摘要
目的探讨柚皮素对炎性刺激时气道上皮细胞黏液高分泌的作用和相关的分子机制。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,构建炎性刺激时气道黏液高分泌模型,分别以柚皮素和活性氧(ROS)清除剂DMTU进行干预,用活性氧试剂盒测定各组细胞中ROS的含量,观察黏蛋白(MUC)5AC、表皮细胞生长因子受体(EGFR)和磷酸化EGFR(P-EGFR)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、DMTU组、柚皮素组和柚皮素+DMTU组。逆转录PCR方法检测各组MUC5ACmRNA、EGFRmRNA的变化;Westernblot法检测EGFR和P-EGFR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。结果不同浓度的HNE处理A549后,细胞内ROS的含量逐渐增加,而柚皮素预处理组与50nmol/L的HNE刺激组相比细胞内ROS的含量明显减少(P<0.01);HNE处理组MUC5AC的mRNA和蛋白的吸光度面积积分值分别为(0.92±0.08)和(152±6)μg/mg,EGFRmRNA和蛋白的吸光度面积积分值分别为(0.91±0.05)和(0.86±0.03)μg/mg,均较对照组([0.70±0.02),(112±4)μg/mg和(0.73±0.10),(0.39±0.03)μg/mg]明显升高(P<0.05),P-EGFR和总EGFR蛋白表达均较对照组显著增加(P<0.01);给予柚皮素预处理后,与HNE刺激组相比,EGFR和P-EGFR表达下调;而柚皮素+DMTU组MUC5ACmRNA和MUC5AC的蛋白较单独DMTU处理组更为明显,其吸光度和面积积分值分别为(0.40±0.03)和(113±2)μg/mg(P<0.01),差异均有统计学意义。结论HNE可刺激气道上皮细胞产生大量ROS,柚皮素可抑制ROS的产生、下调ROS诱导的EGFR表达水平、并部分阻断EGFR磷酸化,从而抑制炎性刺激时气道黏液高分泌的形成。
【Objection】To investigate the effects of Naringenin on airway mucous hypersecretion under inflammation and the relative molecular mechanism. 【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 ROS scavenger DMTU, respectively. The relative content of reactive oxygen species （ROS） were assayed by special kit. The expression of mucin （MUC）5AC, EGFR, P-EGFR 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 DMTU pre-treatment group and a Naringenin ＋ DMTU group. The changes of MUC5ACmRNA and EGFRmRNA were examined by the use of reverse transcriptase-polymerase chain reaction （RT-PCR）. The protein expression changes of EGFR and P-EGFR 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. The data were analyzed with SPSS 13.0 software. Differences between groups assessed for significance by t-test. 【Results】A dose-dependent increasing of ROS production in cells exposed to HNE was detected, after the cells were pre-treated with Naringenin, the content of ROS was decreased significantly as compared with the 50nmol/L HNE group （P 0.01）. The expression levels of MUC5ACmRNA and its protein in the HNE group were 0.92 ± 0.08 and （152 ± 6） μg/mg, and those of EGFR were 0.91 ± 0.05 and （0.86 ± 0.03） μg/mg, both of them increased significantly as compared to those in the control group [0.70 ± 0.02, （112 ± 4） μg/mg and 0.73 ± 0.10, 0.39 ± 0.03] （P 0.05）. The protein expression changes of EGFR and P-EGFR were increased significantly as compared with the control group （P 0.01）. 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＋DMTU group （P 0.01）.【Conclusions】Naringenin inhibited the production of ROS, decreased the level of EGFR by modulating ROS and blocked the phosphorylation of EGFR, and attenuated airway mucous hypersecretion under inflammation.
出处
《中国现代医学杂志》
CAS
CSCD
北大核心
2010年第5期726-731,共6页
China Journal of Modern Medicine
基金
国家自然科学基金(No:30770951)
