Chuangyu Liquid promotes wound healing by modulating an AKT1‑centered regulatory network based on network pharmacology and molecular docking

Technical Summary: Chuangyu Liquid Promotes Wound Healing by Modulating an AKT1‑Centered Regulatory Network

Problem Statement
Impaired wound healing represents a significant clinical challenge, particularly for chronic conditions such as diabetic ulcers, burns, and extensive trauma, which impose substantial economic burdens and reduce patient quality of life due to scarring and functional loss. While Traditional Chinese Medicine (TCM) external therapies, specifically the compound preparation Chuangyu Liquid, have demonstrated clinical efficacy in accelerating wound closure and reducing inflammation, their material basis (active components) and underlying molecular mechanisms remain insufficiently elucidated. This lack of mechanistic understanding hinders the evidence-based application and modernization of the formulation.

Methodology
The study employed a multi-step integrative approach combining chemical profiling, network pharmacology, molecular docking, and in vivo validation:

1. Chemical Profiling: The chemical composition of Chuangyu Liquid was characterized using Ultra-High-Performance Liquid Chromatography–Tandem Mass Spectrometry (UHPLC–MS/MS), identifying 1,158 compounds.
2. Active Constituent Screening: To identify bioactive components likely to be systemically available, the study intersected LC-MS/MS annotations with blood-absorbed constituents curated in the DCABM-TCM database. This filtering process yielded eight candidate active constituents (including Rhein, Physcion, Magnoflorine, and Berbericinine).
3. Network Pharmacology:
   • Potential targets for the eight candidates were predicted using SwissTargetPrediction.
   • Wound healing-related targets were aggregated from five databases (GeneCards, MGI, NCBI, TiRe, TTD), resulting in 1,239 targets.
   • Intersection of candidate targets and wound healing targets identified potential therapeutic targets.
   • A weighted scoring model (integrating compound-target interaction frequency and Protein-Protein Interaction (PPI) network topology) was used to prioritize core targets.
   • Gene Ontology (GO) and KEGG/Reactome enrichment analyses were performed to map biological processes and pathways.
4. Molecular Docking: The binding potential between the eight candidate compounds and the prioritized core targets was evaluated using AutoDock Vina and SwissDock.
5. In Vivo Validation:
   • A mouse full-thickness excisional wound model (C57BL/6 mice) was established.
   • Mice were treated with saline, Kangfuxin Liquid (positive control), or low/medium/high doses of Chuangyu Liquid.
   • Efficacy Assessment: Wound closure rates were measured via photography; histological evaluation (H&E and Masson's trichrome staining) assessed re-epithelialization, granulation tissue, and collagen organization.
   • Mechanistic Validation: Quantitative real-time PCR (qRT-PCR) and Immunohistochemistry (IHC) were used to verify the expression levels of mRNA and protein (AKT and p-AKT) for the predicted core targets (AKT1, STAT3, HIF1A, MMP9) at different healing stages (days 0, 3, 5, 12).

Key Contributions and Results

• Chemical Identification: The study successfully identified eight candidate bioactive constituents that are potentially absorbable in vivo, moving beyond simple chemical profiling to focus on pharmacologically relevant components.
• Target Prioritization: Network analysis identified four core therapeutic targets: AKT1, STAT3, HIF1A, and MMP9. Among these, AKT1 emerged as the primary hub based on its high weighted score and favorable molecular docking interactions with multiple candidate compounds (e.g., Rhein, Physcion, Emodin-8-glucoside).
• Pathway Enrichment: Enrichment analyses indicated that the candidate targets are significantly involved in processes critical to wound healing, including muscle cell proliferation, PI3K-Akt signaling, receptor tyrosine kinase signaling, and extracellular matrix organization.
• In Vivo Efficacy: Chuangyu Liquid demonstrated a dose-dependent acceleration of wound closure. The high-dose group showed the most significant improvement, with faster re-epithelialization, reduced residual wound length, and more orderly collagen deposition compared to controls.
• Mechanistic Validation:
  • qRT-PCR: Confirmed dynamic, time-dependent regulation of Akt1, Stat3, Hif1a, and Mmp9 mRNA expression. Notably, Akt1 expression was elevated during the proliferative phase (Day 5).
  • IHC: Showed that Chuangyu Liquid treatment increased both total AKT and phosphorylated AKT (p-AKT) levels in the epidermis and dermis during the proliferative phase (Day 5). By the remodeling phase (Day 12), expression levels returned toward baseline, suggesting a stage-specific regulatory effect.

Significance and Claims
The paper claims that Chuangyu Liquid promotes cutaneous wound healing through a "multi-component–multi-target–multi-pathway" mechanism, with AKT1 serving as a central regulatory node.

• Mechanistic Insight: The study provides a systematic evidence framework linking the formulation's chemical profile to its phenotypic effects, suggesting that the liquid modulates AKT1-related signaling to balance granulation tissue formation and extracellular matrix remodeling.
• Stage-Specific Regulation: The authors posit that Chuangyu Liquid exerts "appropriate activation" of AKT1 during the proliferative phase to facilitate tissue regeneration, while avoiding sustained overactivation during the remodeling phase, which could otherwise lead to excessive scarring.
• Scientific Basis: These findings offer a mechanistic basis for the ethnopharmacological use of Chuangyu Liquid, supporting its transition from empirical use to a more evidence-based therapeutic strategy.

Limitations and Modesty
The authors explicitly acknowledge limitations, noting that the study was conducted in healthy mice and may not directly extrapolate to chronic wound models (e.g., diabetic ulcers). They also state that the specific fine-tuned regulatory patterns of AKT1 in different cell types require further verification using inhibitors or genetic knockout models. The study presents these findings as a preliminary exploration of the material basis and mechanisms, calling for future multi-layered "drug component–metabolite–target–pathway–phenotype" network construction to better reflect in vivo conditions.