基于mTOR/SIRT1信号通路的补肺益肾方调控肺泡上皮细胞治疗COPD机制研究

Chronic obstructive pulmonary disease (COPD) is a major public health problem worldwide and results in an economic and social burden, and without appropriate pharmacologic therapy. Cell apoptosis and autophagy are one of the principal physiopathologic mechanism of COPD. There is increasing evidence that signalling though the mammalian target of rapamycin (mTOR) / Sirtuin type 1（SIRT1） pathway is of critical importance in cellular senescence and ageing, It is reported that treatment with traditional Chinese medicine (TCM) may do some benefit to the disease. .On the basis of previous research, The aim of the study is to investigate the cellular and molecular mechanisms of TCM. Our work focuses on two parts: ①we preparing animal models of stable COPD, taking rapamycin as the inhibitors, evaluating the therapeutic effect of TCM on animal models by pulmonary function, cytokine expression in blood and BALF, pathology and ultrastructure. By way of immunohistochemistry, TUNEL、qPCR and western blot method, investigating mechanism of Bufei Yishen decoction on COPD by evaluating lung tissue apoptosis and autophagy and related gene and protein expression of mTOR/ SIRT1 pathway.②Culturing of alveolar epithelial cells A549, and preparing containingTCM medicine serum. By activated of cigarette smoke extract and inhibited by rapamycin and sirtinol, the study is to investigate the secretion of cytokines of A549 cells and cell apoptosis and autophagy, and the serum was used to do inhibitory tests in vitro. The related gene and protein of mTOR/SIRT1 pathway were detected by molecular biotechnology such as q-PCR、western blot method. The experiment would investigate the related mechanism of TCM on COPD, and would provide scientific evidence for research of new drugs and TCM clinical application.

慢性阻塞性肺疾病（COPD）严重危害公众健康，目前缺乏理想的治疗方法。细胞凋亡和自噬是COPD的重要病理生理机制，mTOR/SIRT1信号通路在此过程中起着重要作用，基于以往研究，进一步探讨补肺益肾方治疗COPD的作用机制，拟开展：①制备大鼠COPD稳定期模型，以阻断剂雷帕霉素为对照，从肺功能，外周血、肺泡灌洗液细胞因子表达，肺组织病理和超微结构观察补肺益肾方治疗COPD的疗效；采用免疫组织化学、TUNEL、qPCR、WB技术，从肺组织细胞凋亡、自噬及mTOR通路相关基因和蛋白表达，探讨补肺益肾方治疗COPD的作用机制。②以肺泡上皮细胞为研究对象，以雷帕霉素和Sirtinol为阻断剂，采用细胞培养、分子生物学等技术，围绕mTOR信号通路，观察补肺益肾方含药血清对香烟烟雾刺激下肺泡上皮细胞分泌细胞因子及细胞凋亡和自噬等影响。以揭示中医药治疗COPD作用机制，为中医临床应用和新药研发提供依据。

目的：基于mTOR/SIRT1信号通路观察中药补肺益肾方对慢性阻塞性肺疾病（COPD）模型大鼠和CSE诱导的A549细胞凋亡、自噬的影响，探讨补肺益肾方治疗COPD的疗效和作用机制。方法：①建立大鼠COPD稳定期模型，随机分空白组、模型组、补肺益肾组、雷帕霉素组和联合组。WBP法检测大鼠肺功能；显微镜和透射电镜观察肺组织形态学和超微结构；ELISA法检测细胞因子水平；TUNEL法检测细胞凋亡率；免疫组织化学、qPCR、Western blot等技术检测mTOR/SIRT1信号通路相关基因和蛋白表达。②以肺泡上皮细胞A549为研究对象，ELISA法检测细胞因子水平；Annexin V/PI双染色法检测细胞凋亡；qPCR、Western blot等技术检测mTOR/SIRT1信号通路相关基因和蛋白表达。 结果：①COPD大鼠表现出精神倦怠，活动量减少，体重增长减慢，肺功能降低等，各治疗组均可改善上述症状，以联合组改善最明显。②COPD大鼠肺组织表现为肺气肿和小气道重塑等，超微结构上可见呼吸膜不完整、Ⅱ型细胞线粒体减少、嵴消失，各治疗组均可不同程度改善上述情况，以联合组改善最明显。③模型组大鼠血清、BALF中以及CSE刺激A549细胞分泌的1L-1β、IL-4、IL-6、IL-23、TNF-α、FGF、VEGF水平均较空白对照组升高，补肺益肾方和雷帕霉素可不同程度降低上述指标表达。④COPD大鼠肺组织中骨桥蛋白、波形蛋白表达均较空白组升高，而上皮型粘钙蛋白降低，各治疗组均可不同程度降低或升高表达。⑤模型组大鼠和CSE刺激的A549细胞中PI3K、AKT、4EBP1、ULK1、ATG8、ATG10、ATG14、SIRT1、FOXO1、BNIP3、FasL、P21、PEPCK、Caspase3基因和蛋白表达均较空白组显著升高；而mTOR、p70S6K、Bcl-6的基因和蛋白表达较空白组显著降低，补肺益肾方和雷帕霉素均可改善上述指标水平；⑥COPD模型组大鼠和CSE刺激A549细胞凋亡较空白组增加，补肺益肾方和雷帕霉素均不同程度减少细胞凋亡，Sirtinol增加凋亡。结论：补肺益肾方可减轻COPD肺泡上皮细胞凋亡、增加自噬、改善肺功能、减轻肺部炎症反应及肺脏病理损伤，其机制可能与下调mTOR、上调SIRT1，进而调控mTOR/SIRT1信号通路有关。