Piperine attenuated CoCl2-induced oxidative stress and cellular injury in TM3 Leydig cells

Hypoxia impairs the function and viability of TM3 Leydig cells by inducing oxidative stress and disrupting testosterone synthesis. Piperine, a bioactive alkaloid derived from black pepper, possesses antioxidant properties that may protect Leydig cells from hypoxia-induced damage. This study investigated the effects of piperine on cell viability, steroidogenesis, oxidative stress, and gene expression in TM3 Leydig cells exposed to cobalt chloride (CoCl 2 )-induced chemical hypoxia. TM3 Leydig cells were cultured under CoCl 2 -induced chemical hypoxia (a hypoxia-mimetic condition) and treated with different concentrations of piperine. Cell viability was assessed using the MTT assay and lactate dehydrogenase release. Testosterone concentrations in the culture supernatant were quantified. Oxidative stress status was determined by measuring malondialdehyde levels and the activities of the antioxidant enzymes superoxide dismutase and glutathione peroxidase. The steroidogenic gene expression of ( Star , Hsd3b1 , Cyp17a1 , and Cyp11a1 ), Nr5a1 , Hif1a , and MAPK pathway components ( Mapk14 , Mapk3 , and Mapk8 ) were analyzed by real-time PCR. Protein expression levels of HIF-1α, ERK1/2, p38, and JNK were evaluated by Western blot analysis. Piperine showed no cytotoxicity under normoxic conditions. CoCl 2 -induced chemical hypoxia significantly reduced TM3 cell viability, testosterone concentration in the culture supernatant, antioxidant enzyme activities, and steroidogenic gene expression, while increasing LDH release, lipid peroxidation, and HIF-1α expression. Piperine co-treatment significantly attenuated the CoCl 2 -associated reduction in cell viability, oxidative stress, and cellular injury, enhanced antioxidant defenses, and increased testosterone concentration in the culture supernatant and partially attenuated the CoCl 2 -associated reduction in steroidogenic gene expression. Piperine co-treatment was also associated with reduced HIF-1α expression, whereas changes in p38 protein expression were descriptive because protein samples were pooled before Western blot analysis. Among the tested concentrations, 20 µM piperine generally produced the greatest attenuation of CoCl 2 -associated changes. Piperine co-treatment attenuated several cellular and oxidative disturbances associated with CoCl 2 exposure in TM3 Leydig cells and was associated with higher testosterone concentration in the culture supernatant and changes in steroidogenic gene expression. Overall, these findings indicate an association between piperine co-treatment and attenuation of CoCl₂-associated cellular and oxidative disturbances, accompanied by changes in steroidogenic responses.