c-src/NADPH氧化酶1/活性氧对中性粒细胞弹力蛋白酶诱导气道黏蛋白5 AC表达的影响

目的 探讨中性粒细胞弹力蛋白酶(NE)诱导气道黏液高分泌的上游信号调节机制.方法 体外培养人气道BEAS-2B上皮细胞,将细胞分为空白对照组(不加任何刺激)、NE组(NE刺激)、NE+PP2组(NE刺激合并c-src抑制剂PP2)、NE+c-src siRNA组(NE刺激合并c-src siRNA)、NE+Noxl siRNA组NE刺激合并NADPH氧化酶1(Nox1)siRNA]、阴件siRNA对照组(加入阴性对照siRNA)及NE+阴性siRNA组(NE刺激合并阴性对照siRNA)为干预条件.检测干预前后各组细胞巾活性氧含量、NoxI蛋白含量、黏蛋白5AC的蛋白及mRNA水平.用四甲基偶氮唑盐法测定各组细胞活性;活性氧试剂盒测定活性氧相对含量;逆转录PCR法检测各组黏蛋白5AC mRNA水平;ELISA法分析细胞黏蛋白5AC的蛋白相对含量;Western blot法检测Noxl蛋白及c-src蛋白的相对含量.结果 NE组细胞中Nox1蛋白相对含量为0.88±0.12,高于空白对照组的0.32±0.09(t=9.12.P=0.003);活性氧相对含量为0.76±0.09,高于空白对照组的0.18±0.02(t=9.44,P=0.003);黏蛋白5AC蛋白相对含量为0.82±0.09,基因转录水平为0.77±0.05,均高于空白对照组(分别为0.21±0.11和0.18±0.08,t值分别为7.75和6.13,P值分别为0.004和0.006).NE+c-src siRNA组细胞的Nox1蛋白相对含最(0.39±0.08)、活性氧相对含量(0.29±0.05)均低于NE组(t值分别为5.43和5.60,均P=0.007);黏蛋白5AC蛋白相对含量(0.38±0.09)及mRNA水平(0.41±0.04)低于NE组(t值分别为5.28和4.09,P值分别为0.008和0.034).NE+PP2组活性氧相对含量为0.41±0.11,Nox1蛋白相对含量为0.44±0.05,黏蛋白5AC蛋白及mRNA水平分别为0.48±0.08和0.46±0.07,均低于NE组(均P<0.05).NE+Noxl siRNA组中活性氧相对含量(0.19±0.06)、黏蛋白5AC蛋白相对含量(0.31±0.05)及mRNA水平(0.32±0.06)也低于NE组(均P<0.05).结论 c-src/Noxl参与了NE诱导的活性氧活化,是气道上皮细胞黏蛋白5AC合成及分泌的上游信号调节因子.

Effects of c-src/Noxl/ROS on mucinSAC expression induced by neutrophil elastase in airway epithelial cells

Objective To explore the upper signaling pathway in neutrophil elastase(NE)-induced mucin5AC(MUCSAC)production in human BEAS-2B airway epithelial cells.Methods The BEAS-2B airway epithelial cells were cultured and divided into 7 groups:negative control group,NE stimulation group,negative siRNA control group treated with or without NE,NADPH oxidase l(Noxl)siRNA group treated with NE,c-src siRNA group treated with NE.and c-src specific inhibitor PP2 group treated NE.The relative content of reactive oxygen species(ROS)was assayed with a special kit.The levels of MUCSAC protein in culture medium,Nox1 protein,phosphoralyted c-SIC(p-c-src)kinase and MUC5 AC mRNA in culture cells were detected with enzyme-linked immunosorbent assay,Western blot,and RT-PCR,respectively. Results There Was an obvious increase of ROS production(0.76±0.09)in cells exposed to NE,with elevation of Noxl protein(0.88±0.12)and MUC5AC protein production(0.82±0.09)and mRNA expression(0.77 4±0.05),all had significant differences when compared with normal control group,t=6.13-9.44.P<0.01.Noxl siRNA inhibited ROS activity(0.19±0.06),reduced MUC5AC protein (0.31 4±0.05)and mRNA level(0.32 4±0.06),compared with single NE-stimulated group,P<0.01.c-src siRNA and PP2 also showed the salne effects,both of them decreased the NE-induced high activity of ROS,Was 0.29 4±0.05,and 0.41±0.11,respectively,the MUC5AC protein in the c-src siRNA group and PP2 group were 0.38±0.09 and 0.48±0.08,MUC5AC mRNA level were 0.41±0.04 and 0.46 4±0.07,the Noxl protein were 0.39 4±0.08 and 0.44±0.05,all P<0.05,compared with single NE-treated group.Conclusion C-src/Noxl/ROS transduction cascade may be upper signal regulators in NE-mediated MUC5AC expression in BEAS-2B cells.