Study On The Effect Mechanism Of Compound Prescription On Regulating Bacterial Metabolites On Preventing And Treating Non-alcoholic Fatty Liver Disease By Regulating Bacteria-derived Metabolites

Object:Based on the preliminary work basis that Qushi Huayu prescription(QSHY)can effectively prevent and treat non-alcoholic fatty liver disease(NAFLD)and regulate the intestinal flora,based on the modern research on the interaction of the "intestine-liver" axis.To study whether QSHY prescription can prevent and treat NAFLD by regulating bacterial-derived metabolites,in order to reveal the effect mechanism of QSHY prescription to prevent and treat NAFLD,and provide a basis for further drug research and development of QSHY prescription.Methods:1.Metabolite profile changes of NAFLD mice with treatment of QSHY prescriptionC57 mice were randomly divided into control group and model group.The control group was given normal diet and water.High-fat and high-sugar diet was used to model the model group for 14 weeks to induce a NAFLD mouse model.They were randomly divided into model group and model + QSHY group,and QSHY prescription was given for 6 weeks,and the experiment ended in the 20 th week,test the following indicators:(1)Pharmacodynamic indicators,including body weight,glucose detection,serum ALT,TC content,liver TG and liver tissue pathological changes;(2)Detection of 16 s RNA diversity of fecal microorganisms;(3)Quantitative detection of fecal and serum metabolite profiles.2.Screening experiment of effector metabolites in the prevention and treatment of NAFLD mice by QSHY prescription through intestinal flora2.1 Intestinal Bacteria Depletion TestC57 mice were randomly divided into control group and model group.The control group was given normal diet and drinking water.The model adopts a high-fat and highsugar diet.After 12 weeks of establishing the model,they were randomly divided into model group,model+QSHY group,model+antibiotic group and model+antibiotic+QSHY group.The antibiotic group was given antibiotic depletion for2 weeks to induce the intestinal flora depletion NAFLD mouse model,after confirming the successful depletion of the flora,the QSHY prescription was used to intervene for 6weeks,and the experiment was ended at the 20 th week,test the following indicators:(1)Pharmacodynamic indicators,including body weight,glucose detection,serum ALT,TC content,liver TG and liver tissue pathological changes;(2)Detection of 16 s RNA diversity of fecal microorganisms;(3)Quantitative detection of fecal and serum metabolite profiles.2.2 Bacteria transplantation experimentC57 mice were randomly divided into control group,model group,model+QSHY group,model fecal microbiota transplantation recipient group,QSHY prescription fecal microbiota transplantation recipient group,the control group was given normal diet and water,and the rest of the groups were given high-fat and high-sugar diet.QSHY prescription group was given QSHY prescription gavage.Collect the feces of the QSHY prescription group every day for fecal microbiota transplantation of the QSHY prescription recipient group mice,and collect the model group feces every day for fecal microbiota transplantation the model recipient group mice.The experiment was ended at the 12 th week.Observe the following indicators:(1)Pharmacodynamic indicators include liver tissue pathological changes and liver TG content,serum ALT,AST,TC content,glucose tolerance and insulin tolerance test;(2)Detection of 16 s RNA diversity of fecal microorganisms;(3)Quantitative detection of fecal and serum metabolite profiles.3 Validation experiment of QSHY prescription on the treatment of NAFLD with bacterial metabolite IPAC57 mice were randomly divided into a control group and a model group.The control group was given a normal diet and water.The model group was induced with a high-fat and high-sugar diet for 14 weeks.They were randomly divided into a model group,model+IPA group and model+QSHY group.Intervene with IPA and QSHY for 6weeks,and end the experiment in the 20 th week.Observe the following indicators:(1)Pharmacodynamic indicators,including body weight,glucose,insulin,HOMA-IR index,serum ALT,AST,TC content,liver TG content and liver tissue pathological changes;(2)The expression of fat synthesis protein in the liver;(3)The expression of inflammatory factor protein in the liver.Results:1.Metabolite profile changes of NAFLD mice with treatment of QSHY prescriptionQSHY prescription improves liver damage and liver steatosis in NAFLD mice.QSHY corrects the disorder of the intestinal flora of NAFLD mice,inhibits the abundance of Firmicutes at the phylum level,promotes the abundance of Bacteroidota,and inhibits the abundance of bacteria such as Rikenella,Eubacterium＿oxidoreducens＿group,Negativibacillus,UCG-005 at the genus level,and promotes norank＿f＿＿Muribaculaceae,Parabacteroides The abundance of Akkermansia and the genera of these bacteria are also consistent with the previous research results of the research group,which proves the reproducibility of the results.QSHY can correct the disorder of metabolites in serum and feces.Differential metabolite pathways are mainly enriched in branched-chain amino acids and aromatic amino acids.2.Screening experiment of effector metabolites in the prevention and treatment of NAFLD mice by QSHY prescription through intestinal flora2.1 Intestinal bacteria depletion testIntestinal bacteria depletion weakened the therapeutic effect of QSHY prescription on NAFLD mice.Intestinal bacteria depletion weakened the promoting effect of QSHY formula on Muribaculaceae,Parabacteroides,Akkermansia bacteria,and weakened the inhibitory effect on the abundance of Rikenella,Eubacterium＿oxidoreducens＿group,Negativibacillus,UCG-005 and other bacteria.Depletion of intestinal bacteria weakened the inhibitory effect of QSHY formula on branched chain amino acids and aromatic amino acids.2.2 Bacteria transplantation experimentThe transplantation of QSHY prescription flora improved liver steatosis in the recipient mice.The transplantation of QSHY square flora promoted the expression of Muribaculaceae and Eubacterium＿xylanophilum＿group abundance in the recipient group of mice,and reduced the abundance of UCG-005 bacteria.QSHY Fang flora transplantation can up-regulate the abundance of Muribaculaceae in recipient mice and promote the catabolism of tryptophan to IPA.3.Validation experiment of QSHY prescription on the treatment of NAFLD with bacterial metabolite IPAIPA can improve liver damage and steatosis in NAFLD mice,and reduce the expression of liver lipid synthesis proteins and inflammatory factors.Part of the effect of QSHY prescription in preventing and treating NAFLD mice is through the bacterialderived metabolite IPA.Conclusion:1.The intestine is an important target of QSHY prescription for the treatment of NAFLD mice.2.QSHY prescription up-regulates the abundance of Muribculaceae and promotes the catabolism of tryptophan to IPA is one of the effective ways to prevent and treat NAFLD mice.