Efficacy And Mechanic Study Of Jian-Gan-Xiao-Zhi Decoction On Non-Alcoholic Fatty Liver Disease Based On Multi-Omics Techniques

Research objectiveNon alcoholic fatty liver disease(NAFLD)is the largest chronic liver disease in the world,which can develop into diseases such as cirrhosis and liver cancer,posing a serious threat to public health.At present,there is a lack of effective treatment drugs for this disease.It is urgent to seek a safe and effective drug to delay the process of NAFLD.Traditional Chinese medicine(TCM)has a history of thousands of years in the treatment of NAFLD with the rich clinical experience.TCM defines that "deficiency of spleen qi and stagnation of liver qi" are one of the main pathogenesis of NAFLD.The treatment should be based on supplementing qi,strengthening the spleen,soothing the liver and relieving depression.Under the guidance of the pathogenesis theory and clinical practice,our research group has established Jian-Gan-Xiao-Zhi decoction(JGXZ).This prescription consists of twelve herbs,such as Astragalus membranaceus,Rhizoma alismatidis,and Pericarpium citri.The entire formula has the synergistic effect of tonifying the spleen,harmonizing the liver and spleen,restoring spleen weakness,and dispersing liver depression.However,there is a lack of objective basis for the clinical efficacy of JGXZ on NAFLD.A comprehensive evaluation of the therapeutic effect of JGXZ on NAFLD is helpful for its promotion and application.In recent years,with the deepening understanding of the "gut-liver axis",the close relationship between gut microbiota imbalance and NAFLD has gradually been revealed.The change of gut microbiota can aggravate NAFLD inflammatory reaction,oxidative stress,lipid metabolism disorder and other pathological processes by causing abnormal metabolic products.The spleen of TCM is closely related to gut microbiota.The role of gut microbiota in food digestion and decomposition is consistent with the function of the spleen in regulating transportation.The micro balance of gut microbiota is an important factor in maintaining the normal function of spleen transportation.The idea of treating NAFLD by regulating gut microbiota is similar to that of treating the liver and spleen in TCM.Therefore,on the basis of clarifying the clinical efficacy of JGXZ in treating NAFLD,this study further conducted animal experiments,combined with 16S rRNA sequencing and non targeted metabolomics technology,to elucidate the molecular biological mechanism of JGXZ on NAFLD from the perspective of"microbiota metabolism",and provide scientific basis for the treatment of NAFLD with JGXZ.Research contents(1)Clinical observation of the effect of JGXZ on NAFLD(Liver Depression and Spleen Deficiency Type).80 patients with liver depression and spleen deficiency type NAFLD were randomly divided into a treatment group and a control group,with 40 cases in each group.The treatment lasted for 12 weeks.By comparing the liver/spleen CT ratio,liver enzymes,blood lipids,TCM syndrome scores,body weight,waist circumference,BMI,HOMA-IR index,inflammatory factors(IL-6,TNF-α)between the two groups before and after treatment,changes in liver fibrosis indicators(HA,LN,and PCⅢ)to evaluate efficacy;evaluate safety by monitoring general vital signs and blood routine,renal function,urine routine,fecal occult blood,electrocardiogram,etc.(2)The effect of JGXZ on NAFLD model rats.A NAFLD rat model was established using a high-fat diet(HFD).Sixty rats were randomly divided into a control group,a model group,a lovastatin group,a low-dose group of JGXZ,a medium dose group of JGXZ,and a high-dose group of JGXZ.The general condition,serum biochemistry(TC,TG,ALT,AST)and histopathology(H&E staining,Oil Red O staining)of rats in each group were observed;Western blot detection of IRS1,p-IRS1 β-Expression of actin protein;Draw blood glucose time curve using OGTT,calculate AUC,detect FINS and FBS using ELISA,calculate HOMA-IR,and study the effect of JGXZ on insulin resistance;ELISA and qPCR detection of TNF-α,IL-1βand IL-6 in serum and liver tissue to study the effect of JGXZ on inflammatory response;Detect the activity of SOD,GSH-Px and MDA levels in serum and liver tissue,and study the effect of JGXZ on oxidative stress.(3)Based on 16S rRNA sequencing technology,the effects of JGXZ on gut microbiota and intestinal mucosal permeability in NAFLD rats were studied.On the basis of the previous study,16s rRNA sequencing technology was used to analyze and study the effect of JGXZ on alpha diversity and beta diversity of gut microbiota in NAFLD model rats.In addition,the impact of JGXZ intervention on species related abundance at the portal level and genus level in NAFLD rats was analyzed,and PICRUSt analysis was used to predict the differential bacterial metabolic pathway;In addition,occludin and tight junction protein 1(ZO-1)were detected by immunohistochemical staining and Western blot to study the effect of JGXZ on intestinal permeability of NAFLD model rats.(4)The effect of JGXZ on blood metabolites in NAFLD rats based on non targeted metabolomics technology.Collect rat serum for non targeted metabolomics testing,use diversified analysis models(PCA,PLS-DA)to screen for differential metabolites,conduct metabolic pathway enrichment analysis on the MetaboAnalyst 5.0 website for differential metabolites,and analyze the metabolic pathways of differential metabolites based on the KEGG database and relevant literature.(5)Correlation analysis between efficacy indicators and non targeted metabonomics and gut microbiota.Using Spearman correlation analysis to study the correlation between efficacy indicators and 16S rRNA sequencing,and the correlation between non targeted metabolomics and 16S rRNA sequencing.Research results(1)JGXZ can improve the liver function and blood lipids of patients with liver stagnation and spleen deficiency type NAFLD,increase the liver/spleen CT ratio,alleviate TCM syndromes,and improve the therapeutic effect of treating NAFLD;control weight,reduce waist circumference,HOMA-IR index,and reduce insulin resistance;downregulate of inflammatory factors(IL-6,TNF-α)level,reduce inflammation;reduce the levels of HA,LN,and PCIII,and delay liver fibrosis;There were no adverse reactions during the entire course of the treatment.(2)JGXZ reduces the body weight and liver index of NAFLD model rats,while also reducing serum TC,TG,AST,and ALT levels;HE staining showed that JGXZ improved the liver histopathological manifestations of NAFLD rats,such as hepatocyte steatosis,inflammatory cell infiltration,etc.Oil Red O staining showed that JGXZ reduced lipid deposition;reduce the levels of OGTT-AUC,FINS,and HOMA-IR index,inhibit the levels of insulin resistance related proteins IRS1 and p-IRS1 in liver tissue,and alleviate insulin resistance;reduce IL-6,IL-1β and TNF-α level in serum and liver tissue,relative expression levels of Illb,116,and TNF-α genes in liver tissue;Improve the activity of SOD and GSH-Px in serum and liver tissue,reduce MDA levels,and alleviate oxidative stress.(3)JGXZ can down regulate Shannon and Simpson indices of gut microbiota in NAFLD model rats;PCA and phylogenetic tree analysis showed that JGXZ intervention affected the gut microbiota of NAFLD rats β Diversity;Differential microbiota analysis found that the JGXZ can reduce the ratio of Firmicutes/Bacteroidetes at the phylum level,increase the relative abundance of Lactobacillus and Blautia at the genus level,and reduce the relative abundance of Turicibacter,Collins ella,and Roseburia;Further PICRUSt analysis found that the effect of JGXZ on microbial communities is related to the Citrate cycle(TCA cycle),Tryptophan metabolism,amino acid metabolism,Linoleic acid metabolism,Taurine and Hydroxyrine metabolism pathways;In addition,JGXZ can upregulate the expression levels of ZO-1 and OCC proteins in intestinal tissue,improving intestinal mucosal permeability in NAFLD model rats.(4)The PCA analysis results showed that NAFLD rats have some significant changes in endogenous metabolite levels under the role of JGXZ;Based on the PLS-DA model to screen differential metabolites;JGXZ can affect the changes on the levels of metabolic products such as Glyceroylphosphorylethanolamine,Alpha linolenic acid,Phosphatidylethanolamine,Choline,etc.in the serum of NAFLD model rats;The improvement of serum metabolic dysfunction in NAFLD model rats by JGXZ may be related to the Linoleic acid metabolism,alpha linolenic acid metabolism,Tryptophan metabolism,and Glycerophospholipid metabolism pathways.(5)Using Spearman correlation analysis method,combined with 16S rRNA sequencing and non targeted metabonomics,it was found that JGXZ had a significant effect on Linoleic acid metabolism,α-linolenic acid metabolism,Tryptophan metabolism and Glycerophospholipid metabolism pathway,which is related to the abundance of Roseburia,Faecalibarum,Blautia,Collinella,Turicibacter and Lactobacillus in gut microbiota.Conclusion(1)JGXZ is safe and effective in treating NAFLD with liver depression and spleen deficiency;(2)JGXZ might improve NAFLD steatosis via regulating the imbalance of gut microbiota,Linoleic acid metabolism,α-Linolenic acid metabolism,Tryptophan metabolism and Glycerophospholipid metabolism.