出泵(BSEP)mRNA及蛋白表达。结果水蛭高剂量组血清ALT、AST、TBil水平低于模型组,差异均有统计学意义(均P<0.05);水蛭高剂量组与模型组TBA水平比较,差异无统计学意义(P>0.05);水蛭低剂量组血清AST、TBA、TBil水平低于模型组,差异均有统计学意义(均P<0.05),水蛭低剂量组与模型组ALT水平比较,差异无统计学意义(P>0.05);UDCA组血清AST的水平低于模型组,差异有统计学意义(P<0.05),UDCA组与模型组ALT、TBA、TBil水平比较,差异无统计学意义(P>0.05)。与模型组比较,水蛭高、低剂量组大鼠肝脏组织损伤减轻。水蛭高、低剂量组及UDCA组FXR、BSEP、UGT2B4、SHP mRNA表达高于模型组,差异均有统计学意义(均P<0.05)。水蛭高、低剂量组FXR、BSEP、UGT2B4、SHP蛋白表达高于模型组,差异均有统计学意义(均P<0.05),UDCA组与模型组蛋白水平比较,差异无统计学意义(P>0.05)。结论水蛭可以缓解ANIT诱导的肝内胆汁淤积,减轻肝脏组织损伤。水蛭可通过对FXR基因表达上调,促进SHP、UGT2B4、BSEP基因表达上调,抑制胆汁淤积通路来保护肝细胞,为临床治疗肝内胆汁淤积提供有效药物。

Objective To study the regulatory effect of leech on farnesol X receptor(FXR)pathway in rat model of intrahepatic cholestasis.Methods Forty SPF male SD rats aged eight weeks were selected and divided into normal group,model group,ursodeoxycholic acid(UDCA)group,leech high-dose group and leech low-dose group according to random number table method,with eight rats in each group.The UDCA group was intragastrically administered with UDCA solution at a dose of 60 mg/kg,and the leech high-dose and low-dose groups were intragastrically administered with Leech Granules at a dose of 50 mg/kg and 20 mg/kg,once a day,for consecutive seven days,the normal group and the model group were given the same amount of normal saline.Except normal group,rats in other groups were given 100 mg/kgα-isothiocyanate(ANIT)on the fifth day of intragastric administration for model formation.Gastric administration for three days,once a day,the normal group was given the same amount of normal saline.Blood samples were collected from the abdominal aorta 24 h after the last administration,and serum levels of alanine aminotransferase(ALT),ascorbic aminotransferase(AST),total bilirubin(TBil)and total bile acid(TBA)were detected.Hepatic tissue was collected for microscopic examination by HE staining to observe the pathological conditions of the liver.The mRNA and protein expressions of FXR and its downstream small heterodimer chaperone receptor(SHP),uridine diphosphate glucuronide transfer 2B4(UGT2B4)and bile salt output pump(BSEP)were detected by real-time fluorescence quantitative PCR and Western blot.Results The levels of ALT,AST and TBil in serum of leech high-dose group were lower than those in model group,with statistically significant differences(all P<0.05).There was no statistical significance in TBA level between leech high-dose group and model group(P>0.05).The levels of serum AST,TBA and TBil in leech low-dose group were lower than those in model group,with statistically significant differences(all P<0.05).There was no significant difference in ALT level between leech low-dose group and model group(P>0.05).The serum AST level of UDCA group was lower than that of model group,and the difference was statistically significant(all P<0.05).There were no statistically significant differences in ALT,TBA and TBil levels between UDCA group and model group(P>0.05).Compared with model group,liver tissue damage of rats in leech high-dose and low-dose groups was alleviated.The mRNA expressions of FXR,BSEP,UGT2B4 and SHP in leech high-dose and low-dose groups and UDCA group were higher than those in model group,with statistically significant differences(all P<0.05).The protein expressions of FXR,BSEP,UGT2B4 and SHP in leech high-dose and low-dose groups were higher than those in model group,and the differences were statistically significant(all P<0.05).There was no statistically significant difference in protein levels between UDCA group and model group(P>0.05).Conclusion Leech can relieve intrahepatic cholestasis induced by ANIT and alleviate liver tissue damage.Leech can protect liver cells by upregulating FXR gene expression,promoting the upregulation of SHP,UGT2B4 and BSEP gene expression and inhibiting cholestasis pathway,thus providing effective drugs for clinical treatment of intrahepatic cholestasis.