Network pharmacology and in vivo experimental analysis for validating the antifibrotic potential of Punica granatum leaves against skin scleroderma: the role of oxidative stress, inflammation and TGF-β1/Snail 1/p-Smad 3 signaling pathway

Given the significant efficacy of the Punica granatum in enhancing skin health, this research employed network pharmacology and in vivo studies to explore the P. granatum ethanolic leaf extract's (PGEL) mechanism in alleviating skin sclerosis. A model of skin fibrosis brought on by bleomycin (100 µl/rat, sc.) was adopted for experimental validation, where PGEL was orally administered at 200 and 400 mg/kg to rats for 3 weeks. Seventy-three compounds of PGEL were identified by LC/MS/MS belonging to different chemical classes; an organic acid, 12 phenolic acids, 4 polysaccharides, 3 amino acids, 19 gallic acid derivatives,7 ellagic acid derivatives, 14 flavonoides, three anthocyanins, 3 fatty acids and 7 miscellaneous groups. Five compounds (Catechin-3-O-gallate, 3,3'-di-O-methyl-4-O-(xylopyranosyl) ellagic acid, ellagic acid glucoside, valoneic acid dilactone, and vitexin 2-O-gallate) were isolated from the PGEL. Network pharmacological studies clarified that TNF-α, TGF-β1, Snail1, p-Smad3, MMP-9, IL-17 A, and COL1A1 are the core targets for skin fibrosis. PGEL lessened MPO and increased SOD activity in rat's skin, underscoring its antioxidant activity. Furthermore, the TNF-α, IL-17 A and MMP-9 were decreased in PGEL groups. Mechanistic studies revealed that PGEL exerts its anti-fibrotic action by downregulating the TGF-β1, Snail 1, COL1A1, and p-Smad 3 in the skin tissues. Additionally, histopathological analyses informed the decline in dermal alterations and thickness in rats treated with PGEL. Collectively, our results give clear evidence that PGEL work through multi-pathway modulation by targeting the core proteins of inflammation mediated the TGF-β/Snail1/Smad 3 signaling, thereby exerting a therapeutic action on skin fibrosis.