鹰嘴豆芽素A通过调节TLR4/NF-κB/NLRP3信号通路减轻卵清蛋白诱导哮喘大鼠的气道炎症

Biochanin A attenuates ovalbumin-induced airway inflammation in asthmatic rats by regulating TLR4/NF-κB/NLRP3 signaling pathway

关键词:  鹰嘴豆芽素A  Toll样受体4/核因子-κB/NOD样受体蛋白3信号通路  哮喘  气道炎症

Key words:  biochanin A  toll-like receptor 4/nuclear factor-κB/NOD-like receptor protein 3 signaling pathway  asthma  airway inflammation

摘要:

[目的] 探讨鹰嘴豆芽素A（BCA）通过调节Toll样受体4（TLR4）/核因子-κB（NF-κB）/NOD样受体蛋白3（NLRP3）信号通路对卵清蛋白（OVA）诱导哮喘大鼠气道炎症的影响。[方法] 将SD大鼠分为对照（CK）组、模型（Model）组、低剂量BCA组（BCA-L组，25 mg/kg）、高剂量BCA组（BCA-H组，100 mg/kg）、地塞米松阳性对照组（Dex组，1 mg/kg）、BCA-H+LPS（TLR4激活剂）组（100 mg/kg+0.1 mg/kg），每组12只。除CK组，其他组均通过OVA诱导构建哮喘大鼠模型。建模成功24 h后，进行给药处理，给药每日1次，持续10 d。利用动物肺功能测定仪检测大鼠吸气阻力、呼气阻力、肺通气顺应性的变化；吉姆萨（Giemsa）染色检测支气管肺泡灌洗液（BALF）中细胞总数、淋巴细胞数、嗜酸性粒细胞数、中性粒细胞数；酶联免疫吸附剂测定（ELISA）检测BALF中肿瘤坏死因子-α（TNF-α）、白细胞介素-1β（IL-1β）、干扰素-γ（IFN-γ）水平；苏木精-伊红染色法（HE）染色检测大鼠肺组织病理变化；免疫组化检测大鼠肺组织中嗜酸性粒细胞趋化因子（Eotaxin）表达；蛋白质印迹法（Western Blot）检测大鼠肺组织中TLR4、p-NF-κB p65、NLRP3蛋白表达。[结果] 与CK组比较，Model组大鼠吸气阻力、呼气阻力、BALF中细胞总数、淋巴细胞数、嗜酸性粒细胞数、中性粒细胞数、TNF-α、IL-1β、IFN-γ水平升高，肺组织中的支气管及血管周围炎性细胞浸润明显，肺组织中Eotaxin阳性染色面积百分数、TLR4、p-NF-κB p65、NLRP3蛋白表达升高，肺通气顺应性降低（P<0.05）；与Model组比较，BCA-L组、BCA-H组、Dex组对应指标变化趋势与上述相反（P<0.05）；LPS减弱了高剂量BCA对哮喘大鼠气道炎症的改善作用。[结论] BCA可能通过抑制TLR4/NF-κB/NLRP3信号通路减轻OVA诱导哮喘大鼠的气道炎症。

Abstract:

[Objective] To investigate the impact of biochanin A (BCA) on ovalbumin (OVA)-induced airway inflammation in asthmatic rats by regulating Toll-like receptor 4 (TLR4)/nuclear factor-κB (NF-κB)/NOD-like receptor protein 3 (NLRP3) signaling pathway. [Methods] SD rats were divided into control check(CK)group, model group, low-dose BCA group (BCA-L group, 25 mg/kg), high-dose BCA group(BCA-H group, 100 mg/kg), and dexamethasone positive control group(Dex group, 1 mg/kg), and BCA-H+LPS (TLR4 activator) group (100 mg/kg+0.1 mg/kg), 12 rats in each group. Except for the CK group, other groups were induced by OVA to establish the asthmatic rat model. After 24 hours of successful modeling, drug treatment was carried out, and the drug was administered once a day for 10 d. The changes of inspiratory resistance, expiratory resistance and lung ventilation compliance of rats were detected by animal lung function analyzer;Giemsa staining was used to detect the total number of cells, and the numbers of lymphocytes, eosinophils and neutrophils in bronchoalveolar lavage fluid(BALF). ELISA was used to detect the levels of tumor necrosis factor-α (TNF-α), interleukin-1β(IL-1β) and interferon-γ(IFN-γ) in BALF;HE staining was used to detect the pathological changes of rat lung tissue;immunohistochemistry was used to detect the expression of eosinophil chemokine (Eotaxin) in rat lung tissue;Western blot was used to detect the protein expressions of TLR4, p-NF-κB p65 and NLRP3 in rat lung tissue. [Results] Compared with the CK group, inspiratory resistance, expiratory resistance, the total number of cells, the numbers of lymphocytes, eosinophils, neutrophils, the levels of TNF-α, IL-1β, and IFN-γ in the BALF of the Model group increased, and the infiltration of inflammatory cells around the bronchi and blood vessels in the lung tissue was obvious, the percentage of Eotaxin positive staining area, and the protein expressions of TLR4, p-NF-κB p65 and NLRP3 in lung tissue increased, reduced lung ventilation compliance (P<0.05);Compared with the Model group, the corresponding indicators of the BCA-L group, BCA-H group and Dex group had the opposite trends(P<0.05);LPS attenuated the ameliorating effect of high-dose BCA on airway inflammation in asthmatic rats. [Conclusion] BCA may alleviate airway inflammation in OVA-induced asthmatic rats by inhibiting TLR4/NF-κB/NLRP3 signaling pathway.