Comparative Transcriptomic Profiling of Diabetic Wound Responses to Recombinant Periplaneta americana Thymosin Isoforms in Male ob/ob Mice

Background: Preparations derived from Periplaneta americana, such as Kangfuxin Liquid, have been used in China for wound and ulcer management, yet their active constituents remain incompletely characterized. Recombinant P. americana thymosin isoforms (Pa-THYs) have been reported to accelerate wound closure in nondiabetic mice. Their effects and associated transcriptional responses in diabetic wounds remain unclear. Methods: Three Pa-THYs (THY1–3) were topically applied to full-thickness excisional wounds in ob/ob diabetic mice, with thymosin β4 (Tβ4) and PBS used as positive and negative controls. Wound healing was assessed by planimetry, hematoxylin and eosin (H&E) staining, Masson trichrome staining, and CD31 immunohistochemistry. RNA sequencing of wound tissues on days 3 and 8 post-injury was performed, followed by differential gene expression analysis, Gene Ontology and pathway enrichment analysis, protein–protein interaction network construction, marker gene-based cell-type-associated expression scoring, and preranked GSEA. Results: On day 8, mean wound-closure percentages ranged from 95% to 97% across the THY1, THY2, THY3, and Tβ4 groups compared to 81% in the PBS group. At this time point, treated wounds showed more complete epithelial coverage. The CD31-positive area fraction was higher in all active-treatment groups, whereas the collagen volume fraction was higher in the THY2 and THY3 groups than in the PBS group. Among the Pa-THY groups, THY2 showed the largest day-3 DEG set, whereas THY1 and THY3 showed prominent epithelial-differentiation signatures at the later time point. THY3 showed the largest day-8 DEG set and the greatest pairwise DEG overlap with Tβ4. As a complementary threshold-independent analysis, GSEA identified coordinated early metabolic responses, broadly distributed interferon-associated enrichment, and late keratinization enrichment in THY1, THY3, and Tβ4. Conclusions: Topical THY1, THY2, and THY3 improved day-8 wound closure in male ob/ob mice, with accompanying changes in epithelial coverage, CD31-positive area fractions, and collagen staining. The transcriptomic findings indicate shared pathway-level responses together with time- and isoform-associated differences. These results support a further preclinical evaluation of Pa-THYs.