Network Pharmacology, Molecular Docking Analysis and Experiment Validations on Molecular Targets and Mechanisms of the Dispel-Scar Ointment in Scar Treatment.

Abstract

Background: Dispel-Scar Ointment is used in Traditional Chinese Medicine to treat scarred tissue and increasing evidence has shown that DSO is a potent therapeutic; however, its exact mechanism remains unexplored.

Aim of the study: This study explored the molecular mechanisms of action of DSO in scarring using network pharmacology, molecular docking, and experimental validation.

Materials and methods: Public databases were applied to predict the bioactive ingredients and putative targets of DSO against scars. A compounds-targets network was constructed using the Cytoscape software. Molecular docking was performed to verify the correlation between the major positive ingredients and hub targets, visualised using PyMol 2.3. Enrichment analysis was implemented using ClueGo and FunRich to specify the biological capabilities and related pathways of hub targets. SwissADME software was used to predict the ADME capabilities of the protein-related active compounds between DSO and scar in order to analyse the absorption and permeation across cell membranes of DSO. We assessed the skin sensitizer potential of the bioactive compounds of DSO using Pred-Skin computational tool. Experimental validations were conducted to elucidate the influence of DSO on keloid fibroblast cells using the CCK-8, woundscratch, cell reactive oxygen species, and western blot assays.

Results: Network pharmacological analysis of DSO for scar treatment identified 146 ingredients and 1078 gene targets. Major targets included prostaglandin-endoperoxide synthase 2 matrix metallopeptidases and nitric oxide synthase 2. Molecular docking showed MMP2-flavoxanthin, MMP9-luteolin and MMP-9-kaempferol bound best to DSO. ClueGo analysis revealed 29 pathways (p<0.05) and FunRich 345 pathways (p<0.05), mainly toll-like receptor, TGF-β, interleukin- 4/13, glypican, and tumour necrosis factor-related apoptosis-inducing ligand pathways. The results valued by the SwissADME and PreADMET tools illustrated that 12 compounds in DSO were almost permeable through the skin. Pred-Skin computational tool represented that these 12 bioactive compounds reflected skin sensitizer potential. Experimental analysis revealed that DSO could restrain the proliferation and migration of scar fibroblasts and facilitate their apoptosis in a concentration-dependent manner. DSO also decreased TGF-β1, -βR2, pSMAD2, pSMAD3, SMAD4, CoL1a1, and MMP2 expression.

Conclusion: Network pharmacology, molecular docking, and experimental validation showed DSO's feasibility in scar therapy. It may restrain scars through the TGF-β1/SMADs/MMPs signalling pathway, providing a basis for DSO's scar treatment application.