Effects Of TNF-α On The Proliferantion And Differentation Of Adipocyte And Toll Like Receptor-4 And Its Signaling Pathway

Objective: Recent studies have suggested that obesity is a state of chronic low-grade inflammation, obesity tissue could secrete kinds of inflammatory mediators. Our research is to explore the effect of human tumor necrosis factor-α(TNF-α) on the proliferation of human preadipocytes and differentiation of adipocytes,to investigate the effects of tumor necrosis factor-α(TNF-α) on the expression of toll like receptor-4(TLR4) and its signal pathway and the inflammation markers adiponectin and leptin in its downstream signaling pathway of human adipocytes with or without monocytes/macrophages co-cultured in vitro.Methods:(1)we obtained the human preadipocytes by enzyme digestion from abdominal greater omentum of surgery sufferers,.(2)detected The proliferation was detected with CCK-8 after treatment with TNF-αat the concentrations of 0, 0.1, 1, 10, 100ng/ml for 12,24,48 hours.(3)We induced the differentiation by TNF-αat different concentrations on the 8th day, detected the level of differentiation with oil red O. (4)Establish of coculture system for human adipocytes and monocytic cell line THP–1 through transwell,The expressions of TLR4 were detected by FCM for every group.These groups were human adipocytes only,co-culture for human adipocytes with THP–1, the other 4 co-cultures were treated with 10ng/ml TNF-αfor 24 hours,the inhibitors of Mitogen-activated protein kinases (MAPK):SP600125, PD98059 and SB203580 for 1 hour ,then TNF-αfor 24 hours.Meanwhile the resulting cell cultured media supernatant were assayed for accumulation of adiponetin and leptin with enzyme-linked immunosorbent assay (ELISA). Results:(1) TNF-αcan significantly promoted the proliferation of preadipocyte(P<0.05), especially the group treated with the concertration of 10 ng/mL TNF-αfor 24 hours.(2) The differentiation of human adipocytes was reduced by TNF-α(P<0.05), significantly at 100ng/ml. (3)After co-culture of human adipocytes and THP-1, the expression of TLR4 in human adipocytes increased(P<0.05), which increased markedly when treated with 10ng/ml TNF-α. But it decreased after treated with SP600125,PD98059 and SB203580 in the co-culteres(P<0.05).(4) Compared with the group of adipocytes, the levels of adiponectin decreased compared with the group of co-culture(P<0.05),decreased most when treated with TNF-α,and Compared with the group of the co-culture treated with TNF-αonly it increased in the co-cultures treated with SP600125,PD98059 and SB203580(P<0.05). But there was no significant change in leptin.Conclusions:TNF-αcan promoted the proliferation of preadipocyte, but inhibit the differentiation of adipocytes . TLR4 and JNK,ERK of its signaling pathway were activated when the human adipocytes were treated with TNF-α. In the cocultue system there might be cross-talk between adipocytes and THP–1 cells, which influenced the release of inflammation markers.