Investigating Molecular Mechanisms of Qijia Rougan Prescription and Its Key Effect or Ingredients Against Hepatic Fibrosis Based on Macrophage M2 Polarization
10.13422/j.cnki.syfjx.20251012
- VernacularTitle:基于巨噬细胞M2极化研究芪甲柔肝方及其关键效应成分抗肝纤维化的分子机制
- Author:
Li WEN
1
;
Quansheng FENG
1
;
Cen JIANG
1
;
Baixue LI
1
;
Dong WANG
1
;
Jike LI
2
;
Xia LI
1
;
Fei WAN
3
;
Yanfeng ZHENG
3
Author Information
1. College of Basic Medicine， Chengdu University of Traditional Chinese Medicine（TCM）， Chengdu 611137，China
2. Public Health Clinic Center of Chengdu，Chengdu 610061，China
3. College of TCM， Chongqing Medical and Pharmaceutical College， Chongqing 401331，China
- Publication Type:Journal Article
- Keywords:
Qijia Rougan prescription;
hepatic fibrosis;
macrophage M2 polarization;
enoxolone;
berberine
- From:
Chinese Journal of Experimental Traditional Medical Formulae
2026;32(14):155-165
- CountryChina
- Language:Chinese
-
Abstract:
ObjectiveBased on the regulation of macrophage M2 polarization， this study aims to explore the molecular mechanism and action targets of the Qijia Rougan prescription and its key effector ingredients in anti-fibrosis， thereby providing a basis and reference for the development of new drugs for hepatic fibrosis. MethodsA rat model of hepatic fibrosis was established by subcutaneous injection of 40%CCl4， followed by oral administration of Qijia Rougan granules. The volume of collagen fibers was detected using Masson staining， the fibrosis markers Collagen Ⅰ and α-SMA were detected using immunohistochemistry， the proportion of M2 macrophages was detected by flow cytometry. The expression levels of M2 macrophage phenotype markers CD163 and CD206 were detected using immunofluorescence double staining. Western blot was used to detect the levels of the transforming growth factor-β （TGF-β）， platelet derived growth factor subunit B （PDGFB）， interleukin-10 （IL-10）， phosphorylated Janus kinase 1 （p-JAK1）， and phosphorylated signal transducer and activator of transcription 6 （p-STAT6）. Real-time fluorescent quantitative PCR was used to detect the relative expression levels of JAK1， STAT6， Arginase 1（Arg1）， and Fizz1. Based on the theory of serum pharmacology， liquid chromatography-mass spectrometry and WENN analysis were used to obtain the active ingredients of Qijia Rougan prescription. Molecular docking and molecular dynamics simulation were performed to analyze the effector ingredients and their targets. The identified effector ingredients were interfered with IL-4-induced M2 polarization of RAW264.7 macrophage in vitro to validate the targets. ResultsQijia Rougan prescription significantly reduced the content of fibrosis markers α-SMA and Collagen Ⅰ， as well as collagen fiber content （P<0.05）. It decreased the proportion of M2 macrophages and the levels of related cytokines IL-10， TGF-β and PDGFB， and up-regulated the levels of p-JAK1 and p-STAT6 （P<0.05）. A total of 1 214 compounds were identified from Qijia Rougan prescription， medicated serum and blank serum， and 29 ingredients were finalized by Venn analysis， including 15 blood-entry prototypes and 14 drug metabolites. Molecular docking showed that enoxolone and berberine bound more strongly to JAK1， with binding free energies of -9.6 kcal·mol-1（1 cal≈4.184 J） and -9.1 kcal·mol-1， respectively. Molecular dynamics simulations showed that JAK1-enoxolone and JAK1-berberine exhibited stable simulation trajectories within 100 ns， with essentially identical conformations and high protein overlap before and after simulation. Their binding free energies were -25.18 5.0.81 kcal·mol-1 and -27.39 7.0.85 kcal·mol-1， respectively. The number of hydrogen bonds formed between JAK1 and enoxolone ranges from 0 to 5， and most of the time can be maintained at 2-3. In vitro intervention with enoxolone or berberine significantly reduced p-JAK1 and p-STAT6 levels （P<0.05）. ConclusionQijia Rougan prescription inhibits M2 macrophage polarization in hepatic fibrosis. Enoxolone and berberine are the key effector ingredients of Qijia Rougan prescription to inhibit macrophage M2 polarization through targeting JAK1 and modulating the JAK1/STAT6 signaling pathway， thereby ameliorating hepatic fibrosis. This study provides a basis for prescription optimization， clinical application and new drug development， as well as a reference for monolithic anti-hepatic fibrosis research.
