Background: Chuankezhi injection (CKZ), derived from the traditional Chinese herbs Ying-Yang-Huo (Epimedium brevicornu Maxim) and Ba-Ji-Tian (Morinda officinalis F.C. How), has demonstrated remarkable clinical effects in the treatment of asthma. However, the underlying mechanisms remain unclear.

Purpose: This study aims to explore the mechanisms and molecular targets of CKZ in the treatment of asthma, utilizing network pharmacology and molecular biology experiments.

Study design: A combination of network pharmacology and experimental validation was used to identify the specific targets and pathways through which CKZ exerts its effects on asthma. In vitro and in vivo experiments were conducted to further verify the findings.

Methods: Liquid chromatography-mass spectrometry (LC/MS) and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry were employed to identify the components of CKZ. GeneCards was used to gather asthma-related targets, and the STRING online database was utilized to construct protein-protein interaction (PPI) networks. Hub genes were identified from the PPI network and analyzed through Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. In vitro and in vivo experiments were conducted to validate the network pharmacology predictions.

Results: LC/MS and MALDI-TOF analysis revealed that CKZ is rich in flavonoid compounds. Network pharmacological analysis identified TNF, AKT1, IL-6, GAPDH, and SRC as the top five hub genes. GO and KEGG pathway analyses suggested that CKZ's effect on asthma is closely associated with mitochondrial function and the mitogen-activated protein kinase (MAPK) signaling pathway. In vivo and in vitro experiments showed that CKZ treatment alleviated airway inflammation and collagen deposition in asthma mouse models, as demonstrated by HE, PAS, and Masson staining. CKZ also reduced the levels of inflammatory cytokines (IL-4, IL-5, IL-13, and TNF-α) in bronchoalveolar lavage fluid (BALF) and serum. Furthermore, CKZ improved mitochondrial damage and inhibited the activation of the MAPK signaling pathway, as confirmed by Western blotting analysis.

Conclusion: CKZ effectively alleviates airway inflammation and improves airway damage in asthma by inhibiting the MAPK signaling pathway. These findings suggest that CKZ is a promising therapeutic option for asthma treatment.

Keywords: Chuankezhi injection; MAPK signaling pathway; Network pharmacology; Traditional Chinese Medicine (TCM); Treatment; asthma; molecular docking.

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背景: 川 kezhi 注射液（CKZ）源自传统中药淫羊藿（Epimedium brevicornu Maxim）和巴戟天（Morinda officinalis F.C. How），在治疗哮喘方面表现出显著的临床效果。然而，其潜在机制尚不清楚。

目的: 本研究旨在利用网络药理学和分子生物学实验探索 CKZ 治疗哮喘的作用机理及分子靶点。

设计: 采用网络药理学与实验验证相结合的方法，识别 CKZ 对哮喘产生作用的特异性靶点和通路。通过体内外实验进一步验证研究结果。

方法: 利用高效液相色谱-质谱（LC/MS）和基质辅助激光解析飞行时间（MALDI-TOF）质谱技术鉴定 CKZ 的成分。使用 GeneCards 收集哮喘相关靶点，并借助 STRING 在线数据库构建蛋白质-蛋白质相互作用（PPI）网络。从 PPI 网络中识别出枢纽基因，通过基因本体论（GO）富集分析和京都基因与基因组百科全书（KEGG）通路分析进行进一步分析。开展体内外实验验证网络药理学预测结果。

结果: LC/MS 和 MALDI-TOF 分析表明，CKZ 富含黄酮类化合物。网络药理学分析确定 TNF、AKT1、IL-6、GAPDH 和 SRC 是前五名枢纽基因。GO 和 KEGG 通路分析表明，CKZ 对哮喘的作用与线粒体功能和丝裂原活化蛋白激酶（MAPK）信号通路密切相关。体内和体外实验显示，CKZ 治疗减轻了哮喘小鼠模型中的气道炎症和胶原沉积，并通过 HE、PAS 和 Masson 染色得到了证实。此外，CKZ 还降低了支气管肺泡灌洗液（BALF）和血清中炎性细胞因子（IL-4、IL-5、IL-13 和 TNF-α）的水平。进一步地，通过 Western blotting 分析验证了 CKZ 改善线粒体损伤并抑制 MAPK 信号通路激活的作用。

结论: CKZ 可有效减轻哮喘气道炎症和改善气道损伤，其作用机制可能涉及抑制 MAPK 信号通路。这些发现表明 CKZ 是治疗哮喘的一种有前途的疗法选择。

关键词: 川 kezhi 注射液；MAPK 信号通路；网络药理学；传统中药（TCM）；治疗；哮喘；分子对接。