乙酰半胱氨酸对LPS诱导肺泡上皮细胞氧化应激反应迁移和EMT进程的抑制作用机制研究

Mechanism of acetylcysteine's inhibition on LPS-induced oxidative stress response,migration and EMT process of alveolar epithelial cells

目的探讨乙酰半胱氨酸(NAC)对脂多糖(LPS)诱导肺泡上皮细胞A549氧化应激、迁移、上皮间充质转化(EMT)及磷脂酰肌醇3-激酶(PI3K)/丝苏氨酸蛋白激酶(AKT)信号通路的调控作用。方法体外培养肺泡上皮细胞A549,分为NC组(不进行干预)、LPS组(10μg·mL^(-1)LPS)、NAC组(10μg·mL^(-1)LPS+1 mmol·L^(-1)NAC)、A组(10μg·mL^(-1)LPS+100 ng·mL^(-1)PI3K/AKT通路激活剂胰岛素样生长因子(IGF)-Ⅰ)、NAC+A组(10μg·mL^(-1)LPS+1 mmol·L^(-1)NAC+100 ng·mL^(-1)IGF-Ⅰ)和NAC+Y组(10μg·mL^(-1)LPS+1 mmol·L^(-1)NAC+5μmol·L^(-1)PI3K/AKT通路抑制剂LY294002),LPS和NAC分别作用24 h。用细胞计数试剂盒-8(CCK-8)测定细胞活力,丙二醇(MDA)试剂盒检测MDA表达,超氧化物歧化酶(SOD)试剂盒检测SOD表达,划痕法检测细胞迁移率,蛋白免疫印迹(WB)法测定PI3K、AKT、磷酸化(p)-PI3K、p-AKT、E-钙粘蛋白(E-cadherin)、N-钙粘蛋白(N-cadherin)以及波形蛋白(vimentin)的表达。结果根据细胞活力情况选择1 mmol·L^(-1)浓度NAC进行后续实验。与NC组相比,LPS组SOD表达、E-cadherin、p-PI3K和p-AKT蛋白水平降低,MDA表达、细胞迁移率、N-cadherin和vimentin蛋白表达升高,差异有统计学意义(P<0.05);与LPS组相比,NAC组和A组扭转了上述指标的变化,差异有统计学意义(P<0.05);与NAC组相比,NAC+A组中的IGF-I增强了NAC对A549细胞的作用,NAC+Y组中的LY294002削弱了NAC对A549细胞的作用(P<0.05)。结论NAC可抑制LPS诱导的肺泡上皮A549细胞的氧化应激反应和迁移能力,并阻碍其EMT进程,其作用机制可能与PI3K/AKT通路的活化有关。

Objective To explore the regulatory effects of acetylcysteine(NAC)on lipopolysaccharide(LPS)-induced oxidative stress,migration and epithelial interstitial transformation(EMT)of A549 alveolar epithelial cells as well as phosphatidylinositol 3-kinase(PI3K)/serine-threonine protein kinase(AKT)signaling pathways.Methods A549 cells cultured in vitro were divided into NC(no-intervention),LPS(10μg·mL^(-1) LPS),NAC(10μg·mL^(-1) LPS+1 mmol·L^(-1) NAC)and A[10μg·mL^(-1) LPS+1 mmol·L^(-1) NAC+100 ng·mL^(-1) PI3K/AKT pathway activator insulin-like growth factor(IGF)-Ⅰ],NAC+A(10μg·mL^(-1) LPS+1 mmol·L^(-1) NAC+100 ng·mL^(-1) IGF-Ⅰ)and NAC+Y group(10μg·mL^(-1) LPS+1 mmol·L^(-1) NAC+5μmol·L^(-1) PI3K/AKT pathway inhibitor LY294002),LPS and NAC respectively acted for 24 hours.Cell viabilities were measured using cell counting kit 8(CCK-8),MDA and SOD expression detected respectively using propylene glycol(MDA)kit and superoxide dismutase(SOD)kit,cell migration rates detected using scratch method,and expression levels of PI3K,AKT,phosphorylated(p)-PI3K,p-AKT,E-cadherin,N-cadherin and Vimentin measured using Western blotting(WB).Results According to cell viability,1 mmol·L^(-1) concentration of NAC was selected for follow-up experiments.Compared with NC group,SOD expression level and E-cadherin,p-PI3K and p-AKT lowered and MDA expression,cell migration rate and the expression levels of N-cadherin,Vimentin elevated in the LPS,differences were statistically significant(P<0.05);compared with LPS group,a bove-mentioned indexes were reversed in the NAC and A,differences were statistically significant(P<0.05);compared with NAC group,IGF-I enhanced NAC action on A549 cells in the NAC+A and LY294002 weakened NAC action on A549 cells in the NAC+Y(P<0.05).Conclusion NAC could inhibit the oxidative stress response and migration ability of LPS-induced A549 cells and hinder the EMT process,the mechanism may be related to the activation of PI3K/AKT pathway.