麻黄碱对LPS诱导的肺泡上皮细胞A549凋亡、炎症和COX-2基因表达的影响

Effects of ephedrine on LPS induced apoptosis,inflammation and COX 2 gene expression in A549 alveolar epithelial cells in vitro

目的探讨麻黄碱对脂多糖(LPS)诱导的肺泡上皮细胞A549凋亡、炎症和环氧化酶-2(COX-2)基因表达的影响。方法体外培养肺泡上皮细胞A549,采用10μg/ml LPS干预A549细胞,实验分为Control组(未做任何处理)、LPS组(10μg/ml LPS)和Ephedrine组(10μg/ml LPS和800μg/ml麻黄碱)。采用细胞计数试剂盒(CCK-8)和流式细胞术检测3组A549细胞增殖和凋亡能力,蛋白免疫印迹(Western blot)法检测凋亡相关蛋白剪切的含半胱氨酸的天冬氨酸蛋白水解酶3(Cleaved Caspase-3)和Cleaved Caspase-9的表达,酶联免疫吸附法(ELISA)检测细胞上清中肿瘤坏死因子-α(TNF-α)、白介素1β(IL-1β)和白介素6(IL-6)的含量,比色法检测超氧化物气化酶(SOD)活力和丙二醛(MDA)的含量,DCFH-DA荧光探针检测细胞活性氧(ROS)水平,实时荧光定量PCR(qRT-PCR)和Western blot检测3组细胞中COX-2 mRNA和蛋白表达量。结果与LPS诱导的A549细胞相比,麻黄碱能够明显提高LPS诱导的A549细胞增殖能力(P<0.05),增加SOD的活力(P<0.05),减少LPS诱导的A549细胞凋亡(P<0.05),下调Cleaved Caspase-3、Cleaved Caspase-9和COX-2的表达(P<0.05),降低TNF-α、IL-1β、IL-6、MDA含量及ROS水平(P<0.05)。结论麻黄碱能够有效缓解LPS诱导的肺泡上皮细胞A549凋亡、炎症损伤和氧化应激,并下调COX-2基因的表达。

Objective To investigate the effects of ephedrine on lipopolysaccharide(LPS)induced apoptosis,inflammation and cyclooxygenase 2(COX-2)gene expression in alveolar epithelial cells A549 in vitro.Methods The alveolar epithelial cells A549 were cultured in vitro,which were intervened by 10μg/ml LPS.The experiment was divided into control group(without any treatment),LPS group(10μg/ml LPS)and ephedrine group(10μg/ml LPS and 800μg/ml ephedrine).Cell counting kit(CCK 8)and flow cytometry were used to detect the proliferation and apoptosis of A549 cells.Western blot was used to detect the expressions of aspartic acid proteolytic enzyme-3(Cleaved Caspase-3)and Cleaved Caspase-9 which contained cysteine cleaved by apoptosis-related protein.Enzyme linked immunosorbent assay(ELISA)was used to detect the levels of tumor necrosis factor-α(TNF-α),interleukin 1βand interleukin 6(IL-6)in cell supernatant.The superoxide gasification enzyme(SOD)activity and malondialdehyde(MDA)content were detected by colorimetric method.The levels of cell reactive oxygen(ROS)were detected by DCFH DA fluorescent probe.Moreover the expression levels of COX-2 mRNA and protein were detected by real time fluorescence quantitative PCR(qRT PCR)and Western Blot,respectively.Results The ephedrine could significantly increase the proliferation ability of A549 cells induced by LPS(P<0.05),increase the activity of SOD(P<0.05),and reduce the apoptosis of the cells(P<0.05),and down regulate the expression levels of Cleaved Caspase-3,Cleaved Caspase-9 and COX-2(P<0.05),and reduce the levels of TNF-α,IL-1β,IL-6,MDA and ROS(P<0.05).Conclusion Ephedrine can effectively relieve LPS induced apoptosis,inflammation and oxidative stress in A549 alveolar epithelial cells in vitro,and down-regulate the expression levels of COX-2 gene.