胱天蛋白9基因信号通路在大气PM2．5促发巨噬细胞损伤中的作用

Role of CARD9 gene signaling pathway in PM_(2.5)-induced peritoneal macrophage injury

[目的]用小鼠腹腔巨噬细胞原代培养实验探索胱天蛋白9(CARD9)基因信号通路在大气细颗粒物(PM_(2.5))对巨噬细胞影响中可能的作用机制。[方法]取6~8周龄的CARD9敲基因(CARD9^(-/-))雌性小鼠或C57BL/6雌性小鼠,原代培养腹腔巨噬细胞,按照低、中、高剂量组(PM_(2.5)质量浓度分别为50、100、200μg/mL)染毒24 h。测定细胞的存活率和巨噬细胞杀伤功能,测量巨噬细胞上清液中肿瘤坏死因子α(TNF-α)和白细胞介素6(IL-6)的表达水平,以及巨噬细胞炎症相关因子(Dectin-1,IL-6和TNF-α)的m RNA表达水平。[结果]PM_(2.5)处理24 h后,C57BL/6小鼠巨噬细胞的存活率在低、中、高剂量组分别下降到93.35%、90.89%、84.44%;CARD9^(-/-)小鼠下降到95.30%、92.23%、86.04%;但两种小鼠间无差异。PM_(2.5)对C57BL/6小鼠的巨噬细胞产生刺激作用,低、中、高剂量组IL-6浓度分别为(14.95±5.84)、(30.55±2.34)、(47.90±9.90)μg/L,TNF-α分别为(144.34±35.75)、(228.93±46.62)、(351.38±46.82)μg/L;暴露于相同剂量的PM_(2.5)时,除低剂量组IL-6外,CARD9^(-/-)小鼠巨噬细胞上清液中IL-6和TNF-α浓度都明显低于C57BL/6小鼠,差异具有统计学意义(P<0.05)。C57BL/6小鼠巨噬细胞内IL-6与TNF-α的m RNA表达量在PM_(2.5)低剂量组时仅为1.15±0.26和1.24±0.29,到高剂量组则变为2.75±0.68和2.67±0.47。CARD9^(-/-)小鼠巨噬细胞内的IL-6和TNF-α的m RNA水平明显低于相同剂量PM_(2.5)组的C57BL/6小鼠(均P<0.05)。[结论]PM_(2.5)可能通过介导CARD9基因信号通路诱导巨噬细胞释放促炎细胞因子。

[Objective] To observe the role of caspase recruitment domain 9 （CARD9） gene signaling pathway in the effect of fine particulate matters （PM2.5） on peritoneal macrophages.[Methods] Macrophages were extracted through intraperitoneal injection of serum-free medium from CARD9-/-or C57BL/6 female mice aged 6-8 weeks. The peritoneal macrophages were treated with 50, 100, and 200μg/mL PM2.5. After 24h PM2.5 exposure, cell viability and phagocytic capacity were measured. The expression levels of interleukin-6 （IL-6） and tumor necrosis factor-α （TNF-α） in macrophage culture supernatant were determined along with the mRNA relative expression of Dectin-1, IL-6, and TNF-α in macrophages.[Methods] Macrophages were extracted through intraperitoneal injection of serum-free medium from CARD9-/- or C57BL/6 female mice aged 6-8 weeks. The peritoneal macrophages were treated with 50, 100, and 200μg/mL PM2.5. After 24h PM2.5 exposure, cell viability and phagocytic capacity were measured. The expression levels of interleukin-6 （IL-6） and tumor necrosis factor-α （TNF-α） in macrophage culture supernatant were determined along with the mRNA relative expression of Dectin-1, IL-6, and TNF-α in macrophages.[Results] After 24 h PM2.5 exposure, the cell viability of macrophages in the 50, 100, and 200 μg/mL PM2.5 groups decreased to 93.35%, 90.89%, and 84.44% in C57BL/6 mice, and 95.30%, 92.23%, and 86.04% in CARD9-/- mice; there were no differences between C57BL/6 mice and CARD9-/- mice. PM2.5 induced increases of IL-6 and TNF-α in macrophages of C57BL/6 mice, which were （14.95±5.84） μg/L and （144.34±35.75） μg/L in the 50 μg/mL PM2.5 group, （30.55±2.34） μg/L and （228.93±46.62） μg/L in the 100 μg/mL group, and （47.90±9.90） μg/L and （351.38±46.82） μg/L in the 200 μg/mL PM2.5 group. For the same PM2.5 exposure dose, the concentrations of IL-6 and TNF-α in the macrophages of CARD9-/- mice were significantly lower than those of C57BL/6 mice （P 〈 0.05）, except the IL-6 of the 50 μg/mL PM2.5 group. The mRNA expressions of IL-6 and TNF-α in C57BL/6 mouse macrophages treated with 50 μg/mL PM2.5 were 1.15±0.26 and 1.24±0.29 respectively, and 2.75±0.68 and 2.67±0.47 respectively, in those treated with 200 μg/mL PM2.5. The mRNA expressions in CARD9-/- mouse macrophages were significantly lower than those in C57BL/6 mouse macrophages exposed to the same concentration of PM2.5 （all Ps 〈 0.05）. [Conclusion] PM2.5 may induce pro-inflammatory cytokines secretion in macrophages though CARD9 gene signaling pathway.