Antioxidant and anti-apoptotic effects of trigonella foenum-graecum linn. and Cinnamomum cassia on isoniazid-induced hepatotoxicity

Background and Aim: Isoniazid (INH) is one of the main drugs used in the treatment of tuberculosis (TB); however, it can induce hepatotoxicity by enhancing oxidative and endoplasmic reticulum (ER) stress. Trigonella foenum-graecum Linn. (Fenugreek) and Cinnamomum cassia (C. cassia) are known to exert antioxidant and anti-apoptotic effects. This study aimed to evaluate the antioxidant and anti-apoptotic effects of Fenugreek and C. cassia, administered alone or in combination with INH, in an INH-induced hepatotoxicity model established on a mouse hepatocyte cell line (AML12). Material and Methods: The study was conducted in AML12 cells and designed with six groups: Control, INH, Fenugreek, C. cassia, INH + Fenugreek, and INH + C. cassia. The concentrations of INH and plant extracts to be applied were determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay based on cell viability. The doses were calculated using Probit regression and dose–response curves. For the antioxidant response, superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH) levels were measured and normalized to total protein. Apoptosis/ER stress-related Bax, Bcl-2, caspase-3, caspase-12 and TNF-α gene expressions were evaluated by reverse transcription-quantitative real-time PCR (RT-qPCR). Cell death patterns were determined by Annexin V-FITC/PI flow cytometry. Results: IC₅₀ values were 1.689 mg/mL for INH, 0.272 mg/mL for Fenugreek and 0.139 mg/mL for C. cassia. The IC₅₀ value of INH + C. cassia was 0.132 mg/mL, whereas that of INH + Fenugreek was 10.939 mg/mL. SOD levels were elevated only in the INH + C. cassia group (p<0.0001). CAT levels were highest in the INH group (p<0.0001), lower in INH + C. cassia yet still above control (p<0.0001), and close to control in the INH + Fenugreek group. GSH levels were highest in the INH + C. cassia group (p<0.0001) and were also above control in the INH, C. cassia and Fenugreek groups. Bax expression was highest in the Fenugreek group (p<0.0001), while Bcl-2 expression was higher in the INH group than in the Fenugreek (p<0.05) and C. cassia (p<0.01) groups. Caspase-3 expression was highest in the INH group (p<0.0001) and was close to control levels in the INH + Fenugreek group. Caspase-12 expression decreased in the INH + Fenugreek (p<0.0001) and INH + C. cassia (p<0.001) groups. In flow cytometry, necrosis was highest in the INH + Fenugreek group (52.9±0.26%) and lowest in the INH + C. cassia group (0.53±0.06%). Conclusion: Isoniazid increased oxidative and ER stress in AML12 cells, weakened antioxidant defenses and triggered apoptosis. Fenugreek and C. cassia extracts reversed these disturbances in different yet complementary ways: C. cassia likely enhanced an Nrf2-centered antioxidant response, whereas fenugreek modulated inflammation and apoptosis mainly through the TNF-α and caspase-3/caspase-12 axis. When supported by future dose–duration optimization and in vivo studies, our findings suggest that these plant extracts may represent a complementary option in INH-induced hepatotoxicity.