摘要
目的研究前列腺素D2(PGD2)通过D类前列腺素受体2(DP2)对人气道上皮细胞黏液分泌的效应及其作用机制。方法用PGD2刺激人气道上皮16HBE细胞,以DP2拮抗剂AZD6430、DP1拮抗剂BWA868C及三磷酸肌醇蛋白激酶(PI3K)抑制剂LY294002为干预因素,将细胞分为对照组、PGD2刺激组、PGD2刺激+AZD6430组、PGD2刺激+BWA868C组、PGD2刺激+LY294002组。ELISA检测细胞中肿瘤坏死因子(TNF)-α、白介素(IL)-4和IL-13的蛋白含量,激光共聚焦技术检测细胞内黏蛋白(MUC)5AC含量;Western blot检测DP2、DP1、PI3K和磷酸化AKT(p-AKT)表达水平;实时荧光定量PCR检测TNF-α、IL-4、IL-13和MUC5AC mRNA表达水平。结果 PGD2刺激后MUC5AC、TNF-α、IL-4、IL-13、DP2、PI3K和p-AKT表达显著高于对照组(P<0.05);AZD6430拮抗组中MUC5AC、TNF-α、IL-4、IL-13、PI3K及p-AKT表达较PGD2刺激组明显减弱(P<0.05);LY294002组TNF-α、IL-4、IL-13和MUC5AC表达较PGD2组亦显著减弱(P<0.05)。结论 PGD2激活人气道上皮细胞的DP2受体,活化PI3K/AKT引起黏液高分泌。
Objective To investigate the effect of D prostanoid receptor 2（ DP2） on mucin（ MUC） 5AC overexpression induced by prostaglandin D2（ PGD2） and the potential mechanism. Methods The human airway epithelial（ 16HBE） cells were stimulated with PGD2（ 1 μmol/L）. In intervention experiments,cells were pretreated with DP2 antagonist AZD6430,DP1 antagonist BWA868 C and PI3 K specific inhibitor LY294002. Then,PGD2 stimulation was used. Cells were divided into 6 groups： control group（ incubated in DEME / Ham＇s F12 medium without fetal bovine serum）,PGD2 stimulation group,PGD2 stimulation ＋ AZD6430 group,PGD2 stimulation ＋ BWA868 C group,PGD2 stimulation ＋ LY294002 group. The levels of tumor necrosis factor（ TNF）-α,interleukin（ IL）-4and IL-13 protein were tested by real-time PCR and ELISA respectively. MUC5 AC protein level was assessed by immunofluorescence. Western blot was used to detected the level of DP2,DP1,PI3 K and phosphorylation of AKT（ p-AKT）. MUC5 AC mRNA expression was tested by real-time PCR. Results PGD2 significantly increased ofMUC5 AC,TNF-α,IL-4,IL-13,DP2,PI3 K and p-AKT（ P〈0. 05）. Inhibition of DP2 activity,production of TNF-α,IL-4,IL-13,MUC5 AC,PI3K and p-AKT were significantly lower than that of PGD2 stimulation group（ P〈0. 05）. The levels of TNF-α,IL-4,IL-13 and MUC5 AC mRAN and protein expression were also attenuated after treatment with LY294002（ P〈0. 05）. Conclusions DP2 mediates mucus hypersecretion induced through PGD2 via PI3 K / AKT pathway.
出处
《基础医学与临床》
CSCD
2016年第1期24-29,共6页
Basic and Clinical Medicine
基金
国家自然科学基金(31171346
81370111)
关键词
D类前列腺素受体2
黏蛋白
炎性因子
信号传导
D prostagland receptor 2
mucin
inflammatory cytokines
signal transduction
