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

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

  • Phytomedicine. 2025 May:140:156628. doi: 10.1016/j.phymed.2025.156628.
Jiaping Zheng 1 Yan Du 2 Wenbo Shao 1 Jiansheng Li 3 Peng Zhao 4 Qin Zhang 5
Affiliations

Affiliations

  • 1 Henan Key Laboratory of Chinese Medicine for Respiratory Disease, Henan University of Chinese Medicine, Zhengzhou, Henan 450046, China; Collaborative Innovation Center for Chinese Medicine and Respiratory Diseases co-constructed by Henan province & Education Ministry of P.R. China, Zhengzhou, Henan 450046, China.
  • 2 Henan Key Laboratory of Chinese Medicine for Respiratory Disease, Henan University of Chinese Medicine, Zhengzhou, Henan 450046, China; Collaborative Innovation Center for Chinese Medicine and Respiratory Diseases co-constructed by Henan province & Education Ministry of P.R. China, Zhengzhou, Henan 450046, China; Academy of Chinese Medical Sciences, Henan University of Chinese Medicine, Zhengzhou 450000, China.
  • 3 Henan Key Laboratory of Chinese Medicine for Respiratory Disease, Henan University of Chinese Medicine, Zhengzhou, Henan 450046, China; Collaborative Innovation Center for Chinese Medicine and Respiratory Diseases co-constructed by Henan province & Education Ministry of P.R. China, Zhengzhou, Henan 450046, China; Academy of Chinese Medical Sciences, Henan University of Chinese Medicine, Zhengzhou 450000, China; Department of Respiratory Diseases, The First Affiliated Hospital of Henan University of Chinese Medicine, Zhengzhou 450000, China.
  • 4 Henan Key Laboratory of Chinese Medicine for Respiratory Disease, Henan University of Chinese Medicine, Zhengzhou, Henan 450046, China; Collaborative Innovation Center for Chinese Medicine and Respiratory Diseases co-constructed by Henan province & Education Ministry of P.R. China, Zhengzhou, Henan 450046, China; Academy of Chinese Medical Sciences, Henan University of Chinese Medicine, Zhengzhou 450000, China. Electronic address: zhaopeng871@126.com.
  • 5 Henan Key Laboratory of Chinese Medicine for Respiratory Disease, Henan University of Chinese Medicine, Zhengzhou, Henan 450046, China; Collaborative Innovation Center for Chinese Medicine and Respiratory Diseases co-constructed by Henan province & Education Ministry of P.R. China, Zhengzhou, Henan 450046, China; Academy of Chinese Medical Sciences, Henan University of Chinese Medicine, Zhengzhou 450000, China. Electronic address: 2418780049@qq.com.
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.

Keywords

ECC-JHF; H3K18la; HK2; Pulmonary fibrosis; SRC.

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