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 PM2.5-induced peritoneal macrophage injury
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摘要:
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（PM2.5）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（PM2.5质量浓度分别为50、100、200 μg/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mRNA表达水平。
结果 PM2.5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。PM2.5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PM2.5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mRNA表达量在PM2.5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mRNA水平明显低于相同剂量PM2.5组的C57BL/6小鼠（均P < 0.05）。
结论 PM2.5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。
Abstract: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.
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