Hypertrophic scarring (HS) is a fibroproliferative disorder characterized by excessive fibroblast activation, aberrant extracellular matrix (ECM) deposition, and persistent inflammation, often resulting from cutaneous injury. Current treatments exhibit limited efficacy and potential side effects. Artesunate (ART), a well-established anti-malarial drug, has demonstrated anti-fibrotic, anti-inflammatory, and anti-angiogenic properties in various disease models. This study aimed to investigate the therapeutic effects of ART on HS formation using a rabbit ear model and to elucidate the underlying cellular and molecular mechanisms, with a particular focus on fibroblast activation, endothelial-mesenchymal transition (EndMT), and the involvement of the PI3K/AKT/mTOR and TGF-β/Smad signaling pathways.

A hypertrophic scar model was established by creating full-thickness square wounds on the ears of New Zealand rabbits. After wound healing, the scars were locally injected with ART (3 mg/mL or 6 mg/mL), triamcinolone acetonide (TAC, as a positive control), ART combined with the mTOR activator 740 Y-P, or a control solution. Scar protrusion and thickness were measured periodically. In vitro studies utilized human skin fibroblasts (HFBs) and human umbilical vein endothelial cells (HUVECs) stimulated with TGF-β1 to mimic a profibrotic environment. Cells were treated with ART, the PI3K inhibitor LY294002, or 740 Y-P. Cellular assays: CCK-8 for viability, EdU flow cytometry for proliferation, scratch assays for migration. Western blotting, immunofluorescence, and immunohistochemistry (IHC) for key markers including α-SMA, COL1A1, FN1, CD31, VEGF, TGF-β1, IL-10, and components of the PI3K/AKT/mTOR and TGF-β/Smad pathways. Hematoxylin and eosin (H&E), Masson's trichrome, and Picrosirius red staining of scar tissues to evaluate collagen deposition, organization, and typing (Collagen I/III ratio). Analysis of publicly available gene expression datasets (GSE181540, GSE190135) from human HS tissues and fibroblasts.

Local injection of ART significantly reduced HS elevation and thickness in rabbit ears, with effects comparable to the positive control TAC. ART treatment led to a marked improvement in the local immune microenvironment, evidenced by downregulation of pro-fibrotic factors TGF-β1 and VEGF, and the immunomodulator IL-10. Histologically, ART reduced collagen deposition, promoted a more organized collagen fiber structure, and increased the ratio of mature Collagen I to Collagen III. ART significantly inhibited fibroblast activation, as shown by decreased expression of α-SMA, COL1A1, and FN1, and suppressed HFB proliferation and migration in vitro. Crucially, ART effectively inhibited EndMT in both HS tissues and TGF-β1-stimulated HUVECs, demonstrated by a reduction in CD31+α-SMA+ double-positive cells and preservation of endothelial marker CD31. Mechanistically, ART suppressed the activation of both the PI3K/AKT/mTOR and TGF-β/Smad signaling pathways in fibroblasts and HUVECs. The anti-fibrotic effects of ART were reversed by co-treatment with the mTOR activator 740 Y-P in vivoand in vitro, confirming the critical role of PI3K/AKT/mTOR inhibition. Furthermore, ART-induced inactivation of the TGF-β/Smad pathway was partially dependent on the suppression of PI3K/AKT/mTOR, revealing a novel crosstalk between these pathways in the context of HS.

This study demonstrates that ART effectively attenuates hypertrophic scar formation by modulating the immune microenvironment, inhibiting fibroblast activation and proliferation, suppressing EndMT in vascular endothelial cells, and promoting normalized collagen remodeling. The anti-fibrotic actions of ART are mediated through the concurrent suppression of the PI3K/AKT/mTOR and TGF-β/Smad signaling cascades, with inhibition of the former pathway partially contributing to the inactivation of the latter. These findings not only provide new insights into the pathogenesis of HS, highlighting EndMT as a significant source of myofibroblasts, but also underscore the promising potential of ART as a novel, multi-targeted therapeutic strategy for hypertrophic scars and potentially other fibrotic disorders.