miR-223对烟草烟雾诱导的慢性阻塞性肺疾病细胞炎症和凋亡的影响

Effects of miR-223 on cell inflammation and apoptosis in chronic obstructive pulmonary disease induced by cigarette smoke

目的研究微小核糖核酸(miR)-223通过激活核因子-κB(NF-κB)信号通路对烟草烟雾(CS)诱导的慢性阻塞性肺疾病(慢阻肺)小鼠的影响。方法将16只C57BL/6野生型(WT)小鼠分为对照组和模拟组,对照组暴露于空气中,模拟组暴露于CS中构建COPD模型,每组8只。miR-223 KO小鼠10只,每组5只,分别暴露于空气和CS中。检测小鼠肺功能,比较各组小鼠用力肺活量(FVC)、呼气峰值流量(PEF)、吸气阻力(Ri)、潮气量(VT);实时荧光定量PCR实验检测肺组织和肺泡巨噬细胞miR-223水平,检测肺组织中炎症因子肿瘤坏死因子-α(TNF-α)、白介素-6(IL-6)、白介素-1β(IL-1β)和NF-κB mRNA;酶联免疫吸附实验(ELISA)检测肺泡灌洗液中TNF-α、IL-6和IL-1β表达;流式细胞术检测肺组织中细胞凋亡;蛋白质免疫印迹检测NF-κB蛋白和磷酸化NF-κB(p-NF-κB)蛋白表达。结果与空气暴露的小鼠相比,CS暴露的小鼠肺组织和肺泡巨噬细胞中miR-223水平升高。对于暴露于CS的小鼠中,miR-223缺失小鼠的炎症反应和细胞凋亡要明显低于WT小鼠。而且,NF-κB的转录不受miR-223调控,但是miR-223缺失通过降低NF-κB磷酸化抑制NF-κB通路激活。结论miR-223通过激活NF-κB信号加重烟草烟雾诱导的COPD细胞凋亡和炎症反应。

Objective To study the effects of microRNA(miR-223)on cigarette smoke(CS)-induced chronic obstructive pulmonary disease(COPD)in mice by activating the nuclear factor-κB(NF-κB)signaling pathway.Methods Sixteen C57BL/6 wild-type(WT)mice were divided into a control group and a simulated group.The control group was exposed to air,and the simulated group was exposed to CS to construct a COPD model,with 10 mice in each group.Ten miR-223 KO mice,five in each group,were exposed to air and CS,respectively.The pulmonary function of mice was measured,and forced vital capacity(FVC),expiratory peak flow value(PEF),inspiratory resistance(Ri),and tidal volume(VT)were compared.The levels of miR-223 in lung tissue and alveolar macrophages were detected by real-time fluorescence quantitative PCR,and the inflammatory cytokines tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),interleukin-1β(IL-1β)and NF-κB mRNA were detected in lung tissue.The expression of TNF-α,IL-6,and IL-1βin bronchoalveolar lavage fluid was detected by enzyme-linked immunosorbent assay(ELISA).Flow cytometry was used to detect apoptosis in lung tissue.The expression of NF-κB protein and phosphorylated NF-κB(p-NF-κB)protein was detected by Western blot.Results miR-223 levels were elevated in lung tissue and alveolar macrophages of CS-exposed mice compared with air-exposed mice.In mice exposed to CS,the inflammatory response and apoptosis of miR-223-deficient mice were significantly lower than those of WT mice.Moreover,NF-κB transcription is not regulated by miR-223,but miR-223 deletion inhibits NF-κB pathway activation by reducing NF-κB phosphorylation.Conclusion miR-223 aggravates cigarette smoke-induced inflammation and apoptosis of COPD cells by activating NF-κB signaling.