Time-dependent gene expression patterns in epidermal wound healing: Mechanistic insights into Cayratia japonica hydrogel intervention after anal fistula surgery.

ETHNOPHARMACOLOGICAL RELEVANCE: Postoperative wound healing, especially after anal-fistula surgery, poses unique clinical challenges due to the complex anal anatomy and the need for accelerated recovery. Although the ethnomedicinal plant Cayratia japonica (Thunb.) Gagnep. has long been recognized for its wound-healing properties, its molecular basis and therapeutic potential remain poorly defined. Here, an optimized C. japonica hydrogel (CJH) markedly improved clinical outcomes, while temporal multi-omics profiling underscored the necessity of stage-specific therapeutic adaptation. Specifically, CJH-derived Luteolin 7-glucuronide was found to enhance fibroblast proliferation via BMP4 protein engagement and downstream signaling activation, thereby accelerating wound remodeling.

PURPOSE OF RESEARCH: This study developed an innovative CJH and systematically investigated its molecular mechanisms in skin wound healing through integrated multi-omics analyses.

MATERIALS AND

METHODS

CJH was optimized through single-factor screening and characterized by scanning electron microscopy and spectroscopic analyses. A prospective randomized controlled trial (n = 81) was conducted to evaluate its clinical efficacy. Transcriptomic datasets (GSE28914, GSE241132) were analyzed using time-series clustering and underwent a comprehensive weighted gene co-expression network analysis (WGCNA), including gene expression matrix standardization, soft-thresholds selection, modules construction, and module-trait correlation analysis, to identify key temporal gene modules. Single-cell RNA sequencing was further to employed to elucidate cell-type-specific gene expression patterns. Ultra-performance liquid chromatography - electrospray ionization - quadrupole time-of-flight - tandem mass spectrometry (UPLC-ESI-Q-TOF-MS/MS), combined with Global Natural Products Social Molecular Networking (GNPS), was used to identify active compounds, followed by molecular docking and in vitro validation.

RESULTS

CJH significantly improved wound healing compared with control (healing rate 68.5 ± 12.3% vs 45.2 ± 15.7%, P < 0.001) and shortened recovery time (P = 0.031). Multi-omics analysis of public human cutaneous wound datasets identified three phase-associated hubs: OASL for the acute immune phase, TIMP3 for the early repair phase, and BMP4 for the remodeling phase. These findings highlight a stage-specific molecular program in skin wound healing. Among these candidates, BMP4 was prioritized for experimental validation due to its temporal concordance with the clinical treatment window. UPLC-ESI-Q-TOF-MS/MS was used to identify the chemical constituents of CJH, among which Luteolin 7-glucuronide was subsequently predicted by molecular docking to be a key bioactive compound capable of binding BMP4 (binding energy -8.2 kcal/mol). In vitro assays demonstrated that Luteolin 7-glucuronide engaged the BMP4 protein, enhanced its thermal stability, and potentiated downstream Smad signaling, accompanied by increased fibroblast proliferation.

CONCLUSION

CJH enhanced clinical wound healing following anal fistula surgery. Skin-derived temporal programs identify TIMP3, OASL, and BMP4 as stage-associated regulators, while perianal tissue readouts together with in vitro assays support BMP4 protein engagement and pathway activation as a likely mechanism contributing to the observed repair benefits.