DataSheet1_Yiqi Huayu decoction alleviates bleomycin-induced pulmonary fibrosis in rats by inhibiting senescence.ZIP

Overview: In treating pulmonary fibrosis (PF), traditional Chinese medicine (TCM) has received much attention, but its mechanism is unclear. The pharmacological mechanisms of TCM can be explored through network pharmacology. However, due to its virtual screening properties, it still needs to be verified by in vitro or in vivo experiments. Therefore, we investigated the anti-PF mechanism of Yiqi Huayu Decoction (YHD) by combining network pharmacology with in vivo experiments.
Methods: Firstly, we used classical bleomycin (BLM)-induced rat model of PF and administrated fibrotic rats with YHD (low-, medium-, and high-dose). We comprehensively assessed the treatment effect of YHD according to body weight, lung coefficient, lung function, and histopathologic examination. Second, we predict the potential targets by ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) combined with network pharmacology. In brief, we obtained the chemical ingredients of YHD based on the UHPLC-MS/MS and TCMSP database. We collected drug targets from TCMSP, HERB, and Swiss target prediction databases based on active ingredients. Disease targets were acquired from drug libraries, Genecards, HERB, and TTD databases. The intersecting targets of drugs and disease were screened out. The STRING database can obtain protein-protein interaction (PPI) networks and hub target proteins. Molecular Complex Detection (MCODE) clustering analysis combined with enrichment analysis can explore the possible biological mechanisms of YHD. Enrichment analyses were conducted through the R package and the David database, including the Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Ontology (GO), and Reactome. Then, we further validated the target genes and target proteins predicted by network pharmacology. Protein and gene expression detection by immunohistochemistry, Western blot (WB), and real-time quantitative PCR (rt-qPCR).
Results: The results showed that high-dose YHD effectively attenuated BLM-induced lung injury and fibrosis in rats, as evidenced by improved lung function, relief of inflammatory response, and reduced collagen deposition. We screened nine core targets and cellular senescence pathways by UHPLC-MS/MS analysis and network pharmacology. We subsequently validated key targets of cellular senescence signaling pathways. WB and rt-qPCR indicated that high-dose YHD decreased protein and gene expression of senescence-related markers, including p53 (TP53), p21 (CDKN1A), and p16 (CDKN2A). Increased reactive oxygen species (ROS) are upstream triggers of the senescence program. The senescence-associated secretory phenotypes (SASPs), containing interleukin 6 (IL-6), tumor necrosis factor-alpha (TNF-α), and transforming growth factor-β1 (TGF-β1), can further exacerbate the progression of senescence. High-dose YHD inhibited ROS production in lung tissue and consistently reduced the SASPs expression in serum.
Conclusion: Our study suggests that YHD improves lung pathological injury and lung function in PF rats. This protective effect may be related to the ability of YHD to inhibit cellular senescence.

概述：在肺纤维化（Pulmonary Fibrosis, PF）的治疗中，中医药（Traditional Chinese Medicine, TCM）受到了广泛关注，但其作用机制尚未明确。中医药的药理机制可通过网络药理学（Network Pharmacology）进行探索，但由于该方法具有虚拟筛选特性，仍需通过体外（In Vitro）或体内（In Vivo）实验进行验证。因此，本研究通过结合网络药理学与体内实验，探讨了益气化瘀汤（Yiqi Huayu Decoction, YHD）抗肺纤维化的作用机制。

方法：首先，本研究采用经典的博来霉素（Bleomycin, BLM）诱导的大鼠肺纤维化模型，对造模成功的大鼠给予低、中、高剂量的益气化瘀汤干预。本研究通过体重、肺系数、肺功能及组织病理学检查，全面评估益气化瘀汤的治疗效果。其次，本研究结合超高效液相色谱-串联质谱（Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry, UHPLC-MS/MS）与网络药理学预测潜在靶点。具体而言，本研究基于UHPLC-MS/MS与TCMSP数据库获取益气化瘀汤的化学成分；基于活性成分，从TCMSP、HERB及Swiss靶点预测数据库中收集药物靶点；从药物库、Genecards、HERB及治疗靶点数据库（Therapeutic Target Database, TTD）中获取疾病靶点；筛选得到药物与疾病的交集靶点。通过STRING数据库可获取蛋白质-蛋白质相互作用（Protein-Protein Interaction, PPI）网络与核心靶点蛋白；结合分子复合物检测（Molecular Complex Detection, MCODE）聚类分析与富集分析，可探索益气化瘀汤潜在的生物学机制。富集分析通过R包与DAVID数据库完成，涵盖京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）、基因本体（Gene Ontology, GO）及Reactome数据库。随后，本研究进一步验证了网络药理学预测得到的靶点基因与靶点蛋白，通过免疫组化、蛋白质免疫印迹（Western Blot, WB）及实时定量聚合酶链反应（Real-Time Quantitative Polymerase Chain Reaction, rt-qPCR）检测蛋白与基因的表达水平。

结果：结果显示，高剂量益气化瘀汤可有效减轻博来霉素诱导的大鼠肺损伤与肺纤维化，具体表现为肺功能改善、炎症反应缓解及胶原沉积减少。通过UHPLC-MS/MS分析与网络药理学筛选，本研究得到9个核心靶点与细胞衰老（Cellular Senescence）通路。随后，本研究验证了细胞衰老信号通路的关键靶点。WB与rt-qPCR结果表明，高剂量益气化瘀汤可降低衰老相关标志物的蛋白与基因表达，包括p53（TP53）、p21（CDKN1A）及p16（CDKN2A）。活性氧（Reactive Oxygen Species, ROS）水平升高是衰老程序的上游触发因素；衰老相关分泌表型（Senescence-Associated Secretory Phenotypes, SASPs）包含白细胞介素6（Interleukin 6, IL-6）、肿瘤坏死因子-α（Tumor Necrosis Factor-α, TNF-α）及转化生长因子-β1（Transforming Growth Factor-β1, TGF-β1），可进一步加剧衰老进程。高剂量益气化瘀汤可抑制肺组织中ROS的产生，并持续降低血清中SASPs的表达水平。

结论：本研究表明，益气化瘀汤可改善肺纤维化大鼠的肺部病理损伤与肺功能，其保护作用可能与益气化瘀汤抑制细胞衰老的能力相关。