柚皮素和PI3K/PKB信号途径对气道黏液高分泌的影响

Role of naringenin and phosphatidylinositol 3-kinase/Protein kinase B(PI3K/PKB) signal transduction pathway in the airway inflammation

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。

Objection：To investigate the effects of naringenin on airway mucous hypersecretion and on the signal transduction pathway of phosphatidylinositol 3-kinase/protein kinase B（PI3K/PKB）.Methods：The cell model of mucous hypersecretion was made by human lung A549 cell stimulated by human neutrophil elastae（HNE）,and treated with naringenin and LY294002,a blocking agent of PI3K,respectively.The expression of mucin（MUC）5AC,PI3K,PKB,P-PKB and nuclear factor-κB（NF-κB）were detected.The cell activity after naringenin treatment was assessed by methylthiazolyl tetrazolium method.The cells were divided into 5 groups：a negative control group,an HNE treatment group,a naringenin pre-treatment group,a LY294002 pre-treatment group and a naringenin ＋ LY294002 group.The changes of MUC5AC mRNA was examined by the use of reverse transcriptase-polymerase chain reaction（RT-PCR）.The protein expression changes of PI3K,PKB,P-PKB and NF-κB were measured by Western blot.The protein expression changes of MUC5AC were detected by enzyme-linked immunosorbent assay,while the protein morphological changes of MUC5AC were observed by immunofluorescence and confocal laser technology.Results：The expression levels of MUC5ACmRNA and its protein were decreased significantly as compared with the control group.The protein expression changes of PI3K,PKB,P-PKB and NF-κB were increased significantly as compared with the control group.After the cells were pre-treated with naringenin,the above measurements were decreased significantly as compared with the HNE group.The decrease was more significant in the naringenin＋ LY294002 group.Conclusions：Naringenin inhibited activiation of NF-κB and attenuated airway mucous hypersecretion via the signal transduction pathway of PI3K/PKB.