Data Sheet 1_Yizong Tongluo formula attenuates idiopathic pulmonary fibrosis and inflammatory injury by inhibiting HIF-1α/LSH/SCD1-mediated ferroptosis.docx

BackgroundIdiopathic pulmonary fibrosis (IPF) is a progressive disease characterized by chronic inflammation, aberrant tissue remodeling, and hypoxia. In traditional Chinese medicine (TCM), it is classified as Qi deficiency–blood stasis syndrome. The mechanism of Yizong Tongluo Formula (YZTLF), a Traditional Chinese Medicine (TCM) herbal formulation and effective pharmaceutical agent for the clinical treatment of IPF, remains unclear.
MethodsThis study investigated the immunomodulatory and anti-fibrotic mechanisms of YZTLF for this IPF subtype by focusing on the HIF-1α/LSH/SCD1 signaling pathway and ferroptosis-induced inflammatory injury. We began by analyzing clinical samples from IPF patients with Qi deficiency and blood stasis was conducted to assess the signalling axis and ferroptosis markers. Molecular and histological analyzes were performed using TGF-β-treated MRC-5 fibroblast cells and a bleomycin-induced rat model of pulmonary fibrosis.
ResultsClinical analysis revealed a dysregulation of the HIF-1α/LSH/SCD1 axis and altered levels of ferroptosis markers in patients. In vitro, YZTLF significantly downregulated HIF-1α and LSH expression while upregulating SCD1 (p < 0.01). Importantly, the treatment markedly suppressed ferroptosis, as evidenced by reduced intracellular Fe2+ and ACSL4 levels alongside increased Glutathione Peroxidase 4 (GPX4) and GSH expression (p < 0.01). It also inhibited TGF-β-induced fibroblast activation, significantly decreasing α-SMA and Collagen I protein levels (p < 0.01). In vivo, the YZTLF treatment attenuated bleomycin-induced lung injury, reduced inflammatory cell infiltration, preserved alveolar architecture, and reduced collagen deposition, alongside normalizing of HIF-1α/LSH/SCD1 signaling and restoration of antioxidant levels.
ConclusionThese findings indicate that YZTLF mitigates IPF progression in the context of Qi deficiency and blood stasis by suppressing ferroptosis-driven inflammation and remodeling the hypoxic microenvironment via the HIF-1α/LSH/SCD1 pathway. This provides a mechanism-based rationale for its use in this TCM-defined IPF subtype.

背景：特发性肺纤维化（Idiopathic pulmonary fibrosis, IPF）是一种以慢性炎症、异常组织重塑及缺氧为核心特征的进行性疾病。在中医（Traditional Chinese Medicine, TCM）体系中，该病被归为气虚血瘀证范畴。益气通络方（Yizong Tongluo Formula, YZTLF）作为一款中药复方，亦是临床治疗IPF的有效药剂，但其具体作用机制至今尚未阐明。
方法：本研究聚焦于缺氧诱导因子-1α（HIF-1α）/LSH/硬脂酰辅酶A去饱和酶1（SCD1）信号通路及铁死亡（ferroptosis）诱导的炎症损伤，探讨YZTLF针对气虚血瘀型IPF的免疫调节与抗纤维化机制。首先，本研究通过分析气虚血瘀型IPF患者的临床样本，对该信号轴及铁死亡标志物水平进行评估；随后，采用转化生长因子-β（Transforming Growth Factor-β, TGF-β）处理的MRC-5成纤维细胞系，以及博莱霉素诱导的肺纤维化大鼠模型，开展分子与组织学相关分析。
结果：临床分析结果显示，气虚血瘀型IPF患者体内HIF-1α/LSH/SCD1信号轴存在失调现象，且铁死亡标志物水平出现异常改变。体外实验中，YZTLF可显著下调HIF-1α与LSH的表达水平，同时上调SCD1的表达（p < 0.01）。尤为关键的是，该治疗可显著抑制铁死亡进程，具体表现为细胞内Fe²+及长链脂酰辅酶A合成酶4（ACSL4）水平降低，谷胱甘肽过氧化物酶4（GPX4）与谷胱甘肽（GSH）的表达水平升高（p < 0.01）。此外，YZTLF可抑制TGF-β诱导的成纤维细胞活化，显著降低α-平滑肌肌动蛋白（α-SMA）及I型胶原蛋白（Collagen I）的蛋白表达水平（p < 0.01）。体内实验中，YZTLF治疗可减轻博莱霉素诱导的肺损伤，减少炎症细胞浸润，维持肺泡结构完整性，降低胶原蛋白沉积，同时可使HIF-1α/LSH/SCD1信号通路恢复至正常水平，并修复机体抗氧化水平。
结论：本研究结果表明，在气虚血瘀证的病理背景下，YZTLF可通过调控HIF-1α/LSH/SCD1通路，抑制铁死亡介导的炎症反应，并重塑缺氧微环境，从而延缓IPF的疾病进展。该研究为该中医分型的IPF患者临床应用YZTLF提供了基于机制的理论依据。