Clinical And Experimental Study On The Mechanism Of Daifan Powder In The Treatment Of Primary Biliary Cholangitis Based On The Regulation Of FXR And Its Target Genes

Objective:1.Based on clinical observation study,the clinical efficacy and security of Daifan powder in the treatment of primary biliary cholangitis(PBC)were evaluated,and the basis for the clinical application of Daifan powder was provided.2.Through animal experiments,taking ANIT-induced intrahepatic cholestasis rats and Poly I: C-induced PBC mice as research objects,based on the regulation of FXR and its target genes,to explore the hepatoprotective effect of Daifan Powder and its mechanism of action in the treatment of PBC.Methods:1.Clinical Observation Study:Clinical retrospective controlled study was adopted to study the clinical efficacy of Daifan powder in the treatment of PBC patients based on the theory of traditional Chinese medicine.Sixty patients diagnosed with PBC were divided into two groups.The control group received conventional treatment including ursodeoxycholic acid capsules.The treatment method of the treatment group was to add the treatment of Daifan powder on the basis of the control group.After 6 weeks of treatment,the records of adverse reactions in the treatment process of Daifan powder,the efficacy evaluation of Daifan powder were observed,and the indexes of serum liver function and TCM syndrome scores were compared before and after treatment.2.Animal Research:(1)In animal experiment 1,the rat model of intrahepatic cholestasis was established by intragastric administration of?-naphthalene isothiocyanate(ANIT)to male SD rats.The method of decomposing prescriptions study was used.Obeticholic Acid was used as the positive drug and normal rats as the control.Objective To study the effects of Daifan Powder(composed of indigo naturalis and alumen)on 2-hour bile flow rate,serum liver function indexes,liver tissue FXR,BSEP molecules and liver histopathology in rats with intrahepatic cholestasis.To observe the antagonistic effect on intrahepatic cholestasis and liver protection effect of Daifan powder group and decomposing prescriptions groups(indigo naturalis group and alumen group),and then observe the hepato-protective effect of Daifan powder.The levels of ALT,AST,ALP,TBIL,DBIL and TBA in liver function of rats were detected by biochemical assay.The m RNA expression levels of FXR and BSEP in rat liver tissues were detected by q RT-PCR.The protein expression levels of FXR and BSEP in liver tissues were detected by Western-blotting and immunohistochemistry.The pathological changes of rat liver were detected by HE staining.(2)In animal experiment 2,female C57BL/6 mice were intraperitoneally injected with Poly I:C to establish PBC mouse models.Obeticholic Acid was used as the positive drug and normal rats were used as the control group to study the effects of Daifan powder on some indexes of serum liver function,liver tissue FXR,SHP,CYP7A1,BSEP molecules and liver histomathology of PBC mice.To observe the effects of Daifan powder group and its disassociated prescription groups(indigo naturalis group and alumen group)on the key molecule FXR and its target genes in the occurrence and development of PBC,and then evaluate the therapeutic effect of Daifan powder on PBC.The levels of ALT,AST,ALP and ?-GGT were detected by biochemical assay.The m RNA expression levels of FXR,SHP,CYP7A1 and BSEP in liver tissues of mice were detected by q RT-PCR.The protein expression levels of FXR,SHP,CYP7A1 and BSEP in liver tissues were detected by Western-blotting.The expression of FXR protein in liver tissue was detected by immunofluorescent staining.HE staining was used to detect the pathological changes of liver tissue in mice.Results:1.Clinical research results:(1)There was no statistical significance in gender,age and course of disease between the two groups before treatment(P>0.05).Before treatment,there were no significant differences in TCM symptom score and serum biochemical indexes between the two groups(P>0.05).(2)The clinical efficacy of Daifan powder on PBC patients,including complete response,incomplete response,no response and relapse,showed that the clinical efficacy of the treatment group was better than that of the control group(P<0.05).(3)About the effects of Daifan powder on TCM syndrome scores in PBC patients.The results showed that there was no difference in TCM syndrome scores between the treatment group and the control group before treatment(P>0.05).The scores of symptoms of TCM in both groups were significantly decreased after treatment(P<0.01).Compared with the control group,the TCM symptom score of the treatment group was significantly lower than that of the control group(P<0.01).(4)The effects of Daifan powder on liver function indexes in PBC patients.The results showed that compared with before treatment,ALT,AST,ALP,?-GGT,TBIL and TBA were significantly decreased(P<0.01)and ALB was significantly increased(P<0.01)in the two groups after treatment.Compared with the control group,ALT,AST and ALB were not significantly different between the two groups after treatment(P > 0.05).Compared with the control group after treatment,ALP,?-GGT,TBil and TBA in the treatment group were significantly lower than those in the control group after treatment(P<0.05).(5)Observation of adverse reactions.The results showed that no adverse reactions occurred in 60 patients in the treatment group and the control group in case data.2.Animal research results:(1)Experiment 1: Animal Experimental Study on Liver Protection and Gall Benefits of Daifan PowderCompared with the control group,AST,ALT,ALP,TBIL,DBIL and TBA in the model group were abnormally increased(P<0.01).Compared with the model group,AST,ALT,ALP,TBIL,DBIL and TBA in each group were significantly decreased after the intervention of indigo naturalis,alumen,daifan powder and obecholic acid,respectively(P<0.05 or P<0.01).But there was no significant difference in TBIL between the alumen group and the model group after intervention(P>0.05).Compared with the obeticholic acid group,ALT,TBIL and TBA of the Daifan powder group after intervention had no statistical significance(P>0.05).On the effect of bile flow velocity,compared with the control group,the2-hour bile flow velocity of rats in the model group was significantly decreased(P<0.01).After the intervention of indigo naturalis,alumen,daifan powder and obeticholic acid,the bile velocity of rats in each group increased(P<0.05 or P<0.01).The m RNA and protein expressions of FXR and BSEP in rat liver tissue were significantly decreased in model group(P<0.01),and the m RNA and protein expressions of FXR and BSEP were significantly increased after the intervention of indigo naturalis,alumen,daifan powder and obeticholic acid,respectively(P<0.05 or P<0.01).Compared with the obeticholic acid group,there was no significant difference in BSEP m RNA expression in the Daifan powder group(P>0.05).There was no difference in the expression of FXR protein between Daifan powder group and obeticholic acid groups(P>0.05).The results of HE staining showed that the liver tissue of rats in model group showed pathological manifestations of intrahepatic cholestasis.After intervention of obeticholic acid,indigo naturalis,alumen and daifan powder,liver tissue lesions were improved in these groups,and liver tissue lesions were significantly reduced in the obeticholic acid group.(2)Experiment 2: Experimental study on the effect of Daifan powder on PBC model mice based on FXR target gene regulation.Compared with control group,AST,ALT,ALP and ?-GGT in model group were all increased(P<0.01),AST,ALT,ALP and ?-GGT were significantly decreased after the intervention of indigo naturalis,alumen,daifan powder and obeticholic acid,respectively(P<0.01).The m RNA and protein expressions of FXR,SHP,CYP7A1 and BSEP in liver tissue of mice were significantly decreased after modeling(P<0.01),CYP7A1 m RNA and protein expression were significantly increased(P<0.01).The m RNA and protein expressions of FXR,SHP and BSEP were significantly increased after the intervention of obeticholic acid,indigo naturalis,alumen and Daifan powder respectively(P<0.01),CYP7A1 m RNA and protein expression were significantly decreased(P<0.01).In the pathological manifestations of liver tissue,the pathological manifestations of mice in each treatment group were alleviated.The inflammatory lesions in obeticholic acid groups were alleviated most obviously,Daifan powder group,indigo naturalis group and alumen group all reduced to different degrees.Conclusion:1.On the basis of the conventional treatment including ursodeoxycholic acid capsules,the combined treatment with Daifan powder has a significant clinical effect on PBC patients.The addition of daifan powder treatment can significantly reduce the clinical symptoms of patients,significantly improve the response rate of conventional treatment,enhance the effect of conventional treatment.2.Daifan powder has hepatoprotective and gallbladder beneficial effects on ANIT-induced intrahepatic cholestasis model rats,the mechanism of which may be through the activation of FXR/BSEP signaling pathway.3.Daifan Powder can effectively protect the liver of PBC model mice,and the mechanism of action may be related to the regulation of FXR and its target genes.