NALP3炎性复合体及ERK信号通路在氧化应激中的作用及阿魏酸钠的干预机制

Sodium Ferulate Attenuates Oxidative Stressvia Suppressing NALP3 Inflammasome and ERK Signal Pathway

目的探讨氧化应激时人肺上皮细胞(A549)中NALP3炎性复合体、细胞外调节蛋白激酶(ERK)的表达变化,阿魏酸钠(SF)的干预作用及其可能的机制。方法培养A549细胞,分为空白对照组,H2O2刺激组,阿魏酸钠组,caspase-1抑制剂(Z-VAD)组,ERK阻断剂(PD98059)组,阿魏酸钠+H2O2组。其中H2O2刺激组用H2O2 200μmol/L培养细胞2h,阿魏酸钠组用阿魏酸钠400μg/mL处理细胞30 min,Z-VAD组、PD98059组、阿魏酸钠+H2O2组分别用Z-VAD 20μmol/L、PD98059 50μmol/L、阿魏酸钠400μg/mL预处理A549细胞30min后加入H2O2200μmol/L培养2h,采用实时荧光定量PCR检测各组A549中caspase-1、NALP3mRNA的表达,Western blot检测caspase-1、NALP3、磷酸化ERK(p-ERK)、ERK蛋白含量;ELISA法检测各组A549上清液中白细胞介素-1β(IL-1β)含量。结果与空白对照组比较,H2O2可使A549细胞caspase-1、NALP3mRNA及caspase-1、NALP3、p-ERK/ERK蛋白水平表达增强,IL-1β分泌增加(P均<0.05),而阿魏酸钠组与空白对照组差异无统计学意义(P>0.05)。与H2O2刺激组比较,Z-VAD组、PD98059组和阿魏酸钠+H2O2组caspase-1、NALP3mRNA及蛋白表达降低,p-ERK/ERK蛋白表达降低,IL-1β分泌下降(P均<0.05),Z-VAD组或PD98059组与阿魏酸钠+H2O2组间上述作用差异无统计学意义(P>0.05)。结论阿魏酸钠可能通过抑制ERK活化,减少caspase-1、NALP3及IL-1β的表达,从而减轻氧化应激导致的炎症级联反应。

Objective Study the gene and protein expression of NACHT-PYD-containing protein 3 （NALP3） inflammasome and extraeellular regulated protein kinase （ERK）, the intervention effects of sodium feruiate （SF） in human lung epithelial cells A549 under oxidative stress, and to investigate the possible mechanism. Methods Human lung epithelial cells A549 cultured in vitro were divided into 6 groups, including control group, H2O2 （200μmol/L） group, SF group （400 μg/mL）, caspase-1 blockers （Z-VAD） group （Z-VAD 20 μmol/L＋H2O2 200 μmol/L）, ERK blockers （PD98059） group （PD98059 50 μmol/L＋H202 200 μmol/L）, and SF-I-H202 group （SF 400 μg/mL＋H2O2 200 μmol/L）. Fluorescent quantitative real-time PCR （qRT-PCR） was performed to detect the mRNA levels of easpase-1 and NALP3, the expression of caspase-1, NALP3, phosphorylated ERK p-ERK, ERK protein were evaluated by Western blot. The level of interleukin-1 beta （IL-1β） were detected by ELISA. Results Compared with the control group, H2 O2 not only increased the mRNA and protein expression levels of caspase-1 and NALP3 and the protein expression levels of p-ERK/ERK, but also enhanced the secretion of IL-1β in human lung epithelial cells A549 （P〈 0. 05）, while SF group showed no statistic significance of those indicators above （P〈0.05）. The Z-VAD group, the PD98059 group and the SF＋H2O2 group resisted the effects of H2O2 on A549 cells by decreasing the mRNA and protein expressions of easpase-1 and NALP3, and the protein expression of p-ERK/ ERK, as well as reducing the secretion of IL-1β （P〈0.05） ,when compared with the H2O2 group. Conclusion SF may reduce the expression of caspase-1, NALP3 and IL-113 by inhibiting ERK, so as to reduce the inflammation caused by oxidative stress.