| Objective: TM3 cells of mouse testicular leydig cell line were used as the research model to study the interaction mechanism of autophagy/Nrf2 signaling pathway in the oxidative damage of testicular mesenchymal cells caused by manganese,so as to provide theoretical and experimental basis for the prevention and treatment of occupational population with manganism.Methods: 1.TM3 cells were exposed to 0 ?M(Blank control group,C),100 ?M(Low dose group,L),200 ?M(Medium dose group,M),and 300 ?M(High dose group,H)of manganese for 24 h,respectively.And cells morphology was observed.Cells viability was determined by MTT colorimetry,intracellular ROS level was detected by DCFH-DA probe,intracellular MDA content was detected by TBA,total SOD activity was detected by WST-8,Total GPx activity was measured with total GPx detection kit,apoptosis rate was detected by flow cytometry after Annexin V/PI double staining,and intracellular autophagy was observed by transmission electron microscopy.Western blot was used to detect the expression of autophagy-related proteins p-Beclin1,p62,LC3?/?,Nrf2 signaling pathways related proteins Nrf2,NQO-1,HO-1 and apoptosis related proteins Active caspase-3,Bax and Bcl-2 levels.2.TM3 cells were exposed to 300 ?M manganese for 24 h after pretreatment with autophagy agonist 0.1 ?M with rapamycin for 2 h(RH group).Cells were exposed to 300 ?M manganese for 24 h after pretreatment with autophagy inhibitor si RNA-Beclin1(40 n M)for 5h and continued culture for 24 h(BH group).Then,cell morphology was observed,ROS levels in cells were detected by DCFH-DA probe,MDA content in cells was detected by TBA,total SOD activity was detected by WST-8,total GPx activity was determined by GPx detection kit,and apoptosis rate of cells was detected by flow cytometry after Annexin V/PI double staining.Using Western blot technique to detect the expression of autophagy related protein p62,LC3 ?/?,Nrf2 signaling pathways related proteins Nrf2,NQO-1,HO-1 and apoptosis related proteins Active caspase-3,Bax and Bcl-2 levels.3.TM3 cells were exposed to 300 ?M of manganese for 24 h after pretreatment with Nrf2 signaling pathway agonist TBHQ(10 ?M)for 2 h(TH group).Cells were exposed to 300 ?M of manganese for 24 h after 5 h of pretreatment with Nrf2 signaling pathway inhibitor si RNA-Nrf2(40 n M)and continued culture for 24 h(NH group).Then,cell morphology was observed,ROS levels in cells were detected by DCFH-DA probe,MDA content in cells was detected by TBA,total SOD activity was detected by WST-8,total GPx activity was determined by GPx detection kit,and apoptosis rate of cells was detected by flow cytometry after Annexin V/PI double staining.Using Western blot technique to detect the expression of Nrf2 signaling pathways related proteins Nrf2,NQO-1,HO-1,autophagy related protein p-Beclin1,p62,LC3?/?and apoptosis related proteins Active caspase-3,Bax and Bcl-2 levels.Results: 1.Compared with C group,manganese induced decreased activity and morphological changes of TM3 cells in a dose-dependent manner.M and H group, compared with C group,the intracellular ROS,MDA content increased(P<0.05)and the total activity of SOD,GPx were decreased(P<0.05),The apoptosis rate increased(P<0.05),and promote apoptosis related proteins Active caspase-3 and Bax expression level increased with the increase of Mn concentration(P<0.05),the antiapoptotic protein expression level of Bcl-2 is gradually reduced(P<0.05).Compared with C group,the number of autophagic lysosomes increased with the increase of manganese concentration.p-Beclin1 expression level and LC3?/? ratio of M,H group were increased(P<0.05),while compared with C group,the expression level of p62 protein increased first and then decreased.Compared with C group,the expression of Nrf2 and its downstream effectant proteins HO-1 and NQO-1 increased and then decreased after manganese exposure,while the expression level of HO-1 protein increased first and then decreased,but their expression level was higher than that of C group after manganese exposure(P<0.05).2.Compared with H group,the ROS content of RH group was slightly reduced(P>0.05),and the activity of total SOD increased(P<0.05).Compared with H group,the content of ROS and MDA in BH group increased(P>0.05),while the activity of total SOD and GPx decreased slightly(P>0.05).Compared with H group,the apoptosis rate and the expression levels of Active caspase-3 and Bax proteins of the RH group were decreased,while the Bax protein level of BH group was slightly increased(P>0.05),but the apoptosis rate was significantly increased(P<0.05),the expression of Bcl-2 protein was significantly increased compared with H group and RH group(P<0.05).Compared with H group,the expression level of HO-1 protein increased in RH group,while the expression level of HO-1 protein in BH group decreased significantly(P<0.05).The expression of Nrf2 protein was not statistically significant among the experimental groups(P>0.05).3.Compared with H group,the ROS content of TH group was significantly reduced(P<0.05),and the activity of total SOD was significantly increased(P<0.05),the content of ROS and MDA in NH group increased significantly compared with H group(P<0.05),while the activity of total SOD and total GPx decreased.Flow cytometry showed that compared with H group,the apoptosis rate of TH group was reduced,while NH group was significantly increased(P<0.05).Compared with H group,the expression level of Active caspase-3 in TH group decreased,and the expression level of Bcl-2 increased significantly(P<0.05),while the expression level of Active caspase-3 in NH group increased,and the expression level of Bcl-2 increased,but it was not statistically significant(P>0.05).Compared with H group,LC3?/? ratio of TH group decrease(P>0.05),p-Beclin1,p62 protein expression level increased(P>0.05),however,compared with H group,the expression levels of p-Beclin1 and p62 proteins in NH group were significantly reduced(P<0.05).Conclusion: 1)Manganese-induced oxidative damage in TM3 cells activates autophagy and Nrf2 signaling pathways.2)Autophagy at a given dose plays a protective role in manganese-induced oxidative damage to TM3 cells.3)Nrf2 signaling pathway plays a protective role in manganese-induced oxidative damage of TM3 cells4)Autophagy and Nrf2 signaling pathways interact with each other in the oxidative damage induced by manganese in TM3 cells. |
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