自噬调控脂多糖诱导的巨噬细胞炎症因子水平

Autophagy modulates the levels of inflammatory cytokines in macrophages induced by lipopolysaccharide

目的探讨细胞自噬调控对脂多糖(LPS)刺激巨噬细胞引发炎症反应的影响及其分子机制。方法分离小鼠骨髓间充质干细胞,经过诱导分化获得骨髓来源的巨噬细胞,通过LPS刺激巨噬细胞建立炎症反应细胞模型。利用绿色荧光蛋白-微管相关蛋白1轻链3(GFP-LC3)质粒转染观察自噬体的形成;利用3-甲基腺嘌呤(3-MA)和雷帕霉素(Rap)分别抑制和促进细胞自噬,研究自噬干预对LPS诱导巨噬细胞炎症反应的影响;实时定量PCR检测LC3B、肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β)、IL-6和IL-12p40的mRNA水平,Western blot法检测LC3B、核因子κB(NF-κB)、NF-κB抑制蛋白α(IκBα)的蛋白水平。结果 LPS刺激巨噬细胞2、4、6 h,诱导巨噬细胞LC3B mRNA和蛋白高表达,巨噬细胞中自噬体增加。与单独LPS刺激相比,3-MA可抑制细胞自噬,显著增加炎症因子TNF-α、IL-1β、IL-6和IL-12p40 mRNA表达,而Rap处理减少自噬数量,降低TNF-α、IL-1β、IL-6和IL-12p40 mRNA水平;自噬抑制后,可通过降低IκBα和增加p-NF-k Bp65的表达而影响NF-κB信号通路,进而调节炎症细胞因子表达。结论自噬可调控LPS刺激巨噬细胞引起的炎症反应。

Objective To analyze the effect of autophagy on inflammatory response in macrophages induced by lipopolysaccharide （LPS） and investigate its molecular mechanism. Methods Bone marrow mesenchymal stem cells, which were separated from the femora of mice, were cultured and induced to differentiate into primary macrophages in vitro. The inflammatory cell model was established by stimulating the primary macrophages with LPS. Autophagy was inhibited by 3-methyladenine （3-MA） or promoted by rapamycin. Green fluorescent protein-microtubule associated protein 1 light chain 3 （GFP-LC3） plasmid was used to transfect primary macrophages and the percentage of cells with GFP-LC3 puncta were counted in the different groups. The mRNA levels of LC3B, tumor necrosis factor α （TNF-α）, interleukin 1β （IL-1β） , IL-6 and IL-12p40 were detected by real-time quantitative PCR, and the protein levels of LC3B, nuclear factor κB （NF-κB） and IκBα were determined by Western blotting. Results LC3B mRNA and protein expression levels were gradually up-regulated and the autophagosomes increased in the macrophages 2, 4 and 6 hours after treated by LPS. Compared with only LPS treatment group, autophagy inhibition by 3-MA pretreatment promoted the mRNA expressions of inflammatory cytokines including TNF-a, IL-115, IL-6 and IL-12p40, and the autophagy induction by rapamycin pretreatment suppressed TNF-α, IL-1β, IL-6 and IL-12p40. Meanwhile, 3-MA or rapamycin pretreatment further regulated the protein expressions of IκBα and p-NF-kBp65 induced by LPS in macrophages. Conclusion Autophagy can suppress the LPS-induced inflammatory response in macrophages by regulating NF-κB signaling pathway.