Standardized Brazilian Green Propolis Extract Attenuates Isoproterenol-Induced Cardiac Fibrosis Through Modulation of TGF-β1/Smad, NF-κB, PI3K/Akt, and Nrf2/HO-1 Signaling in Rats

Background Cardiac fibrosis is a major pathological feature of chronic cardiovascular diseases and heart failure, characterized by excessive extracellular matrix deposition, persistent inflammation, oxidative stress, and progressive ventricular remodeling. Despite advances in cardiovascular therapeutics, effective pharmacological strategies capable of directly attenuating myocardial fibrosis remain limited. Brazilian green propolis contains numerous bioactive compounds with potent antioxidant and anti-inflammatory properties, although its molecular effects on cardiac fibrosis have not been comprehensively elucidated. Objective This study investigated the cardioprotective effects of standardized Brazilian green propolis extract (EPP-AF®) against isoproterenol-induced cardiac fibrosis in rats and explored the underlying molecular mechanisms involving the TGF-β1/Smad, NF-κB, PI3K/Akt, and Nrf2/HO-1 signaling pathways. Methods Forty adult male Wistar rats were randomly assigned to four groups (n = 10/group): Control, Cardiac Fibrosis (isoproterenol), Isoproterenol + EPP-AF® (100 mg/kg/day), and Isoproterenol + EPP-AF® (200 mg/kg/day). Cardiac fibrosis was induced by subcutaneous administration of isoproterenol (5 mg/kg/day) for seven consecutive days. Following fibrosis induction, EPP-AF® was administered orally for 28 days. Cardiac function was evaluated using transthoracic echocardiography. Histopathological alterations were assessed by hematoxylin and eosin, Masson's Trichrome, and Picrosirius Red staining. Immunohistochemistry, ELISA, quantitative real-time PCR, and Western blot analyses were performed to evaluate fibrosis-related proteins, inflammatory cytokines, oxidative stress biomarkers, and intracellular signaling pathways. Results EPP-AF® significantly improved cardiac function, increasing left ventricular ejection fraction from 48.9 ± 3.8% in untreated fibrotic animals to 67.5 ± 4.2% in the high-dose group (P < 0.001). Histological analysis demonstrated a marked reduction in myocardial collagen deposition, with collagen volume fraction decreasing from 27.3 ± 3.1% to 8.4 ± 1.8% (P < 0.001). Immunohistochemical analysis revealed significant reductions in TGF-β1, α-SMA, Collagen I, Collagen III, and Fibronectin expression following EPP-AF® treatment. ELISA demonstrated substantial suppression of TNF-α, IL-1β, IL-6, MCP-1, and TGF-β1, accompanied by restoration of IL-10 levels. Gene expression analysis confirmed downregulation of COL1A1, COL3A1, CTGF, Smad2, and Smad3, while Western blotting demonstrated inhibition of the TGF-β1/Smad, NF-κB, and PI3K/Akt signaling pathways together with activation of the Nrf2/HO-1 antioxidant pathway. Correlation analysis showed that collagen volume fraction was strongly associated with impaired cardiac function and activation of profibrotic molecular markers. Conclusion Standardized Brazilian green propolis extract (EPP-AF®) significantly attenuated experimental cardiac fibrosis by suppressing inflammation, oxidative stress, fibroblast activation, and extracellular matrix remodeling through coordinated modulation of the TGF-β1/Smad, NF-κB, PI3K/Akt, and Nrf2/HO-1 signaling pathways. These findings identify Brazilian green propolis as a promising multitarget therapeutic candidate for preventing pathological myocardial remodeling and provide a strong preclinical foundation for future translational and clinical investigations.