Studies On The Toxic Of Nickel And The Protective Effects Of Procyanidin On Reproduction Of Male Rat

The major objectives of the present study were to determine whether nickel sulfate (Ni)-induced reproductive damage occurs via apoptosis and oxidative stress and to examine the expression of Bax, Caspase-3 and c-kit and their effects on Ni exposure. This study also explored the protective effects of grape seed Procyanidin extract (GSPE) against Ni toxicity in the testes. Wistar rats were treated with normal saline, Ni alone (1.25,2.50, and 5.00 mg/kg/day), and Ni (2.50 mg/kg/day) plus GSPE (50 and 100 mg/kg/day). Thus, sperm motility, testicular apoptosis, oxidative stress, and the expression of related proteins were investigated to explore their possible mechanisms. The results were showed as follow:1. During the 30 day treatment period, body weight and relative testes weight did not show significant changes among the control and experimental groups. No deaths occurred in any of the groups.2. Significant increases in curvilinear velocity (VCL) and decreases in linearity (LIN), straightness (STR), and density of spermatozoa (p) in the 2.50 and 5.00 mg Ni groups were observed compared with the control group (p<0.05). The results also showed significant increases in mean angular deviation (MAD) in the 2.50 mg Ni group and decreases in average path velocity (VAP) and straight line velocity (VSL) in the 5.00 mg Ni group compared with the control group (p<0.05). No significant differences in amplitude of lateral head displacement (ALH), beat cross frequency (BCF), and wobble (WOB) were found between the control group and Ni groups. All GSPE groups displayed significant decreases in VCL and MAD and increases in STR and p compared with the 2.50 mg Ni group (p<0.05). Moreover, LIN and WOB significantly increased in the 100 mg GSPE group (p<0.05).3. A significant decrease in the percentage of S-phase cells was observed in the 2.50 and 5.00 mg Ni groups compared with the control group. The percentage of GO/G1-and G2/M-phase cells showed relative increases. The rate of testicular cell apoptosis in all Ni groups was higher than the control group, but only the 2.50 and 5.00 mg Ni groups demonstrated significant increases compared with the control group. The addition of GSPE resulted in an increase in the percentage of S-phase cells and a decrease in the rate of apoptosis, indicating that GSPE may maintain a normal testicular cell cycle and decrease the incidence of apoptosis. 4. Ni treatment significantly decreased glutathione peroxidase (GSH-Px) and catalase (CAT) activity and increased malondialdehyde (MDA) and hydrogen peroxide (H2O2) content in the testes of rats, especially in the 2.50 and 5.00 mg Ni groups (p<0.05). A significant increase in the activity of GSH-Px was observed in the 50 mg GSPE group compared with the 2.50 mg Ni group (p< 0.05). A significant decrease in MDA content was observed in the 100 mg GSPE group compared with the 2.50 mg Ni group (p<0.05). Significant decreases in H2O2 content were observed in all GSPE groups compared with the 2.50 mg Ni group (p<0.05).5. The analysis of enzyme activity in testicular tissue isolated from control and experimental animals revealed a significant increase in nitric oxide (NO) content and inducible nitric oxide synthase (iNOS) activity in all three Ni groups (p<0.05). However, the levels in the 5.00 mg Ni group were much higher than in the other two Ni groups (p<0.05).Significant increases in total nitric oxide synthase (TNOS) activity were found in the 5.00 mg Ni group compared with the control group and the other two Ni groups (p<0.05). A significant decrease in iNOS activity and a decrease in NO content were observed in the two GSPE groups compared with the 2.50 mg Ni group (p<0.05).6. Western blot analysis showed that the expression of Bax and Pro-caspase-3 protein increased in the 2.50 and 5.00 mg Ni groups compared with controls (p<0.05). At the same time, Pro-caspase-3 protein increased significantly and cleaved-caspase-3 protein expression occured from 1.25 to 5.00 mg Ni groups. Furthermore, the expression of cleaved-caspase-3 protein increased in a dose-dependent manner in all three Ni groups. GSPE significantly reduced Bax and Pro-and cleaved-caspase-3 expression in the testes of rats exposed to 2.50 mg Ni (p<0.05).7. Western blot analysis showed that the expression of c-kit increased in the 2.50 and 5.00 mg Ni groups compared with controls (p<0.05). GSPE significantly reduced c-kit expression in the testes of rats exposed to Ni (p<0.05).Collectively, These results demonstrate the following:(1) Ni exhibits reproductive toxicity in rats by decreasing sperm at concentrations of 2.50 and 5.00 mg; (2) intratesticular oxidative stress, c-kit overexpression and apoptosis play pivotal roles in reproductive damage induced by Ni; and (3) GSPE enhances sperm motility by downregulating c-kit expression and offsetting the apoptosis and oxidative stress induced by Ni by directly decreasing MDA and NO, scavenging H2O2, and downregulating Bax, Pro-and cleaved-Caspase-3 protein expression.