基于调控PTEN/PI3K/Akt信号转导通路抑制炎症反应补肺益肾方治疗COPD的作用机制研究

Chronic obstructive pulmonary disease (COPD) is a major cause of chronic morbidity and mortality throughout the world. Chronic inflammation plays the pivotal role in pathogenesis of COPD. The therapeutic mechanism of Bufei Yishen granules(BYF), a special prescription for lung-kidney qi deficiency syndrome with confirmed curative effects, is yet illusive on COPD. Phosphatase and Tensin Homolog Deleted from Chromosome 10 (PTEN), which is down-regulated in patients with COPD in the lung, is a well-described negative regulator of PI3K, leading to the inactivation of downstream target Akt. Our preliminary study proved that BYF could significantly increased the PTEN protein levels, which was reduced by the cigarette smoke extract (CSE) in COPD rats lung tissues. In addition, BYF could activate the PPAR-γ signaling, and elevate the expressions of PPAR-γ at RNA level and the phosphorylation of the protein in COPD rats. We next aim to explore how BYF inhibit the inflammation of COPD. First, the activation of NF-κB would be detected by EMSA in BEAS-2B cells treated with PI3K inhibitor wortmannin after CSE exposure. Second, PTEN-/- BEAS-2B cells treated with Rosiglitazone were used to further confirm that the anti-inflammation effects of BYF for COPD via PTEN/PI3K/Akt/IKKβ signal pathway in PPAR-γ dependent manner. In addition, the key amino acid site and interaction domain between PPAR-γ and PTEN promoter would be screened by dual-luciferase assay. This study provides the basis for mechanisms of BYF on COPD, and as reseach continues, provide us a new therapeutic drug targets.

本项目以COPD是一种慢性炎症疾病为出发点，探究PTEN在补肺益肾方（BYF）治疗COPD中的作用。预实验发现BYF可明显提高COPD大鼠肺组织中PTEN的表达，抑制PI3K/Akt信号转导通路激活。本项目拟采用COPD大鼠及BEAS-2B细胞烟雾模型，利用分子生物学技术检测BYF 对PI3K/Akt 信号通路及其下游炎症因子的影响；通过PI3K特异性抑制剂渥曼青霉素处理细胞，采用EMSA法检测NF-κB活性，明确BYF对PI3K/Akt/IKKβ的调控作用；采用CRISPR/Cas9系统构建PTEN缺失细胞系，PPAR-γ激动剂罗格列酮刺激后考查BYF对PTEN的调控是否依赖PPAR-γ；采用截短或点突变技术结合双荧光素酶检测技术筛选PPARγ与PTEN启动子结合的结构域及关键氨基酸位点。探讨补肺益肾方调控PTEN/PI3K/Akt通路治疗COPD的慢性炎症机制，为其防治提供新靶点。

慢性炎症伴随慢性阻塞性肺疾病（COPD）的发生与发展，10号染色体缺失与张力蛋白同源物磷酸酯酶（PTEN）活性降低和PI3K/Akt信号转导异常激活在该环节中起着关键作用。本项目基于确证的补肺益肾方治疗COPD临床疗效，从激活PTEN以调控PI3K/Akt信号转导角度探讨补肺益肾组分方抑制COPD炎症反应的机制。通过研究得出以下结论：①补肺益肾组分方可显著改善COPD大鼠症状，调控PTEN/PI3K/Akt信号转导：补肺益肾组分方高、中剂量和氨茶碱均可改善香烟熏吸联合细菌感染诱导COPD大鼠的肺组织病理、肺功能、炎细胞浸润；下调炎症相关细胞因子水平；补肺益肾组分方还可上调肺组织中PTEN 表达水平，下调p-PI3K、p-Akt、p-P65表达水平，说明补肺益肾组分方的抗炎作用可能与调控PTEN/PI3K/Akt有关。②补肺益肾组分方及其组分配伍可通过调控PTEN/Akt/PI3K抑制BEAS-2B细胞炎症反应：以香烟烟雾诱导的BEAS-2B细胞炎症模型为研究对象，补肺益肾组分方及其组分配伍（补肾、补气和补肾补气）均可上调PTEN表达水平；下调PI3K、Akt、IκBα、NF-κB（P65）的磷酸化水平；减少炎症相关细胞因子分泌，并且补肺益肾组分方不同组分配伍对炎症反应的抑制作用有所差异，以补肾补气组分配伍最佳。采用CRISPR-Cas9技术构建PTEN敲除（PTEN-/-）的BEAS-2B细胞系，PTEN敲除后PI3K、Akt、IκBα和P65磷酸化水平显著升高；P65大量入核，且补肾补气组分配伍不可逆转上述作用，表明PTEN在补肺益肾组分方调控PI3K/Akt/NF-κB抑制炎症反应中起着关键作用。.综上，本研究证实了补肺益肾组分方可通过调控PTEN/PI3K/Akt信号转导通路抑制炎症反应，为补肺益肾方临床应用及进一步研究提供理论依据。