基于TLR-4/NF-κB信号通路探讨益气活血法治疗内毒素性急性肺损伤的分子机制

Acute lung injury (ALI) is one of the common clinical critical diseases, which identified with the features of difficult treatment and high mortality. Sepsis induced by lipopolysaccharide (LPS) is an important inducement of ALI. Toll like receptor 4 (TLR-4) is considered to be an important receptor mediated by LPS signal transduction. It is known that the TLR-4 targeted intervention for ALI occurrence and development has been the hot point in the recent research. Our previous studies found that the YiQiHuoXue therapy had protective effects on ALI in rats.The current study was designed to evaluate the alveolar and interstitial pulmonary ultrastructural changes of LPS induced ALI rats model by YiQiHuoxue Decoction treatment. The biological methods, including immunohistochemical, ELISA, western-blot and RT-PCR were applied to evaluate the changes of TLR-4, IRAK-M, NF- κB and inflammatory factor levels, which are all components of the TLR-4/NF-κB signal pathway. The study focused on the changes of TLR-4 and the inhibition of inflammatory cascade. The mRNA, protein, and tissue pathology methods were applied to detect the above mentioned factors and indictors. The traditional Chinese medicine (TCM) has the superiority of multi-target, multi-links, high cost-effect, and mild adverse effects. The aim of this study was to clarify the potential therapeutic target and molecular rationale of TCM on the treatment of ALI.

急性肺损伤（Acute lung injury,ALI）为常见危重症，治疗困难、病死率高。Toll样受体4（Toll like receptor 4,TLR-4）是介导LPS诱发脓毒症及ALI的重要原因。以TLR-4为靶点干预ALI的发生、发展是近年研究热点。我们既往研究发现益气活血法对ALI大鼠肺具有保护作用。本研究制备LPS诱导的ALI大鼠模型，应用益气活血中药进行干预，观察用药前后肺泡及肺间质超微结构变化，采用免疫组化、ELISA、Western-blot及RT-PCR技术检测TLR-4/NF-κB信号通路中TLR-4、IRAK-M、NF-κB、炎症因子等变化。以TLR-4为核心，从抑制炎症级联反应角度出发，在mRNA、蛋白及病理组织水平上探讨益气活血法改善ALI肺组织炎症的作用靶点与机理，发挥中医药多层次、多环节优势，探索治疗靶点、分子机制及量效关系，为临床治疗ALI提供理论依据。

背景：急性肺损伤(ALI)为临床常见急危重症，病死率可高达40%-50%。通过既往研究，我们认为“气虚血瘀”是ALI基本病理状态，采用益气活血中药防治ALI。ALI的发生发展与TLR-4/ NF-κB信号通路活化密切相关。本实验基于此信号通路进一步探讨益气活血法对LPS诱导ALI大鼠防治作用和分子机制。内容：①模型研究：LPS组气管内滴注LPS制备ALI模型；测定血气分析、W/D比值、BALF中蛋白含量，观察肺病理。②防治研究：造模前3天中药Ⅱ组中药灌胃；后3天中药Ⅰ、Ⅱ组中药灌胃，对照组予以地塞米松溶液灌胃；测定大鼠W/D比值，BALF总蛋白及肺病理学改变。③机制研究：造模前后3天中药组每天以中药灌胃；于末次灌胃2h后处死大鼠，检测肺TLR-4、IRAK-1、NF-κB蛋白含量及mRNA表达量，BALF中炎症因子TNF-α、IL-1β、IL-6的浓度。结果：①模型组PaCO2、W/D比值、BALF中蛋白浓度均高于正常组，PaO2低于正常组；光镜下造模组肺泡结构破坏，肺间隔增厚、充血水肿，肺泡腔内大量炎性粒细胞浸润。②与模型组比较，药物干预组W/D比值、BALF中蛋白浓度及病理学评分均降低；中药Ⅱ组W/D比值、病理学评分与对照组无差异，且低于中药Ⅰ组，中药Ⅱ组BALF蛋白浓度高于对照组，低于中药Ⅰ组。③模型组和中药组BALF中TNF-α、IL-1β、IL-6浓度均高于正常组，与模型组比较，中药组浓度降低；与正常组相比，模型组和中药组肺中TLR-4、IRAK-1、NF-κB mRNA和蛋白表达均升高，而中药组低于模型组。结论：①通过暴露式气管内滴注LPS能够成功诱导ALI大鼠模型。②黄芪-丹参配伍对LPS致ALI大鼠具有防治作用，可改善肺组织的病变程度，减轻炎症反应，早期药物干预有助于改善ALI的预后。③黄芪-丹参可通过抑制TLR-4/NF-κB信号通路发挥对ALI的防治作用。