Effects Of TNF-α On The Toll Like Receptor-4(TLR4) And Its Signaling Pathway Of Monocyte/Macrophage Cells With Or Without Human Adipocytes Co-cultured

Background and objective:Obesity has been identified as an epidemic disease in the world over the recent years. It has been associated with the activation of inflammation.The infiltration of fat by macrophages increases, which are an important source of inflammation in obese adipose tissue. The study was to investigate the effects of tumor necrosis factor-α(TNF-α) on the expression of toll like receptor-4(TLR4) and its signaling pathway of monocytes/macrophages with or without human adipocytes co-cultured in vitro, to explore the effection and molecular mechanism of TLR4 and its signaling pathway in the relationship between inflammation and obesity.Methods: (1)THP-1 cells were stimulated with TNF-αat different concentrations (0,0.1,1,10,20 ng/ml) for 12, 24 and 48 hours, the proliferation of which was observed by inverted phase contrast microscope and determined by Cell counting kit 8 (CCK8). (2)THP-1 cells were stimulated with TNF-αat different concentrations for 24 hours,the expression of TLR4 in THP-1 cells detected with Flow cytometry (FCM). Meanwhile, total RNA was extracted and the expression of TLR4 and Myd88 at mRNA levels were measured by Reverse transcription-polymerase chain reaction (RT-PCR). (3) Establish of coculture system for monocytic cell line THP–1 and human adipocytes through transwell, then treat with 10ng/ml TNF-α,JNK inhibitor SP600125,ERK inhibitor PD98059 and P38 inhibitor SB203580. Meanwhile,culture THP-1 and human adipocytes respectively. The expression of TLR4 was detected by FCM. Then, for every group, the resulting cell cultured media supernatant were assayed for accumulation of adiponetin, leptin and IL-6 with enzyme-linked immunosorb -ent assay (ELISA).Results:(1) TNF-αcould stimulate the proliferation of THP-1 cells in a time and dose dependent manner, and its maximum effect appeared at 10ng/ml for 24 hours. (2) The result of FCM showed that TNF-αcould promote the expression of TLR4 in THP-1 cell significantly, especially the concentraion of 10ng/ml. And it also could upregulated the expression of TLR4 and Myd88 at mRNA levels.(3) After co-culture of THP-1 and human adipocytes, the expression of TLR4 in THP-1 cells increased, which increased markedly when treated with 10ng/ml TNF-α. But it decreased after the JNK,ERK,P38 inhibitor added. (4) Compared with control, the levels of IL-6 increased after coculture, but there was no significant change of resistin. IL-6 and resistin increased when treated with TNF-α,while it decreased with JNK, P38 inhibitor added.Conclusions:TLR4 and its signaling pathway was activated when the THP-1 cells were treated with TNF-α. In the cocultue system, there might be cross-talk between THP–1 cells and adipocytes, which influenced the expression of TLR-4 and the relea -se of inflammation markers, while JNK, ERK, P38 inhibitor could decrease the expression of TLR4 and inflammation markers, they might become the new therapy targets for obesity.