Effective-compounds of Jinshui Huanxian Formula acts as an SRC inhibitor to inhibit HK2-mediated H3K18 lactation and improve pulmonary fibrosis

IF 6.7 1区医学 Q1 CHEMISTRY, MEDICINAL

Phytomedicine Pub Date : 2025-03-08 DOI:10.1016/j.phymed.2025.156628

Jiaping Zheng , Yan Du , Wenbo Shao , Jiansheng Li , Peng Zhao , Qin Zhang

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Abstract

Background

The Active Ingredient Composition of Jinshui Huanxian Formula (ECC-JHF) consists of five active ingredients: icariin, isoliquiritigenin, nobiletin, peimine, and paeoniflorin, which demonstrate notable therapeutic effects on pulmonary fibrosis.

Purpose

Inhibition of glycolysis has been demonstrated to be effective in treating experimental idiopathic pulmonary fibrosis (IPF). This research seeks to explore the impact of aerobic glycolysis on the mitigation of pulmonary fibrosis through ECC-JHF.

Methods

A pulmonary fibrosis mouse model was generated through the administration of bleomycin (Bleomycin). The degree of pulmonary fibrosis was analyzed through hematoxylin and eosin (H&E) staining as well as Masson's trichrome staining. Western Blot (WB), Immunofluorescence (IF), and real-time quantitative PCR (Q-PCR) assay for fibroblast activation markers and glycolysis-related genes in lung tissues. The Lactic Acid (LA) Content Assay Kit was employed to quantify lactate concentrations in lung tissues and fibroblast cultures. Immunoprecipitation (IP) was applied to detect lactylated modified protein levels, and mass spectrometry (MS) was used to analyze lactate substrate profiles in fibroblasts. WB was employed to detect the lactate modification level of histone H3K18 (H3K18la). The targets of ECC-JHF were analyzed using network pharmacology, while molecular docking and cellular enthusiasm transfer analysis (CETSA) examined the binding of ECC-JHF to SRC. The influence of ECC-JHF on SRC activation was assessed using WB. SRC small interfering RNA (siSRC) was designed and transfected into L929 cells to validate the function of SRC in the inhibition of fibroblast activation by ECC-JHF.

Results

In BLM-induced pulmonary fibrosis mice, ECC-JHF significantly reduced alveolar inflammation and collagen deposition. In lung tissues and fibroblasts, ECC-JHF notably inhibited the expression of HK2, lactate levels, and lactylated modifying proteins. IP-MS and WB analyses showed that ECC-JHF significantly reduced H3K18la levels. Network pharmacology analysis, molecular docking and CETSA results indicated that SRC serves as a key target for ECC-JHF. siSRC effectively mitigated the impact of ECC-JHF on the expression of HK2, levels of H3K18la, and the activation of fibroblasts.

Conclusion

ECC-JHF may improve pulmonary fibrosis by inhibiting SRC activation, blocking HK2-mediated lactate production, down-regulating H3K18la levels, and inhibiting fibroblast activation. Our results serve as a significant reference for the advancement of ECC-JHF and the exploration of IPF.

Abstract Image

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研究背景金水环仙方（ECC-JHF）的有效成分由5种有效成分组成：冰片苷、异冰片苷、金钗素、芍药苷和芍药苷，这5种有效成分对肺纤维化有显著的治疗作用。研究目的抑制糖酵解对治疗实验性特发性肺纤维化（IPF）有效。本研究试图探讨有氧糖酵解对通过 ECC-JHF 缓解肺纤维化的影响。方法通过服用博莱霉素（Bleomycin）建立肺纤维化小鼠模型。通过苏木精和伊红（H&E）染色以及马森三色染色分析肺纤维化程度。对肺组织中的成纤维细胞活化标志物和糖酵解相关基因进行 Western Blot (WB)、免疫荧光 (IF) 和实时定量 PCR (Q-PCR) 检测。乳酸（LA）含量检测试剂盒用于定量检测肺组织和成纤维细胞培养物中的乳酸浓度。免疫沉淀（IP）用于检测乳酸修饰蛋白的水平，质谱（MS）用于分析成纤维细胞中乳酸底物的概况。利用WB检测组蛋白H3K18（H3K18la）的乳酸修饰水平。利用网络药理学分析了ECC-JHF的靶标，而分子对接和细胞热传递分析（CETSA）则检验了ECC-JHF与SRC的结合。ECC-JHF 对 SRC 活化的影响是通过 WB 评估的。结果在 BLM 诱导的肺纤维化小鼠中，ECC-JHF 能显著减少肺泡炎症和胶原沉积。在肺组织和成纤维细胞中，ECC-JHF明显抑制了HK2、乳酸水平和乳酸修饰蛋白的表达。IP-MS 和 WB 分析表明，ECC-JHF 能显著降低 H3K18la 水平。网络药理学分析、分子对接和 CETSA 结果表明，SRC 是 ECC-JHF 的一个关键靶点。siSRC 能有效减轻 ECC-JHF 对 HK2 表达、H3K18la 水平和成纤维细胞活化的影响。我们的研究结果为 ECC-JHF 的发展和 IPF 的探索提供了重要参考。

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来源期刊

Phytomedicine 医学-药学

CiteScore

10.30

自引率

5.10%

发文量

670

审稿时长

91 days

期刊介绍： Phytomedicine is a therapy-oriented journal that publishes innovative studies on the efficacy, safety, quality, and mechanisms of action of specified plant extracts, phytopharmaceuticals, and their isolated constituents. This includes clinical, pharmacological, pharmacokinetic, and toxicological studies of herbal medicinal products, preparations, and purified compounds with defined and consistent quality, ensuring reproducible pharmacological activity. Founded in 1994, Phytomedicine aims to focus and stimulate research in this field and establish internationally accepted scientific standards for pharmacological studies, proof of clinical efficacy, and safety of phytomedicines.