Effects Of Selenium On Apoptosis And Oxidative Stress Of Sheep Leydig Cells Induced By Lead

The objective of this study was to study the reproductive toxicity of lead on Leydig cells of sheep,the effects of selenium on apoptosis and oxidative stress of Leydig cells induced by lead,and the effects of selenium and lead on Nrf2 signaling pathway in Leydig cells of sheep,so as to provide theoretical reference for the study of selenium antagonizing reproductive toxicity of lead with Nrf2 as the target.Jinzhong sheep aged 5-8 months were selected as the experimental objects.The Leydig cells were isolated and purified by collagenase I.The cells were cultured in groups after stable growth,and the purity of cells was identified by 3β-HSD staining.The experiment was divided into two parts.In the first part,sheep Leydig cells were cultured for 24 h in control group,Pb group(40 μmol/L),Se group(2 μmol/L)and Se+Pb group.The second part was pretreated with Nrf2 inhibitor ML385(5 μm)for 12 h,and then cultured in control group,Pb group,Se group,Se+Pb group,control+ML385 group,Pb+ML385 group,Se+ML385group and Se+Pb+ML385 group for 24 h.Cell viability was measured by CCK-8 method,ROS level was measured by DCFH-DA probe,MDA level was measured by TBA method,m RNA and protein expression of SOD gene,apoptosis related genes(Caspase3,Caspase8 and Bax),Nrf2 pathway and downstream genes(Nrf2,HO-1,NQO1 and γ-GCS)were detected by q RT PCR and western blotting.The test results and analysis are as follows:(1)Compared with the control group,the cell viability of Pb group was significantly decreased(P<0.05),and that of Se+Pb group was significantly higher than that of Pb group(P<0.05).(2)Compared with control group,the levels of ROS and MDA in Pb group were significantly increased(P<0.05),ROS level was significantly decreased in Se group(P<0.05),MDA level was decreased but not significantly(P>0.05),ROS and MDA levels in Se+Pb group were significantly lower than those in Pb group(P<0.05).The expression of SOD in Se+Pb group was significantly higher than that in control group(P<0.05).The expression of SOD in Se+Pb group was significantly higher than that in Pb group(P<0.05).(3)The m RNA and protein expression trends of Caspase3,caspase8 and Bax were consistent,that is,the expression levels of Caspase3,caspase8 and Bax were the highest in Pb group,the lowest in Se group,and significantly lower in Se+Pb group than in Pb group(P<0.05).(4)The m RNA and protein expressions of Nrf2 and downstream genes HO-1,NQO1 and γ-GCs in Pb group and Se group were significantly higher than those in control group(P<0.05),and the m RNA and protein expression of Pb group was significantly higher than that of Se group(P<0.05).(5)After pretreatment with ML 385,the protein expression levels of Nrf2 and downstream genes HO-1,NQO1 and GCLC were all down regulated in each group,with the decrease of cell viability,the increase of ROS level and the increase of protein expression of apoptosis related genes.In conclusion,40 μmol/L lead exposure caused oxidative damage to Leydig cells,inhibited their proliferation and promoted apoptosis.Lead can highly activate Nrf2 pathway for its own antioxidant defense response;2 μmol/L selenium can activate Nrf2 signaling pathway by regulating antioxidant activity and apoptosis related genes to antagonize oxidative stress of Leydig cells caused by lead and alleviate cell apoptosis;Nrf2 pathway is involved in regulating apoptosis of Leydig cells in sheep.