| Objectives:Da-Chai-Hu Decoction(DCH)can be used to treat Nonalcoholic fatty liver disease(NAFLD)to relieve Shaoyang disorder and purge internal heat knot.However,its mechanism is still unclear.Recent studies have shown that gut microbiota can affect metabolism and participate in the process of NAFLD.In this study,16 S r RNA sequencing and untargeted metabolomics were used to explore the potential mechanism of DCH on NAFLD,to provide the experimental basis and reference for further clinical application.Methods:In this study,we first established NAFLD model rats fed with high-fat diet and treated them with DCH.The therapeutic effect of DCH on NAFLD model rats was observed by measuring the body weight,liver index,blood lipid,liver function,and liver histopathology of rats in each group.Blood glucose,area under the curve(AUC)of oral glucose tolerance test and fasting insulin were detected in each group.The effects of DCH on blood glucose and insulin resistance in NAFLD model rats were observed.The oxidative stress indexes were detected to observe the effect of DCH on the antioxidant capacity of NAFLD rats.Secondly,16 S r RNA sequencing was used to detect the composition of intestinal microflora in rats in each group.Alpha diversity analysis was used to calculate the microbial community diversity in each group of samples,and Beta diversity analysis was used to calculate the microbial community similarity between each group of samples.The species-related abundances at the level of gut microbiota and genera in rats of each group were analyzed.PICRUSt analysis was used to predict possible pathways associated with gut microbiota at the genus level.In addition,the effect of DCH on serum metabolites of NAFLD was studied based on serum untargeted metabolomics.The metabolites related to NAFLD in serum were identified by principal component analysis(PCA)and partial least squares discriminant analysis(PLSDA).Metabo Analyst website was used to analyze the metabolic pathway enrichment of differential metabolites,and KEGG was selected as a database to screen important differential metabolic pathways.Finally,Spearman correlation analysis was used to analyze the relationship between physiological data,serum untargeted metabolomics,and gut microbiota.Results:(1)Our results show that DCH has therapeutic effect on NAFLD model rats.Compared with the control group,the body weight of the model group increased significantly(P < 0.01).Compared with the model group,the body weight of DCH group decreased significantly(P <0.01).Compared with the control group,the liver index in the model group was significantly higher(P < 0.01).Compared with the model group,the liver index of DCH group decreased significantly(P < 0.05).Compared with the control group,the rats in the model group had dyslipidemia and liver function injury,which showed that the levels of TC,TG,and the activities of ALT and AST in serum were significantly higher than those in the control group(P < 0.01 respectively);Compared with NAFLD model rats,DCH significantly reduced the levels of TC,TG and the activities of ALT and AST in serum(P < 0.01,respectively).The results of HE staining and oil red O staining of liver tissue showed that there were obvious hepatocyte steatosis and lipid deposition in the liver of the model group compared with the control group.DCH can effectively improve the above pathological changes after treatment.DCH can also reduce the levels of blood glucose and insulin resistance in NAFLD model rats.Compared with the control group,the levels of AUC of OGTT,FINS and HOMA-IR in the model group were significantly higher(P < 0.01 respectively);Compared with the model group,AUC of OGTT,FINS and HOMA-IR levels in DCH group were significantly lower(P < 0.01,respectively).Concurrently,DCH can improve the antioxidant level of NAFLD model rats.Compared with the control group group,the activities of SOD and GSH-Px in NAFLD model rats decreased significantly,and the level of MDA increased(P < 0.01 respectively);Compared with the model group,the activity of SOD(P < 0.05)and GSH-Px(P < 0.01)in DCH group increased,and the level of MDA(P < 0.01)decreased.(2)16S r RNA sequencing showed that DCH could regulate the state of gut microbiota in NAFLD model rats.Alpha diversity and beta diversity analysis showed that there were significant differences in gut microbiota among control group,model group and DCH group.Further analysis of the relative abundance of flora showed that the ratio of Firmicutes to Bacteroidetes in the model group was higher than that in the control group(P < 0.01)at the phylum level.After DCH treatment,the ratio of Firmicutes to Bacteroidetes decreased compared with the model group(P < 0.05).At the genus level,the relative abundance of Romboutsia,Bacteroides,Lactobacillus,Akkermansia and Turicibacter decreased significantly in the model group(P < 0.01,P < 0.01,P < 0.01,P < 0.01 and P < 0.05,respectively),and the relative abundances of Lachnoclostridium,unidentified＿Enterobacteriaceae,Allobaculum and Enterococcus increased significantly(P < 0.05,P < 0.05,P < 0.01 and P < 0.01,respectively).Compared with the model group,DCH treatment increased the relative abundance of Romboutsia,Bacteroides,Lactobacillus,Akkermansia and Turicibacter(P < 0.01,P < 0.05,P< 0.01,P < 0.05 and P < 0.05,respectively),and decreased the relative abundance of Lachnoclostridium and unidentified＿Enterobacteriaceae(P < 0.01 and P < 0.05,respectively).In addition,PICRUSt analysis found that pentose phosphate,glycine/serine/threonine,arachidonic acid and glycerophospholipid metabolism pathways may be related to DCH improving the disorder of gut microbiota in NAFLD model rats.(3)Serum untargeted metabolomics found that DCH could improve the blood metabolites of NAFLD model rats.Multivariate analysis of PCA and PLS-DA showed that there were significant differences in serum metabolites among control group,model group and DCH group.Further analysis of the differential metabolites showed that compared with the control group,the levels of Phosphodylcholine(PC),Rumenic acid,Linoleic acid,Eicosapentaenoic acid,Lthreonine,Gluconic acid and Lacto-N-tetraose decreased,and the levels of L-proline,L-lysine,L-isoleucine,L-valine,L-arginine,L-leucine,Glycocholic acid,Uric acid,Creatinine,Stearic acid,Ursodeoxycholic acid,Phosphotidylethanolamine(PE),L-tryptophan,12(R)-HETE,5-HPETE and Glycine increased.Compared with the model group,the contents of Linoleic acid,PC,L-threonine and Rumenic acid in DCH group increased,while the contents of L-isoleucine,L-valine,L-arginine,L-leucine,Stearic acid,Indolearic acid,THTC,12(R)-HETE,5-HPETE,Glycine,Uric acid and PE decreased.Through the pathway enrichment analysis of these differential metabolites in serum,the results show that DCH can improve the disorder of serum metabolic function in NAFLD model rats,which may be related to linoleic acid metabolism,glycine/serine/threonine,glycerol phospholipid metabolism,tryptophan metabolism and arachidonic acid metabolic pathway.(4)Using Spearman correlation analysis method,combined with 16 S r RNA sequencing and untargeted metabolomics,it was found that the regulatory effect of DCH on arachidonic acid,glycine/serine/threonine and glycerol phospholipid metabolic pathway was related to the abundance of Romboutsia,Bacteroides,Lactobacillus,Akkermansia,Lachnoclostridium and Enterobacteriaceae in gut microbiota.Conclusions:(1)DCH has therapeutic effect on NAFLD.(2)DCH can regulate the relative abundance of Romboutsia,Bacteroides,Lactobacillus,Akkermansia and Turicibacter in the intestine of NAFLD model rats.These gut microbiota may play a role through the metabolism of pentose phosphate,glycine/serine/threonine,arachidonic acid and glycerol phospholipids.(3)DCH can affect the levels of Phosphatidylcholine,Rumenic acid,Linoleic acid,Eicosapentaenoic acid,L-threonine and other metabolites in the serum of NAFLD model rats.These metabolites are related to linoleic acid metabolism,glycine/serine/threonine metabolism,glycerol phospholipid metabolism,tryptophan metabolism and arachidonic acid metabolic pathway.(4)DCH regulates the metabolic pathways of serum arachidonic acid,glycine/serine/threonine and glycerol phospholipids,which may be related to the influence of Lactobacillus,Enterococcus and lachnoclostridium flora. |
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