Study On The Effect And Mechanism Of Spleen-invigorating And Expectorating Method By Regulating Intestinal Flora And Bile Acid Metabolism To Improve Dyslipidemia

Purpose:1.By observing the effects of strengthening the spleen and expelling phlegm therapy on blood lipid levels,TCM syndrome scores and serum expression levels of metabolic regulator fibroblast growth factor 19 in dyslipidemia patients with pixu tanzhuo.Analyze the structural changes of gut microbiota in normal and high-fat populations,dyslipidemia patients before and after therapy,measure the content of the main active ingredients(Gynostemma Pentaphyllin A,Verbascum isoflavone glucoside,Astragaloside IV,Salvianolic acid B,Curcumin,Ferulic acid,lotus leaf base)in traditional Chinese medicine compound,Gynostemma saponin A,mullein isoflavone glucoside,astragaloside IV and other active ingredients in refined prescription.Master the clinical efficacy,investigate the pharmacological mechanismand and biological mechanism of strengthening the spleen and expelling phlegm method to lower blood lipids.2.By observing the effective of strengthening the spleen and expelling phlegm therapy on blood lipid level of SD rat hyperlipidemia model,changes in liver lipid deposition,expression levels of lipid metabolism pathway related proteins(Arnesoid X Receptor(FXR)?Fibroblast Growth Factor 15(FGF15)?Fibroblast Growthfactor Receptor 4(FGFR4)?Small Heterodimer Partner(SHP)?Cholesterol 7?-Hydroxylase(CYP7A1)),serum-bound bile acid content and effective of intestinal gut mircrobiota.Investigate the correlation between the changes of conjugated bile acid content and the structure of gut microbiota in rats.Further expound the effect and mechanism of strengthening the spleen and expelling phlegm therapy on regulating lipid metabolism according to liver-bile acid-enteric axis.Material and method:1.Clinical part: A total of 240 subjects with normal and dyslipidemia patient of pixu tanzhuo were enrolled in the Affiliated Hospital of Liaoning University of Traditional Chinese Medicine.According to the order of consultation,the patients were randomly divided into normal group,model group,traditional Chinese medicine compound group and refined prescription group,with 60 cases in each group.All groups were given health education,the non-dyslipidemia group and the dyslipidemia group are not subject to other interventions.The traditional Chinese medicine compound group was given Chinese medicine compound granules 100 ml warm water three times a day and the refined group was given refined prescription 100 ml warm water three times a day,the course of treatments was 2 months.The levels of serum cholesterol(TC),triglycerides(TG),low-density lipoprotein cholesterol(LDL-C),and high-density lipoprotein cholesterol(HDL-C)were measured by a fully automatic biochemistry analyzer,evaluation of Chinese Medicine Syndrome Efficacy of Patients before and after Treatment with Chinese Medicine Syndrome Scale,Enzyme-linked immunosorbent assay was used to determine the expression level of FGF19 in the serum of subjects,16 Sr RNA high-throughput sequencing technology was used to detect the structural changes of gut mircrobiota stool samples of subjects.The content of main active ingredients in granules of Chinese traditional medicine compound and refined prescription were measured by High Performance Liquid Chromatography.2.Basic part: SPF male SD rats was selected as experimental animals,a total of 24.The rats were first labeled according to their weight,and then random numbers were used to randomly divide the rats into blank groups(6)and high-fat feed groups(18).The modeling method adopts the currently recognized classic high-fat feed feeding modeling method,after evaluating the model successfully,the successful animal models are randomly divided into 3groups:Model group(6 animals),Chinese medicine compound group(6 animals),refined prescription group(6 animals).The blank group and the model group were orally administered with normal saline,the traditional Chinese medicine compound group was orally administered with traditional Chinese medicine granules and refined prescription group was orally administered with refined prescription granules for 4 weeks.At the end of the experiment,Oil Red O Staining was used to observe liver lipid deposition,and HE staining was used to observe liver morphological changes.Real-time quantitative PCR and Western blot were used to detect the expression levels of FXR,FGFR4,CYP7A1,SHP and FGF15 of each group of rats.UHPLC-MS/MS were used to quantitatively detect changes in bile acid GCA,GCDCA,TCA,TDCCA and T-?-MCA in each group of rats.16 Sr RNA high-throughput sequencing technology was used to detect the structural changes of gut microbiota in rat stool samples.Results:1.A total of 240 subjects were enrolled this time,according to the criteria for shedding and rejection,60 cases of non-dyslipidemia group,55 cases of control group,58 cases of traditional Chinese medicine compound group and refined prescription group were included.During the observation process,6 cases of adverse reactions occurred in the traditional Chinese medicine compound group,5 adverse reactions occurred in the refined prescription group.There were no abnormal changes in blood,urine routine,stool routine,aspartate aminotransferase,alanine aminotransferase,urea nitrogen,creatinine,and electrocardiogram before and after treatment in the two groups of patients.2.Dyslipidemia patients with pixu tanzhuo treated with traditional Chinese medicine and refined prescriptions had significantly improved blood lipid levels and TCM syndrome scores compared with those before treatment,with statistical differences(P<0.01).The curative effect of blood lipids and the total effective rate of TCM syndrome scores of the traditional Chinese medicine compound group and the refined prescription group were significantly better than those of the model group with statistical differences(P<0.01).The compound group had a slightly higher effective rate than the refined group,with no statistical difference(P>0.05).However,the effective rate of TCM syndrome points in the compound prescription group is higher than that in the refined prescription group,and the difference is statistically significant(p<0.05).3.The levels of Fgf19 in the serum of patients in the traditional Chinese medicine compound group and the refined prescription group decreased after treatment,and there were significant statistical differences(P<0.01).After treatment,the Fgf19 content in the peripheral blood of the model group was slightly reduced,the difference was not statistically significant(P>0.05).4.The gut microbiota diversity and species abundance of the high-fat population were significantly reduced compared with the normal population,with statistical differences(P<0.05).Beneficial bacteria are reduced,enterobacter overgrowth,and gut microbiota structure was destroyed.Abundance and diversity of the patients in traditional Chinese medicine compound group and refined prescription group had increased significantly between samples after administration and the species composition ratio at each classification level changed,and the uniformity and richness of the community composition are higher than before the medication.The abundance of microorganisms(Bacteroides,Clostridium,Enterococ cus)encoding bile salt hydrolase(BSH)decreased,with statistical difference(P<0.05).The intestinal probiotic bacteria Lactobacillus were increased in the Chinese medicine compound group after administration,with statistical difference(P < 0.05),the gut microbiota balance was restored.5.The traditional Chinese medicine compound mainly exerts lipid-lowering effects through active ingredients such as Gynostemma Pentaphyllin A,Verbascum isoflavone glucoside,Astragaloside IV,Salvianolic acid B,Curcumin,Ferulic acid and lotus leaf base.The content of the seven active ingredients varies greatly,with the content of Gynostemma pentaphyllum A being the highest and the content of curcumin the lowest.The refined prescription mainly used Gypenosides A,Mullein isoflavone glucoside,Astragaloside IV and other active ingredients to exert lipid-lowering effects.The content of the three active ingredients is higher than that of the traditional Chinese medicine compound,of which the content of Gynostemma pentaphyllum A is the highest and the content of Astragaloside A is the lowest.6.Compared with the model group,the blood lipid levels of the Chinese herbal compound and refined prescription groups were significantly lower than those of the model group(P<0.01).Hepatocyte steatosis ?lipid deposition and the hepatocyte morphology recovered were alleviated in both traditional Chinese medicine compound group and refined prescription group compared with the model groupand.7.The expressions of FXR,FGF15,FXR,FGFR4,and SHP related to the FXR signaling pathway in rats in the Chinese herbal compound and refined prescription groups were significantly lower than those in the model group,and the differences were statistically significant(P<0.01).The expression of CYP7A1 increased significantly,and the difference was statistically significant(P<0.01).8.Compared with the model group,the combined bile acids of the blank group,compound group and refined group(glycine chenodeoxycholic acid,glycocholic acid,taurochenodeoxycholic acid,tauro-?-ratcholic acid,Taurocholic acid)concentration increased,there was a statistical difference(P <0.05).9.The structure of gut microbiota in high-fat rats was changed,enterobacteria was overgrowth.Compared with the model group,inter-sample flora in the traditional Chinese medicine compound group and the refined prescription group have significant differences,and the proportion of species composition at each classification level changes.The proportion of species composition at each classification level changes,the intestinal probiotic bacteria Lactobacillus were increased,with statistical differences(P<0.05),and the uniformity and richness of the community composition are higher than the model group.Intestinal flora balance has been restored,The above results are consistent with the clinical changes in gut microbiota of patients.At the same time,the abundance of microorganisms related to bile salt hydrolase(BSH)(Bacteroides,Clostridium,Enterococcus,Bacillus)decreased,with statistical difference(P<0.05).The concentration of conjugated bile acids(Glycochenodeoxy-cholic acid,Glycocholic acid,Taurochenodeoxychol cholic acid,Tauro?-Muricholic acid,Taurocholic acid)increased,with statistical differences(P<0.05).Conclusion:1.The strengthening the spleen and expelling phlegm therapy can significantly reduce blood lipid levels and improve the syndrome of traditional Chinese medicine in patients with spleen deficiency and phlegm dyslipidemia.2.The traditional Chinese medicine compound and refined prescription mainly exerts lipid-lowering effects through active ingredients such as Salvianolic acid B,Curcumin,Ferulic acid,Stroganine,Gypenosides A,Verbascumin,Glucoside,and Astragaloside IV.3.The refined formula is equivalent to the traditional Chinese medicine compound in reducing blood lipid levels in clinic,and the traditional Chinese compound compound is superior to the refined compound in terms of the overall efficacy of traditional Chinese medicine syndrome and the promotion of probiotic growth in patients' gut microbiota.4.Invigorating the spleen and removing phlegm can regulate the blood lipid level of high-fat rats,improve the morphology of liver tissue cells,and reduce the generation of lipid droplets in liver cells.5.Spleen and expectorant method can restore the abundance and diversity of intestinal flora in patients with spleen deficiency and phlegm-turbid dyslipidemia,and adjust the uniformity and richness of community composition.Down-regulate the abundance of bacteria associated with bile salt hydrolase(BSH),increase the abundance of lipid-regulating intestinal probiotics Lactobacillus,and ameliorate the intestinal flora of patients.6.The spleen-removing and phlegm-reducing method can reduce the concentration of conjugated bile acid,regulate the expression of related genes and proteins of the FXR signaling pathway to inhibit the negative feedback effect of the FXR pathway,and promote the metabolic process of the conversion of cholesterol to bile acid.7.The strengthening the spleen and expelling phlegm therapy inhibits FXR signaling pathway to reduce lipids by regulating intestinal flora and bile acid metabolism.