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Article Open Access

Orcinol glucoside ameliorates pulmonary fibrosis by suppressing hyaluronic acid synthesis and macrophage M2 polarization via targeting hyaluronic acid synthase 2

  • Authors:
    • Caizi Li
    • Xinglinzi Tang
    • Xiaoru Luo
    • Xin Lai
    • Jing Yang
    • Zheng Xu
    • Gulizeba Muhetaer
    • Yizi Xie
    • Xiufang Huang
    • Hang Li
  • View Affiliations / Copyright

    Affiliations: Central Laboratory, Shenzhen Bao'an Chinese Medicine Hospital, Guangzhou University of Chinese Medicine, Shenzhen, Guangdong 518133, P.R. China, The Second Clinical Medical School, Guangzhou University of Chinese Medicine, Guangzhou, Guangdong 511436, P.R. China, The First Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou, Guangdong 510405, P.R. China
    Copyright: © Li et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 93
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    Published online on: February 10, 2026
       https://doi.org/10.3892/ijmm.2026.5764
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Abstract

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive lung disorder characterized by unexplained fibrosis and limited therapeutic options, highlighting the urgent need for innovative treatments. Hyaluronic acid (HA), which is upregulated in IPF and correlates with disease severity, plays an undefined role in its pathogenesis. Hyaluronic acid synthase 2 (HAS2), a key enzyme in HA production, has an unclear function in IPF progression, particularly regarding its involvement in macrophage polarization. Understanding this mechanism is essential for identifying novel therapeutic targets and developing effective drugs for IPF. The present study investigated the roles of HAS2 and HA in IPF and identified potential therapeutic agents. Transcriptomic analysis revealed HAS2 as a critical IPF‑associated gene in patient samples, bleomycin (BLM)‑induced mouse models, and transforming growth factor β1 (TGF‑β1)‑induced myofibroblasts. Single‑cell RNA sequencing further confirmed the fibroblast‑specific upregulation of HAS2 in fibrotic lungs. Experimental validation showed elevated HAS2 expression and HA accumulation in fibrosis models. HA facilitated macrophage M2 polarization and TGF‑β1 secretion through CD44‑dependent STAT6 activation, with CD44 inhibition blocking this effect. Knockdown of HAS2 in fibroblasts decreased HA release and impaired their ability to promote M2 polarization, suggesting that fibroblast‑derived HA drives this process. High‑throughput virtual screening, coupled with absorption, distribution, metabolism and excretion (ADME) profiling, identified orcinol glucoside (OG) as a potential HAS2 inhibitor, which was validated through surface plasmon resonance, cellular thermal shift assays, and molecular dynamics simulations. OG suppressed HA synthesis in TGF‑β1‑induced and HAS2‑overexpressing myofibroblasts in a dose‑dependent manner, inhibiting M2 polarization induction. In vivo, OG reduced collagen deposition, HA, and TGF‑β1 levels in BLM‑induced fibrotic mice. These findings established HAS2 as a central pathogenic factor in IPF and suggested OG as a promising therapeutic candidate, providing a novel approach for IPF treatment by targeting HA synthesis and macrophage polarization.
View Figures

Figure 1

Cross-species transcriptomics and
single-cell resolution analysis reveal fibroblast-specific
elevation of HAS2 expression in pulmonary fibrosis. (A) Analysis of
gene expression profiles from the GSE110147 dataset in the GEO
database identified DEGs in patients with IPF (n=22) compared with
healthy controls (n=11), with upregulated genes (red) and
downregulated genes (green) indicated. (B) Transcriptome sequencing
was conducted in a BLM-induced pulmonary fibrosis mouse model to
evaluate DEGs by comparing fibrotic mice with normal controls (n=3
for each group), with upregulated genes (red) and downregulated
genes (green) indicated. (C) NIH/3T3 cells were stimulated with
TGF-β1 to induce myofibroblast transition and transcriptome
sequencing was performed to assess DEGs, comparing myofibroblasts
to fibroblasts (n=3 for each group). (D) A comprehensive analysis
combining data from human, mouse, and cell models to identify key
upregulated genes markedly associated with IPF. (E) UMAP plot of
76,995 quality-filtered cells clustered into 32 distinct
populations. (F) UMAP visualization annotating clusters into 10
major cell types, with relative cell quantities displayed.
Populations include B cells, endothelial cells, epithelial cells,
fibroblasts, mast cells, mononuclear phagocytes, mural cells,
neutrophils, plasmacytoid dendritic cells, and T cells. (G)
Comparative UMAP plots demonstrating conserved spatial distribution
of annotated cell types across saline-treated controls and
BLM-induced fibrotic lungs (n=3 for each group). (H) Heatmap of
cell type-specific HAS2 expression, highlighting significant
upregulation exclusively in fibroblast populations of fibrotic
lungs. HAS2, hyaluronic acid synthase 2; GEO, Gene Expression
Omnibus; DEGs, differentially expressed genes; IPF, idiopathic
pulmonary fibrosis; BLM, bleomycin; UMAP, Uniform Manifold
Approximation and Projection.

Figure 2

Elevated HAS2 expression and HA
accumulation in murine and cellular fibrosis models. (A) Schematic
of the experimental design for the time-course study. C57BL/6J mice
received a single intratracheal dose of BLM or saline and were
sacrificed at the indicated time points (days 0, 3, 5, 7, 10, 14
and 21) for sample collection (n=5 for per group). (B)
Representative western blot images (left panel) and densitometric
quantification (right panel) showing the protein expression levels
of COL1A1 and HAS2 in lung tissues across the time course.
β-tubulin served as the loading control (n=4 for per group). (C)
ELISA quantification of HA levels in BALF at different days
post-BLM injury (n=5 for per group). (D) Schematic diagram
illustrating the workflow for the isolation and culture of primary
mouse lung fibroblasts from BLM-treated fibrotic mice. (E) Western
blotting analysis of HAS2 and COL1A1 protein expression in primary
lung fibroblasts isolated from control (saline) and BLM-induced
fibrotic mice. Representative blot images (left panel) and
densitometric quantification of protein levels normalized to
β-tubulin are shown (n=3 for each group). (F) Schematic diagram
depicting TGF-β1-induced transition of NIH/3T3 fibroblasts into
myofibroblasts. (G) COL1A1, ACTA2, and HAS2 mRNA expression in
NIH/3T3 cells was detected using PCR after 5 ng/ml TGF-β1
administration for 12 h (n=3-4 for each group). (H) HAS2 protein
expression in NIH/3T3 was detected using western blotting after 5
ng/ml TGF-β1 administration for 24 h (n=3 for each group). (I)
ELISA was used to quantify HA concentrations in the culture media
of myofibroblasts (n=6 for each group). *P<0.05,
**P<0.01, ***P<0.001. HAS2, hyaluronic
acid synthase 2; HA, hyaluronic acid; BLM, bleomycin; BALF,
bronchoalveolar lavage fluid; COL1A1, Collagen type I α 1 chain;
ACTA2, actin alpha 2, smooth muscle.

Figure 3

HAS2 in fibroblasts mediates
macrophage M2 polarization. (A) Western blotting of HAS2 expression
in NIH/3T3 fibroblasts transduced with lentiviral vectors carrying
shRNA targeting HAS2. Scramble shRNA and untreated cells were used
as controls. (B) ELISA quantification of HA levels in supernatants
collected 24 h after TGF-β1 stimulation of NIH/3T3 fibroblasts
transduced with HAS2-targeting or scramble shRNA vectors (n=3 for
each group). (C) Schematic workflow: Conditioned medium from
TGF-β1-stimulated, HAS2-knockdown NIH/3T3 fibroblasts was used to
culture RAW264.7 macrophages for 24 h, followed by flow cytometric
analysis of macrophage polarization. (D) Flow cytometry was used to
measure the expression levels of CD206 (FITC) and CD86 (PE) (n=3
for each group). ***P<0.001. HAS2, hyaluronic acid
synthase 2; shRNA, short hairpin RNA; HA, hyaluronic acid; FITC,
fluorescein isothiocyanate; PE, phycoerythrin.

Figure 4

HA from myofibroblasts induces
macrophage M2 polarization via the CD44/STAT6 axis. (A) RAW264.7
macrophages were stimulated with HA for 24 h, and flow cytometry
was performed to assess the expression of CD206 (FITC) and CD86
(PE) (n=3 for each group). (B) The content of TGF-β1 in macrophages
following HA treatment over 48 h was quantified using ELISA (n=3
for each group). (C) Western blotting analysis of p-STAT6 and total
STAT6 expression at various time points following 100 μg/ml
HA stimulation (n=4 for each group). (D) Flow cytometric analysis
of CD86 and CD206 expression in macrophages after 24-h treatment
with 100 μg/ml HA and CD44 inhibitor at indicated
concentrations (n=3 for each group). (E) TGF-β1 quantification by
ELISA in culture supernatants (n=3 for each group). (F) Western
blotting analysis of p-STAT6 and total STAT6 in macrophages
following 2-h treatment with HA and CD44 inhibitor at varying
concentrations (n=3 for each group). (G) Western blotting analysis
of p-STAT6 and total STAT6 in macrophages following 2-h treatment
with HA and STAT6 inhibitor (AS1517499) at varying concentrations
(n=4 for each group). (H) Schematic workflow: Conditioned medium
from TGF-β1-stimulated NIH/3T3 fibroblasts was used to culture
RAW264.7 macrophages for 24 h. Where indicated, RAW264.7 cells were
co-treated with a CD44 inhibitor or a STAT6 inhibitor. Macrophage
polarization was subsequently analyzed by flow cytometry. (I) Flow
cytometry was used to measure the expression levels of CD206 (FITC)
and CD86 (PE) (n=3 for each group). *P<0.05,
**P<0.01, ***P<0.001. HA, hyaluronic
acid; FITC, fluorescein isothiocyanate; PE, phycoerythrin; p-,
phosphorylated.

Figure 5

High-throughput virtual screening and
target validation experiments identifies OG as a natural HAS2
inhibitor. (A) Flowchart outlining the high-throughput virtual
screening process. (B) The three-dimensional structure of human
HAS2 was obtained from the AlphaFold database (AlphaFold ID:
AF-Q92819-F1). (C) The radar map shows that OG met the ADME
criteria. (D) The molecular formula of OG is illustrated. (E) SPR
experiment was conducted to determine the binding affinity of OG to
HAS2, providing quantitative data on binding kinetics. (F) CETSA
assay was performed to confirm the direct interaction between OG
and HAS2, showing enhanced protein stability upon OG treatment (n=3
for each group). (G) The cytotoxicity of OG was assessed using
CCK-8 assays on NIH/3T3 and HFL-1 cell lines following a 48-h
treatment at various concentrations (n=3-6 for each time point).
(H) ELISA assays were performed to quantify HA levels in the
supernatant of TGF-β1-stimulated NIH/3T3 and HFL-1 cells treated
with OG for 48 h (n=3 for each group). (I) Lentiviral vectors were
used to overexpress HAS2 in NIH/3T3 fibroblasts, with western
blotting conducted to confirm HAS2 expression levels (n=6 for each
group). (J) OG was administered to both HAS2-overexpressing NIH/3T3
cells and control cells without overexpression. After a 48-h
treatment with OG, ELISA was used to measure the HA content in the
culture medium (n=3 for each group). *P<0.05,
**P<0.01, ***P<0.001. OG, glucoside;
HAS2, hyaluronic acid synthase 2; ADME, absorption, distribution,
metabolism and excretion; SPR, surface plasmon resonance; CETSA,
cellular thermal shift assay.

Figure 6

MDS indicates the binding stability
of OG and HAS2. (A) The lowest energy binding position of OG and
HAS2. (B and C) Hydrogen bond interactions at the lowest energy
binding position between OG and HAS2. (D) Hydrogen bond analysis
between OG and HAS2. (E) RMSF analysis of the bound complex of OG
and HAS2. (F) RMSD trajectory analysis of the bound complex of OG
and HAS2. (G) Rg analysis of the bound complex of OG and HAS2. (H)
SASA analysis of the bound complex of OG and HAS2. (I) Frames at
the minimal free energy landscape for the bound complex of OG and
HAS2. MDS, molecular dynamics simulation; OG, glucoside; HAS2,
hyaluronic acid synthase 2; RMSF, root-mean-square fluctuations;
SASA, solvent accessible surface area.

Figure 7

OG exerts an anti-pulmonary fibrosis
effect by impairing myofibroblast-mediated macrophage M2
polarization. (A) Schematic workflow: Fibroblast treatment with OG
during TGF-β1 stimulation, followed by conditioned medium
collection and macrophage culture for downstream assays. (B) TGF-β1
release from macrophages measured by ELISA (n=3 for each group).
(C) Flow cytometric analysis of CD206 and CD86 expression in
macrophages cultured in conditioned medium. The bar graph
quantifies the proportion of CD206+CD86−
cells (M2-like macrophage subpopulation; n=3 for each group). (D)
Animal experimental protocol. (E) A BLM-induced mouse model was
established to assess the anti-fibrotic effects of OG.
Histopathological alterations in lung tissues from different groups
were assessed using H&E and Masson's trichrome staining. Scale
bars: 500 μm; magnification, ×40. (F) Western blotting
analysis to measure the expression levels of fibrotic markers,
COL1A1 and α-SMA, in lung tissues (n=4 for each group). (G) The
levels of HA in mouse serum were quantified by ELISA. (H) The
levels of TGF-β1 in mouse serum were quantified by ELISA (n=6 for
each group). (I) OG concentrations in serum and lung tissues after
14-day oral administration. Left: serum concentrations of OG across
treatment groups. Right: lung tissue concentrations of OG,
expressed as a percentage of tissue weight (n=3 for each group).
*P<0.05, **P<0.01,
***P<0.001. OG, glucoside; BLM, bleomycin; COL1A1,
Collagen type I α 1 chain; α-SMA, α-smooth muscle actin; H&E,
hematoxylin and eosin.
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Copy and paste a formatted citation
Spandidos Publications style
Li C, Tang X, Luo X, Lai X, Yang J, Xu Z, Muhetaer G, Xie Y, Huang X, Li H, Li H, et al: Orcinol glucoside ameliorates pulmonary fibrosis by suppressing hyaluronic acid synthesis and macrophage M2 polarization via targeting hyaluronic acid synthase 2. Int J Mol Med 57: 93, 2026.
APA
Li, C., Tang, X., Luo, X., Lai, X., Yang, J., Xu, Z. ... Li, H. (2026). Orcinol glucoside ameliorates pulmonary fibrosis by suppressing hyaluronic acid synthesis and macrophage M2 polarization via targeting hyaluronic acid synthase 2. International Journal of Molecular Medicine, 57, 93. https://doi.org/10.3892/ijmm.2026.5764
MLA
Li, C., Tang, X., Luo, X., Lai, X., Yang, J., Xu, Z., Muhetaer, G., Xie, Y., Huang, X., Li, H."Orcinol glucoside ameliorates pulmonary fibrosis by suppressing hyaluronic acid synthesis and macrophage M2 polarization via targeting hyaluronic acid synthase 2". International Journal of Molecular Medicine 57.4 (2026): 93.
Chicago
Li, C., Tang, X., Luo, X., Lai, X., Yang, J., Xu, Z., Muhetaer, G., Xie, Y., Huang, X., Li, H."Orcinol glucoside ameliorates pulmonary fibrosis by suppressing hyaluronic acid synthesis and macrophage M2 polarization via targeting hyaluronic acid synthase 2". International Journal of Molecular Medicine 57, no. 4 (2026): 93. https://doi.org/10.3892/ijmm.2026.5764
Copy and paste a formatted citation
x
Spandidos Publications style
Li C, Tang X, Luo X, Lai X, Yang J, Xu Z, Muhetaer G, Xie Y, Huang X, Li H, Li H, et al: Orcinol glucoside ameliorates pulmonary fibrosis by suppressing hyaluronic acid synthesis and macrophage M2 polarization via targeting hyaluronic acid synthase 2. Int J Mol Med 57: 93, 2026.
APA
Li, C., Tang, X., Luo, X., Lai, X., Yang, J., Xu, Z. ... Li, H. (2026). Orcinol glucoside ameliorates pulmonary fibrosis by suppressing hyaluronic acid synthesis and macrophage M2 polarization via targeting hyaluronic acid synthase 2. International Journal of Molecular Medicine, 57, 93. https://doi.org/10.3892/ijmm.2026.5764
MLA
Li, C., Tang, X., Luo, X., Lai, X., Yang, J., Xu, Z., Muhetaer, G., Xie, Y., Huang, X., Li, H."Orcinol glucoside ameliorates pulmonary fibrosis by suppressing hyaluronic acid synthesis and macrophage M2 polarization via targeting hyaluronic acid synthase 2". International Journal of Molecular Medicine 57.4 (2026): 93.
Chicago
Li, C., Tang, X., Luo, X., Lai, X., Yang, J., Xu, Z., Muhetaer, G., Xie, Y., Huang, X., Li, H."Orcinol glucoside ameliorates pulmonary fibrosis by suppressing hyaluronic acid synthesis and macrophage M2 polarization via targeting hyaluronic acid synthase 2". International Journal of Molecular Medicine 57, no. 4 (2026): 93. https://doi.org/10.3892/ijmm.2026.5764
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