Danshen/Huanglian-loaded gelatin/Bletilla striata gum dressings accelerate wound healing in anal fistula by promoting angiogenesis through TGF-β/Smad pathway activation.

Abstract

Anal fistula (AF) is a common anorectal disease that significantly impairs patients' quality of life. Dressings were frequently used for therapeutic wound healing. In this study, Danshen/Huanglian-loaded gelatin/Bletilla striata gum (DH-GB) dressings were prepared. The aim was to investigate its therapeutic effects and potential mechanism in AF. DH-GB, Danshen-loaded gelatin/Bletilla striata gum (D-GB), and GB dressings were prepared. AF models were constructed using wire surgery method, with dressing treatment for two weeks. Wound healing rate and blood flow were detected during this period. HE staining, MASSON staining, and immunohistochemistry for Angiopoietins-1 (Ang-1) expression were performed. In vitro, rat primary skin flbroblast cells (PRSFs) were randomized into Control, immersion solution of dressings (Danshen/Huanglian), Danshen/Huanglian + small interfering RNA-negative control (si-NC), and Danshen/Huanglian + siRNA targeting transforming growth factor-β (si-TGF-β) groups. Cell viability and cell proliferation were assessed by CCK8 and EdU assay. Additionally, both in vivo and in vitro, ELISA for VEGF levels and Western blot for TGF-β/Smad pathway-related expression were conducted. In AF rats, DH-GB treatment up-regulated wound healing rate, collagen deposition, and blood flow, increased VEGF, Ang-1, TGF-β, p-Smad2/Smad2, p-Smad3/Smad3, and p-Smad4/Smad4 expression, and improved pathological damage. Notably, DH-GB dressings exhibited enhanced efficacy in promoting wound healing compared to D-GB and GB dressings. Moreover, in rat PRSFs, Danshen/Huanglian promoted cell viability, cell proliferation, and enhanced VEGF, TGF-β, p-Smad2/Smad2, p-Smad3/Smad3, and p-Smad4/Smad4 expression, which were reversed by si-TGF-β. Our study revealed that DH-GB dressings promoted AF wound healing by stimulating angiogenesis through up-regulating TGF-β/Smad pathway, offering scientific foundation for its clinical application in AF.