宣肺通腑法调控mTOR/HIF-1α/VEGF信号通路治疗急性肺损伤的机制研究

The acute lung injury remains a difficult problem in the field of modern seriously injured medicine. The epidemiologic statistics indicate that the fatality rate of this hurt still reaches as high as 30-50%. Therefore studies on its pathogenesis focusing on a new treatmen, has the vital significance in the early treatment of the acute lung damages. Researches suggest that the imbalance of the inflammatory response is the essential reason of acute lung injury and it is difficult to obtain satisfactory results for relying solely on anti-inflammatory medication ALI. On the basis of the research of inflammation reaction in the acute lung injury imbalances and TLR4-NF-kB pathway, the project has studied the mechanism of Xuanfeitongfu therapy for LPS-induced acute lung injury by regulating mTOR/HIF-1ɑ/VEGF signaling pathway through the establishment of ALI model, PMVEC cells and IMMVEC cells and RAW264.7 cellsin vitro model induced by LPS., offered experimental groundwork and theory basis to cure ALI, and revealed the scientific connotation of "the lung and the large intestine exterior and interior" in Chinese medicine by the mTOR/HIF-1 ɑ/VEGF signaling pathway.

急性肺损伤是现代危重医学的一大难题。流行病学资料表明其病死率仍高达30-50％。因此研究其发病机制，针对新的治疗靶点对于急性肺损伤的早期治疗有重要意义。研究认为炎症反应的失衡是急性肺损伤的本质原因，单纯依靠抗炎药物治疗急性肺损伤难以取得满意效果。本项目在研究急性肺损伤炎症反应失衡及TLR4-NF-kB通路的基础上，通过建立内毒素肺损伤体内模型和LPS诱导鼠肺血管内皮细胞、肠黏膜内皮细胞、RAW264.7细胞体外模型,研究宣白承气汤调控mTOR/HIF-1α/VEGF信号通路治疗急性肺损伤的作用机制，为宣肺通腑法治疗急性肺损伤提供科学依据和理论基础，从mTOR/HIF-1α/VEGF信号通路揭示中医“肺与大肠相表里”的科学内涵。

本研究认为炎症反应失衡的急性肺损伤的本质原因，临床数据及前期实验表明宣肺通腑法及其代表方宣白承气汤可改善急性肺损伤并具有良好的抗炎效果，结合前期研究我们推测宣白承气汤可通过调控mTOR/HIF-1α/VEGF信号通路治疗急性肺损伤并发挥抗炎作用。本项目重点观察了宣白承气汤或其拆方宣肺方、通腑方组大鼠肺、肠组织HE病理染色，肺组织干湿重测定，支气管肺泡灌洗液TNF-α、IL-1β 和 IL-6蛋白的含量，确定了宣白承气汤或其拆方能抑制LPS诱导的ALI炎症反应，并利用网络药理学结合高通量测序，筛选出mTOR、HIF-1α、VEGF等相关通路，用Western blot和免疫组化法检测肺、肠组织PI3K、Akt、mTOR、HIF-1α和VEGF蛋白表达，用RT-qPCR法检测肺泡巨噬细胞EIF4e、EIF4ebp1、Rps6kb1 mRNA表达。在用HPLC-GC/MS方法获得宣白承气汤主要活性成分后，我们用LPS刺激PMVECs、IMMVECs、RAW264.7细胞，检测促炎因子的表达和释放以及相关蛋白和基因表达，并用siRNA转染HIF-1α，观察VEGF的表达变化。此外我们增加了通腑方的主要有效成分大黄素通过mTOR/HIF-1α/VEGF途径抗ALI炎症反应的作用。我们的结果证实，宣白承气汤在动物和细胞水平上均可以抑制mTOR/HIF-1α/VEGF信号通路及其关键靶基因，并且动物实验中肺、肠组织相关实验结果趋近一致，这为宣白承气汤改善ALI炎症反应提供了强有力的证据，也为“肺肠相关”理论提供了实验依据。