青蒿琥酯对香烟烟雾诱导气道上皮屏障损伤的防护作用及机制

Protective Effect and Mechanism of Artesunate on Airway Epithelial Barrier Dysfunction Induced by Cigarette Smoke

目的探讨青蒿琥酯(ART)对气道上皮屏障损伤的防护作用及其机制。方法细胞实验,将人支气管上皮细胞(HBEC)分为对照组(A组,常规培养),香烟烟雾提取物(CSE)组(B组,2.5%CSE处理24 h),ART低、中、高剂量组(C1组、C2组、C3组,1,10,100μmol/L,予2.5%CSE刺激前1 h加入ART共孵育),制备上清液,采用酶联免疫吸附(ELISA)法检测细胞肿瘤坏死因子-α(TNF-α)和白细胞介素6(IL-6)水平,采用流式细胞术检测细胞凋亡水平,通过测定跨上皮细胞电阻(TEER)考察上皮屏障完整性,采用免疫印迹法检测细胞间连接蛋白ZO-1、E-cadherin、表皮生长因子受体(EGFR)的表达水平。动物实验,将25只BALB/c小鼠随机分为对照组(a组,等体积生理盐水),香烟烟雾(CS)组(b组,等体积生理盐水),ART低、中、高剂量组(c_(1)组、c_(2)组、c_(3)组,25,50,100 mg/kg)。使小鼠持续暴露于CS环境(每天2次,每次1 h,持续8周),以建立气道上皮屏障损伤小鼠模型。各组小鼠每日首次CS刺激前1 h腹腔注射相应药物或生理盐水。采用免疫组化染色观察肺组织中细胞连接蛋白ZO-1,E-cadherin和EGFR的表达情况。结果与B组比较,C_(2),C_(3)组细胞TNF-α和IL-6水平显著降低(P<0.05),C_(1),C_(2),C_(3)组细胞凋亡率显著降低(P<0.05),C_(2),C_(3)组细胞TEER值显著升高(P<0.05),ZO-1和E-cadherin蛋白表达水平显著升高,EGFR表达水平显著降低(P<0.05)。与b组比较,c_(3)组小鼠肺组织结构明显改善,ZO-1和E-cadherin蛋白表达水平显著升高,EGFR表达水平显著降低。结论ART能抑制CSE诱导的气道炎性反应和上皮细胞凋亡,改善气道上皮屏障损伤。其作用机制可能与抑制EGFR表达有关。

Objective To investigate the protective effect and mechanism of artesunate(ART)on the airway epithelial barrier dysfunction.Methods In the cell experiment,human bronchial epithelial cells(HBECs)were divided into the control group(group A,conventional culture),the cigarette smoke extract(CSE)group[group B,2.5%CSE treatment for 24 h],the ART low-,medium-and high-dose groups(groups C_(1),C_(2)and C_(3),1,10,100μmol/L,adding ART for co-incubation 1 h before 2.5%CSE stimulation).Supernatant was prepared to detect the cellular tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)levels by the enzyme-linked immunosorbent assay(ELISA).Cell apoptosis was detected by the flow cytometry,epithelial barrier integrity was investigated by the determination of trans-epithelial electrical resistance(TEER),expression levels of intercellular junction proteins ZO-1,E-cadherin and epidermal growth factor receptor(EGFR)were detected by the Western blot.In the animal experiment,25 BALB/c mice were randomly divided into the control group(group a,equal volume physiological saline),the cigarette smoke(CS)group(group b,equal volume physiological saline),the ART low-,medium-and high-dose groups(groups c_(1),c_(2)and c_(3),25,50,100 mg/kg).The mice were exposed to CS for eight weeks(twice a day,1 h each time)to establish models of airway epithelial barrier dysfunction.Each group was intraperitoneally injected with corresponding drugs or physiological saline 1 h before the first CS stimulation everyday.The expression of intercellular junction proteins ZO-1,E-cadherin and EGFR in lung tissue were observed by the immunohistochemistry staining method.Results Compared with those in the group B,the TNF-αand IL-6 levels in the groups C_(2)and C_(3)significantly decreased(P<0.05);the apoptosis rate in the groups C_(1),C_(2)and C_(3)significantly decreased(P<0.05);the TEER in the groups C_(2)and C_(3)significantly increased(P<0.05);the expression levels of ZO-1 and E-cadherin proteins significantly increased,and the EGFR expression level significantly decreased(P<0.05).Compared with those in the group b,the lung tissue structure in the group c3 significantly improved,the expression levels of ZO-1 and E-cadherin proteins significantly increased,and the EGFR expression level significantly decreased.Conclusion ART can inhibit CSE-induced airway inflammatory response and epithelial cell apoptosis,improve airway epithelial barrier dysfunction,and its mechanism may be related to the inhibition of EGFR expression.