火把花根保护油酸肺损伤肺通透性屏障的作用机制研究

Both current studies and clinical observations were confirmed that colquhounia root had multiple pharmacological effects against inflammatory mediators and cytokines to alleviate inflammatory exudation. Our previous studies showed that colquhounia root could significantly decrease pulmonary permeability of rats with oleic acid-induced ALI and attenuate pulmonary edema, but the mechanism of it remains largely unknown. Down-regulated expression of alveolar epithelial pulmonary and microvascular endothelial tight junction protein Claudin family and ZO-1 is one of main factors resulted in pulmonary hyperpermeability of ALI. We assume that colquhounia root can decrease pulmonary permeability by up-regulating the expression of the tight junction protein Claudin family and ZO-1. The damage model was induced by treating oleic acid-induced ALI of rats with TNF. Using immunohistochemical, immunofluorescent and western blotting methods, the effects of colquhounia root on alveolar epithelial pulmonary and microvascular endothelial tight junction protein Claudin-2、Claudin-4、Claudin-5 and ZO-1 is to be determined both in vivo and in vitro to investigate its potential pathway.

目前研究和临床观察均证实火把花根具有拮抗炎症介质和细胞因子，减轻炎症渗出的多重药理效应，我们研究已表明火把花根可明显降低油酸致大鼠急性肺损伤（ALI）肺屏障通透性、减轻肺水肿，从而对ALI有治疗作用，但分子机制尚不明确。而肺泡上皮和微血管内皮细胞表达的紧密连接蛋白组分Claudin家族和ZO-1表达的下调是ALI肺通透性增高主要因素之一。我们设想火把花根通过上调紧密连接蛋白组分Claudin家族和ZO-1表达，降低肺屏障通透性。本研究拟通过建立油酸致大鼠ALI模型及TNF处理体外培养大鼠肺泡上皮和微血管内皮细胞损伤模型，采用免疫组化、免疫荧光和免疫印记方法，从在体水平和离体细胞多层面研究火把花根对肺泡上皮和微血管内皮细胞Claudin-2、Claudin-4、Claudin-5和ZO-1蛋白表达的影响, 明确火把花根对ALI肺屏障通透性干预的分子机制，并探讨其可能的细胞内信号调节通路。

急性肺损伤（ALI）是一种临床常见的危重病，目前尚无有效的治疗措施。主要病理特征为炎症因子损伤肺泡上皮及内皮细胞，使肺通透性屏障功能破坏，导致高通透性肺水肿及透明膜形成。目前研究和临床观察均证实中药火把花根具有拮抗炎症介质和细胞因子，减轻炎症渗出的多重药理作用。前期研究已证实火把花根具有保护油酸致大鼠ALI的作用，本研究旨在探讨其保护作用的分子机制。本研究建立了油酸致大鼠ALI动物模型，火把花根处理ALI大鼠后其肺湿/干重比值、肺通透性指数及肺损伤评分均较对照组明显降低，同时，ALI组大鼠的肺紧密连接蛋白Claudin-2、Claudin-5及ZO-1的水平明显降低，火把花根明显上调了Claudin-2、Claudin-5及ZO-1的表达水平，说明火把花根通过上调肺紧密连接蛋白的表达，减轻肺损伤。另外。本研究建立了TNF-α处理体外培养大鼠肺泡上皮和肺微血管内皮细胞损伤模型，结果显示损伤组细胞的增值抑制率明显升高，并呈剂量-时间依赖关系，损伤组凋亡率也较正常对照组明显升高，火把花根组细胞的增值抑制率较损伤组降低，凋亡率明显降低。说明火把花根可以抑制TNF-α诱导的细胞凋亡。同时，实时荧光定量PCR及免疫印迹显示TNF-α组细胞间紧密连接蛋白Claudin-4、Claudin-5及ZO-1的mRNA水平及蛋白表达量明显降低， 火把花根组明显上调了紧密连接蛋白mRNA及蛋白的表达水平，从细胞水平证实了火把花根降低肺泡上皮及内皮细胞的通透性及保护ALI的分子机制。同时，我们测定了TNF-α及火把花根干预后磷酸化p115RhoGEF及活化RhoA的水平与正常对照组相比并无明显差异，说明RhoA信号通路可能并不参与TNF-α诱导的ALI过程。