The Role Of Testosterone In Inflammatory Cytokines Expression And Its Molecular Mechanisms In Mouse

PART ONE:The effects of androgen upon the production of inflammatory factors in the co-cultured system of mouse macrophages RAW264.7cells and3T3-L1adipocytes in vitro[Objectives]To build a model of co-cultured system of mouse macrophages RAW264.7and3T3-L1adipocytes in vitro, and to explore the effects of androgen upon the production and expression of inflammatory factors in th co-cultured system of mouse macrophages RAW264.7and3T3-L1adipocytes in vitro.[Method]Building a model of co-cultured system of mouse macrophages RAW264.7and3T3-L1adipocytes in vitro by a transwell. After that, we treat the cells with normal physical level and high level testosterone for different times. The celluar express ion(mRNA) of tumor necrosis factor-a(TNF-a)、Interleukin6(IL-6)and monocyte chemoattractant protein-1(MCP-1) were measured by Real Time-PCR, and their protein expression in cell culture supernatant were measure by ELISA.[Results]1. Building a model of co-cultured system of mouse macrophages RAW264.7and3T3-L1adipocytes in vitro susccessfully.2. The effect of testerone on IL-6、MCP-1、TNF-a in cell culture supermatant: The production of IL-6、MCP-1and the expression of IL-6mRNA and MCP-1mRNA were signicantly increased after incubated with testosterone compared to co-cultured cells without testosterone (P<0.05). The level in co-cultred cells treated with testosterone were significantly increased after24hours compared with12hours (P<0.05).And there was no statistically significant difference in cells treated with testosterone after24hours and48hours(P>0.05). What’s more, cells treated with10-5mol/L testosterone produce much more IL-6、MCP-1compared with10-7mol/L and10-9mol/L(P<0.05). The level is much more higher than mouse RAW264.7cultured individually and3T3-L1adipocytes cultured individually (P<0.05) after treatde with10-5mol/L testerone24hours. The production of TNF-a and the expression of TNF-a mRNA were signicantly increased after incubated with testosterone compared to co-cultured cells without testosterone (P<0.05). The level of TNF-a protein and the expression of TNF-a mRNA in co-cultred cells treated with testosterone were significantly increased after24hours compared with12hours (P<0.05).What’s more, cells treated with10mol/L testosterone produce much more TNF-a compared with10-5mol/L and10-9mol/L (P<0.05). And cells treated with10-5mol/L testosterone produce much more TNF-a compared with10-9mol/L(P<0.05). The level of TNF-a and the TNF-a mRNA is much more higher than cells cultured individually(P<0.05).[Conelution]Testosterone can promote IL-6、MCP-1、TNF-a and the expression of mRNA in co-cultured system of mouse macrophages RAW264.7and3T3-L1adipocytes in vitro with both time and the effect. PART TWO:Key signaling molecules participated in testosterone promoting cytokine production in co-cultured system of mouse macrophages RAW264.7and3T3-L1adipocytes[Objectives]In the previous study,we found that testosterone can promote IL-6、 MCP-1、TNF-a in co-cultured system of mouse macrophages RAW264.7and3T3-L1adipocytes. In this part, we investigated if it was through the activation of signaling molecules NF-κB, that testosterone promote cytokine production in co-culturod system of mouse macrophages RAW264.7and3T3-L1adipocytes.[Method]In this part, we chosen a testerone dose of10-mol/L as working concentration for it showed the most significant effect on cytokine expression in previous study. l.NF-κB were detected by westerning blotting.2. cells were pretreated with or without a NF-κB inhibitor PDTC(100um,1hour) before testoterone incubation, the expression of TNF-a、 IL-6and MCP-1were detected by ELISA.[Result]The expression of NF-κB p-p65was signicantly increased after treated with testosterone for24hours (P<0.05) than those without testosterone.The level of the TNF-a、IL-6and MCP-1in PDTC+T group were signicantly lower than those in T group but higher than those in blank control group (P<0.05). And the level of the TNF-a、IL-6and MCP-1in PDTC group had no signicance difference than those in blank control group(P>0.05).[Conclusions]Testosterone can promote the expression of TNF-a、IL-6and MCP-lby activating NF-κB signal pathways in co-cultured system of mouse macrophages RAW264.7and3T3-L1adipocytes.