Abstract
Background: Myocardial fibrosis (MF) results from excessive collagen deposition in the cardiac interstitium, causing structural and functional cardiac impairments that underlie multiple cardiovascular diseases. Grape seed oil (GSO), rich in various bioactive fatty acids, demonstrates established cardiovascular benefits, yet its potential mechanisms against MF remain incompletely elucidated. This study was designed to investigate the inhibitory effects of bioactive components from GSO on TGF-β1-induced fibrosis in cardiac fibroblasts (CFs) and to elucidate the underlying molecular mechanisms. Methods: GSO was obtained using supercritical CO(2) extraction technology. Initially, the anti-fibrotic activity of GSO was evaluated in vitro: a fibrosis model was established by inducing cardiac fibroblasts with TGF-β1 (10 ng/mL for 48 h), followed by treatment with 20% (v/v) GSO. Subsequently, the bioactive constituents of GSO were identified by Gas Chromatography-Mass Spectrometry (GC-MS). Network pharmacology approaches were employed to predict its potential therapeutic targets and associated signaling pathways. Molecular docking simulations were then performed to validate the binding interactions between the key bioactive components and the core targets obtained from enrichment analysis. Finally, the predicted core pathway was experimentally verified by Western blot analysis. Results: In vitro experiments demonstrated that 20% GSO treatment significantly downregulated TGF-β1-induced fibrotic markers at both transcriptional (MMP9, MMP2, Col1a1) and protein (TGF, Col I/III, α-SMA) levels (p < 0.01). GC-MS analysis identified nine fatty acids in GSO, including palmitic acid and linolenic acid. Network pharmacology revealed interactions between these compounds and 357 myocardial fibrosis-related targets. Molecular docking confirmed strong binding affinities (below -5.0 kcal/mol) of key components (heptadecanoic acid, palmitic acid) to core targets (MMP-9, PTGS2, MAPK3). Western blot analysis further verified that GSO significantly inhibited the expression of PI3K-AKT pathway-related proteins (p < 0.01). Conclusions: The fatty acids in GSO (linolenic acid, palmitic acid) attenuate myocardial fibrosis by inhibiting the PI3K/AKT signaling pathway and downregulating key fibrotic markers. These findings establish a novel theoretical foundation for the treatment of myocardial fibrosis and highlight the potential value of grape industry byproducts in cardiovascular therapeutics.