基于热敏通道TRPV1/TRPA1探讨清肺排毒汤对急性肺损伤模型小鼠的影响

Effect of Qingfei Paidu Decoction on Acute Lung Injury Model Mice Based on TRPV1/TRPA1 Heat-sensitive Channel

摘要:

目的 探讨清肺排毒汤对热敏通道瞬时受体电位香草酸受体1/瞬时受体电位锚蛋白1（TRPV1/TRPA1）及炎症反应的相关机制及影响。方法 将80只8周龄C57BL/6小鼠按体质量随机分为正常组、模型组、地塞米松组（5 mg·kg^-1）、清肺排毒汤低、中、高剂量组（14.865、29.73、59.46 g·kg^-1），每组12只。除正常组外，其余组采用气道滴注法予每只小鼠20 μL（1×10^-3 g·kg^-1）构建小鼠急性肺损伤（ALI）模型。给药组在造模后1 h给药，间隔24 h后再次给药，造模后36 h取肺组织，用双肺湿/干质量比（W/D）、苏木素-伊红（HE）染色、酶联免疫吸附测定法（ELISA）、蛋白免疫印迹法（Western blot）观察并检测肺组织病理形态学变化、炎性细胞因子肿瘤坏死因子-α（TNF-α）、白细胞介素-6（IL-6）的表达水平、热敏通道TRPV1、TRPA1蛋白、核转录因子-κB（NF-κB）、炎症通路中NF-κB抑制蛋白α（IκBα）及二者磷酸化蛋白的表达，并计算磷酸化蛋白与总蛋白比值。结果 与正常组比较，模型组小鼠肺组织严重损伤，肺毛细血管通透性增高，肺泡毛细血管充血扩张，肺泡完整结构被破坏，肺泡壁增厚，大量炎性细胞与红细胞浸润，肺水肿显著加重，TNF-α、IL-6、TRPV1、TRPA1、磷酸化NF-κB p65/NF-κB p65、磷酸化IκBα/IκBα表达显著升高（P<0.01），全肺W/D显著升高（P<0.01）；与模型组比较，地塞米松与清肺排毒汤低、中、高剂量组能明显改善肺水肿，TNF-α、IL-6、TRPV1、TRPA1及肺组织NF-κB p65、IκBα磷酸化蛋白/总蛋白表达明显下降（P<0.05，P<0.01），全肺W/D明显下降（P<0.05，P<0.01）。结论 清肺排毒汤对LPS-ALI小鼠具有抗炎保护作用，能有效减轻炎症、利水消肿，其机制可能与调节TRPA1、TRPV1的表达，抑制NF-κB通路活化有关。

Abstract:

Objective To investigate the mechanism and effect of Qingfei Paidu decoction on transient receptor potential vanilloid-1/Transient receptor potential ankyrin1 （TRPV1/TRPA1） based on heat-sensitive channel and inflammatory response.Method According to body weight， 80 8-week-old C57BL/6 mice were randomly divided into the normal group， model group， dexamethasone group （5 mg·kg^-1）， and low-dose， medium-dose， and high-dose groups of Qingfei Paidu decoction （14.865， 29.73， 59.46 g·kg^-1）， with 12 mice in each group. In addition to the normal group， the other groups were administered 20 μL （1×10^-3 g·kg^-1） to each mouse by airway infusion to establish the acute lung injury （ALI） model. In the administration group， the drug was given 1 h after modeling and again after an interval of 24 h. The lung tissue was taken 36 h after modeling. Double lung wet/dry weight ratio（W/D）， hematoxylin-eosin （HE） staining， enzyme-linked immunosorbent assay （ELISA）， and Western blot were used to observe and detect the pathological changes of lung tissue， expression levels of inflammatory cytokine tumor necrosis factor-α （TNF-α） and interleukin-6 （IL-6）， and expressions of TRPV1 and TRPA1 proteins in heat-sensitive channel， nuclear factor kappa-B （NF-κB）， inhibitor of NF-κB （IκBα） in inflammatory pathway， and phosphorylated proteins. The phosphorylated protein/total protein ratio was calculated.Result Compared with that in the normal group， the lung tissue of mice in the model group was seriously damaged， and pulmonary capillary permeability increased. Alveolar capillary congestion and dilation destroyed the complete structure of the alveolar， and the alveolar wall thickened. A large number of inflammatory cells and red blood cells were infiltrated， and pulmonary edema was significantly aggravated. The expressions of TNF-α， IL-6， TRPV1， TRPA1， phosphorylated NF-κB p65/NF-κB p65， and phosphorylated IκBα/IκBα were significantly increased （P<0.01）， and the whole lung W/D was significantly increased （P<0.01）. Compared with the model group， the dexamethasone group and low-dose， medium-dose， and high-dose groups of Qingfei Paidu decoction could significantly improve pulmonary edema. TNF-α， IL-6， TRPV1， TRPA1， lung tissue NF-κB p65， and IκBα phosphorylated protein/total protein ratio decreased significantly （P<0.05， P<0.01）. The whole lung W/D also decreased significantly （P<0.05， P<0.01）.Conclusion Qingfei Paidu decoction has anti-inflammatory and protective effects on LPS-ALI mice， which can effectively reduce inflammation， induce diuresis， and alleviate edema. Its mechanism may be related to the regulation of the expression of TRPA1 and TRPV1 and the inhibition of the activation of the NF-κB pathway.