【摘要】 目的:研究栀子酸(geniposidic acid,GPA)对α-萘异硫氰酸酯(α-naphthyl isothiocyanate,ANIT)诱导胆汁淤积大鼠胆汁酸代谢小分子异源二聚体伴侣受体(small heterodimer partner,SHP)与肝受体同源物1(liver receptor homologue1,LRH-1)信号通路的调节。方法:将50只SD大鼠随机分为5组,分别为空白组、ANIT组、ANIT+GPA 100 mg/kg组(AG100组)、ANIT+GPA 50 mg/kg组(AG50组)、ANIT+GPA 25 mg/kg组(AG25组),每组10只,连续给药10 d,空白组和ANIT组用生理盐水灌胃,ANIT+GPA各组给予GPA,给药的第8天,除空白组外,其余各组用ANIT(65 mg/kg)灌胃1次造模,继续给药至第10天,测血清总胆固醇(total cholesterol,TC)、三酰甘油(triglyceride,TG)和总胆汁酸(total bile acids,TBA)的含量;分离并培养大鼠原代肝细胞(rat primary hepatocytes,RPH),将RPH分为空白组、40μmol/L ANIT组、40μmol/L ANIT+GPA 4.00,1.00,0.25 mmol/L组(A4G,A1G和A0.25G组),real-time RT-PCR检测RPH中SHP和胆汁酸合成关键酶胆固醇7α羟化酶(cytochrome P450 family 7 subfamily A member 1,CYP7a1) mRNA转录水平,蛋白印迹法检测SHP和CYP7a1蛋白表达;沉默实验中,将RPH分为空白对照组、阴性转染组、siRNA-LRH-1组(ZR组)、siRNA-LRH-1+GPA 4.00,1.00,0.25 mmol/L组(ZR4G、ZR1G、ZR0.25G组),检测SHP,CYP7a1和LRH-1蛋白和基因的表达;过表达实验中,将RPH又分为空白对照组、阴性转染组(空白转染siRNA-阴性组)、LRH-1过表达组(LRH-1过表达质粒,OE组)、LRH-1过表达质粒+GPA 4.00,1.00,0.25 mmol/L组(OE4G,OE1G,OE0.25G组),检测SHP,CYP7a1和LRH-1蛋白和基因的表达。结果:在体实验中,与空白对照组比较,ANIT组的TC和TBA均显著升高(均P<0.01)、TG无差别;与ANIT组比较,AG100组和AG50组的TC和TBA均显著降低(均P<0.01)。RPH实验中,与空白对照组比较,ANIT组中SHP的蛋白和基因水平均显著降低(均P<0.01),CYP7a1的蛋白和基因水平均显著升高(均P<0.01);与ANIT组比较,A4G和A1G组的SHP基因和蛋白水平均显著升高(均P<0.01),A0.25G组SHP基因和蛋白水平均升高(P<0.05),A4G和A1G组的CYP7a1基因和蛋白水平均显著降低(均P<0.01)。LRH-1沉默实验中,与阴性转染组比较,ZR组中CYP7a1和LRH-1蛋白和基因均显著抑制(均P<0.01),SHP无变化;与ZR组比较,ZR4G,ZR1G和ZR0.25G组SHP基因和蛋白水平均显著升高(均P<0.01),LRH-1和CYP7a1无显著变化。LRH-1过表达实验中,与阴性转染组比较,OE组的CYP7a1和LRH-1蛋白和基因均显著抑制(均P<0.01),SHP无变化;与OE组比较,OE4G和OE1G组SHP基因和蛋白水平均显著升高(均P<0.01),OE0.25G组SHP基因显著升高(P<0.05),LRH-1和CYP7a1无显著变化。结论:RPH中LRH-1的过表达能上调CYP7a1的活性,GPA可以改善ANIT诱导的胆汁淤积大鼠的生物化学水平和肝脏病理,其机制可能与GPA激活大鼠RPH中SHP造成LRH-1的消耗来降低CYP7a1的表达有关。更多还原

【Abstract】 Objective:To explore the effect of geniposidic acid(GPA)on the signal pathway of small heterodimer dimer receptor(SHP)and liver receptor homologue 1(LRH-1)in cholestasis rats induced by alpha-naphthalene isothiocyanate(ANIT).Methods:Fifty SD rats were randomly divided into five groups:a blank group,an ANIT group,an ANIT+GPA(100 mg/kg)group,an ANIT+GPA(50 mg/kg)group,and an ANIT+GPA(25 mg/kg)group(n=10 in each group).The GPA were intragastrically given to rats for 10 days,and the control group and the ANIT group were given normal saline.At the eighth day of administration,all rats except the blank group were given 65 mg/kg ANIT once until the tenth day.After the last administration,serum total cholesterol(TC),triglyceride(TG)and total bile acids(TBA)were measured.The primary hepatocytes(RPH)were isolated from normal rats and cultured.The cells were divided into a blank group,an ANIT(40 Vmol/L)group,an ANIT(40μmol/L)+GPA(4.00 mmol/L)group(A4.00 G group);an ANIT(40μmol/L)+GPA(1.00 mmol/L)group(A1.00 G group),and an ANIT(40 μmol/L)+GPA(0.25 mmol/L)group(A0.25 G group).The mRNA transcription levels of SHP and cholesterol 7 alpha hydroxylase(CYP7 A1)in RPH were detected by real-time-PCR,and the protein levels of SHP and CYP7 a1 were detected by Western blotting.In the LRH-1 silence experiment,the RPH were divided into a blank group,a negative transfection group,a siRNA-LRH group(ZR group),a siRNA-LRH+GPA(4.00 mmol/L)group(ZR4.00 G group),a siRNA-LRH+GPA(1.00 mmol/L)group(ZR1.00 G group)and a siRNA-LRH+GPA(0.25 mmol/L)group(ZR0.25 G group).The protein and mRNA levels of SHP,CYP7 a1,LRH-1 were detected.In the over-expression experiment,the RPH were also divided into a blank group,a negative transfection group,a LRH-1 over-expression plasmid group(OE group),a LRH-1 over-expression plasmid+GPA(4.00 mmol/L)group(OE4.00 G group),a LRH-1 over-expression plasmid+GPA(1.00 mmol/L)group(OE1.00 G group),and a LRH-1 over-expression plasmid+GPA(0.25 mmol/L)group(OE0.25 G group).The protein and mRNA levels of SHP,CYP7 al and LRH-1 were detected.Results:Compared with the blank control group,TC and TBA were significantly increased(both P<0.01)in the ANIT group,but there was no difference in TG;compared with the ANIT group,the contents of TC and TBA in the AG100 and AG50 groups were significantly reduced(all P<0.01).Compared with the blank control group,the proteins and mRNA levels of SHP were significantly decreased(P<0.01),while CYP7 a1 were dramatically increased(P<0.01)in the ANIT group;compared with the ANIT group,the proteins and mRNA levels of SHP in the A4.00 G group and the A1.00 G group were significantly increased(both P<0.01),while the levels of CYP7 a1 proteins and mRNA levels were evidently decreased in the A4.00 G and A1.00 G groups(both P<0.01).Compared with the negative transfection group;the proteins and mRNA levels of CYP7 a1 and LRH-1 were dramatically restrained(all P<0.01),while there was no change in SHP in the ZR group;compared with the ZR group,the proteins and mRNA levels of SHP were significantly increased(all P<0.01),while LRH-1 and CYP7 al were not changed in the ZR4.00 G;ZR1.00 G and ZR0.25 G groups.Compared with the negative transfection group,the protein and mRNA levels of CYP7 al and LRH-1 were significantly suppressed in the OE group(all P<0.01).Compared with the OE group, the protein and mRNA levels of SHP were evidently increased in the OE4 G and OE1 G groups(all P<0.01),while LRH-1 and CYP7 al were not changed in the OE4 G,OE1 G and OE0.25 G groups.Conclusion:The over-expression of LRH-1 in RPH can up-regulate the mRNA and protein levels of CYP7 al.GPA can improve the biochemical and liver pathology of ANIT-induced cholestasis rats,which maybe related to the decrease of CYP7 al by activating SHP through LRH-1 in RPH.更多还原