Targeting TGF-β1/MMP-9/α-SMA Signaling Axes Using Piperine and Galangin in Bleomycin Induced Pulmonary Fibrosis Model

Abstract

Aims: To evaluate and compare the anti-fibrotic efficacy of galangin and piperine alone and in combination against bleomycin-induced pulmonary fibrosis in C57BL/6 mice, using biochemical (hydroxyproline, TGF-β1), histological (Masson’s trichrome), and immunohistochemical (MMP-9, α-SMA) endpoints. Study Design: Controlled, randomized, experimental animal study with seven parallel groups. Place and Duration of Study: Department of Veterinary Pharmacology and Toxicology, College of Veterinary Science, Hyderabad, India. Experiments were conducted under IAEC approval using a 21-day protocol. Methodology: Fifty male C57BL/6 mice were allocated into seven groups, including control, bleomycin, galangin, piperine, and their combinations. After 21 days of treatment, lung tissues were analyzed for hydroxyproline, TGF-β1, collagen (Masson’s trichrome), and MMP-9 and α-SMA (IHC). Data were statistically evaluated using one-way ANOVA followed by Tukey’s test (P < 0.05). Results: BLM significantly increased hydroxyproline vs control (171.41±6.46 vs 27.35±2.57 pg/mg protein; P<0.05) and TGF-β1 (129.24±2.87 vs 16.61±2.09 pg/mg; P<0.05). Treatment reduced both biomarkers vs BLM: GA (hydroxyproline 110.87±2.84; TGF-β1 64.17±2.87), PIP (97.68±3.22; 58.73±2.56), and combination (65.82±5.22; 42.15±2.41), with the combination superior to monotherapies (all P<0.05). Per se GA and PIP (groups 3–4) did not differ from control for either marker (P>0.05). Masson’s trichrome showed marked collagen deposition in BLM lungs, attenuated to mild (GA, PIP) and very mild (combination) fibrosis. IHC demonstrated strong MMP-9 and α-SMA expression in BLM lungs, reduced with GA or PIP and minimal with combination therapy. Conclusion: Galangin and piperine mitigate bleomycin-induced lung fibrosis, with combination therapy yielding the greatest improvement across biochemical, histological, and immunohistochemical endpoints. Benefits likely arise from dampening profibrotic signaling (TGF-β1/α-SMA/MMP-9) alongside antioxidant/anti-inflammatory actions, supporting these phytochemicals particularly in combination as promising candidates for anti-fibrotic intervention.