Role Of ROS In Nickel-induced Rat Leydig Cells Apoptosis Via Endoplasmic Reticulum Stress And Mitochondrial Apoptotic Pathway In Vitro

Objectives To investigate the relationship between the ROS level and cell apoptosis of rat Leydig cells induced by nickel sulfate(NiSO4), and explore the role of ROS in nickel-induced Leydig cells apoptosis via endoplasmic reticulum stress and mitochondrial apoptotic pathway in vitro. Methods(1) The rat Leydig cells were separated by 0.25% collagenase and purified by Percoll liquid continuous gradient and attachment culture. The purity of the cells was identified by 3β-HSD staining.(2) The logarithmic phase of purified Leydig cells were treated by NiSO4 with different concentration of 0, 250, 500 and 1000 μmol/L for 0, 6, 12 and 24 h, respectively. The cells survival rates and the activity of LDH in culture supernatant was assessed by MTT and lactate dehydrogenase release assay.(3) Before treated with NiSO4, the cells were intervened by the free radical scavenger of NAC and TEMPO. The ROS level was detected with DCFH-DA fluorescence staining. The nucleus morphological changes were detected with DAPI staining assay, and the cells apoptosis was analyzed by Flow Cytometry. The content of protein such as GRP78, GADD153, Caspase-12, Bak, Cyt C, Caspase-9 and Caspase-3 were evaluated by Western blot. Results(1) The purity coefficient of Leydig cells can reach more than 90%, which was identified by 3β-HSD after attachment culture for 24 h.(2) Compared with control group, the cell survival rates were significantly decreased with the increase of NiSO4 concentration(250, 500 and 1000 μmol/L) and treatment time(6, 12 and 24h) and the activity of LDH in culture supernatant was significantly increased(P<0.05).(3) Compared with control group, the ROS level was significantly increased in exposure group treated with NiSO4 500 μmol/L for 12h(NiSO4 group). Compared with NiSO4 group, the level of ROS declined in NAC group and TEMPO group respectively(P<0.05). After DAPI staining, the Leydig cells became smaller, and cell nucleus appeared some creases and condensed chromatins as well as there were some fragments of the nucleus and apoptotic body in NiSO4 group, but above phenomenon can be reversed by NAC and TEMPO. Compared with control group, cell apoptosis increased significantlyin NiSO4 group(P<0.05), but which can be inhibited by NAC and TEMPO respectively.(4)Compared with control group, the protein expression levels of GRP78, GADD153, Caspase-12, Bak, Cyt C, Caspase-9 and Caspase-3 significantly increased in NiSO4 group(P<0.05), which can be reversed by NAC and TEMPO respectively. Conclusions NiSO4-induced Leydig cells damage may be associated with ROS generation and apoptosis. NiSO4 upregulated protein levels of Endoplasmic Reticulum stress and mitochondrial apoptosis pathway, can be inhibited by radical scavenger NAC or TEMPO, this suggest that ROS play an important role in nickel-induced Leydig cells apoptosis via endoplasmic reticulum stress and mitochondrial apoptotic pathway.