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。