Zhu Jie-ping, Su Chun-lin, Chen Min, Lin Jin-fang
Obstetrics and Gynecology Hospital, Fudan University, Shanghai, China.
Zhonghua Yi Xue Za Zhi. 2009 Sep 22;89(35):2500-3.
To investigate the role of androgen in TNF-alpha and MCP-1 expression in RAW264.7 macrophage and its molecular mechanism.
(1) RAW264.7 macrophage was treated with 10(-9) mol/L or 10(-7) mol/L testosterone (T) and then subjected to the measurement of: 1) the cellular expression of TNF-alpha and MCP-1 mRNA by RT-PCR; 2) the expression of TNF-alpha and MCP-1 in cell culture supernatant by ELISA; (2) The expression of phospho-NF-kappaB after treatment with T was measured by Western blot. (3) Cells were pre-incubated with 10(-4) mol/L PDTC (an inhibitor of NF-kappaB) for 1 hour, followed by T treatment. Expression of mRNA and supernatant levels of TNF-alpha and MCP-1 were measured by RT-PCR and ELISA.
(1) 1) After a 6 h treatment with 10(-9) mol/L or 10(-7) mol/L T, the expression of TNF-alpha mRNA increased by 1.78 and 1.87 folds, MCP-1 by 1.58 and 1.66 folds respectively (all P < 0.05). 2) Incubated with both concentration of T for 6 h showed no significant changes of supernatant levels of TNF-alpha and MCP-1. After a 24 h treatment, the levels of TNF-alpha and MCP-1 increased significantly (all P < 0.05) while more significant increase was found in 10(-7) mol/L T group (P < 0.05). (2) The expression of phospho-NF-kappaB (ser276) increased significantly after cells were treated with 10(-7) mol/L T for 30 min (P < 0.05) and peaked at 60 min. (3) With 1 h PDTC pre-incubation, T (10(-9) mol/L or 10(-7) mol/L) treatment for 6 h led to a lower mRNA expression and 24 h led to lower supernatant levels of TNF-alpha and MCP-1 than those without (P < 0.05). However, both cellular and supernatant expression of TNF-alpha and MCP-1 with PDTC pre-incubation were still higher than those of blank controls (all P < 0.05, except for TNF-alpha in 10(-9) mol/L T treatment).
Testosterone can increase TNF-alpha and MCP-1 expression in RAW264.7 macrophage in vitro. Activation of cellular NF-kappaB by testosterone may be one of underlying molecular mechanisms.
探讨雄激素对RAW264.7巨噬细胞中肿瘤坏死因子-α（TNF-α）和单核细胞趋化蛋白-1（MCP-1）表达的作用及其分子机制。
（1）用10⁻⁹mol/L或10⁻⁷mol/L睾酮（T）处理RAW264.7巨噬细胞，然后进行以下检测：1）通过逆转录聚合酶链反应（RT-PCR）检测TNF-α和MCP-1 mRNA的细胞表达；2）通过酶联免疫吸附测定（ELISA）检测细胞培养上清液中TNF-α和MCP-1的表达；（2）用蛋白质免疫印迹法检测T处理后磷酸化核因子κB（phospho-NF-κB）的表达；（3）细胞先用10⁻⁴mol/L PDTC（NF-κB抑制剂）预孵育1小时，然后进行T处理。通过RT-PCR和ELISA检测TNF-α和MCP-1的mRNA表达及上清液水平。
（1）1）用10⁻⁹mol/L或10⁻⁷mol/L T处理6小时后，TNF-α mRNA表达分别增加1.78倍和1.87倍，MCP-1分别增加1.58倍和1.66倍（均P<0.05）。2）用两种浓度的T孵育6小时，TNF-α和MCP-1上清液水平无显著变化。处理24小时后，TNF-α和MCP-1水平显著升高（均P<0.05），而10⁻⁷mol/L T组升高更显著（P<0.05）。（2）用10⁻⁷mol/L T处理细胞30分钟后，磷酸化NF-κB（ser276）表达显著增加（P<0.05），并在60分钟达到峰值。（3）预孵育1小时PDTC后，用T（10⁻⁹mol/L或10⁻⁷mol/L）处理6小时导致mRNA表达降低，处理24小时导致TNF-α和MCP-1上清液水平低于未处理组（P<0.05）。然而，预孵育PDTC后TNF-α和MCP-1的细胞和上清液表达仍高于空白对照组（均P<0.05，10⁻⁹mol/L T处理的TNF-α除外）。
睾酮可在体外增加RAW264.7巨噬细胞中TNF-α和MCP-1的表达。睾酮激活细胞NF-κB可能是其潜在的分子机制之一。
