Clinical And Experimental Study Of Leech And Its Extracts For Intrahepatic Cholestasis

Part I Clinical observation of leech treatment of intrahepatic cholestatic liver disease Objective: To investigate the effects of leech on serum alkaline phosphatase(ALP),gamma-glutamyl transferase(GGT),and total bilirubin(TBil)levels in patients with intrahepatic cholestasis liver disease,and their effect on clinical symptoms of patients.Methods: Forty patients with intrahepatic cholestatic liver disease were selected and randomly divided into two groups:treatment group and control group,with 20 patients in each group.Conventional treatment of cholestatic liver disease was given to20 patients in the control group,and 20 patients in the treatment group.In addition to conventional treatment,patients were treated with leech granules(3g/day)for 2 weeks.The levels of ALP,GGT and TBil were measured after 2 weeks,and the clinical symptoms before and after treatment were compared.Results: After 2 weeks of treatment,the levels of ALP,GGT and TBil in the two groups of patients were significantly lower than before treatment,and the symptoms of fatigue and pruritus were improved than before treatment(P <0.05);the levels of ALP、GGT and TBil in the treatment group were higher than those of the control The level in the treatment group was reduced(P <0.05).The improvementof pruritus and fatigue symptoms in the treatment group was better than that in the control group.Conclusion: Leech can significantly reduce the levels of ALP,GGT and TBil in the serum of patients with intrahepatic cholestasis liver disease,and effectively improve the symptoms of pruritus and fatigue in patients.It provides a clinical basis for the treatment of intrahepatic cholestasis liver disease by Tongluo drugs.Part 2 Effects of Leech Extract-Hirudin on Intrahepatic Models of Intrahepatic CholestasisObjective: This study was carried out to investigate the effect of leech extract-hirudin on modle of intrahepatic cholestasis in vivo and vitro.Method: In vivo,fivty male Sprague-Dawley rats were randomly divided into normal group,model group,ursodeoxycholic acid(UDCA)group(60 mg/kg),hirudin treatment group(84 u/kg),hirudin treatment group(63 u/kg).hirudin treatment group(42 u/kg).The UDCA group was intragastrically administered with a corresponding concentration of 0.005 m L/g body weight for 7 days,once a day;and the hirudin treatment group was administered subcutaneously at different concentration for 7 days,once a day;except for the normal group,the other groups were given 100 mg/kg ANIT on the 5th day of gavage.The model was administered by gavage once a day for3 days.In vitro,(Z)-Guggulsterone was used to stimulate the L02cells(0.05μmol/ml),with or without different concentrations of hirudin(2 u/ml,4 u/ml and 8 u/ml)for 24 h.Hepatic tissue was taken for HE microscopic examination to observe the pathological condition of rat liver;FXR,SHP,UGT2B4,BSEP mRNA and protein expressions were detected by real-time fluorescent quantitative PCR and Western blot test.And immunohistochemistry(IHC)was used to analyze the expression of FXR.Results: Compared with the normal group,liver injury was severe in the model group,and the expressions of FXR,SHP,UGT2B4,BSEP protein and mRNA were reduced(P <0.05,P <0.01);Compared with the model group,the hirudin group can improve liver tissue damage,and promote the expression of FXR,SHP,BSEP and UGT2B4 proteins and mRNA in vivo and vitro.Conclusion: Hirudin can alleviate intrahepatic cholestasis,reduce liver tissue damage.The hirudin can up-regulate the expression of FXR gene,promote the up-regulation of SHP,UGT2B4,and BSEP genes,and inhibit the cholestasis pathway to protect liver cells,Provide directions and drug screening for clinical application of Huoxuetongluo method in the treatment of intrahepatic cholestasis.