ATF3-MKP-1通路在慢性氧化应激所致支气管哮喘激素抵抗中的作用及机制研究

激素抵抗(SR)性哮喘由于机制不明一直是临床治疗难题。课题组前期研究证实慢性氧化应激(OS)阻抑丝裂原蛋白激酶磷酸酶(MKP)1表达/活性与SR有关，但具体阻抑机制不明。文献及预实验提示转录激活因子（ATF）3可上调MKP-1的表达/活性，而前期研究证实慢性OS可抑制ATF3的表达，后者则被发现与SR哮喘有关。据此推测慢性OS通过下调ATF3表达，进而抑制MKP-1表达/活性，从而引起SR。为此，我们拟利用OVA联合臭氧构建SR哮喘模型以及功能补强实验，验证ATF3对MKP-1的调控及该通路在慢性OS所致SR中的作用；通过ATF3-/-哮喘模型、双向调控细胞ATF3和MKP-1表达，体内外验证ATF3-MKP-1通路对激素抑炎效应的调控；利用凝胶迁移实验、双荧光素酶基因报告系统、免疫共沉淀等手段从分子水平探讨ATF3对MKP-1表达/活性的调控机制；从而为SR哮喘的防治提供新思路和新靶点。

Asthmatic patients with steroid resistance (SR) are difficult to be controlled, mostly due to its related mechanisms remaining illusive. We have previously demonstrated that chronic oxidative stress (OS)-induced inhibition on the expression of mitogen kinase phosphatase 1 (MKP-1) is involved in the pathogenesis of SR, but the inhibitory mechanisms are not known. Literature and our preliminary experiment indicated potential promoting effect of activating transcription factor 3 (ATF3) on the expression of MKP-1 in airway epithelial cells. We previously demonstrated that chronic OS inhibited the expression of ATF3 in airway epithelial cells, while the expression of ATF3 was found to be downregulated in lungs of SR asthmatic patients. Therefore, we hypothesized that chronic oxidative stress might inhibit the expression of ATF3, which leads to the downregulation of MKP-1 expression in lungs, and hence causes SR. To prove this hypothesis, we plan to explore the influence of ATF3 on the pulmonary expression of MKP-1 and their involvements in the development of SR, utilizing a steroid-resistant asthma model established with repeated ozone exposures. Using as an allergic asthma model that established with ATF3-/- mice and by in vitro tuning the expression of ATF3 and MKP-1 bidirectionally in airway epithelial cells, we plan to demonstrate the direct influence of ATF3-MKP-1 pathway on the anti-inflammatory action of glucocorticoids. The fundamental mechanisms of ATF3 regulating the expression of MKP-1 is also a part of this research plan, which will employ some techniques, including electrophoretic mobility shift assay, dual-luciferase reporter assay system, and immunoprecipitation assays. The completion of this study will offer new thoughts as well as targets on the prevention and treatment of steroid resistant asthma.