Study On Anti-inflammatory Mechanism Of Tea Polyphenols In Macrophage-adipocyte Co-culture System

Objective: To establish a co-culture system of adipocytes and macrophages using transwell co-culture plates to analyze the anti-inflammatory effects of tea polyphenols and to explore the anti-inflammatory mechanism.And explore its anti-inflammatory mechanism from the AMPK signaling pathway Methods: 1、Processing and grouping: establish 4 treatment groups,there were the mature adipocyte group(N group),TNF-α treatment group(T group),macrophage-adipocyte co-culture group(R group),and LPS-induced macrophages-Adipocyte co-culture group(L group).The mature adipocyte group was induced by addition of 0.5 mmol/L IBMX,1μmol/L DEX,and 10 μg/mLInslulin to the precursor adipocytes,48 h later,the addition of10 μg/mL Inslulin in fetal bovine culture medium induces the differentiation of precursor adipocytes,after adipocyte differentiation and maturation,0 μg/mL,50 μg/mL and 100μg/mL tea polyphenols were added;in the macrophages-adipocyte co-culture group,mature and differentiated adipocytes and macrophages were co-cultured in Transwell co-culture plates for 24 hours and then 0 μg/mL,50 μg/mL and 100 μg/mL tea polyphenols were added at different concentrations;in the LPS-induced macrophage-co-culture group,mature and differentiated adipocytes were co-cultured with LPS-induced macrophages in Transwell co-culture plates for 24 h and then 0 μg/mL,50 μg/mL and 100 μg/mL tea polyphenols were added at different concentrations.2、Each group was added with different concentrations of tea polyphenols for 48 h,and then each well was added with 1000 μl of the lysate in the RNA extraction kit,the lysed cells are received in a previously labeled RNase-free EP tube,the gene expression levels of pro-inflammatory factors,anti-inflammatory factors,and inflammatory enzymes in adipocytes of each treatment group were detected by a fluorescent quantitative PCR method.3、After the cells were differentiated and matured,200 μl of PIPA lysate containing 2 μl of the protease/phosphatase inhibitor mixture was added,the phosphorylation levels of AMPK,Sirt1,NF-кB(p65),Foxo3 a,P53(THr172)and C-JUN(S73)related signal pathways in adipocytes of each group were detected by Western blotting.Result:1、The gene expression levels of IL-1α,IL-1β,IL-6,IL-12,COX-2,and iNOS were significantly increased in the T0,R0,and L0 groups compared with the N0 group(p< 0.05).After the addition of tea polyphenols with different concentrations,the expression levels of IL-1α,IL-1β,IL-6,IL-12,COX-2 and iNOS inflammatory enzymes were significantly decreased(p<0.05)and with the increase of the concentration,the expression level of the gene decreases more obviously.2、The N0 group had high expression levels of IL-4 and IL-10 genes,Comparison of T0 group,R0 group and L0 group with N0 group,the expression levels of IL-4 and IL-10 genes were decreased(p<0.05).The expression levels of IL-4 and IL-10 were significantly increased after the addition of different concentrations of tea polyphenols(p<0.05),and with the increase in concentration,the increase is more pronounced.3、The phosphorylation of each target was significantly inhibited in T0 group,R0 group and L0 group compared with N0 group;the phosphorylation of AMPK was enhanced in N50 and N100 compared with N0,and N100 is more effective than N50;compared with T0 group,the phosphorylation of AMPK,Sirt1,NF-κB(p65),Foxo3 a,P53and C-JUN was inhibited in T50 and T100,after adding tea polyphenols,phosphorylation of AMPK,Sirt1,NF-κB(p65),Foxo3 a,P53 and C-JUN increased significantly,and the effect of T100 is more obvious than that of T50;R50,R100 compared to R0,Phosphorylation of AMPK was higher in R50 and R100 than in R0,and the effect of R100 is more obvious than that of R50;L50,L100 compared to L0,Phosphorylation of AMPK was up-regulated in L50 and L100 groups,and L100 group was significantly up-regulated compared to L50 group.Only the phosphorylation of each target was inhibited,and there was no difference in total protein among the groups.CONCLUSION: Tea polyphenols have a certain resistance to inflammation,can play an anti-inflammatory role through the AMPK/Sirt1 pathway,and play a role by reducing gene expression of related pro-inflammatory factors and elevating gene expression ofanti-inflammatory factors,and with the concentration of tea polyphenols Increased anti-inflammatory effects;macrophages after LPS induced lipopolysaccharide,can better stimulate fat cells secrete pro-inflammatory factors.