Effects Of Xuebijing On Immunometabolism Of The Regulatory T Cell In Rat Induced By Lipopolysaccharide By AMPK Signaling Pathway

Objective:In this experiment,Treg cells,isolated from the spleen tissue of SD rats,were activated by lipopolysaccharide(LPS)to imitate the immune state in sepsis in vitro.After treatment with Xuebijing,the level of the function and the metabolic biomarkers of Treg cells were measured.The regulatory effect of Xuebijing on immune function and metabolism of Treg cells was clarified through observation of Teff proliferation and differentiation and helper T cell differentiation drift in co-culture.What’s more,adenylate activated protein kinase(AMPK)agonist was added to Treg cells which were pre-treated with Xuebijing in vitro.The contribution of AMPK in the process of how Xuebijing regulate the immune funtion of Treg cells in sepsis was eluciated by measurement of the expression of Treg cell metabolites and AMPK signaling pathway related proteins.Methods:1.CD4~+CD25~+Treg and CD4~+CD25~-Teff cells in normal SD rats were sorted by MACS,and the cell purity was measured by flow cytometry.In vitro,isolated Treg cells were divided into control group,anti-CD3/CD28 group,anti-CD3/CD28+LPS group,anti-CD3/CD28+LPS+XBJ group,and were cultured for 72h.2.CTLA-4/Foxp3 expression was measured using FCM,and the concentration of IL-10/TGF-βin Treg cells were measured using ELISA.3.The proliferation of Teff cells was detected by CFSE staining after Treg and Teff were co-cultured for 68h,and the level of IL-2,IL-4 and IFN-γin supernatant was determined by ELISA method.4.The level of CPT-1 and GLUT1 of Treg and the level of intracellular reactive oxygen(ROS)were measured using FCM.5.Isolated Treg cells were divided into control group,anti-CD3/CD28 group,anti-CD3/CD28+LPS group,anti-CD3/CD28+LPS+A-769662 group,anti-CD3/CD28+LPS+XBJ,and anti-CD3/CD28+LPS+XBJ+A-769662 group,then repeated the above experiments.The expression of p-AMPKαand AMPKαprotein in each group was detected by WB method.Results:1.In anti-CD3/CD28+LPS group,the expression of CTLA-4/Foxp3 and the concentration of IL-10/TGF-βwere the highest,the ability of supressing Teff proliferation was the most significant.The ratio of IFN-γ/IL-4 was the lowest in anti-CD3/CD28+LPS group.In anti-CD3/CD28+LPS+XBJ(20μl/ml),the expression of CTLA-4 and Foxp3 in Treg cells and the level of IL-10 and TGF-βin supernatant were significantly decreased(P<0.05).the divide%of CD4~+CD25~-Teff cells and the ratio of IFN-γ/IL-4 in anti-CD3/CD28+LPS+XBJ group were significantly higher than in CD3/CD28+LPS group(P<0.05).2.The expression of CPT-1 and GLUT1 in anti-CD3/CD28+LPS group were significantly higher than in other groups(P<0.05),no difference was observed beteween anti-CD3/CD28+LPS+XBJ group and anti-CD3/CD28 group.The content of ROS in Treg cells was 1014.333±49.136 in anti-CD3/CD28+LPS group,significantly higher than in other groups(P<0.05).The content of ROS in Treg cells was near normal in anti-CD3/CD28+LPS+XBJ(20μl/ml)group.3.Compared with anti-CD3/CD28 group,the expression of p-AMPKαin anti-CD3/CD28+LPS group was significantly increased(p<0.05)and the expression of AMPKαphosphorylation was significantly reduced in anti-CD3/CD28+LPS+XBJ(20μl/ml)group(P<0.05).Compared with anti-CD3/CD28+LPS+XBJ(20μl/ml)group,the level of AMPKαphosphorylation was significantly increased in anti-CD3/CD28+LPS+XBJ+A-769662 group(P<0.05).Conclusion:1.XBJ was effectively alleviate the immune suppression of Treg in sepsis in vitro.2.In vitro,the main metabolic pathway of Treg cells are FAO and glycolysis.XBJ can prevent mitochondrial from damage in sepsis through effectively blocking two metabolic pathway,and reducing the content of ROS in cells.3.XBJ improved the immune metabolism of regulatory T cell by restricting AMPKαphosphorylation.