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目的:探讨柚皮素对炎症反应时气道上皮细胞黏液高分泌的影响。方法:通过人中性粒细胞弹性蛋白酶(HNE)刺激人肺腺癌细胞A549,构建炎症反应时气道黏液高分泌模型,分别以柚皮素和磷脂酰肌醇3-激酶(PI3K)抑制剂LY294002进行干预,观察黏蛋白5AC(MUC5AC)、PI3K、PKB、P-PKB及核因子-κB(NF-κB)的表达。培养细胞经甲基偶氮唑盐法测定活性后分为对照组、HNE处理组、LY294002组、柚皮素组和柚皮素+LY294002组。逆转录PCR方法检测各组MUC5ACmRNA变化;Western blot法检测PI3K、PKB、P-PKB和NF-κB蛋白的表达;酶联免疫吸附测定法观察MUC5AC蛋白表达的变化,并用细胞免疫激光共聚焦显微镜观察柚皮素作用前后黏蛋白的分布。结果:HNE处理组MUC5AC的mRNA和蛋白的吸光度面积积分值较对照组明显降低;PI3K、PKB、P-PKB和NF-κB蛋白表达均较对照组明显升高;给予柚皮素预处理后,与HNE刺激组相比均明显下调;而柚皮素+LY294002组MUC5ACmRNA和蛋白均较单独LY294002处理组更为明显。结论:柚皮素可通过抑制PI3K/PKB信号途径,抑制NF-κB的活化,从而发挥抑制气道黏液高分泌的作用。
Objective: To investigate the effect of naringenin on mucus hypersecretion in airway epithelial cells during inflammatory reaction. Methods: Human lung adenocarcinoma A549 cells were stimulated with human neutrophil elastase (HNE) to establish a model of airway mucus hypersecretion induced by inflammatory reaction. Naringenin and phosphatidylinositol 3-kinase (PI3K) inhibitors LY294002 to observe the expression of MUC5AC, PI3K, PKB, P-PKB and NF-κB. The cultured cells were divided into control group, HNE treatment group, LY294002 group, naringenin group and naringenin + LY294002 group after the activity was measured by methyzolamine salt method. The changes of MUC5AC mRNA and protein were detected by reverse transcription-polymerase chain reaction (RT-PCR), the protein expressions of PI3K, PKB, P-PKB and NF-κB were detected by Western blotting and the expression of MUC5AC protein by enzyme-linked immunosorbent assay Distribution of mucins before and after naringenin action. Results: The area integral value of MUC5AC mRNA and protein in HNE treatment group was significantly lower than that in control group. The protein expressions of PI3K, PKB, P-PKB and NF-κB in HNE treatment group were significantly higher than those in control group. After naringenin pretreatment, Compared with HNE-stimulated group, the expression of MUC5AC mRNA and protein in naringenin + LY294002 group was significantly lower than that in LY294002-treated group. Conclusion: Naringenin can inhibit the airway mucus hypersecretion by inhibiting the PI3K / PKB signal pathway and inhibiting the activation of NF-κB.