干扰STAT3基因对哮喘气道损伤的影响

摘要

目的:探讨信号转导及转录激活因子3(STAT3)在哮喘气道损伤中的作用及相关机制.方法:首先,通过Western blot检测哮喘患者的支气管黏膜组织中STAT3的表达及活化情况;其次,以支气管上皮细胞为研究对象,检测屋尘螨抗原P1(Derp1)刺激对STAT3的表达及活化的影响;再次,采用shRNA干扰支气管上皮细胞中STAT3的表达,CCK-8实验检测细胞活力,同时检测细胞培养液中肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β)和IL-6的含量来反映细胞的炎症反应,检测细胞中丙二醛(MDA)的含量及超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)的活性来反映细胞氧化应激水平;流式细胞术检测细胞凋亡情况.结果:哮喘患者的支气管黏膜组织中p-STAT3的蛋白水平显著上升,Derp1刺激可显著上调支气管上皮细胞中p-STAT3的蛋白水平.沉默STAT3可抑制TNF-α、IL-1β和IL-6释放,降低MDA的含量,升高SOD和GSH-Px的活性,并抑制细胞凋亡,提高细胞活力(P<0.05).结论:沉默STAT3可减轻尘螨诱导的气道损伤,其作用机制可能是通过抑制JAK/STAT信号通路的激活,抑制支气管上皮细胞分泌炎症细胞因子及细胞内氧化应激反应,进而减少细胞凋亡.%AIM:To investigate the effect of signal transducer and activator of transcription 3 (STAT3) on air-way injury in asthma and its mechanism .METHODS:The expression and activation of STAT 3 in bronchial mucosa tissues of asthmatic patients were measured by Western blot .The expression and activation of STAT 3 in bronchial epithelial cells pretreated with Dermatophagoides pteronyssinus antigen P1 (Derp1) were estimated.Bronchial epithelial cells were trans-fected with STAT3 shRNA.STAT3 expression was measured by Western blot .The cell viability was detected by CCK-8 as-say.The concentrations of TNF-α, IL-1βand IL-6 were detected by ELISA .The content of malondialdehyde ( MDA) and the activity of superoxide dismutase ( SOD) and glutathione peroxidase ( GSH-Px) were also determined for evaluating the status of oxidative stress .The cell apoptosis was analyzed by flow cytometry .RESULTS:The protein level of p-STAT3 was significantly up-regulated in both bronchial mucosa of asthmatic tissues and bronchial epithelial cells pretreated with Derp 1. Knockdown of STAT3 inhibited the releases of TNF-α, IL-1βand IL-6, decreased the content of MDA and enhanced the activity of SOD and GSH-Px with the suppression of cell apoptosis ( P <0.05 ) .CONCLUSION: Down-regulation of STAT3 attenuates Derp1-induced the airway injury .The mechanism may involve that knockdown of STAT3 suppresses the activation of JAK/STAT signaling pathway , the release of asthma-related inflammatory cytokines and oxidative stress in bronchial epithelial cells , thus inhibiting cell apoptosis .