Effect of compound Yindan decoction on alpha-naphthylisothiocyanate-Induced acute intrahepatic cholestasis in rats
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摘要
OBJECTIVE:To investigate the therapeutic mechanism of compound Yindan decoction(CYD)in a rat model of acute intrahepatic cholestatic(AIC).METHODS:A total of 108 adult male rats were randomly divided into control(n= 18)and AIC groups(n=90).AIC was induced in rats using alpha-naphthylisothiocyanate(ANIT)(75 mg/kg,10 mL/kg in corn oil,p.o.).Then,90 AIC rats were randomly divided into five groups:a control group(n = 18),a CYD high dose group(n = 18),a CYD middle dose group(n= 18),a CYD low dose group(n= 18),and a ursodeoxycholic acid(UDCA)group(n = 18).According to sampling time,each group was subdivided into three subgroups:24 h(n=6),48 h(n=6),and 72 h groups(n = 6).The CYD-high,-middle and-low groups were orally administered 24.48,12.24,and 6.12 g·kg~(-1)·d~(-1) modified CYD,respectively,while the model group was given 20 mL/kg of body weight of distilled water once a day.The UDCA group was given 67.5 mg·kg~(-1)·d~(-1) UDCA once a day.Radioimmunity assay was used to detect the activity of alanine aminotransferase(ALT),aspartate aminotransferase(AST),alkaline phosphatase(ALP),gamma-glutamyl transpeptidase(GGT)and the levels of total bilirubin(TBil)and indirect biliruin(DBil)in rats.Reverse transcription quantitative polymerase chain reaction(qRT-PCR),Western blot analysis,and immunohistochemistry were used to detect multidrug resistance-associated protein 2(MRP2)expression.In vitro,HepG2 hepatocellular carcinoma cells were treated with CYD medicated serum at a concentration of 15 mol/L.MRP2 and retinoid X receptor alpha(RXRa)expression was analyzed by qRT-PCR and Western blotting.RESULTS:Serum levels of ALT,AST,GGT,ALP,TBil,and DBil were significantly reduced in the CYD and positive drug groups compared with the control group(P<0.05 and P<0.01,respectively).Pathological changes in rat liver tissues at 72 h in the CYD-high and-medium dose groups and positive drug group were not significant compared with the control group.CYD and UDCA treatment ameliorated ANIT-induced biliary epithelial cell proliferation.Neutrophil infiltration was rare and little focal necrosis was observed in lobules in the CYD-high and-medium dose groups and UDCA group at 72 h.Compared with the control group,the expression of MRP2 mRNA and MRP2 protein in the liver tissue of the CYD groups was significantly increased(P<0.05 and P<0.01,respectively).MRP2 expression and RXRa nuclear receptor mRNA and protein levels in the CYD groups were significantly increased compared with the control and UDCA groups(P<0.01).CONCLUSION:CYD can alleviate cholestasis in ANIT-induced AIC rats,and the mechanism underlying this action might involve increases in ALT,AST,GGT,ALP,TBil,and DBil and upregulation of MRP2 and RXRa mRNA and protein levels.
OBJECTIVE:To investigate the therapeutic mechanism of compound Yindan decoction(CYD)in a rat model of acute intrahepatic cholestatic(AIC).METHODS:A total of 108 adult male rats were randomly divided into control(n= 18)and AIC groups(n=90).AIC was induced in rats using alpha-naphthylisothiocyanate(ANIT)(75 mg/kg,10 mL/kg in corn oil,p.o.).Then,90 AIC rats were randomly divided into five groups:a control group(n = 18),a CYD high dose group(n = 18),a CYD middle dose group(n= 18),a CYD low dose group(n= 18),and a ursodeoxycholic acid(UDCA)group(n = 18).According to sampling time,each group was subdivided into three subgroups:24 h(n=6),48 h(n=6),and 72 h groups(n = 6).The CYD-high,-middle and-low groups were orally administered 24.48,12.24,and 6.12 g·kg~(-1)·d~(-1) modified CYD,respectively,while the model group was given 20 mL/kg of body weight of distilled water once a day.The UDCA group was given 67.5 mg·kg~(-1)·d~(-1) UDCA once a day.Radioimmunity assay was used to detect the activity of alanine aminotransferase(ALT),aspartate aminotransferase(AST),alkaline phosphatase(ALP),gamma-glutamyl transpeptidase(GGT)and the levels of total bilirubin(TBil)and indirect biliruin(DBil)in rats.Reverse transcription quantitative polymerase chain reaction(qRT-PCR),Western blot analysis,and immunohistochemistry were used to detect multidrug resistance-associated protein 2(MRP2)expression.In vitro,HepG2 hepatocellular carcinoma cells were treated with CYD medicated serum at a concentration of 15 mol/L.MRP2 and retinoid X receptor alpha(RXRa)expression was analyzed by qRT-PCR and Western blotting.RESULTS:Serum levels of ALT,AST,GGT,ALP,TBil,and DBil were significantly reduced in the CYD and positive drug groups compared with the control group(P<0.05 and P<0.01,respectively).Pathological changes in rat liver tissues at 72 h in the CYD-high and-medium dose groups and positive drug group were not significant compared with the control group.CYD and UDCA treatment ameliorated ANIT-induced biliary epithelial cell proliferation.Neutrophil infiltration was rare and little focal necrosis was observed in lobules in the CYD-high and-medium dose groups and UDCA group at 72 h.Compared with the control group,the expression of MRP2 mRNA and MRP2 protein in the liver tissue of the CYD groups was significantly increased(P<0.05 and P<0.01,respectively).MRP2 expression and RXRa nuclear receptor mRNA and protein levels in the CYD groups were significantly increased compared with the control and UDCA groups(P<0.01).CONCLUSION:CYD can alleviate cholestasis in ANIT-induced AIC rats,and the mechanism underlying this action might involve increases in ALT,AST,GGT,ALP,TBil,and DBil and upregulation of MRP2 and RXRa mRNA and protein levels.
OBJECTIVE:To investigate the therapeutic mechanism of compound Yindan decoction(CYD)in a rat model of acute intrahepatic cholestatic(AIC).METHODS:A total of 108 adult male rats were randomly divided into control(n= 18)and AIC groups(n=90).AIC was induced in rats using alpha-naphthylisothiocyanate(ANIT)(75 mg/kg,10 mL/kg in corn oil,p.o.).Then,90 AIC rats were randomly divided into five groups:a control group(n = 18),a CYD high dose group(n = 18),a CYD middle dose group(n= 18),a CYD low dose group(n= 18),and a ursodeoxycholic acid(UDCA)group(n = 18).According to sampling time,each group was subdivided into three subgroups:24 h(n=6),48 h(n=6),and 72 h groups(n = 6).The CYD-high,-middle and-low groups were orally administered 24.48,12.24,and 6.12 g·kg~(-1)·d~(-1) modified CYD,respectively,while the model group was given 20 mL/kg of body weight of distilled water once a day.The UDCA group was given 67.5 mg·kg~(-1)·d~(-1) UDCA once a day.Radioimmunity assay was used to detect the activity of alanine aminotransferase(ALT),aspartate aminotransferase(AST),alkaline phosphatase(ALP),gamma-glutamyl transpeptidase(GGT)and the levels of total bilirubin(TBil)and indirect biliruin(DBil)in rats.Reverse transcription quantitative polymerase chain reaction(qRT-PCR),Western blot analysis,and immunohistochemistry were used to detect multidrug resistance-associated protein 2(MRP2)expression.In vitro,HepG2 hepatocellular carcinoma cells were treated with CYD medicated serum at a concentration of 15 mol/L.MRP2 and retinoid X receptor alpha(RXRa)expression was analyzed by qRT-PCR and Western blotting.RESULTS:Serum levels of ALT,AST,GGT,ALP,TBil,and DBil were significantly reduced in the CYD and positive drug groups compared with the control group(P<0.05 and P<0.01,respectively).Pathological changes in rat liver tissues at 72 h in the CYD-high and-medium dose groups and positive drug group were not significant compared with the control group.CYD and UDCA treatment ameliorated ANIT-induced biliary epithelial cell proliferation.Neutrophil infiltration was rare and little focal necrosis was observed in lobules in the CYD-high and-medium dose groups and UDCA group at 72 h.Compared with the control group,the expression of MRP2 mRNA and MRP2 protein in the liver tissue of the CYD groups was significantly increased(P<0.05 and P<0.01,respectively).MRP2 expression and RXRa nuclear receptor mRNA and protein levels in the CYD groups were significantly increased compared with the control and UDCA groups(P<0.01).CONCLUSION:CYD can alleviate cholestasis in ANIT-induced AIC rats,and the mechanism underlying this action might involve increases in ALT,AST,GGT,ALP,TBil,and DBil and upregulation of MRP2 and RXRa mRNA and protein levels.
引文
1 Chinese medical association liver disease branch,Chinese medical association digestive diseases branch,Chinese medical association infectious Diseasess branch.The consensus of diagnosis and treatment on cholestasis liver disease(2015).Zhong Hua Lin Chuang Gan Bing Za Zhi2015;31(12):1989-1999.
2 Yang HL,Wu CQ,Guang YM,et al.The research progress of the mechanism of intrahepatic bile accumulation.Du Li Xue Za Zhi 2007;21(1):65-68.
3 Sun FX,Wang JM,Liu L.Clinical observation of 30 cases of acute cholestasis hepatitis treated by compound yindan decoction.Zhong Guo Zhong Xi Yi Jie He Za Zhi 2015;35(3):310-313.
4 Sandusky GE,Mintze KS,Pratt SE,et al.Expression of multidrug resistance-associated protein2(MRP2)in normal human tissues and carcinomas using tissue microarrays.Histopathology 2010;41(1):65-74.
5 Kullak-Ublick GA,Baretton GB,Oswald M,et al.Expression of the hepatocyte canalicular multidrug resistance protein(MRP2)in primary biliary cirrhosis.Hepatol Res2002;23(1):78-82.
6 Boyer JL.Nuclear receptor ligands:rational and effective therapy for chronic cholestatic liver disease?Gastroenterology 2005;129(2):735-740.
7 Chen Q.Experimental methodology of pharmacological research in Traditional Chinese Medicine.2nd ed.Shanghai:Shanghai science and technology Publishing House,2006:302-305.
8 Kossor DC,Meunier PC,Handler JA,et al.Temporal relationship of changes in hepatobiliary function and morphology in rats followingα-naphthylisothiocyanate(ANIT)administration.Toxicol Appl Pharm 1993;119(1):108-114.
9 Tang JW,Rui J.The research progress of MRP2 in cholestasis.Fang She Mian Yi Xue Za Zhi 2009;22(5):501-503.
10 Decaens C,Durand M,Grosse B,et al.Which in vitro models could be best used to study hepatocyte polarity?Biol Cell 2008;100(7):387-398.
11 Bohan A,Chen WS,Denson LA,et al.Tumor necrosis factor?-dependent up-regulation of Lrh-1 and Mrp3(Abcc3)reduces liver injury in obstructive cholestasis.J Biol Chem 2003;278(38):36688-36698.
12 Boyer JL.Nuclear receptor ligands:rational and effective therapy for chronic cholestatic liver disease?Gastroenterology 2005;129(2):735-740.
13 O'Leary JG,Pratt DS.Cholestasis and cholestatic syndromes.Curr Opin Gastroen 2007;23(3):232-236.
14 Halilbasic E,Claudel T,Trauner M.Bile acid transporters and regulatory nuclear receptors in the liver and beyond.JHepatol 2013;58(1):155-168.
15 Lin LC,Wu CC,Yeh HI,et al.Downregulated myocardial connexin 43 and suppressed contractility in rabbits subjected to a cholesterol-enriched diet.Lab Invest 2005;85(10):1224-1237.
2 Yang HL,Wu CQ,Guang YM,et al.The research progress of the mechanism of intrahepatic bile accumulation.Du Li Xue Za Zhi 2007;21(1):65-68.
3 Sun FX,Wang JM,Liu L.Clinical observation of 30 cases of acute cholestasis hepatitis treated by compound yindan decoction.Zhong Guo Zhong Xi Yi Jie He Za Zhi 2015;35(3):310-313.
4 Sandusky GE,Mintze KS,Pratt SE,et al.Expression of multidrug resistance-associated protein2(MRP2)in normal human tissues and carcinomas using tissue microarrays.Histopathology 2010;41(1):65-74.
5 Kullak-Ublick GA,Baretton GB,Oswald M,et al.Expression of the hepatocyte canalicular multidrug resistance protein(MRP2)in primary biliary cirrhosis.Hepatol Res2002;23(1):78-82.
6 Boyer JL.Nuclear receptor ligands:rational and effective therapy for chronic cholestatic liver disease?Gastroenterology 2005;129(2):735-740.
7 Chen Q.Experimental methodology of pharmacological research in Traditional Chinese Medicine.2nd ed.Shanghai:Shanghai science and technology Publishing House,2006:302-305.
8 Kossor DC,Meunier PC,Handler JA,et al.Temporal relationship of changes in hepatobiliary function and morphology in rats followingα-naphthylisothiocyanate(ANIT)administration.Toxicol Appl Pharm 1993;119(1):108-114.
9 Tang JW,Rui J.The research progress of MRP2 in cholestasis.Fang She Mian Yi Xue Za Zhi 2009;22(5):501-503.
10 Decaens C,Durand M,Grosse B,et al.Which in vitro models could be best used to study hepatocyte polarity?Biol Cell 2008;100(7):387-398.
11 Bohan A,Chen WS,Denson LA,et al.Tumor necrosis factor?-dependent up-regulation of Lrh-1 and Mrp3(Abcc3)reduces liver injury in obstructive cholestasis.J Biol Chem 2003;278(38):36688-36698.
12 Boyer JL.Nuclear receptor ligands:rational and effective therapy for chronic cholestatic liver disease?Gastroenterology 2005;129(2):735-740.
13 O'Leary JG,Pratt DS.Cholestasis and cholestatic syndromes.Curr Opin Gastroen 2007;23(3):232-236.
14 Halilbasic E,Claudel T,Trauner M.Bile acid transporters and regulatory nuclear receptors in the liver and beyond.JHepatol 2013;58(1):155-168.
15 Lin LC,Wu CC,Yeh HI,et al.Downregulated myocardial connexin 43 and suppressed contractility in rabbits subjected to a cholesterol-enriched diet.Lab Invest 2005;85(10):1224-1237.