The Role Of Testosterone In Inflammatory Cytokines Expression And Its Molecular Mechanisms In Mouse Macrophages RAW264.7

Partâ… The role of testosterone in TNF-αand MCP-1 expression in RAW264.7 macrophagesObjectives The pathogenesis of low-grade chronic inflammatory condition in Polycystic Ovary Syndrome(PCOS) is unclear.The objective of this study is to investigate the influence of testosterone on inflammatory cytokines production in a mouse macrophages cell line RAW264.7.Methods Cells were treated with normal physical level and high level testosterone for different times.The cellular expression(mRNA) of tumor necrosis factor-α(TNF-α) and monocyte chemoattractant protein-1(MCP-1) were measured by RT-PCR,and their protein expression in cell culture supernatant were measured by ELISA.The time- and dose-effect of testosterone were also investigated.Results 1.The effect of testosterone on TNF-αand MCP-1 mRNA expression in RAW264.7 macrophages.By incubated with physical level testosterone(10^-9M) and high level testosterone(10^-7M) for 6 hours,the expression of TNF-αand MCP-1 mRNA were significantly up-regulated(all p＜0.05).After incubated with testosterone for 24 hours,the expression of TNF-∝mRNA were also up-regulated(all p＜0.05) but were lower than those incubated for 6 hours(p＜0.01,p＜0.05).The expression of TNF-αmRNA were also up-regulated(all p＜0.05).2.The effect of testosterone on TNF-αand MCP-1 production in cell culture supematant:(1) the time effect of testosterone on TNF-αand MCP-1 production:Cells were incubated with physical level testosterone(10^-9M) and vehicle of testosterone,DMSO(controls) for 0～24 hours.Levels of TNF-a and MCP-1 were significantly increased after incubated with testosterone for 12 hours(all p＜0.05),though without apparent differences comparing to those of controls.Level of TNF-αincreased further and significantly higher than that of controls after treated with testosterone for 24 hours(1331.8±26.43 pg/ml v.s. 893.0±73.23 pg/ml,p＜0.05),so was MCP-1(212.72±8.09 pg/ml v.s.119.34±15.50 pg/ml,respectively,p＜0.05).(2) the dose effect of testosterone on TNF-αand MCP-1 production:Cells were incubated with DMSO(control),physical level(10^-9M) and high levels(10^-7M and 10^-9M) testosterone for 24 hours.The production of TNF-αand MCP-1 in cell culture supernatant were significantly increased after treatment with all levels of testosterone(all p＜0.05).TNF-αlevels were much higher in culture supematant of high-level testosterone(10^-7M and 10^-9M) treated cells than in physical-level testosterone-treated cells(p＜0.05 and p＜0.01).Level of MCP-1 in 10^-5M testosterone group was lower than that of 10^-7M group(p＜0.05).Level of TNF-αin 10^-9M testosterone group was also lower than that of 10_⁷M group,but without statistical significant(p＞0.05).Conclusions Testosterone can promote TNF-αand MCP-1 production in mouse macrophages RAW264.7 with both time- and dose-effect.Partâ…¡Key signaling molecules participated in testosterone promoting cytokine production in RAW264.7 macrophagesObjectives In the previous study,we found that testosterone can promote TNF-αand MCP-1 production in RAW264.7 macrophages.In this part,we investigated if it was through the activation of signaling molecules,NF-κB and ERK1/2,that testosterone promoted cytokine production in RAW264.7 macrophages.Methods In this part,we chosen a testosterone dose of 10^-7M as working concentration for it showed the most significant effect on cytokine expression in previous study.1.Cells were incubated with testosterone for different time and the expression of total NF-κB p65(t-p65),phospholated NF-κB p65 Ser²⁷⁶(p-p65),total ERK1/2(t-ERK1/2),and phospholated ERK1/2(p-ERK1/2) were detected by Western blotting.2.RAW264.7 macrophages were pretreated with or without a NF-κB inhibitor PDTC(100 uM,for 1 hour) or a MEK inhibitor PD98059(50 uM, for 2 hours) before testosterone incubation.The expression of t-p65,p-p65,t-ERK1/2, and t-ERK1/2 were detected by Western blotting.3.Cells were pretreated with or without PDTC and/or PD98059 before testosterone incubation for 6 or 24 hours(as PDTC and/or PD98059+T groups).Cells treated with PDTC and/or PD98059 only served as inhibitor controls(as PDTC and/or PD98059 groups),with testosterone only as testosterone control(T group),and with DMSO only as blank control.The expression of TNF-αand MCP-1 mRNA and levels in cell culture supernatant were detected by RT-PCR and ELISA respectively.Results 1.(1) With the extension of incubation time with testosterone,the expression of p-p65 of RAW264.7 macrophages was significantly increased.The expression of p-p65 was significantly increased after treated with testosterone for 30 min(p＜0.05), and reached to a peak level after 60 min treatment(2.49±0.40 times of 0min control, p＜0.01).It turned to decrease after 180 min treatment but was still significantly higher than 0min control after 360 min(all p＜0.05).(2) The expression of p-ERK1/2 of RAW264.7 macrophages was significantly increased with the extension of incubation time with testosterone.The peak level of p-ERK1/2 was reached after 30 min incubation(4.05±0.32 times of 0min control,p＜0.05).2.In comparison with that without PDTC pretreatment(incubation with testosterone for 1 hour only),the expression of p-p65 was significantly decreased in cells with 100 uM PDTC pretreatment for 1 hour(p＜0.01).In compassion with that without PD98059 pretreatment(incubation with testosterone for 30 min only),the expression of p-ERK1/2 was significantly decreased in cells with 50 uM PD98059 pretreatment for 2 hours(p＜0.05).3.(1) The cellular expression of TNF-αand MCP-1 mRNA in PDTC+T group were significantly lower than those in T group(all p＜0.05),but were higher than those in blank control(all p＜0.05).The production of TNF-αand MCP-1 in cell culture supernatant in PDTC+T group were significantly lower than those in T group and higher than those in blank control(all p＜0.05).(2) Both the mRNA expression and the levels in cell culture supernatant of TNF-αand MCP-lin PD98059+T group were significantly lower than those in T group and higher than those in blank control(all p＜0.05).(3) Both the mRNA expression and the levels in cell culture supernatant of TNF-αand MCP-1 in PDTC+PD98059+T group were significantly lower than those in T group(all p＜0.05),without significant difference in comparison with those in blank control(all p＞0.05).Conclusions Testosterone can promote the expression of TNF-αand MCP-1 by activating both NF-κB and ERK1/2 signal pathways in RAW264.7 macrophages.