| Type 2 diabetes mellitus(T2DM)is a complicated endocrine and metabolic disease characterized by hyperglycemia and dyslipidemia,including varying degrees of insulin resistance,pancreatic cell dysfunction,low-grade inflammation,intestinal microflora imbalance,and other endocrine disorders under the interaction of genetic and environmental factors.Currently,most patients have to require oral medications and injectable therapies for glycemic control.Oral drugs,especially traditional Chinese medicine(TCM),are bound to contact with intestinal microbiota,and their therapeutic effect is closely related to the role of gut bacteria.At present,most relevant studies have focused on the regulatory effect of TCM on intestinal flora,while there are few reports on the target intestinal bacteria that play a key role responded to the TCM treatment.Astragalus Membranaceus,Ganoderma lucidum,Inonotus obliquus and Momordica charantia L.have all been reported to regulate blood glucose.In previous studies,we have found that the combination of the four herbs into a Chinese medicine formula Huang-Qi-Ling-Hua-San(HQLHS)can help metformin and other drugs to improve the symptoms of T2 DM patients.However,its mechanism of action was not clear.In this study,a model of T2 DM induced by a high fat diet(HFD)and streptozotocin(STZ)was used.Through multiple rounds of animal experiments,including TCM formula feeding,fecal microbiota transplantation(FMT),and live bacteria feeding,which aims to explore the mechanism of HQLHS in improving hyperglycemia and hyperlipidemia and its potential target intestinal flora.This study includes the following three parts:1.Preliminary study on the mechanism of HQLHS improving T2 DM.In this part,the T2 DM model induced by high-fat diet(HFD)and streptozotocin(STZ)was used,taken metformin as a positive control,to explore the potential mechanism of HQLHS in treating T2 DM.Results showed that HQLHS reduced fasting blood glucose and fasting insulin levels,improved insulin resistance,increased the accumulation of liver glycogen,and down-regulated the expression of key gluconiogenesis enzymes(Pepck and G6pc);The levels of TG,T-CHO,LDL-C and HDL-C were regulated towards normal levels;The AST and ALT levels were lowered;The activities of SOD,CAT and GSH-PX were increased and the content of MDA was decreased.Besides,HQLHS decreased the expression of Tnf-α,Il-6,Il-1β and Mcp-1 in colon,thereby reducing intestinal inflammation.The results of 16 S r DNA sequencing of cecal contents indicated that HQLHS regulated the structure of intestinal flora,reducing the relative abundance of Blautia,Klebsiella,Ruminiclostridium＿5 and Family＿XIII＿AD3011＿group,while raising the abundance of Turicibacter,Alistipes,Bifidobacterium,Romboustia,Ruminococcaceae＿UCG-009 and uncultured＿bacterium＿f＿Christensenellaceae.Correlation analysis showed that the intestinal bacteria enriched by HQLHS were negatively correlated with abnormal blood glucose and lipid metabolism.It is preliminarily inferred that HQLHS can improve T2 DM by regulating intestinal flora,reducing inflammation and alleviating oxidative stress.2.The exploration of the role of beneficial bacteria enriched by HQLHS in improving T2 DM.In this part,FMT method was used to transplant the feces of mice after HQLHS intervention into T2 DM mice,which verified that HQLHS can improve blood glucose and lipids by regulating intestinal flora,especially by increasing beneficial bacteria.In addition to improving the physiological indexes related to blood glucose and blood lipid,q PCR results showed that FMT down-regulated the expression of the key enzymes of hepatic gluconeogenesis Pepck and G6 pc,and the expression of adipogenesis Ppar2.FMT up-regulated the expression of the rate-limiting enzyme Cyp7a1 in the liver while down-regulated the m RNA levels of Fxr and Fgf15 in ileum.FMT also reduced the expression of glucose transporters Sglt1 and Glut2 in ileum.The ELISA results showed that after FMT intervention,the content of LPS in serum was decreased,and the expression of LPS recognition receptor Tlr4,Nf-κb,Il-1β,Il-6,Tnf-α in liver and colon were significantly decreased.Intestinal flora analysis indicated that certain intestinal bacteria such as Bifidobacterium,Turicibacter,Alistipes,Romboutsia and Christensenella could be stably enriched by HQLHS.3.The study on the effect of Christensenella spp.on T2 DM and its potential mechanism.In this part,C.minuta DSM 22607(CM)and C.timonensis DSM 102800(CT)were administered to T2 DM mice,respectively The findings of this part are as follows:(1)Christensenella spp.reversed T2DM-related physiological indicators,including fasting blood glucose,fasting insulin,oral glucose tolerance,blood lipids and liver glycogen.CM and CT increased the level of GLP-1 in serum and the expression of its precursor gene Gcg in ileum,suggesting that Christensenella spp.could promote GLP-1 production.In addition,Christensenella spp.increased antioxidant levels in diabetic mice.CM notably increased the activities of SOD and GSH-PX,while CT significantly increased the activities of CAT,SOD and GSH-PX.Both groups reduced the content of MDA in serum.(2)Christensenella spp.improved the expression of genes related to glucose and lipid metabolism and transport in liver and intestine.Both CM and CT significantly reduced the expression of gluconeose-related genes G6 pc,Pepck and adipogenesis gene Pparγ2 in liver.Christensenella could alter the bile acid cycle by regulating the expression of Cyp7a1 in the liver and the expression of Fxr and Fgf15 in the small intestine.CM inhibited the expression of Glut2 and Sglt1 in the small intestine,thus inhibiting the absorption of glucose in the intestine.(3)Christensenella spp.regulated factors associated with inflammatory response induced by diabetes.Both CM and CT reversed the expression of related genes in LPS/TLR4 pathway,including the expression of Tlr4,Nf-κb,Tnf-α,Il-6 and Il-1β in liver and colon.In addition,Christensenella spp.up-regulated the intestinal barrier by increasing the expression of colonic tight junction proteins Zo-1 and Claudin-1.Christensenella spp.increased the expression of antimicrobial peptides Reg3γ and Defa in ileum of diabetic mice,thereby regulating intestinal homeostasis.(4)Christensenella spp.regulated the structure of intestinal flora.CM significantly increased the relative abundance of Bifidobactetium,Phascolarctobacterium and Collisella.The relative abundance of Muribaculum,Ruminiclostridium＿5 and Lachnospiraceae＿FCS020＿group decreased significantly.CT significantly increased the relative abundance of Bifidobactetium,Phascolarctobacterium and Turicibacter,and significantly decreased the relative abundance of Muribaculum.(5)Christensenella spp.treatment regulated liver and intestinal metabolism.The differential metabolites of liver in CM group were ethanol,fatty acid residue,mono-triglyceride,triglyceride,choline,glucose-1 phosphate,glucose,glycerophosphorine and canine uric acid.The differential metabolites of liver in CT group were ethanol,fatty acid residue,mono-triglyceride,triglyceride,glucose-1phosphate,glucose,taurine,leucine,isoleucine,valine,canine uric acid,tryptophan and tyrosine.The intestinal differential metabolites in CM group were butyric acid,choline,glycerophosphatidylcholine,horse uric acid,inositol,creatine,ethanol,glutamic acid,isoleucine,dog uric acid,phenylalanine,threonine,tyrosine and valine.The intestinal differential metabolites in CT group were acetic acid,butyric acid,horse uric acid,creatine,ethanol,dog uric acid and threonine.Metabolic pathway analysis showed that both CM and CT significantly affected starch and glucose metabolism pathways and tricarboxylic acid cycle in liver,CM significantly regulated cysteine and methionine metabolism pathways in ileum,and CT significantly regulated phosphatidylinositol metabolism pathway in ileum.In conclusion,the study used HFD and STZ-induced diabetic model to explore the mechanism of HQLHS in improving hyperglycemia based on gut microbiota.The experimental results have showed that the TCM formula plays a therapeutic role by enriching beneficial gut bacteria.Among them,Christensenella taxon was identified and screened as a significant biomarker in response to HQLHS intervention,and it was verified by live bacteria feeding that Christensenella was the target gut bacteria of HQLHS.At the same time,the potential mechanism of Christensenella spp.in improving blood glucose and blood lipid was preliminarily explored.It is reflected in promoting the secretion of GLP-1 in the small intestine,improving the antioxidant capacity of the body,inhibiting hepatic gluconeogenesis,inhibiting the absorption of glucose in the small intestine,enhancing the intestinal barrier,reducing the inflammatory response induced by LPS,and regulating liver and intestinal metabolites. |
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