Study On The Molecular Mechanism Of Colonic GLP-1secretion And M2 Polarization Induced By Milk-derived Composite Probiotics Based On Intestinal Microbiota

Objective: There is a close relationship between the intestinal microbiota and type ?diabetes(T2D).The imbalance of intestinal microbiota can lead to an increase of the intestinal permeability,thus making the lipopolysaccharides and other toxic substances more easily enter the blood through the intestinal mucosal system,and then enter the insulin-sensitive organs,and make these organs in inflammation and insulin resistance,and accelerate the development of diabetes.Probiotics have the anti-diabetic effect by regulating intestinal microbiota.Previous studies have preliminarily confirmed that milk-derived composite probiotic fermented camel milk can effectively reduce the glucose and lipid in streptozotocin-induced diabetic rat,which is related to the upregulation of glucagon-like peptide-1(GLP-1)secretion.However,the anti-diabetic effect and the molecular mechanism of the milk-derived composite probiotics are still unclear.First,the study aimed to explore the anti-diabetic pharmacodynamic effect of milk-derived composite probiotics.Second,explore the effect of milk-derived composite probiotics on the intestinal microbiota in db/db mice.Third,explore the possible molecular mechanism of the milk-derived composite probiotics on the GLP-1 secretion and M2 polarization in db/db mice.Methods: Part one:The C57BL/Ks mice were the normal group and the db/db mice were the T2 D model group.The experiment was randomly divided into six groups: the normal group,the model group,the metformin group,the liraglutide group,the low dose of milk-derived composite probiotic group and the high dose of milk-derived composite probiotic group.All groups were operated in parallel for six weeks.The kits were used to determine blood glucose(FBG,OGTT,AUC,Hb A1 c,C peptide)and blood lipids(TG,TC,HDL-C,LDL-C)parameters,and the HE staining was used to observe the morphology of pancreas,liver,and adipose tissue,thus verifying the pharmacodynamic effect of milk-derived composite probiotics on diabetes mellitus.Part two: the kit was used to extract the total DNA in the feces,and the agarose gel electrophoresis and nucleic acid protein analyzer were respectively used to detect the total DNA purity and the concentration of total DNA,and the q RT-PCR was used to construct the standard curve of target bacteria and detect the concentration of target bacteria,thus illuminating the regulatory effect of milk-derived compound probiotics on intestinal microbiota.Part three(1): 1)The gas chromatography was detected the levels of acetic acid,propionic acid and butyric acid.The Elisa kit,q RT-PCR and western blot were respectively used to determine the plasma GLP-1 level,GLP-1R m RNA and GLP protein,and the q RT-PCR was used to determine the m RNA expression of pre-glucagon(GCG),pro-hormone converting enzyme 1/3(PC1/3),G protein-coupled receptor 43(GPR43),GPR41 and GLP-1R m RNA in the colon,thus verifying whether the milk-derived compound probiotics can promote GLP-1 secretion by upregulating the m RNA expression of GPR43,GPR41,GCG,PC1/3;2)Western blot and immunohistochemistry were used to determine the expression of inflammatory factors(IL-1?,TNF-?,IFN-?,NF-?B,p-NF-?B,ICAM-1,VCAM-1),and the Elisa kits were used to determine the serum anti-oxidant enzymes(SOD,CAT,GSH/GSSG)activity,and the immunohistochemistry was used to detect the insulin expression in pancreas,thus verifying whether the milk-derived composite probiotics can improve pancreas function through anti-inflammatory and antioxidant effects;3)Western blot and immunohistochemistry were used to determine expression of the apoptosis-related proteins(Bax,Bcl-2,Caspase-3,PI3K/AKT),thus verifying the milk-derived compound probiotics can inhibit pancreas tissue apoptosis by activating PI3K/AKT signal pathway.Part three(2): 1)The kits were used to determine the lipopolysaccharide(LPS)level in feces and serum,and the immunohistochemistry was used to detect the expression of inflammatory factors(ICAM-1,VCAM-1,NF-?B,p-NF-?B),and the q RT-PCR and immunohistochemistry were used to determine the expression of tight junction protein and mucin(Claudin-1,Occludin-1,ZO-1,Mucin-2),thus verifying whether the milk-derived composite probiotics can improve the function of intestinal barrier by reducing LPS production,inhibiting the expression of inflammatory factors and increasing the expression of tight junction protein and mucin;2)the q RT-PCR was used to determine the expression of anti-oxidant enzymes(CAT?SOD1?GR?GSH-Px and so on),thus verifying whether the milk-derived composite probiotics can inhibit the colon oxidative stress injuryby upregulating the anti-oxidant enzymes activity;3)The q RT-PCR and western blot were used to detect the expression of M1 polarizing factors(IL-1?,TNF-?,IFN-? and so on),M2 polarizing factors(IL-10,Arg-1,TGF-? and so on)and TLRs/My D88/NF-?B,thus verifying whether the milk-derived composite probiotics can induce colon M2 polarization through TLRs/My D88/NF-?B signal pathway.Results: Part one:1)The milk-derived composite probiotics significantly reduced blood glucose by reducing FBG and Hb A1 c level,increasing C-peptide level and improving OGTT;2)The milk-derived composite probiotics significantly reduced blood lipid by decreasing the levels of TG,TC and LDL-C;3)HE staining showed that the milk-derived composite probiotics significantly improved the morphology of pancreas,liver and adipose tissue in db/db mice.Part two: 1)The milk-derived composite probiotics significantly increased short-chain fatty acid bacteria in db/db mice,including Lactobacillus,Bifidobacterium,Roseburia,Clostridium leptum,Prevotella;2)The milk-derived composite probiotics significantly decreased the contents of Firmicutes/Bacteroidetes,Gram-negative bacteria and Escherichia coli;3)The milk-derived composite probiotics had no effect on total bacteria and Gram-positive bacteria and Enterococcus faecalis,Enterococcus faecium.Part three(1): 1)The milk-derived composite probiotics promoted GLP-1 secretion through upregulating activity of GPR43 and GPR41 by increasing propionic acid and butyric acid level,and increasing the m RNA expression of GCG,PC1/3;2)The milk-derived composite probiotics can improve pancreas function through anti-inflammatory and antioxidant effects;3)The milk-derived composite probiotics inhibited pancreas tissue apoptosis by activating PI3K/AKT signal pathway.Part three(2): 1)The milk-derived composite probiotics improved the function of intestinal barrier by reducing LPS production,inhibiting the expression of inflammatory factors and increasing the expression of tight junction protein and mucin;2)The milk-derived composite probiotics inhibited the colon oxidative stress injury by increasing the m RNA expression of the anti-oxidant enzymes(CAT,SOD1,GSH-Px)and decreasing m RNA expression of i NOS and COX-2;3)The milk-derived composite probiotics induced colon from M1 proinflammatory polarization to M2 anti-inflammatory polarization by inhibiting TLRs/My D88/NF-?B signal pathway.Conclusion: 1)The milk-derived composite probiotics has the anti-diabetic pharmacodynamic effect by reducing blood glucose and blood lipid and improving the morphology of pancreas,liver and adipose tissue;2)The milk-derived composite probiotics significantly improved pancreas function by increasing the levels of short-chainfatty acid-producing bacteria,propionic acid and butyric acid,promoting GLP-1 secretion through upregulating the m RNA expression of GPR43,GPR41,GCG,PC1/3,and inhibiting pancreas tissue apoptosis through activating PI3K/AKT signal pathway,thus increasing insulin secretion;3)The milk-derived composite probiotics significantly improved intestinal barrier function decreasing the level of Gram-negative bacteria and LPS,increasing the colonic anti-oxidant enzymes activity,inhibiting inflammatory factors expression,upregulating tight junction protein and mucin expression,and induced colon M2 polarization by inhibiting TLRs/My D88/NF-?B signal pathway.