Inulin And Lycium Barbarum Polysaccharides Ameliorate Inflammatory State Of Diabetes By Activating Gut Mucosal Intraepithelial γδ T Cells In Rats

Objective To study the effects and mechanisms of inulin(INU)and lycium barbarum polysaccharides(LBP)on improvement of type 2 diabetes mellitus(T2DM)via modulating gut microbiota and activating TLR2~+γδIELs in rats.Methods 1.Six-week-old male Sprague-Dawley(SD)rats were fed a high-fat diet for 4weeks and then intraperitoneally injected with 35 mg/kg streptozotocin(STZ)to induce diabetic rat model,then diabetic rats were randomly divided into 4 groups(10 rats/group):diabetes group(T2DM),diabetes+INU intervention group(T2DM+INU),diabetes+LBP intervention group(T2DM+LBP)and diabetes+INU+LBP intervention group(T2DM+INU+LBP).2.During the intervention,body weight(once a week)and fasting blood glucose(once every 2 weeks)were regularly monitored.The feces of rats in each group were collected after8 weeks of treatment.16S r RNA hypervariable sequencing was used to detect the diversity and abundance of gut microbiota among diverse groups.Quantification fecal short chain fatty acids(SCFAs)were analyzed by gas chromatography-mass spectrometry(GC-MS).3.After 8 weeks of treatment with INU and LBP,rats were sacrificed under anaesthesia;plasma/serum and tissue samples were collected to detect associated indications.(1)Plasma glycated hemoglobin(GHb)in each group was measured using the enzyme linked immunosorbent assay(ELISA);The levels of serum triglyceride(TG)and total cholesterol(TC)in each group were assessed using AU 400 automatic biochemical analyzer.(2)Rat plasma lipopolysaccharide(LPS)levels were detected by limulus amebocyte lysate test.(3)The plasma levels of interleukin(IL)-1β,IL-6,IL-17A,IL-10 and tumor necrosis factor(TNF)-αwere measured by rats inflammation panel.(3)Flow cytometry was used to detect the proportions ofγδintestinal intraepithelial lymphocytes(IELs),Toll like receptor(TLR)2~+γδIELs,and the contents of myeloid-derived suppressor cells(MDSCs)in peripheral blood and spleen of rats in each group.(4)The localization expressions of ZO-1,occludin and TLR2~+γδIELs of rats were observed by immunofluorescence.(5)Western blot was performed to examine the expression of tight junction proteins(ZO-1,occludin)and TLR2 in rat intestine.(6)The correlations among gut microbiota and SCFAs,TLR2~+γδIELs,ZO-1 and plasma inflammatory factors were analyzed.Results 1.After 8 weeks of INU and LBP interventions,the body weight,blood glucose,GHb,blood lipid,plasma LPS,IL-1β,IL-6,TNF-αand IL-17A levels of T2DM rats were significantly decreased,while the concentrations of plasma anti-inflammatory cytokine IL-10was significantly increased.The proportions ofγδIELs,TLR2~+γδIELs and MDSCs in all treatment groups were significantly higher than those in the untreated group.2.Sequencing analysis of intestinal flora showed that at the phylum level,compared with T2DM group,the relative abundances of Bacteroidetes and Cyanobacteria were increased in all treatment groups,while the relative abundances of Deferribacteres and Tenericutes were decreased.At the genus level,compared to T2DM group,the relative abundances of Bifidobacterium,Lactobacillus and Alistipes in three treated groups were increased,while the relative abundances of Desulfovibrio and Blautia were decreased.In addition,the GS-MS results indicated that compared to T2DM group,the contents of SCFAs(acetic acid,propionic acid,butyric acid and valeric acid)in fecal stool of rats in all intervention groups were notably increased.3.Immunofluorescence histochemistry results:The expression levels of ZO-1,occludin and TLR2~+γδIELs in intestinal tissues were increased in three treated groups,compared to T2DM group.Western blot results showed that compared with T2DM group,the protein expressions of ZO-1 and TLR2 in intestinal tract of rats were increased in all treated groups.4.Correlation analysis showed that the relative abundances of Bacteroidetes,Bifidobacterium and Lactobacillus were positively correlated with the levels of SCFAs,TLR2~+γδIELs,ZO-1,and negatively correlated with pro-inflammatory factors.Conclusion Dietary INU and LBP treatments ameliorated chronic inflammation of diabetes via modulating gut microbiota,SCFAs and enhancing gut barrier by activating gut mucosalγδT cells via TLR2,which provides experimental data for the prevention and treatment of clinical diabetes.