miR-21调控PTEN/Akt/mTOR信号通路诱导上皮-间质转化在肺纤维化中的作用及补阳还五汤干预机制研究

As a refractory disease of respiratory medicine, pulmonary fibrosis belongs to the category of "lung paralysis" in traditional Chinese medicine, its pathogenesis is Qi deficiency and blood stasis. The research group used Buyang Huanwu decoction, which has the effect of tonifying qi, removing blood stasis and dredging collaterals, to treat pulmonary fibrosis and achieved satisfactory curative effect. The previous study found that Buyang Huanwu Decoction not only effectively relieved the degree of alveolitis and pulmonary fibrosis in bleomycin-induced pulmonary fibrosis in rats, but also inhibited the expression of CTGF, p-AKt protein and mRNA in rats, and inhibited TGF-β1. Induction of A549 cells EMT and PI3K, AKt protein expression. Studies have confirmed that miR-21 as a PTEN negative regulatory gene may induce EMT through the regulation of PTEN/AKT/mTOR pathway plays an important role in the formation of fibrosis. Combining with the work foundation, this study proposes a scientific hypothesis that the preventive action of Buyang Huanwu decoction on pulmonary fibrosis may be related to the intervention of EMT which is induced by miR-21-regulated PTEN/Akt/mTOR pathway. Through the animal experiments and in vitro cell experiments, using immunology and molecular biology techniques to explore the mechanism of Buyang Huanwu decoction’s intervention of EMT which is induced by miR-21-regulated PTEN/Akt/mTOR pathway, and provides new ideas and methods for the clinical treatment of pulmonary fibrosis.

肺纤维化为呼吸内科难治性疾病，属中医“肺痹”，病机是气虚血瘀。据此，课题组运用益气化瘀通络方剂补阳还五汤治疗肺纤维化，取得了满意疗效。前期研究发现补阳还五汤有效缓解博莱霉素诱导的肺纤维化大鼠肺泡炎及肺纤维化程度，抑制大鼠的CTGF、p-AKt蛋白及mRNA的表达，并抑制TGF-β1诱导的A549细胞EMT及PI3K、AKt蛋白表达。已有研究证实miR-21作为PTEN负性调控基因，可能通过调节PTEN/AKT/mTOR通路诱导EMT在纤维化形成中起重要作用。结合工作基础，本研究提出补阳还五汤干预肺纤维化的作用可能与干预miR-21调控的PTEN/Akt/mTOR通路诱导EMT有关的科学假说，拟借助动物实验及体外细胞实验，运用免疫学及分子生物学技术，探讨补阳还五汤干预miR-21调控的PTEN/Akt/mTOR通路诱导EMT的作用机制,为中医治疗肺纤维化提供新的思路与方法。

肺纤维化是间质性肺疾病的最终病理结局，其典型特征是肺间质中成纤维细胞分泌胶原，细胞外基质过度沉积，上皮-间质转化且普遍伴有转化生长因子-β及多种炎症介质的表达增加。根据临床表现及病理特征认为本病属于中医“肺痹”范畴，病机是气虚血瘀。据此，课题组提出运用益气化瘀通络方剂补阳还五汤防治肺纤维化。.本研究在前期研究的基础上，进一步研究补阳还五汤干预肺纤维化的作用及机制。通过动物实验发现，补阳还五汤能明显抑制博莱霉素诱导大鼠血清中的HYP、ColⅠ、ColⅢ 表达水平（P＜0.01）。补阳还五汤能明显抑制博来霉素诱导大鼠肺组织中的Vimentin、α-SMAmRNA， 增强E-cadherin mRNA表达（P＜0.01），抑制肺纤维化大鼠肺组织中的 miR-21蛋白表达（P＜0.01），p-Akt、 p-mTOR蛋白的表达降低，但无统计学意义（P＞0.05 ），增加PTEN蛋白表达（P＜0.05）。.通过体外细胞实验发现，补阳还五汤能使A549细胞TGF-β1诱导E-cadherin蛋白荧光颜色加深，Vimentin、ColⅠ蛋白荧光颜色变浅；补阳还五汤能够抑制IGF-I诱导的A549细胞p-PI3K的表达（P＜0.05），抑制SC79诱导的A549细胞p-PI3K和p-Akt的蛋白的表达（P＜0.05）。 PTEN基因沉默后Vimentin 表达增强，E-cadherin 表达减轻，补阳还五汤通过影响PTEN基因沉默后的A549细胞Vimentin 及E-cadherin 表达，从而干预PTEN基因沉默后的EMT。补阳还五汤还能抑制PTEN基因沉默后的A549细胞的PTEN、AKT、p-AKT、PI3K、p-PI3K、mTOR的表达（P＜0.05或P＜0.01）；补阳还五汤能够抑制miR-21 mimic转染的A549细胞Vimentin、α-SMA、PTEN、COL-1mRNA，提升E-cadherin、PTENmRNA（P＜0.05）。补阳还五汤还能抑制mIR-21mimic转染A549细胞的p-PI3K和p-mTOR蛋白的表达，增加PTEN蛋白表达。.综上，益气活血化瘀的经典方剂补阳还五汤具有抗肺纤维化作用，其作用机制可能是通过调控miR-21、PTEN/Akt/mTOR 信号通路从而抑制 EMT 延缓了肺纤维化的进程。本研究对中医药防治肺纤维化提供新的思路与方法