维生素A调节PI3K/Akt通路抑制哮喘气道上皮凋亡的机制研究

Asthma contributes to significant increases in the numbers of patients and presents a marked economic burden, seriously threatening children's health. Glucocorticoids (GCs) are the first-line drugs for asthma treatment but appear to be imperfect therapies because they lead to airway epithelial apoptosis and injure airway wall, which might weaken the treatment of GCs on asthma and even lead to refractory asthma. Vitamin A (VA) deficiency is common in asthmatic patients. We have found that VA exhibits combined effects with GCs on the relief of airway hyperresponsiveness and the decrease of airway epithelial apoptosis. PI3K/Akt signal pathway is critical for inhibiting cell apoptosis, and it might be regulated by both GCs and VA. We suppose that GCs might inhibit PI3K/Akt pathway and further increase airway epithelial apoptosis, but VA might attenuate the inhibition of GCs on PI3K/Akt pathway and further decrease excessive apoptosis of airway epithelial cells in asthma. In this study, we will conduct a further study both in vitro and in vivo trying to answer the following questions: firstly, whether VA has anti-apoptosis effect on airway epithelial cells? Secondly, whether can VA activate PI3K/Akt signaling pathway and further weaken apoptosis-promoting effect of GCs on airway epithelial cells? If VA supplementation activates PI3K/Akt signaling pathway and further inhibits airway epithelial apoptosis, it might neutralize the side effects of GCs on airway epithelial cells. It might provide new methods for the asthma therapy, which might reduce the recurrence rate and mortality of asthma.

哮喘严重威胁儿童健康。糖皮质激素（GCs）是目前治疗哮喘最有效的一线药物，但它在控制哮喘气道炎症的同时也诱导了气道上皮凋亡，可能削弱哮喘疗效，甚至导致难治性哮喘。维生素A（VA）缺乏在哮喘中比较普遍，我们发现VA可协同GCs降低哮喘小鼠气道高反应性，并可能抑制气道上皮凋亡。PI3K/Akt通路是抑制细胞凋亡的关键通路。我们推测GCs抑制PI3K/Akt通路从而促进气道上皮凋亡；而VA可能活化PI3K/Akt通路，削弱GCs对后者的抑制作用。本课题立足临床，通过细胞培养和小鼠动物实验，从形态、功能和分子水平多方面探讨：①论证VA对气道上皮的抑凋亡作用；②论证VA通过活化PI3K/Akt信号通路，进而削弱GCs对哮喘气道上皮的促凋亡作用，在 GCs 治疗哮喘时补充VA，抑制哮喘气道炎症的同时抑制气道细胞过度凋亡，为提高哮喘治疗效果，降低哮喘复发率和死亡率提供新的思路和方法。

哮喘严重威胁儿童健康。糖皮质激素（GCs）是目前治疗哮喘最有效的一线药物，但它在控制哮喘气道炎症的同时也诱导了气道上皮凋亡，可能削弱哮喘疗效，甚至导致难治性哮喘。维生素A（VA）缺乏在哮喘中比较普遍，我们发现VA可协同GCs降低哮喘小鼠气道高反应性，并可能抑制气道上皮凋亡。PI3K/Akt通路是抑制细胞凋亡的关键通路。我们推测GCs抑制PI3K/Akt通路从而促进气道上皮凋亡；而VA可能活化PI3K/Akt通路，削弱GCs对后者的抑制作用。本课题立足临床，通过细胞培养和小鼠动物实验，从形态、功能和分子水平多方面探讨：①论证VA对气道上皮的抑凋亡作用；②论证VA通过活化PI3K/Akt信号通路，进而削弱GCs对哮喘气道上皮的促凋亡作用，在 GCs 治疗哮喘时补充VA，抑制哮喘气道炎症的同时抑制气道细胞过度凋亡，为提高哮喘治疗效果，降低哮喘复发率和死亡率提供新的思路和方法。. 体外研究发现，10 uM 地塞米松（dexamethasone, Dex）和/或全反式维甲酸（ATRA）24小时干预人支气管上皮（16HBE）细胞，相对于对照组，TUNEL流式细胞术发现Dex使16HBE细胞核DNA断裂比例增加；Annexin V/PI双染法发现Dex使16HBE细胞Annexin V阳性蛋白上调，JC-1检测提示Dex使16HBE细胞线粒体膜电位下降、Western blot检测提示Dex使16HBE细胞凋亡标志蛋白Caspase-3和Cleaved-Caspase3的水平均上调。以上指标，ATRA组与对照组无明显差异；较单独Dex组，ATRA联合Dex则使以上指标下降，但仍高于对照组。.体内研究发现，Dex组哮喘小鼠气管上皮病理损伤较asthma组加重，同时肺组织E-cadhein表达较asthma组降低，Caspase3及Cleaved-Caspase3表达较asthma组升高。VA组AHR较asthma组降低，但BALF细胞计数、肺病理学检测结果与Dex组无明显差异。Dex+VA组AHR低于Dex组，且气道上皮损伤较Dex组明显减轻，同时肺组织E-cadherin表达较Dex组增加，caspase3及Cleaved-Caspase3表达较Dex组降低。.总之，Dex可能引起哮喘气道上皮凋亡，VA可能减轻这一副作用，具体机制仍需进一步研究。