Silybum Marianum Oil Attenuates Carbon Monoxide-Induced Pancreatic Bcl-2 Overexpression: An Age-Dependent Immunohistochemical Study

Abstract

Chronic low-level carbon monoxide (CO) exposure is an escalating environmental hazard, yet its impact on the apoptotic balance of the exocrine and endocrine pancreas, and the modulating role of natural antioxidant oils, remain poorly defined. We evaluated the anti-apoptotic protein Bcl-2 in the rat pancreas after chronic CO exposure and after correction with Silybum marianum (milk thistle) seed oil, and how chronological age conditions this response. Outbred male rats aged 3 and 12 months were allocated to control, CO (0.01–0.05 mg/L in air, 2 months) and CO plus intragastric S. marianum oil (14 days) groups. Bcl-2 was detected by immunohistochemistry and quantified as the labelling index (percentage of positive-stained area) using QuPath digital image analysis (n=10 per subgroup). Data were analysed by two-way analysis of variance on log-transformed values with Tukey post-hoc tests and confirmed by non-parametric procedures. Chronic CO markedly increased pancreatic Bcl-2 positivity relative to age-matched controls (3-month: 5.12→23.91%, 4.7-fold; 12-month: 1.19→24.15%, 20.3-fold; p<0.001), indicating an intense but morphologically insufficient compensatory anti-apoptotic response. S. marianum oil significantly lowered Bcl-2 toward baseline (3-month: 11.08%; 12-month: 15.17%; p<0.001 versus CO), with a significant group×age interaction (p<0.001) reflecting less complete normalization in older animals. Pancreatic Bcl-2 over-expression tracks the severity of CO-induced oxidative–hypoxic injury in an age-dependent manner, and S. marianum oil restores the apoptosis–survival balance, supporting its use as an adjunct pancreatoprotective agent.