LPS Promotes The Secretion Of Inflammatory Cytokines In Macrophages By Enhancing Adipophilin Expression Through The ERK1/2-PPAR? Pathway

[Objective] To clarify the role of adipophilin in LPS-induced inflammation, and explore the relationship between adipophilin and type M1 macrophages.[Methods] Firstly, we observed the perturbations of the adipophilin level in LPS-induced RAW264.7 macrophages, and detected protein level of adipophilin, i NOS and Arg-1 by Western Blot. Then, the secretion levels of pro-inflammatory cytokines, including TNF-?, MCP-1 as well as IL-6 were detected by ELISA. Afterwards, cells were pre-treated with PD98059(ERK1/2 inhibitor) and T0070907(PPAR? inhibitor) for 6 hours, respectively, and then treated with 100ng/ml LPS for 12 h. Western Blot were used to measure the protein levels of adipophilin, i NOS and PPAR?, and ELISA was used to detect the secretion levels of TNF-?, MCP-1 and IL-6. Finally, by means of retroviral vector transfection, we constructed the RAW264.7 cells which can stably silence the expression of adipophilin, then treated the cells with LPS. At last, the levels of some cytokines were measured.[Results] After RAW264.7 macrophages were treated with LPS of different concentrations for 12 hours, the protein level of adipophilin was increased dose-dependently, with the maximum effect at 100ng/ml. Besides, cells treated with LPS for various time showed a trend from rise to decline as to the protein level of adipophilin, TNF-?, MCP-1 and IL-6, and the highest levels were observed at 12 hours. In addition, cells treated with PD98059 presented lower levels of adipophilin, PPAR?, TNF-?, MCP-1 as well as IL-6. Meanwhile, treatment with T0070907 did not influence the activity of ERK1/2, whereas the levels of adipophilin, TNF-?, MCP-1 and IL-6 were significantly augmented. Moreover, after LPS was used to treat the adipophilin-silent cells, compared to non-silent group, the ERK1/2 activity and protein level of PPAR? were not influenced, whereas the levels of TNF-?, MCP-1 and IL-6 were significantly reduced. In addition, during the whole experiment, we detected that the trend of adipophilin levels were almost consistent with the levels of M1 macrophages markers i NOS and TNF-?, the effect of which did not be influenced by PD9805, indicating that adipophilin is closely related to M1 macrophages. [Conclusions] LPS can promote the protein level of adipophilin through ERK1/2-PPAR? pathway whereby it enhances the secretion levels of TNF-?, MCP-1 and IL-6.