Study On The Mechanism Of Mongolian Medicine Wuwei Qingzhuo San Against Hyperlipidemia Based On The Axis Of "Gut Microbiota-bile Acid-liver Metabolism"

Hyperlipidemia is a disorder of blood lipid metabolism,which refers to the abnormal increase of serum Total cholesterol(TC),total triglyceride,TG,low-density lipoprotein cholesterol(LDL-C)and other lipids.The adult blood lipid survey in China reveals that incidence of dyslipidemia is 40.4%,and it is increasing by years.Abnormal elevation of serum cholesterol will lead to pathological phenomena such as blood lipid deposition,vascular stricture or occlusion lesions,and arteriosclerosis,which is an important cause of cardiovascular and cerebrovascular diseases.At present,Statins are the main drugs for lowering blood lipids,but there are side effects such as liver and kidney toxicity and rhabdomyolysis.Therefore,it is necessary to develop new lipid-lowering drugs.Wuwei Qingzhuo San(WQS),a traditional Mongolian prescription,is included in the Pharmacopoeia of the People’s Republic of China(2015 edition,Part I).The Mongolian name is"Tonglaga-5".It is composed of Pomegranate,Safflower,Amomum cardamomum and Piper longum,and has the functions of promoting digestion and warming the stomach.Modern clinical studies have found that WQS can treat hyperlipidemia,coronary heart disease,angina pectoris and other cardiovascular diseases,especially for dyslipidemia caused by improper diet,but its specific mechanism is unknown.Therefore,this study established a hyperlipidemia mouse model to explore the effect of WQS intervention on mice liver metabolism,the influence of WQS on mice intestinal microenvironment,aiming to reveal the mechanism of WQS in cholesterol-lowering efficacy by regulating gut microbiota.The main research contents are divided into the following three parts:Part Ⅰ:Pharmacodynamics study of WQS in preventing and treating mice hyperlipidemia and its effect on liver metabolismObjective:To evaluate the preventive and therapeutic effect of WQS on hyperlipidemia mice model,and to observe the effect of WQS intervention on endogenous liver metabolism.Methods:40 C57BL/6J mice were randomly and evenly divided into 5 groups,with 8 mice in each group.Except the control group,which was given normal maintenance diet,the other groups were given high-fat diet for 4 weeks.The hyperlipidemia mice were regrouped into model group(Model,0.1%sodium carboxymethylcellulose solution,10g/kg/day),simvastatin group(ST,5mg/kg/day),low-dose Wuwei Qingzhuo Group(WQL,0.598g/kg/day),high-dose Wuwei Qingzhuo group(WQH,3.588g/kg),body weight and food intake were measured every week,after 6 weeks of administration,mice were anesthetized after the experiment,blood was collected to detect blood lipids;liver function and oxidation-reduction state were determined by Elisa;H&E staining was used to detect the main organs pathological morphology.In addition,UPLC-Q/TOF-MS metabolomics technology combined with bioinformatics analysis was used to observe the mouse liver metabolic profile after WQS administration.finally,the UPLC-MS/MS targeted metabolomics technology was used to explore the changes of serum bile acids.Results:1.The lipid-lowering efficacy of WQS was confirmed.The hyperlipidemic mice model was successfully established.Compared with the Model mice,the serum TC,TG and LDL-C contents of the mice after WQS administration were significantly reduced(P<0.01),the content of liver TC and TG were decreased(P<0.05);the accumulation of liver lipid droplets was reduced,the liver morphology was improved.The liver function index MDA decreased significantly(P<0.01),and no obvious pathological abnormalities were found in the heart,lung,kidney,and brain.2.The liver metabolism profile analysisBased on orthogonal partial least squares discriminant analysis(OPLS-DA)multivariate statistics,the results showed that there were obvious metabolic differences among the control group,model group and the WQS group.After standard screening of the importance projected variable(VIP)value and univariate analysis P<0.05,it was found that compared with the Model group,34 metabolites were down-regulated and 9 metabolites were up-regulated in the WQS group,mainly including bile acids,organic acids and their derivatives.Combined with multi-database analysis,bile acids were identified as the key differential metabolites,3.Changes in serum bile acid profile.The serum bile acids of mice were measured to obtain 14 kinds of bile acids after filling in the missing data in a right way,including seven primary bile acids and seven secondary bile acids.There are nine bile acids with more than half of the available data:cholic acid(CA),β-murocholic acid(β-MCA),taurine-β-murocholic acid(T-β-MCA),taurine Ursodeoxycholic acid(TUDCA),taurocholic acid(TCA),taurochenodeoxycholic acid(TCDCA),taurohyodeoxycholic acid(THDCA),allocholic acid(ACA)and deoxycholic acid(DCA),Among them,TUDCA was significantly increased(P<0.01)and ACA was decreased(P<0.05)in WQS group.Conclusion:WQS can effectively reduce serum and liver lipid levels in hyperlipidemia mice,improve liver function without toxic side effects;its lipid-lowering effect is related to hepatic bile acid metabolism.TUDCA play an important role in cholesterol-lowering effect.Part Ⅱ:The regulatory effect of WQS on gut microbiota in hyperlipidemic mice Objective:To explore the changes of gut microbiota after WQS administration in hyperlipidemic mice,and to analyze the correlation between gut microbiota and bile acids metabolismMethods:The cecal contents of mice in the Control,Model and WQS groups were collected,and the compositions of gut microbiota were revealed by Illumina NovaSeq high-throughput sequencing of bacterial 16S rDNA.Alpha diversity analysis,Beta diversity analysis,and LEfSe differences were performed combined with multivariate statistical methods and species annotation.The changes in the type and quantity of mice intestinal flora at different levels were obtained.According to the screening criteria of LDA>2,P<0.05 in the Lefse analysis results,the species with significant differences between Model mice and mice after WQS intervention were determined.Results:Compared with mice in Control group,the Alpha diversity and richness of gut microbiota in Model mice decreased;The ratio of Firmicutes/Bacteroidetes abundance increased while this anormal Alpha diversity was reversed after WQS treatment;Beta diversity analysis found that there were significant differences in the bacterial community structure among the three groups.After the Lefse differential species analysis,it was found that at the phylum level,the abundance of 13 bacteria,including Ruminococcus torques group、Lachnoclostridium and Anaerotruncus with disrupting intestinal barrier,pro-inflammatory,and LPS-producing opportunistic pathogens,were significantly increased in the high-fat mouse flora.While the colonies in WQS group were dominated by 21 bacteria,such as Bilophila and Roseburia,which were beneficial to correcting glucose and lipid metabolism disorders.At the phylum level,TUDCA was positively correlated with F(P<0.01)and negatively correlated with B(P<0.01).Conclusion:WQS could modulate the gut microbiota disturbance caused by high-fat diet,and affect endogenous TUDCA metabolism by upregulating the ratio of Firmicutes/Bacteroidetes abundance.Part Ⅲ:The mechanism of WQS in cholesterol lowering by regulating FXR-PPARα-Cyp7A1 pathwayObjective:To clarify the changes of liver proteins after WQS intervention,and to further explore the mechanism of WQS in the effect of lipid-lowering.Methods:The mice liver proteome expression differences were analyzed and determined by using label-free DIA proteomics methods.The differential proteins were annotated by GeneOntology(GO),the pathway enrichment was analysed by Kyoto Encyclopedia of Gene and Genomes(KEGG)and Reactome database.The protein network interaction was completed by PPI database.The genes and proteins expression in FXR-PPARα-Cyp7A1 pathway were determined by RT-PCR,IHC,IF and Western blot detection.Results:1.Hepatic proteomics profileAfter 6 weeks of WQS intervention,123 proteins were up-regulated,and 148 proteins were down-regulated in the liver of hyperlipidemic mice.After multi database annotation analysis,it was found that the body metabolism was mainly concentrated in the processes of carbon metabolism,glucose metabolism,cholesterol metabolism and protein transport.Ppar signing pathway was selected as the key regulatory pathway for lipid reduction.2.FXR-PPAR α-CYP7A1 pathwayCompared with mice in model group,the mRNA expression of CD36、PPARα and FXR in WQS group were significantly down-regulated(P<0.01),the expression of CYP7A1 increased(P<0.05)in IHC detection.WB experiment showed that after WQS intervention,the expression of FXR,PPARaand CYP7A1 protein increased(P<0.05,P<0.01)in mice liver.Conclusion:WQS affects cholesterol metabolism by regulating FXR-PPAR αCYP7A1 pathwaySummary:WQS has a good effect on the prevention and treatment of mice hyperlipidemia.Mechanisms are as follows:after the intervention of WQS,the structure of intestinal flora is changed and the rich proportion of Firmicutes/Bacteroidetes is increased,which upregulate the level of TUDCA with the activation of liver FXR,following the enhanced expression of PPAR α,at next,CYP7A1 enzyme in downstream is promoted to accelerate the transform of excess serum cholesterol to bile acids.