State Key Laboratory of Traditional Chinese Medicine Syndrome/Department of Respiratory Disease, The Second Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangdong Provincial Hospital of Chinese Medicine, Guangdong Provincial Academy of Chinese Medical Sciences, Guangzhou, China; State Key Laboratory of Dampness Syndrome of Chinese Medicine, The Second Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou, China; Guangdong-Hong Kong-Macau Joint Lab on Chinese Medicine and Immune Disease Research, Guangzhou, China.
Zhuhai Hospital of Integrated Traditional Chinese and Western Medicine, China.
Phytomedicine. 2024 Dec;135:156090. doi: 10.1016/j.phymed.2024.156090. Epub 2024 Oct 3.
Asthma is a prevalent long-term inflammatory condition that causes airway inflammation and remodeling. Increasing evidence indicates that epithelial-mesenchymal transition (EMT) holds a prominent implication in airway reconstruction in patients with asthma. Flavonoids obtained from Chinese Materia Medica (CMM), such as Luteolin (Lut), exhibit various beneficial effects in various asthma models. Lut has been shown to mitigate various asthma symptoms, including airway inflammation, hyperresponsiveness, bronchoconstriction, excessive mucus production, pulmonary autophagy, and neutrophilic asthma. However, whether flavonoids can suppress EMT-associated airway remodeling in asthma and the fundamental mechanisms involved remain unclear, with no studies specifically addressing Lut in this context.
To evaluate the inhibition of airway remodeling in asthma by Lut and its potential mechanisms, while examining the significance of β-catenin in this process through cellular and animal studies.
A BEAS-2B cell model stimulated by lipopolysaccharide (LPS) was established in vitro. Wound closure and Transwell assays were utilized to assess the cellular migratory ability. EMT- and fibrosis-related markers in LPS-stimulated cells were evaluated using RT-qPCR and western blotting. The status of the β-catenin/E-cadherin and β-catenin destruction complexes was evaluated using western blotting, immunofluorescence (IF) staining, and co-immunoprecipitation (Co-IP) analysis. The regulatory function of Lut in β-catenin-dependent EMT was further validated by β-catenin overexpression with adenovirus transduction and siRNA-mediated knockdown of β-catenin. Moreover, the counts of different types of bronchoalveolar lavage fluid (BALF) inflammatory cells from mice with asthma induced by ovalbumin (OVA) were evaluated in vivo using Congo red staining. Hematoxylin and eosin (H&E), Masson's trichrome, and periodic acid-Schiff (PAS) staining were used to evaluate collagen deposition, mucus production, and inflammation in murine lung tissues. Western blotting and immunohistochemistry (IHC) assays were used to assess EMT- and fibrosis-related markers in the lung tissues in vivo.
Six naturally derived flavonoids, including Lut, attenuated cell migration and prevented EMT in LPS-treated BEAS-2B cells. Moreover, Lut suppressed TGF-β1, MMP-9, fibronectin (FN), and α-smooth muscle actin (α-SMA) levels in LPS-stimulated BEAS-2B cells. Additionally, Lut downregulated the levels of β-catenin by modulating the β-catenin/E-cadherin and β-catenin destruction complexes, highlighting the pivotal role of β-catenin in EMT inhibition by Lut in LPS-stimulated BEAS-2B cells. Furthermore, Lut suppressed airway inflammation and attenuated EMT-associated airway remodeling through β-catenin blockade in OVA-induced asthmatic mice. The bronchial wall thickness notably reduced from 37.24 ± 4.00 μm in the asthmatic model group to 30.06 ± 4.40 μm in the Lut low-dose group and 24.69 ± 2.87 μm in the Lut high-dose group.
According to our current understanding, this research is the first to reveal that Lut diminishes airway remodeling in asthma by inhibiting EMT via β-catenin regulation, thereby filling a research gap concerning Lut and flavonoids. These results provide a theoretical basis for treating asthma with anti-asthmatic CMM, as well as a candidate and complementary therapeutic approach to treat asthma.
哮喘是一种常见的慢性炎症性疾病，会导致气道炎症和重塑。越来越多的证据表明，上皮-间充质转化（EMT）在哮喘患者的气道重建中具有重要意义。从中药中提取的黄酮类化合物，如木樨草素（Lut），在各种哮喘模型中表现出多种有益作用。Lut 已被证明可减轻各种哮喘症状，包括气道炎症、高反应性、支气管收缩、过度黏液产生、肺自噬和中性粒细胞性哮喘。然而，黄酮类化合物是否能抑制哮喘中与 EMT 相关的气道重塑，以及其中涉及的基本机制尚不清楚，也没有专门针对 Lut 的研究。
通过细胞和动物研究，评估 Lut 对哮喘气道重塑的抑制作用及其潜在机制，并研究β-连环蛋白在这一过程中的意义。
建立脂多糖（LPS）体外刺激 BEAS-2B 细胞模型。采用划痕愈合和 Transwell 实验评估细胞迁移能力。采用 RT-qPCR 和 Western blot 检测 LPS 刺激细胞中 EMT 和纤维化相关标志物。采用 Western blot、免疫荧光（IF）染色和免疫共沉淀（Co-IP）分析评估β-连环蛋白/E-钙黏蛋白和β-连环蛋白破坏复合物的状态。通过腺病毒转导过表达β-连环蛋白和 siRNA 介导的β-连环蛋白敲低进一步验证 Lut 在β-连环蛋白依赖性 EMT 中的调节作用。此外，采用刚果红染色法在体内评估卵清蛋白（OVA）诱导哮喘小鼠不同类型支气管肺泡灌洗液（BALF）炎症细胞的计数。采用苏木精和伊红（H&E）、Masson 三色和过碘酸-Schiff（PAS）染色评估小鼠肺组织中的胶原沉积、黏液产生和炎症。采用 Western blot 和免疫组化（IHC）检测体内肺组织中 EMT 和纤维化相关标志物。
六种天然黄酮类化合物，包括 Lut，可减轻 LPS 处理的 BEAS-2B 细胞迁移并预防 EMT。此外，Lut 可降低 LPS 刺激的 BEAS-2B 细胞中 TGF-β1、MMP-9、纤维连接蛋白（FN）和α-平滑肌肌动蛋白（α-SMA）水平。此外，Lut 通过调节β-连环蛋白/E-钙黏蛋白和β-连环蛋白破坏复合物来下调β-连环蛋白水平，突出了β-连环蛋白在 Lut 抑制 LPS 刺激的 BEAS-2B 细胞 EMT 中的关键作用。此外，Lut 通过阻断β-连环蛋白抑制气道炎症和减轻 OVA 诱导的哮喘小鼠 EMT 相关气道重塑。支气管壁厚度从哮喘模型组的 37.24±4.00μm显著降低至 Lut 低剂量组的 30.06±4.40μm和 Lut 高剂量组的 24.69±2.87μm。
根据我们目前的认识，这项研究首次揭示 Lut 通过调节β-连环蛋白抑制 EMT 来减轻哮喘中的气道重塑，从而填补了 Lut 和黄酮类化合物研究的空白。这些结果为使用抗哮喘中药治疗哮喘提供了理论依据，也是治疗哮喘的候选和补充治疗方法。
