脂氧素A_4通过p38 MAPK及Nrf2通路调控气道炎症反应

Lipoxin A_4 regulation of LPS induced pro-inflammatory responses through inhibiting activation of p38 MAPK and activating Nrf2 pathway

目的:研究脂氧素A_4(LXA_4)对脂多糖(LPS)诱导的人正常支气管上皮细胞炎症反应的抑制作用及其机制。方法:将对数生长期的BEAS-2B细胞分为3组。对照组:不做任何处理;LPS组:100 ng/mL LPS刺激24 h;LPS+LXA_4组:100 nmol/L LXA_4预处理30 min,加入100 ng/mL LPS刺激24 h。qPCR法检测IL-6、IL-1β、血红素加氧酶-1(HO-1)、醌氧化还原酶(NQO-1)mRNA表达水平;流式细胞术测定胞内活性氧(ROS)水平;谷胱甘肽(GSH)试剂盒检测GSH水平。为进一步了解LXA_4的作用机制,Western blot法检测各处理组p38的磷酸化水平以及Nrf2的核转位及磷酸化水平。结果:与对照组相比,LPS组IL-6、IL-1β mRNA以及胞内ROS表达水平升高(P<0.05),HO-1 mRNA水平下降(P<0.01),p38磷酸化水平上升(P<0.01),胞核中Nrf2相对表达量下降(P<0.01),总Nrf2磷酸化水平下降(P<0.05)。经LXA_4干预后,与LPS组相比,除上述改变逆转外(P<0.05),NQO-1和GSH水平显著上升(P<0.05)。结论:LXA_4可减轻LPS引起的BEAS-2B细胞炎症反应并促进炎症消退,其机制可能一方面与抑制p38 MAPK通路,减少促炎因子的分泌有关;另一方面与增强Nrf2的核转位以及磷酸化,减轻氧化应激损伤有关。

Objective： To investigate the effect and mechanism of Lipoxin A4（LXA4） on inflammatory response induced by lipopolysaccharide（LPS） in BEAS-2 B cells. Methods： Cultured BEAS-2 B cells in logarithmic growth phase were divided into 3 groups： control group（group A, non-treatment）, LPS group（group B, incubated with 100 ng/mL LPS for 24 h）, LPS＋LXA4 treatment group（group C, pretreated with 100 nmol/L LXA4 for 30 min and incubated with 100 ng/mL LPS for 24 h）. The m RNA levels of IL-6, IL-1β, heme oxygenase（HO-1） and NAD（P） H： quinone oxidoreductase（NQO-1） were detected by q PCR. The expression of reactive oxygen species（ROS） was detected by flow cytometric analysis. Glutathione（GSH） was measured by a GSH assay kit. Moreover, we investigated the effects of LXA4 on LPS-induced phosphorylation of p38 mitogen-activated protein kinases（MAPK） as well as nuclear translocation and phosphorylation of Nrf2. Results： Compared with group A, the mRNA expressions of IL-6, IL-1β and the level of ROS in group B were significantly increased（P〈0.05）, while the mRNA level of HO-1 in cells was significantly decreased（P〈0.01）. Nuclear translocation and phosphorylation of Nrf2 were reduced（P〈0.05） and phosphorylation of p38 MAPK was significantly increased（P〈0.01） after LPS stimulation. In contrast, in LXA4 treatment group, the above changes were reversed（P〈0.05）; besides, GSH activity and the mRNA level of NQO-1 were elevated（P〈0.05）. Conclusion： LXA4 may attenuateLPS-induced pro-inflammatory responses in BEAS-2 B cells which is probably associated with reduced secretion of pro-inflammatory cytokine via inhibiting p38 MAPK pathway and alleviated oxidative stress through activating Nrf2.