Change In Gut Microbiota Is Correlated With Alterations In The Surface Molecule Expression Of Monocytes After Roux-en-Y Gastric Bypass Surgery In Obese Type 2 Diabetic Patients

Object:Type 2 diabetes mellitus（T₂DM）is a low-grade chronic inflammatory disease associated with elevated cytokines.To date,the underlying molecular mechanism is not well understood.Roux-en-Y gastric bypass（RYGB）is an effective surgical procedure for the treatment of T₂DM.The postoperative relief rate of diabetes is more than 80%,but the mechanism of relieving T₂DM is not clear.Except of reduction intake,weight loss,increased intestinal hormone secretion,regulation of vagal tension and other factors,It is growing concern gut microbiota,It is also play a role in RYGB postoperative.The aim of this study was to investigate the correlation between changes in gut microbiota and the expression of monocyte surface molecules in obese T₂DM patients who underwent Roux-en-Y gastric bypass（RYGB）surgery.To investigate whether changes in gut microbiota lead to the reversal of low-grade chronic inflammation by activation of monocytes,To clarify clearly the role of gut microbiota in the occurrence and development and reverse of obesity T₂DM.Methods:Twenty-four T₂DM patients were enrolled.7mL heparinized blood and fresh fecal samples were collected in the morning of the RYGB surgery and six months after the surgery.100μL peripheral blood was incubated for 30 minutes with 10μL FITC-labeled CD14 antibody,10μL PerCP-Cy5.5-labeled CD16 antibody,10μL PE-labeled CCR₂antibody,APC-labeled CX₃CR₁ antibody or 10μL isotype control antibody.the red blood cells were lysed by lysing solution.The average fluorescence intensity of CCR₂ and CX₃CR₁ on the surface of monocytes was detected by flow cytometry.100μL peripheral blood was incubated with 50 ng/m L Phorbol-12-Myristate-13-Acetate（PMA）,1μg/m L ionomycin,and 1μg/mL brefeldin for 4 h.Subsequently,the red blood cells were lysed by lysing solution.Then the cells were labeled with 10μL FITC mouse anti-human CD14antibody and 10μL Per CP-Cy5.5 mouse anti-human CD16 antibody.Next,the cells labeled intracellularly with 10μL PE mouse anti-human TNF-αantibodies and 10μL APC mouse anti-human IL-6 antibody,Flow cytometry was used to detect intracellular cytokines.200μL mononuclear cells isolated were taken.The cells were labeled with 10μL FITC-mouse anti-human CD14 mAb and 10μL PerCP-Cy5.5 mouse anti-human CD16antibody.Next,10μL PE-mouse anti-human TLR₄ and 10μL PE-mouse anti-human TLR₂were added respectively.The protein expression positive rate of TLR₄ and TLR₂ and MFI were detected by flow cytometry.peri-pheral blood mononuclear cells（PBMCs）were separated from the blood using lymphocyte separation medium.PBMCs were seeded and cultured in 24-well plates.100μL aliquots of suspension containing 5×10⁵ cells/mL were placed in the upper wells.100ng/mL MCP-1,100ng/mL IP-10,1ng/m L RANTES and a chemokine-mix were placed in the lower wells of the chemotaxis chamber.Monocytes in the lower chamber were quantified after 150 minutes at 37°C,the chemotaxis index was calculated.200 mg of fresh fecal samples was suspended in 1.6 m LBuffer ASL Mixed and centrifuged and filtered then frozened at-20°C.0.05 g of each standard strain DNA and total bacterial DNA were extracted from the frozen fecal samples according to the bacterial genomic DNA extraction kit instructions.The specific bacterial DNA was used as a template for specific PCR amplification to obtain the target 16SrRNA gene fragment,PCR products were purified and ligated with pMD18-T vector.The right fragment was inserted into the plasmid by sequencing,The DNA concentration was detected by a NanoDrop1000 spectrophotometer.The content of target bacterial DNA in fecal samples was detected by RT-PCR.Spearman correlation analysis was used to analyze the correlation between changes in gut microbiota and expression of surface molecules in monocytes.Results:To compare RYGB after 6 months and preoperative,Changes in the distribution of mononuclear cells:The percentage of NCM(CD14^dimCD16⁺)and IM（CD14⁺CD16⁺）in peripheral blood was significantly decreased（P<0.05）,and the percentage of CM（CD14⁺CD16^-）was significantly increased（P<0.05）.After RYGB the levels of TNF-αin IM（CD14⁺CD16⁺）cells were significantly decreased（P<0.05）);the levels of IL-6 in CM（CD14⁺CD16⁺）and IM（CD14⁺CD16⁺）cells were significantly decreased（P<0.05）).In addition,the expression of TLR₄ on the surface of CM（CD14+CD16-）and IM（CD14⁺CD16⁺）was significantly decreased（P<0.05）,peptidoglycan and lipopeptide receptor TLR₂ expression was not statistically significant（P>0.05）.Mononuclear cell migration capacity assessment:The expression of chemokine receptor CCR₂ in IM（CD14⁺CD16⁺）was significantly decreased（P<0.05）and the expression of chemokine receptor CX₃CR₁ in three mononuclear subpopulations were significantly decreased（P<0.05）The chemotactic factors of monocyte chemotactic factor MCP-1,RANTES and chemokine were decreased,and the ability of monocyte migration was significantly decreased after RYGB.Changes in gut microbiota after RYGB:the abundance of intestinal Bacteroidetes phyla was significantly increased after RYGB.The numbers of Bifidobacterium were a signifcant increase after RYGB.the numbers of Escherichia were reduced after RYGB.Correlation between intestinal flora and mononuclear cell subsets:The decrease of Bifidobacterium was negatively correlated with the decrease of NCM percentage（r=-0.477 P<0.05）,There was a negative correlation between the increase of Bifidobacterium and the decrease of CCR₂ expression in IM（r=-0.5817 P<0.05）and the decrease of CX₃CR₁ expression（r=-0.478 P<0.05）.The decrease in the number of Escherichia was positively correlated with the decrease of TNF-αexpression in IM mononuclear cells（r=0.442 P<0.05）.The decrease in the number of Escherichia was positively correlated with the decrease of LPS receptor TLR₄ on the IM surface（r=0.425 P<0.05）.Conclusions:RYGB signifcantly affected the phenotypes of monocytes in obese T₂DM,with a decrease in the inflammatory subpopulations of monocytes;In parallel with the alterations in the phenotypes,the function of monocyte migration and proinflammatory cytokines of the monocytes subsets were also signifcantly declined after RYGB in the obese T₂DM patients;A significant change of gut microbiota after RYGB was detected,mainly with an increase in Bifdobacterium and Bacteroidetes but a decrease in Escherichia;Spearman’s rank correlation coeffcient analysis revealed a signifcant correlation between the shift of gut microbiota and expression of surface molecules in monocytes.This study,for the first time,demonstrated a link between the changes in gut microbiota and alterations in both phenotypes and functions of monocytes after RYGB,which may decrease metabolic endotoxemia,This may contribute to the elimination of chronic inflammation of obese T₂DM.