| Objective:Observe the effects of Erchen Decoction on the phenotypes,especially lipid metabolism disorders,and intestinal flora and its metabolite short chain fatty acids(SCFAs)in ZDF(Zucker diabetic fatty)rats,a spontaneous obesity model,determine the potential correlation between host phenotypes and bacterial targets,in order to further explore the mechanism of Erchen Decoction in improving obesity and related metabolic diseases via intestinal flora.Methods:The chemical compounds of Erchen Decoction were detected.ZDF rats were taken as the research object and were randomly divided into model(Z),Erchen Decoction low(EC-L),medium(EC-M),and high dose(EC-H)treatment groups,with Zucker lean rats as the control(L),a total of 5 groups.The intervention with Erchen Decoction in rats ranged from 5 to 9 weeks of age.The specific researches were as follows:1.To evaluate the effect of Erchen Decoction on the obesity phenotypes of ZDF rats,the body weight,abdominal circumference,food intake and fecal water content of five groups of rats were detected weekly from 5 weeks to 9 weeks old.Insulin tolerance test(ITT)was performed at 9 weeks old and fasting serum insulin(FSI)and homeostasis model assessment-insulin resistance(HOMA-IR)were determined at the end of the experiment to evaluate the level of insulin sensitivity.The blood lipids including total cholesterol(TC),high-density lipoprotein cholesterol(HDL-C),low-density lipoprotein cholesterol(LDL-C),triglycerides(TG)and fat body ratio,including perirenal,epididymal and total fat body ratio were detected to observe the level of lipid metabolism in rats.The levels of alanine aminotransferase(ALT),aspartate aminotransferase(AST),blood urea nitrogen(BUN),and creatinine(Cr)were determined to observe the liver and kidney functions of rats.2.To evaluate the effect of Erchen Decoction on lipolysis in WAT in ZDF rats,hematoxylin-eosin(HE)staining and western blotting of epididymal adipose tissue of control,model,and optimal treatment(EC-M)groups were used to observe the effect of Erchen decoction on the cell morphology,insulin receptor substrates 1(IRS1)/protein kinase B(AKT)/protein kinase A(PKA)/hormone sensitive lipase(HSL)signaling pathway,and the expression of adipose triglyceride lipase(ATGL)in white adipose tissue(WAT),and it was verified by testing serum free fatty acids(FFA).3.To evaluate the effect of Erchen Decoction on the intestinal flora of ZDF rats and its metabolite SCFAs,the feces of control,model and optimal treatment(EC-M)groups of rats were collected at the end of adaptive feeding(5 weeks old)and before the end of the experiment(9 weeks old)for intestinal flora 16S rRNA gene sequencing,and the intestinal flora metabolites SCFAs in colon and cecum content of three groups were detected at the end of the experiment.The regulation of Erchen Decoction on the intestinal flora and its metabolite SCFAs were observed,the bacterial targets were detected and correlated with the host phenotypes.Results:1.The effects of Erchen Decoction on phenotypes of ZDF rats:The rats in Z group showed obesity.The weekly body weight,abdominal circumference,food intake and fecal water content were significantly higher than those of L.At 9 weeks of age,there were obvious insulin resistance and lipid metabolism disorders in Z group.After the treatment of Erchen Decoction,body weight,abdominal circumference,insulin resistance and lipid metabolism disorders of ZDF rats were significantly improved and no side effects on liver and kidney function.Interestingly,Erchen Decoction also reduced the fecal water content of ZDF rats.2.The effects of Erchen Decoction on lipolysis in WAT of ZDF rats:The adipocytes numbers decreased,area increased in the same field of view,and p-IRS1/IRS1,p-PKA/PKA,and pHSL/HSL were increased,p-AKT/AKT and ATGL was decreased in WAT of Z groups.The serum FFA concentration increased.The treatment of Erchen Decoction improved the morphology of adipocytes and reduced serum FFA.The improvement of WAT lipolysis function might be related to the regulation of the activity of the IRS 1/AKT/PKA/HSL signaling pathway.3.The effects of Erchen Decoction on the intestinal flora and its metabolite SCFAs of ZDF rats:In the structure of the intestinal flora,there was no difference in α diversity among groups at 5 weeks of age,while β diversity had significant separation in Z group than that.of L.At 9 weeks of age,α diversity of Z group was significantly higher than that of Z,and the separation of βdiversity was more obvious.After the treatment of Erchen Decoction,α diversity of ZDF rats was reduced,the β diversity was changed and could be significantly distinguished.In the composition of intestinal flora,the development of obesity caused changes in the composition of flora,and the regulation of Erchen Decoction made the composition of the flora of ZDF rats more similar to those of the L group.The results of differential flora analysis based on genus level showed that Prevotella,Blautia,Ruminococcus,Holdemania,Coprobacillus,and Akkermansia were the biomarkers for Erchen Decoction intervention.In the function of the intestinal flora,three groups of rats were separated at 9 weeks of age.Erchen Decoction made the function of the intestinal flora of ZDF rats and the L groups more similar in the PCI dimension.There were 8 significantly different signaling pathways among groups.Among them,the abundance of the insulin signaling pathway(ko04910)was significantly positively correlated with Prevotella,Blautia,Ruminococcus,Holdemania and Coprobacillus.Compared with L groups,the content of SCFAs in colon and cecum of ZDF rats changed.Propionic,butyric and isovaleric acid were significantly increased,isobutyric and 3hydroxyvaleric acid were significantly decreased.The treatment of Erchen Decoction significantly reduced the content of propionic acid and there was a tendency to adverse butyric,isovaleric,isobutyric and 3-hydroxyvaleric acid to normal levels.Correlation analysis showed that Prevotella,Blautia,Holdemania,and propionic acid were significantly positively correlated with host phenotypes,especially lipid metabolism disorders,which might be the target of Erchen Decoction in improving obesity.Conclusion:Erchen Decoction can significantly improve obesity,insulin resistance and lipid metabolism disorders,improve adipocytes morphology and reduce the release of FFA from excessive lipolysis of WAT to circulation by intervening the activity of IRS1/AKT/PKA/HSL signaling pathway.In addition,Erchen Decoction has an impact on the composition of obesity-related flora and the content of its metabolite SCFAs,especially the reduction of the relative abundance of Prevotella,Blautia,Holdemania and the content of the metabolite propionic acid,which are closely related to host phenotypes,especially the improvement of insulin signaling and lipolysis in WAT.This study provides new insights into the role of Erchen Decoction in regulating lipid metabolism via intestinal flora,and suggests the important role of insulin signal transduction in the regulation of lipid metabolism,which will help to further clarify the mechanism of Erchen Decoction in improving obesity. |
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