类固醇受体辅活化子-3蛋白在巨噬细胞炎症反应中的作用研究

Effect of steroid receptor coactivator - 3 on inflammatory response in peritoneal macrophages

目的探讨类固醇受体辅活化子（SRC）-3蛋白在脂多糖（LPS）诱导腹腔巨噬细胞（peritoneal macrophages，PMφ）炎症反应中的作用。方法健康SPF级野生型（SRC-3＋/＋）小鼠、SRC-3基因敲除（SRC-3-/-）小鼠各5只，分别分离和原代培养PMφ，并分为SRC-3＋/＋组和SRC-3-/-组。收集并调整细胞浓度为1×10^6／mL，接种于6孔板，1mL／μL，给予10μg/mL LPS刺激，分别在刺激前（N）、刺激后4h和12h收集培养上清和PMφ。测定培养上清中TNF-α、IL-1β、IL-6、高迁移率族蛋白B1（HMGB1）和NO的浓度，PMφ的p65、c—Jun和iNOS的表达及NF—κB和AP-1的DNA结合活性。结果PMO经10μg／mLLPS刺激4h和12h后，培养上清液中分泌的TNF-α、IL-1β、IL-6和NO均显著上升，但在相应时间点SRC-3-/-组显著低于SRC-3＋/＋组，当LPS刺激4h后，两组PMO培养上清液中HMGBl的表达水平与刺激前比较差异无统计学意义（P〉0．05），刺激12h后SRC-3＋/＋组有所升高，而SRC-3-/-组与刺激前比较差异无统计学意义（P〉0．05）；LPS刺激4h和12h后PMφ的p65和C—Jun表达显著增加，在相应时间点SRC-3-/-组的p65显著高于SRC-3＋/＋组，而SRC-3-/-组C—Jun却显著低于SRC-3＋/＋组；LPS刺激4h和12h后，SRC-3＋/＋组的iNOS表达逐渐增加，而SRC-3-/-组未见变化；LPS刺激4h和12h后NF—κB和AP-1的DNA结合活性均显著增高，在相应时间点SRC-3-/-组均显著低于SRC-3＋/＋组。结论SRC-3可能通过调节iNOS、NF—κB和AP-1的表达及活性来调控PMφ的炎症反应。

Objective To investigate the effect of steroid receptor coactivator （SRC） -3 on inflammatory response induced by lipopolysaccharide （LPS） in peritoneal macrophages （PMφ）. Methods PMφ were isolated and primarily cultured from healthy SPF - grade wild type （ SRC - 3 ＋/ ＋ ） mice and SRC -3 knockout （SRC -3 -/- ） mice, and were divided into SRC -3 ＋/＋ group and SRC - 3 -/- group respectively. After collected and adjusted the cell concentration into 1 ×106/mL, PMφ were cultured in 6 well plates, 1 mL/well, and stimulated by 10 μg/mL LPS. The concentration of TNF-α, IL- 1β, IL- 6, high mobility group protein B1 （HMGB1） and NO were determined in the culture supernatant, and the expressions of p65, c - Jun and iNOS and the DNA binding activity of NF - κB and AP- 1 were examined in PMφ at pre -stimulus （N）, 4 h and 12 h after LPS stimulation. Results The concentration of TNF - α, IL - 1β, IL - 6 and NO in the supernatant were increased significantly at 4 h and 12 h after LPS stimulation in SRC -3 ＋/＋ group and SRC -3 -/- group, but in the corresponding time points, they were lower in the SRC -3-/- group than those in SRC -3＋/＋ group. After LPS stimulation, there were no significant differences at 4 h between two groups about the levels of HMGB1, but it was increased in the SRC -3 ＋/＋ group and no significant difference was observed at 12 h. The expressions of p65 and c - Jun were significantly increased at 4 h and 12 h in two groups, but in the corresponding time points, the p65 expression was markedly higher while the c - Jun expression was obviously lower in SRC - 3 -/- group than those of SRC - 3 ＋/＋ group. At 4 h and 12 h, the iNOS expression was increased gradually in SRC- 3 ＋/＋ group, while no change was observed in SRC -3 -/- group. The DNA binding activity of NF - KB and AP - 1 was significantly increased at 4 h and 12 h, but in the relative time points, it was significantly lower in SRC -3-/- group than that of SRC -3 ＋/＋ group. Conclusion The inflammatory response of PMφ could be regulated by SRC - 3 protein, which the mechanisms are probably due to the expression and activity of iNOS, NF - κB and AP - 1.