| BackgroundCholestasis may result either from a functional defect in bile formation at the level of the hepatocyte or from an impairment in bile secretion and flow at the bile duct level and results in the intrahepatic and systemic accumulation of bile acids.Reduced expression and function of bile acid transporter(Mrp2 and Bsep)play a key role in the pathogenesis of cholestasis and can result in or maintain cholestasis.Oldenlandia,a genus of dicotyledonous plants in the family Rubiaceae,is clinically used in the treatment of dysentery and jaundice.This study investigated the effects of the ethanol extract of oldenlandia(BA)on choleresis,the expression of hepatic transporters(Mrp2 and Bsep),and chronic/fulminant hepatitis.ObjectiveThe aim of the study is to investigate the cholagogue effect of BA and the expression of Mrp2/Bsep of rats.Method1.Biliary excretion of BA and biochemical parameter.Animals were randomly divided into 4 experimental groups,namely:(1)Control rats,receiving only the vehicle(0.5%CMC-Na),(2)BA-treated rats,which were orally administered BA(doses of 0.5 g/kg body wt)for 10 consecutive days,(3)BA-treated rats,which were orally administered BA(doses of 1.0 g/kg body wt)for 10 consecutive days,(4)BA-treated rats,which were orally administered BA(doses of 2.0 g/kg body wt)for 10 consecutive days.Surgical procedures were made on the day after the last(tenth)dose of BA was administered.Animals were anesthetized with a single dose of urethane(1 g/kg body wt,intraperitoneally),and maintained under this condition throughout the experiment.Bile samples were collected and bile flow rate was also measured.The biochemical parameter(including alanine aminotransferase(ALT),aspartate aminotransferase(AST),alkaline phosphatase(ALP)and total bile acid(TBA)),total bilirubin(TB),total bile acid(TBA),and reduced glutathione(GSH)from bile were determined using assay kits.The mRNA and protein levels of Mrp2 and Bsep were determined by real-time quantitative PCR,Western blotting and immunohistochemistry analysis,respectively.In vivo bilirubin disposal.With the same method,rats were intravenously injected with bilirubin solution(20 mg/kg)via the tail vein on the tenth day of BA treatment.After the injection,blood and bile were collected over a 120-min period.Urine was collected over a 180-min period using a metabolic cage.The total bilirubin concentration in plasma,bile,and urine was measured by the assay kits.2.Biliary excretion of BA and biochemical parameter induced by ANIT(a-naphthyl isothiocyanate).Animals were randomly divided into 6 experimental groups,namely:(1)Control(peanut oil-treated)rats,receiving only the vehicle,(2)ANIT-treated rats,which were administered with ANIT at the dose of 60 mg/kg body wt,at the fifth dose of 0.5%CMC-Na,(3)BA +ANIT rats,which were administered with ANIT and BA(doses of 0.5 g/kg body wt),(4)BA + ANIT rats,which were administered with ANIT and BA(doses of 1L 0 g/kg body wt),(5)BA + ANIT rats,which were administered with ANIT and BA(doses of 2.0 g/kg body wt),(6)positive control rats,which were administered with ANIT and ursodeoxycholic acid(dose of 50 mg/kg body wt).Surgical procedures were made on the day after the last(tenth)dose of BA.Animals were anesthetized with a single dose of urethane(1 g/kg body wt,intraperitoneally),and maintained under this condition throughout the experiment.Bile samples were collected and bile flow rate was also measured.The biochemical parameter(including ALT,AST,ALP,TBA),total bilirubin(TB),total bile acid(TBA)from bile and reduced glutathione(GSH)from bile and liver were determined using assay kits.Liver samples were stained with hematoxylin and eosin which examined the pathological changes of hepatic tissue.3.Biliary excretion of BA and biochemical parameter induced by EE(Ethinylestradiol).Animals were randomly divided into 6 experimental groups,namely:(1)Control(propylene glycol-treated)rats,receiving only the vehicle,(2)EE-treated rats,which were administered daily with EE at the dose of 5 mg/kg body wt,subcutaneously,for 5 consecutive days,(3)BA + EE rats,which were coadministerd with EE and BA(doses of 0.5 g/kg body wt),(4)BA + EE rats,which were coadministerd with EE and BA(doses of 1.0 g/kg body wt),(5)BA + EE rats,which were coadministerd with EE and BA(doses of 2.0 g/kg body wt),(6)positive control rats,which were administered with EE and ursodeoxycholic acid(dose of 50 mg/kg body wt),subcutaneously,for 5 consecutiye days.Surgical procedures were made on the day after the last(tenth)dose of BA was administered.Animals were anesthetized with a single dose of urethane(1 g/kg body wt,intraperitoneally),and maintained under this condition throughout the experiment.The biochemical parameter,bile flow,TBA,TB,TBA,GSH from bile,the mRNA and protein levels of Mrp2 and Bsep were determined as above.In vivo bilirubin disposal.With the same method,rats were intravenously injected with bilirubin solution(20 mg/kg)via the tail vein on the tenth day of BA treatment.After the injection,blood and bile were collected over a 120-min period.Urine was collected 'over a 180-min period using a metabolic cage.The total bilirubin concentration in plasma,bile,and urine was measured by the assay kits.Results1.The biliary concentration of TB,TBA,GSH and the bile flow are obviously increased(P<0.01),the proportion of liver and the serum concentrations of ALT,AST,ALP,TB,TBA were no apparent change(P>0.05),suggesting no obvious liver toxicity.Moreover the genes expression of Bsep and Mrp2 were up-regulated(P<0.01)after a 10 day treatment with BA.In vivo bilirubin disposal,the clearance of TB in the serum,bile and urine were significantly increased(P<0.01)after a 10 day treatment with BA.2.Compared with the model group,the serum concentrations of ALT,AST,ALP,TB,TBA were diminished(P<0.01)while the biliary concentration of TB,TBA,GSH were increased(P<0.01)after treatment with BA.In addition the pathology showed BA could prevent the impairment in the pathological changes of hepatic tissue.3.Compared with the model group,the bodyweight,the bile flow and the biliary concentrations of TB,TBA were increased(P<0.01)and the serum concentrations of ALT,AST,ALP,TB,TBA were obviously decreased(P<0.01),the genes expression of Bsep and Mrp2 were up-regulated in the treatment groups administrated BA(P<0.01).In vivo bilirubin disposal,compared with the model group,the clearance of TB in the serum,bile and urine were increased(P<0.01).ConclusionBA has cholagogue effect in cholestatic rats by strengthening the activation of GSH,up-regulating Bsep and Mrp2 in the liver to improve the function of the liver and relieve the toxicity of bile acid. |
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