Resveratrol attenuates hydrogen peroxide-induced oxidative stress in TM3 Leydig cells in vitro

The objective of present study was to investigate in vitro protective potential of resveratrol in TM3 Leydig cells with induced oxidative stress using hydrogen peroxide (H₂O₂). Leydig cells experiencing oxidative stress exhibit reduced activities in androgens production, and become hypofunctional with age, which is also related to growing oxidative stress, while resveratrol has received growing attention as a cytoprotective agent. TM3 mouse Leydig cells were cultivated during 24 h in the presence of resveratrol (5, 10, 25, 50 and 100 μM) alone, or in combination with H₂O₂ (300/600 μM) to induce oxidative stress. Mitochondrial activity was evaluated using MTT test, triple assay was used in order to assess cell viability parameters, intracellular generation of superoxide was determined by the nitroblue-tetrazolium assay, and quantification of steroid hormones was performed by the enzyme- linked immunosorbent assay. Resveratrol alone treatment led to the most significantly improved values of all tested parameters in the cells of experimental group with addition of 10 μM of resveratrol in comparison to the control group. In the case of cells with induced oxidative stress (300 μM H₂O₂) resveratrol administration resulted in significantly increased (P < 0.05) metabolic activity, as well as cell membrane integrity at concentration 10 μM. Significantly improved (P < 0.001) lysosomal activity showed cells treated with 5 and 10 μM of resveratrol, and the level of both measured hormones was significantly higher (P < 0.05) in cells supplemented with 10 μM of resveratrol. Significant decline of superoxide radical production was observed in all experimental groups in comparison to the control exposed to H₂O₂ alone. With respect to cells exposed to higher concentration of H₂O₂ (600 μM)_, results showed positive effect of resveratrol only in biosynthesis of both androgens with significant increased values in experimental group treated with 5 μM (P < 0.05) and 10 μM (P < 0.01) of resveratrol, in addition, in the case of testosterone we recorded significant higher (P < 0.05) values in cells with addition of 25 and 50 μM resveratrol when compared to H₂O₂ control. More specific and systematic research focused especially on androgen biosynthesis is necessary related to the biological activity of resveratrol in male reproductive system due to inconsistent results of studies.