Anti-inflammatory Mechanism Of Tea Polyphenols On 3T3-L1 Adipocytes Via AMPK/SIRT1 Signal Pathway

Inflammation is an important defensive process of the human body against the invasion of exogenous viruses,it is a kind of self protection form when the body is stimulated by external stimuli.Inflammation is normally beneficial for human body,but when exceeding body needs,it will be accompanied by some chronic diseases.According to reports in recent year,the production of inflammatory factors is a process of energy consumption.Adipocytes not only can store large amounts of energy,but also an active endocrine organ.The pathogenes of many chronic diseases is closely related to the number and volume of adipocytes.As hydroxyl compounds of potentially curative,TP can play a regulatory role on the induced disease,so it attracts more attention.In this paper,the anti-inflammatory mechanism of TP via AMPK/SIRT1 in 3T3-L1 adipocytes was studied.It will provide a new idea and drug target for TP on the adipocytes anti-flammatory and inflammation-related metabolic diseases.The results are as follows: 1.Effects of different concentrations of TP on gene expression of cytokinesThe inflammatory model of 3T3-L1 adipocytes was established by 10ng/mltumor necrosis factor-??TNF-??every plate in 37 ?,5%CO₂ cell incubator for 24h?except the control group?.The culture medium was removed and washed with PBS for one time,the cells were cultured with medium containing 0,10,25?50?100 ?g/ml TP for 48 h.Collecting cells to determination the expression level of m RNA for proinflammatory factors?anti-inflammatory factors and inflammatory enzyme.The results show that compared with the control group,TNF-? could significantly increase the expression levels of IL-6,IL-1?,IL-1?,IL-12,COX-2,i NOS,decrease the expression of IL-4 and IL-10.After adding differentconcentrations of TP,IL-6,IL-1?,IL-1?,IL-12,COX-2,i NOS gene expressionlevel decreased gradually,IL-4 and IL-10 gene expression level increased gradually.2.Effects of different concentrations of TP on AMPK/SIRT1 pathway and downstream target in adipocytesAfter the differentiation and maturation of 3T3-L1 adipocytes,we used Western blot techniques to determination the effect of different concentration TP on the expression of AMPK,SIRT1,NF-?B and Foxo3 a protein,comparison ofdifferent treatment groups AMPK?Thr172?,SIRT1?Ser27?,NF-?B-P65?Ser276?,Foxo3a?S253?changes of phosphorylation level.The results show that comparedwith the control group,TNF-? treatment group AMPK,SIRT1,NF-?B,Foxo3 a total protein had no significant difference,AMPK?Thr172?,SIRT1?Ser27?,NF-?B-P65?Ser276?,Foxo3a?S253?phosphorylation expression decreased obviously,after different concentrations of TP intervention phosphorylation expression gradually increase.3.Effects of activators and inhibitors on m RNA expression in 3T3-L1 adipocytesAfter adipocytes differentiated completely,the cells were cultured with medium containing 100?g/ml TP,100?g/ml TP+1m M AMPK activator?AICAR?,100?g/ml TP+20?M AMPK inhibitor?Compound C?,100?g/ml TP+ 40?M SIRT1 activator?Resveratrol?,100?g/ml TP +20m M SIRT1 inhibitor?Nicotinamide?,100?g/ml TP + 10?M NF-?B signal blocking agent?PDTC?for 48h?not add any drug as the blank control group?.Cells were collected to determine the expression level of m RNA for flammatory factors and inflammatory enzyme.RT-PCR results have demonstrated:compared with the control group,TNF-? could significantly increase the expression levels of IL-6,IL-1?,IL-1?,IL-12,COX-2,i NOS,decrease the expression of IL-4 and IL-10.TP can efficiently reduce IL-6,IL-1?,IL-1?,IL-12,COX-2,i NOS gene expression and increase IL-4 and IL-10 gene expression.Adding the medium contaning 100?g/ml TP+1m M AICAR and 100?g/ml TP +40?M Resveratrol can activate AMPK and SIRT1 activity,the effect is better than TP alone.Adding the medium containing 100?g/ml TP+20?M Compound C and 100?g/ml TP+20m M Nicotinamide didn't have a significant effect than TP alone.4.Effects of activators and inhibitors on AMPK/SIRT1 pathway and downstream targetAfter the differentiation and maturation of 3T3-L1 adipocytes,we used Western blot techniques to determine the expression of different treatment groups AMPK,SIRT1,NF-?B,Foxo3 a,p53,c-Jun protein,with comparison of different treatment groups AMPK?T hr172?,SIRT1?Ser27?,NF-?B-P65?Ser276?,Foxo3a?S253?,p53?ser15?,c-Jun?S73?chang es of phosphorylation level.The results show that compared with the control group,TNF-? treatment group AMPK,SIRT1,NF-?B,Foxo3 a,p53,c-Jun total protein had no signif icant difference,AMPK?Thr172?,SIRT1?Ser27?,NF-?B-P65?Ser276?,Foxo3a?S253?phosp horylation expression decreased obviously,p53?ser15?,c-Jun?S73?phosphorylation expressi on increase obviously.After 100?g/ml TP intervention,AMPK?Thr172?,SIRT1?Ser27?,NF-?B-P65?Ser276?,Foxo3a?S253?phosphorylation expression gradually increase,p53?ser15?,c-Jun?S73?phosphorylation expression gradually decrease.Adding the medium contaning 100?g/ml TP +1m M AICAR and + 100?g/ml TP + 40?M Resveratrol,the effect is better tha n TP alone.Adding the medium containing 100?g/ml TP+20?M Compound C and 100?g/ml TP+20m M Nicotinamidedidn't have a significant effect than TP alone.