Clinical And Experimental Study On The Treatment Of Liver Fibrosis With Chaiqi Yigan Recipe

China is a major liver disease country with a high prevalence of many liver diseases,and liver fibrosis is receiving increasing attention as an important area of liver disease research and treatment.Liver fibrosis is a pathological change resulting from abnormal connective tissue proliferation and excessive deposition of extracellular matrix caused by liver repair from liver injury.Liver fibrosis is present in most chronic liver diseases and is the key step in the progression of chronic liver disease to cirrhosis,which can even progress continuously to cirrhosis and liver cancer,posing a serious threat to the life and health of patients.Investigating the etiology and pathogenesis of liver fibrosis and finding effective therapeutic targets are of great clinical significance to stop the progression of the disease and maintain the life and health of patients with liver fibrosis.In recent years,the pathological and molecular biological mechanisms of liver fibrosis and clinical treatment techniques have made great progress.However,the efficacy of Western medicine for anti-fibrosis is still inexact and there is a lack of effective treatment means.Chinese medicine has definite advantages in the treatment of liver fibrosis,and a variety of proprietary Chinese medicines with registered indications for liver fibrosis have been marketed.Chaiqiyigan formula(CQYG)was created by mentor Professor Hu Shiping,and is an experienced formula for the treatment of liver fibrosis in chronic hepatitis B.Preliminary clinical and experimental studies have found that it has good efficacy in liver fibrosis,but its mechanism is still unclear.Professor Hu is currently the Secretary of the Party Committee of Shenzhen Hospital of Beijing University of Chinese Medicin,a famous Chinese medicine practitioner in Guangdong Province,a mentor of the National Grassroots Master Teacher Training Program,a local leading talent in Shenzhen,the "Most Beautiful Chinese Medicine Practitioner of South Guangdong".He has been engaged in the clinical and scientific research of Chinese medicine for more than 30 years,specializing in the treatment of liver diseases with the combination of Chinese medicine and Western medicine,and has formed a unique academic thought system.ObjectiveExploring the efficacy and mechanism of CQYG in the treatment of liver fibrosis by combining clinical retrospective study,network pharmacology prediction and animal experiments to provide a basis for further development of the prescription.Besides,summarizing the academic thought "Tui Chen Zhi Xin" of Professor Hu and its application in the formulation of CQYG.Methods1.Clinical research parts:Throughing a retrospective study method,case data of patients diagnosed with chronic hepatitis B in the outpatient and inpatient departments of the Department of Hepatology,Shenzhen Hospital of Beijing University of Chinese Medicine between January 1,2018 and February 3,2021 were collected.Patients with chronic hepatitis B fibrosis with liver depression and spleen deficiency who met the criteria of this study were screened.Patients were divided into regular treatment group and CQYG group according to the medications they were taking.The indexes before and after 12 months of treatment were collected from all patients,main indicators included:liver stiffness measurement(LSM),fibrosis-4 index(FIB-4),aspartate aminotransferase and platelet ratio index(APRI),hepatitis B virus surface antigen(HBsAg),and hepatitis B virus(HBV-DNA)quantification.Secondary indicators included:alanine transaminase(ALT),glutamate transaminase(AST),total bilirubin(TBiL),glutamyltransferase(GGT),albumin(ALB).All testing indicators were compared within and between groups before and after the treatment.2.Network pharmacology:To screen the active ingredients of CQYG and possible targeted proteins for liver fibrosis treatment,databases such as TCMSP,TCMID,Swiss Target Prediction,OMIM,Gene Cards,etc.were retrieved."drug-component-target-disease" network and protein interaction PPI network were constructed,respectively.topological analysis was performed to screen the key pharmacodynamic molecules and core targets assisted with Cytoscape and String database.Molecular docking for the active components in CQYG was predicted via Autodock vina.GO and KEGG enrichment analysis were used for the possible protein targets based on R language.3.Animal experiments:C57BL/6 male mice were randomly categorized into control group(CTL),liver fibrosis model group(Model),CQYG low-dose treated group(CQYG-L),CQYG high-dose treated group(CQYG-H)and silymarin treated group(Silymarin).The liver fibrosis mouse model was established via injected intraperitoneally with olive oil containing 15%CCl4 twice a week for 8 weeks.Mice in the CQYG-L and CQYG-H groups were administrated 0.37 g·kg-1·d-1 and 0.74 g·kg-1·d-1 of CQYG(i.g),respectively,and silymarin group were given 100 mg·kg-1·d-1(i.g).At the end of the experiment,HE,Sirius red and Masson staining were exploited to evaluate the structural changes and collagen fiber deposition in each group;measurement of serum AST and ALT by automated biochemical analyzer;MDA,SOD,GSH-Px and Hyp in liver tissue were determined by ELISA;detection of ?-SMA,Collagen I and Vimentin expression in liver tissues by immunohistochemistry;detection of Ki67+and Lgr5+cells in liver tissues by immunofluorescence.Real-time PCR and Western blot was performed to detect mRNA and protein in NF-?B,TGF-?/Smad and Wnt/?-Catenin signaling pathway in liver tissue.Results1.Clinical research parts:One hundred and one patients in RT group were collected aging from 22 to 76 years old,and 102 in the CQYG group were obtained aging from 27 to 67 years old;There was no statistical difference in the age ratio between the two groups(P>0.05).(1)No significant difference exhibited(P>0.05)in LSM,FIB-4 and APRI between the two groups before and after treatment,but there was a declining trend for LSM in both groups after treatment compared with that before treatment.(2)There was no marked difference in the comparison of HBV-DNA and HBsAg between the two groups before and after treatment(P>0.05);HBsAg was significantly lowered in the CQYG group compared with RT after treatment(P<0.05),and HBV-DNA quantification and HBsAg in the CQYG group showed falling trend after treatment compared with that before treatment.(3)The sera liver function indexes(ALT,AST,GGT,TBiL,ALB)fell within the normal range before and after treatment in both groups,and there was no significant difference between the groups comparing liver function after treatment(P>0.05);there was no notable difference before and after treatment in the control group(P>0.05).2.Network pharmacology part:Total of 121 active ingredients and 257 corresponding targets of CQYG for the treatment of liver fibrosis were obtained,and 14 key pharmacodynamic molecules and 28 core targets were selected.The results of molecular docking showed that the top 10 core targets and key potent molecules had good binding activities.The results of GO and KEGG mainly involved biological processes such as inflammatory response,oxidative stress,fibroblast proliferation,endothelial cell proliferation,regulation of lipid metabolism,angiogenesis,liver regeneration and TNF signaling pathway,Th17 cell differentiation signaling pathway,IL-17 signaling pathway,PI3K/Akt signaling pathway,Wnt signaling pathway,NF-?B signaling pathway,etc.3.Animal experimental part:Compared with the model group,the liver morphology and collagen deposition were better in each CQYG group and silymarin group than the model group,and the ALT and AST levels declined significantly in both silymarin group and CQYG-H mice(P<0.01),and the levels of MDA and Hyp in liver tissues of CQYG-H group were significantly lower than those of the model group(P<0.05),the levels of SOD and GSH-Px were significantly higher than those of the model group(P<0.05).The protein expression levels of p-NF-?B p-65,TNF-?,IL-6 and IL-1? proteins in liver tissues were significantly lower in the CQYG group compared with the model group(P<0.05).The immunohistochemical results showed that the?-SMA,Collagen I and Vimentin positive expression areas were less in the CQYG and silymarin groups than in the model group;the immunofluorescence results showed that the number of Ki67+cells and Lgr5+cells were both increased in the Chaiqi Yi Liver Formula group compared with the model group;The m RNA levels of Collagen I,TGF-?,TNF-?,and IL-1?in CQYG-H liver tissues,as well as Collagen I,?-SMA,TIMP-1,TGF-?,p-Smad2/3,Smad2/3,Smad4,Wnt3a,?-Catenin,and p-NF-?B protein expression were down-regulated compared with the model group,while MMP-9 and Smad7 protein expressions were significantly up-regulated(P<0.05).Conclusions1.Clinical parts:In clinical studies,it was found that CQYG combined with conventional treatment had a tendency to reduce liver hardness values,HBV-DNA and HBsAg levels in patients with chronic hepatitis B fibrosis,and was superior to regular treatment alone in reducing HBsAg quantification.2.Network pharmacology:This study suggested that CQYG may act on the targets of mitogen-activated protein kinase MAPK1/3/8,proto-oncogene tyrosine kinase Src,activator protein Jun,as well as TNF signaling pathway,Th17 cell differentiation signaling pathway,IL-17 signaling pathway,PI3K/Akt signaling pathway,HIF-1 signaling pathway,Rapl signaling pathway,Wnt signaling pathway,NF-?B signaling pathway to regulate liver inflammatory response,oxidative stress,cell proliferation,liver regeneration and other biological processes to treat liver fibrosis.3.Animal experiment:CQYG significantly attenuated carbon tetrachloride-induced liver fibrosis in mice,and its mechanism of action may attenuate oxidative stress,as well as inhibit NF-?B-mediated inflammatory signaling pathway,downregulate the release of inflammatory cytokines IL-6,TNF-?,IL-1?,reduce liver inflammation,and through regulating TGF-?/Smad,Wnt3a/?-catenin signaling pathway.inhibits the activation of hepatic stellate cells,regulates the balance of MMP-9 and TIMP-1 activity,reduces the synthesis of extracellular matrix ?-SMA,Collagen I,Hyp,and promotes ECM degradation-related.4.The thought of "Tui Chen Zhi Xin":The core of Professor Hu Shiping's academic thought of "Tui Chen Zhi Xin" is to comply with the human body's own trend of healthy Qi and removing evil Qi and the respective metabolic processes of Qi,blood and body fluid,to assist the human body to naturally remove evil Qi and promote the healing of diseases.In the treatment of liver fibrosis,through "Tui Chen Zhi Xin",strengthen the power of Qi transformation and regulate the rise and fall of Qi,so that while the pathological products are eliminated,the body's Qi,blood and fluids can be produced normally.