氯喹对金黄色葡萄球菌激活的RAW264.7巨噬细胞表达TNF-α的影响

The influence of chloroquine on the expression of TNF-α by SAC-stimulated RAW264.7 cells

目的分析氯喹(CQ)对灭活金黄色葡萄球菌(SAC)刺激活化的RAW264.7巨噬细胞表达肿瘤坏死因子α(TNF-α)的影响,探讨其对革兰氏阳性菌引起的炎症反应的潜在作用及机制。方法采用细胞内细胞因子染色法分析胞内TNF-α的表达,利用流式微球阵列法检测细胞培养上清中TNF-α的表达,应用定量RT-PCR检测TNF-α的mRNA表达水平,免疫印迹法分析NF-κB信号通路的活化以及TNF-α前体的表达。结果 CQ明显抑制SAC激活的RAW264.7巨噬细胞分泌可溶性TNF-α,而细胞内TNF-α表达水平提高。定量RT-PCR显示CQ对TNF-α的mRNA水平没有影响;与此相一致,CQ对调控其转录的关键信号通路NF-κB p65的磷酸化水平影响不大。然而,免疫印迹分析表明,CQ使SAC诱导的Mr26 000和28 000 TNF-α前体蛋白的表达水平显著升高。结论 CQ通过抑制TNF-α从膜结合的Mr26 000和28 000前体蛋白转变为可溶性成熟蛋白的加工过程而抑制该炎症细胞因子的分泌,从而发挥其抗炎效应。

This study aimed to explore the potential anti-inflammatory effect and underlying action mechanism of ebloroquine （CQ） by using Gram-positive bacteria （inactivated Staphylococcus aureus cells, SAC）- activated RAW264.7 macrophages as a model. The expression of intraeellular TNF-α protein was evaluated by intracellular cytokine staining, and TNF-α expression in the culture supernatant was detected by cytometric bead array. Quantitative RT-PCR was used to detect the mRNA levels of TNF-α. The levels of the pro-TNF-α and the activation of NF-κB pathway were evaluated by Western blot. Our results showed that CQ significantly inhibited the secretion of TNF-α by SAC-activated RAW264.7 cells, but enhanced the intraeellular expression of TNF-α. At mRNA level, as compared to SAC stimulation alone, CQ pretreatment did not change TNF-α expression in SAC- stimulated cells, consistent with this, CQ had minimal effect on the levels of NF-κB phosphorylation. However, the 26 kDa and 28 kDa pro-TNF-α levels were markedly increased by pretreatment of CQ. Altogether, these results demonstrated that CQ inhibit TNF-α secretion through suppression of the processing of membrane-bound 26 kDa and 28 kDa pro-TNF-α into soluble mature TNF-α, thus exhibiting its anti-inflammatory activity.