IN VITRO EVALUATION OF HESPERIDIN AS A HEPATOPROTECTIVE AGENT AGAINST CYCLOPHOSPHAMIDE-INDUCED OXIDATIVE AND INFLAMMATORY INJURY
Abstract
Objective: The objective of the study is to investigate the hepatoprotective effects of hesperidin against cyclophosphamide-induced oxidative stress and inflammatory injury in HepG2 cells, with particular emphasis on modulation of the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) antioxidant pathway and the nuclear factor-κB (NF-κB)-mediated inflammatory signaling pathway. Methods: HepG2 cells were allocated to control, hesperidin (50 μM), cyclophosphamide (5 mM), cyclophosphamide plus hesperidin (25 μM), and cyclophosphamide plus hesperidin (50 μM) groups. Since cyclophosphamide requires metabolic activation, treatments were performed in the presence of a rat liver S9 metabolic activation system for 24 h. Hepatocellular injury was assessed by measuring aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), total protein, antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase, and glutathione), and malondialdehyde (MDA). The expression of NFE2L2, HMOX1, NQO1, RELA, tumor necrosis factor (TNF), and interleukin-6 (IL-6) was evaluated by reverse transcription-quantitative polymerase chain reaction, while Nrf2, HO-1, p-NF-κB p65, TNF-α, and IL-6 protein expression was determined by Western blotting. Data were analyzed using one-way analysis of variance followed by Tukey’s post hoc test, with p<0.05 considered statistically significant. Results: Cyclophosphamide enhanced the expressions of AST, ALT, LDH, MDA, and pro-inflammatory genes and proteins but decreased the expression of total protein, endogenous antioxidant defenses, and Nrf2/HO-1-related genes and proteins. Hesperidin caused concentration-dependent amelioration in biochemical injury and inflammatory signaling, with higher efficacy of recovery at 50 μM. Conclusion: Hesperidin protected HepG2 cells from cyclophosphamide-induced oxidative and inflammatory damage in part by enhancing antioxidant levels, by stimulating Nrf2/HO-1 signaling, and by reducing NF-κB-related activities.