| AimsTo investigate the effect of Gardenia Jasminoides(Zhi-zi)aqueous extraction17?-ethynylestradiol(EE)-induced cholestasis and jaundice in rats,and preliminarily explore its molecular mechanism via bilirubin and bile acids relevant transporter-enzyme systems.Methods A total of 40 male Wistar rats(200-220 g)were randomly divided into normal group,model group,Zhi-zi group and ursodeoxycholic acid(UDCA)group(n=10).Rats in model group,Zhi-zi group and UDCA group were administered subcutaneously with 17?-ethynylestradiol(EE,5 mg/kg/d)for the first 7 days,and then 3 mg/kg 17?-ethynylestradiolfor another 7 days to establish intrahepatic cholestasis and jaundice rat model.Rats in the normal group were injected subcutaneously with propylene glycol as control.During the model establishment,rats in the control and model group were also given saline(i.g.,bid)for 14 days;while rats in Zhi-zi group were orally administrated with Gardenia Jasminoides(Zhi-zi)aqueous extract(3 g/kg/d,equals to15 g/kg/d crude herb,clinical equivalent dose,bid)for 14 days;and rats in UDCA group were orally given UDCA(60 mg/kg,bid,i.g.)for 14 days.After the final drug administration,5 rats in each group were selected randomly to collect feces and urine for 12 hours,bile was also collected during 1 hour by biliary cannulation.The rest rats in each group(n=5)were fasting for 12 hours,and then anesthetized by injecting 10% ulatan intraperitoneally.Blood samples were collected in abdominal aorta,and then the rats were sacrificed to collect liver,kidney and ileum tissue samples.These tissue samples were used to determine:(1)the level of aspartate transaminase(AST),alanine aminotransferase(ALT)and alkaline phosphatase(ALP)as well as the concentration of total bile acids(TBAs),total bilirubin(TBIL)and direct bilirubin(DBIL)of serum;(2)alterations in liver histopathology;(3)excretion of TBIL,DBIL,IBIL and TBAs in billiary,fecal and urinary;(4)TBAs and the concentration of 12 individual bile acids(BAs)in liver and ileum;(5)m RNAexpression of bilirubin and BAs-related transporters,enzymes and nuclear receptors in liver,ileum and kidney.Results1.Alterations in serum transaminase levels and liver histopathology: compared with the normal rats,the serum level of ALT,TBIL and TBAs were significantly increased in model rats(P<0.05);compared with the model rats,serum level of ALT in Zhi-zi-treated rats was decreased to the normal level(P < 0.05);the TBIL level was significantly increased(P < 0.05);and decreased trend was also observed in TBAs level after Zhi-zi treatment,but no statistical difference was found.Compared with the UDCA-treated rats,serum level of ALT and ALP in Zhi-zi-treated rats was all markedly decreased.Moreover,compared with the normal rats,alterations in liver histopathology in model rats involved bile duct dilatation,inflammatory cell infiltration,and mild steatosis;compared with the model group,the histopathological alterations of liver in Zhi-zi group and UDCA group were alleviated,with hepatocyte lesion remission and mild inflammatory cells infiltration.2.Alternation of bilirubin in vivo:(1)compared with the normal group,serum TBIL was significantly increased by 1.3 times with a marked increase of DBIL in the model group;the billary excretion of TBIL,DBIL and IBIL were increased remarkably(P<0.05);fecal excretion of IBIL as well as urinary excretion of TBIL,IBIL,and DBIL were all significantly increased in model group(P<0.05).(2)compared with the model group,serum TBIL in Zhi-zi group was significantly increased with DBIL increased by 56% and IBIL increased by 3 folds(P<0.05);no significant difference was found in the bile or feces excretion of bilirubin;but urine bilirubin excretion during 12 hours in Zhi-zi group was markedly increased(P<0.05).(3)Compared with the model group,no significant difference was observed in bilirubin level of serum,feces and urine in UDCA group.3.Alternation of bile acids in vivo:(1)Compared with the normal group,serum TBAs level in model group was significantly increased(P<0.05);no significant difference was found in the TBAs level of liver;but the hepatic concentration of GCA,CDCAs,CDCA,TCDCA,GLCA and GCDCA were all significantly increased(P < 0.05)with DCAs(mainly TDCA)and TLCA significant decreases(P<0.05);TBAs level in ileum was decreased by 50%with CA,CAs,TCA,DCA,DCAs and TCDCA significant decreases(P < 0.05)and GCDCA,LCA and GLCA significant increases(P<0.05);TBAs level in kidney was increased by 9 folds(P<0.05);and TBAs in bile and urine were all significantly increased(P < 0.05).(2)Compared with modelgroup,no significant differences of TBAs in serum,liver,ileum,bile and feces was observed in Zhi-zi group;butt CDCA,GCDCA,CDCAs in liver were significantly decreased(P<0.05);GCA,GCDCA and GLCA in ileum were significantly decreased(P<0.05)in Zhi-zi group.An elevated trend of TBAs was observed in kidney of Zhi-zi-treated rats,but no significant difference was found.Urine TBAs excretion in Zhi-zi group was elevated by 1.3 folds(P<0.05).(3)Compared with the model group,there were no significant differences of TBAs in serum,liver,ileum,kidney,feces and urine in the UDCA treated group,but bile TBAs excretion in UDCA treated group was increased markedly(P<0.05).Alterations in the m RNA expression of transporters,enzymes and nuclear receptors in the liver,ileum and kidney:(1)compared with normal group,the m RNA expression of Oatp2,Ugt1a1,Cyp3a2 and Fgf15 in the liver were all significantly decreased in the model group(P<0.05),but the m RNA expression of Mrp3,Bsep,Cyp7a1,Cyp27a1 and Shp were all significantly increased(P<0.05).Compared with the model group,the hepatic m RNA expression of Ntcp,Bsep,Cyp7a1,Cyp27a1 and Shp were all significantly decreased in Zhi-zi group(P<0.05);but the m RNA expression of Ugt1a1 was significantly increased.The hepatic Mrp3 m RNA expression in UDCA group was significantly decreased when compared to that of the model group(P<0.05).(2)compared with the normal group,the m RNA expression of Ost?/?and Fxr of ileum were all significantly up-regulated in the model group(P<0.05),but Fgf15 was significantly down-regulated(P<0.05).Compared with the model group,the m RNA expression of Fxr of ileum in Zhi-zi group was markedly decreased,and the expression of Fgf15 was decreased in UDCA group(P<0.05).(3)compared with the normal group,the m RNA expression of Mrp3 and Mrp4 of kidney were all significantly increased in model group,(P<0.05),but the m RNA expression of Asbt and Ost? were all markedly decreased(P<0.05)..Compared with the model group,Mrp3 m RNA expression of kidney was up-regulated in Zhi-zi group,and the m RNA expression of Ost?/?,Mrp2,Mrp3 and Mrp4 were all strongly increased(P<0.05)with 6-fold increase in Mrp2 and 1.5-fold increase in Mrp4.Additionally,compared with the model group,the m RNA expression of Ost? and Mrp3 of kidney were all significantly increased in UDCA group(P<0.05).Conclusion1.EE can indcue intrahepatic cholestasis and jaundice in rats,the underlying mechanisms may have correlation with(1)decreased expression of hepatic bilirubin metabolism enzyme Ugt1a1;(2)increased BAs accumulation and primary bile acid synthesis via elevating the expression of hepatic BAs biosynthetic enzymes Cyp7a1 and Cyp27a1.2.Jaundice-removing effect of Zhi-zi in EE-induced jaundice rats was related to the increased urinary excretion of bilirubin,the underlying mechanisms involves:(1)up-regulation of hepaticmetabolic enzyme Ugt1a1 to promote unconjugated bilirubin change into conjugated bilirubin,which is easily excreted via kidney;(2)increased renal excretion of bilirubin via up-regulating the expression of the efflux transporter Mrp2 that expressing at the apical membrane of the renal tubular epithelium;(3)decreased reabsorption of bilirubin from urine via down-regulating the expression of Mrp3 at the basolateral membrane of renal tubular epithelium.3.Zhi-zi aqueous extract decreasd the accumulation of BAs in EE-induced intrahepatic cholestasis in rats,its cholagogue mechanism has close correlation with(1)significantly increased excretion of BAs in urine via up-regulated expression of Mrp2 and Mrp4 at the brush border membrane of renal epithelial cells;(2)reduced synthesis of primary BAs in liver via down-regulated expression of Cyp7a1 and Cyp27a1 that play critical roles in the biosynthesis of BAs in liver. |
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