The Role Of NRF2 Signaling Pathway In The Oxidative Damage Of TM3 Cells In Manganese

Objective:To investigate the role and possible mechanism of NRF2/KEP1/ARE signaling pathway in Mn-induced murine testicular stromal cells?TM3 cells?injury,as a mechanism for the reproductive toxicity of manganese and biology of NRF2/KEP1/ARE signaling pathways The function and the role of the pathway in the disease and the mechanism of action provide new experimental evidence,providing new ideas and approaches for follow-up research and the development of new drugs and the treatment of diseases.Methods:Through cell culture,the cells were divided into the following groups:1.blank control group?MnCl₂:0?mol/L,group C?2.low-dose manganese staining group?MnCl₂:100?mol/L,group L?,3.middle-dose manganese staining group?MnCl₂:200?mol/L,M group?,4.high-dose manganese staining group?MnCl₂:300?mol/L,H group?;5.agonist control group?TBHQ:10?mol?/L,TC group)6.Agonist+low dose group?TBHQ:10?mol/L+MnCl₂:100?mol/L,TL group?,7.Agonist+middle dose group?TBHQ:10?mol/L+MnCl₂:200?mol/L,TM Group?8.Agonist+high dose group?TBHQ:10?mol/L+MnCl₂:300?mol/L,TH group?;9.Inhibitor control group?ATRA:10?mol/L,RC group?,10.Inhibitor+low dose Group?ATRA:10?mol/L+MnCl₂:100?mol/L,RL group?11.Inhibitor+middle dose group?ATRA:10?mol/L+MnCl₂:200 umol/L,RM group?,12.Inhibitor+high dose group?ATRA:10?mol/L+MnCl₂:300?mol/L,RH group?;the time of Mn staining was 24 hours.The morphology of the cells was observed by inverted phase contrast microscopy.The cell viability was measured by MTT assay,the apoptosis rate was measured by Annnexin V/PI double staining method,the ROS level was measured by DCFH-DA probe,the intracellular MDA content was measured by TBA method,and the Western Blot and RT-Qpcr technique were measured Intracellular expression of NRF2,HO-1,GSTpi,SOD,NQO1 and other proteins and genes.Results:1.MTT assay found that the difference in cell viability between the three dose groups at 24h,48h and 72h was statistically significant?P<0.05?.The higher manganese dose compared with the lower the cell survival rate.There was no significant difference in cell survival rate between the C group and the RC group?P>0.05?.Compared with the control group,the survival rate of TM3 cells gradually decreased with the increase in the dose of manganese,and the difference was statistically significant?P<0.05?and dose-effect relationship;RL,RM,RH group compared with RC group,the survival rate of RL,RM,RH group cells also gradually decreased,the difference was statistically significant?P<0.05?There was a dose-effect relationship,and the survival rates of the cells in the ATRA group were decreased compared with the RL,RM,and RH groups?P<0.05?;Compared with the group,the survival rate of the TM group and the TH group increased,the difference was statistically significant?P<0.05?,2.Inverted phase contrast microscope was used to observe different concentrations of manganese?0?mol/L,100?mol/l,200?mol/L,300?mol/L?and ATRA?10?mol/L?,TBHQ?10?mol/L?.After treatment with TM3 cells for 1.5 h and then with different concentrations of manganese,the morphology of TM3 cells changed.Adhered cells in groups C and RC were tightly adherent,and the cells were plump and well differentiated.The proliferating cells were connected to each other in a single layer and the cytoplasm spread out.Prominence.With the increase of the dose of manganese,the cells gradually retracted and rounded,the cells floated,and gradually produced cell debris.After being treated with ATRA and reinfected,it was found that compared with the group without ATRA,the floating cells were more prominent,the cells retracted and rounded,and the cell debris generated increased;the cells in groups C and TC were tightly adherent,well differentiated,and full of cell bodies.The proliferating cells were connected into a single layer of mesh,with the cytoplasm spread out and protruding out of the protrusions,among which the TC group had a better cell state.With the increase of the concentration of manganese,the above changes were more obvious.After treatment with an agonist and then staining with manganese,it was found that compared with the group without the agonist,the cells were concentrated and round and the suspended cells were not obvious,and the amount of cell debris generated was not as large as Plus agonist group more 3.In the two-variable flow scatter plot,compared with the C group,there was no significant difference in the apoptotic rate between RC group and TC group?P>0.05?,L,M,H,RL,RM,RH,Apoptotic rate increased in TL,TM,and TH groups?P<0.05?.Compared with L,M,and H groups,the apoptotic rate of RL,RM,and RH groups increased,and the difference was statistically significant.Significance?P<0.05?,but the apoptotic rates of TL,TM,and TH groups were all decreased?P<0.05?.Compared with RC group,the apoptotic rates of RL,RM,and RH groups were all increased.The difference was statistically significant?P<0.05?.Compared with TC group,the apoptotic rate of TL,TM,and TH groups increased,the difference was statistically significant?P<0.05?;RL,RM,RH and Compared with TL,TM,and TH groups,the apoptotic rate of RL,RM,and RH groups increased,and the difference was statistically significant.4,DCFH-DA probe detection of intracellular ROS levels found that different concentrations of manganese and pre-stained TBHQ,ATRA and then stained manganese in TM3 cells,compared with the C group,L,M,H,TL,TM,TH group cells The levels of ROS were significantly higher in TH and H groups than in TM and M groups were higher than those in TL and L groups?P<0.05?.Compared with L,M,and H groups,TL,TM The ROS content in the TH group was significantly lower?P<0.05?.The ROS content in the RL,RM,and RH groups increased,and the difference was statistically significant?P<0.05?.5.The intracellular MDA level was detected by the TBA method.It was found that MDA levels in cells of L,M,H,TL,TM,and TH groups were significantly higher than those in C group,among which TH and H were significantly increased after different concentrations of manganese,pre-dye TBHQ,and ATRA,and then manganese staining were applied to TM3cells.The difference was statistically significant?P<0.05?between the group above the TM and the M group and the L group?P<0.05?.Compared with the M and H groups,the MDA content in the TM and TH groups was significantly lower and the difference was statistically significant?P<0.05?.<0.05);Compared with L and M groups,MDA content in RL and RM groups were significantly higher?P<0.05?.6.Western Blot technique was used to detect Nrf2 in each group.The expression levels of HO-1,NQO1,SOD,and GSTpi proteins were significantly lower in the RL,RM,and RH groups than in the L,M,and H groups,and the differences were found in the expression levels of Nrf2,HO-1,NQO1,SOD,and GSTpi.There was statistical significance?P<0.05?.Compared with the inhibitor control group,the expression levels of Nrf2,HO-1,NQO1,SOD and GSTpi protein in RL,RM,and RH groups gradually increased with the increase of manganese concentration.The difference was statistically significant?P<0.05?.Compared with groups M and H,the expression levels of Nrf2,HO-1,NQO1,SOD and GSTpi in TM and TH groups were all increased?P<0.05?,and the difference was statistically significant.Compared with TC group,TM The expression levels of Nrf2,HO-1,NQO1,SOD,GSTpi in the TH group increased with the concentration of Mn,and their expression levels also gradually increased.The difference was statistically significant?P<0.05?.There was no statistically significant difference between the other groups.7.RT-Qpcr method was used to determine the changes of mRNA expression of Nrf2,HO-1,NQO1,SOD,GSTpi and other genes in TM3 cells treated with manganese and after treatment with inhibitors and agonists.TM3cells were stimulated with manganese.There was no significant difference in the expression of Nrf2 mRNA in each group?P>0.05?.Compared with group C,the expression of HO-1 mRNA was increased in TC,TL,TM,and TH groups,and the difference was statistically significant?p<0.05?,the expression of HO-1 m RNA in RC,RL,RM and RH groups was decreased,the difference was statistically significant?p<0.05?;compared with C,L,M,H group,TC,TL,The expression of HO-1 mRNA in TM and TH groups increased,the difference was statistically significant?p<0.05?.The expression of HO-1mRNA in RC,RL,RM,and RH groups decreased,and the difference was statistically significant?p<0.05?.NQO1 mRNA expression was only in RC,RL and RM groups compared with C,L and M groups.The expression of NQO1 mRNA in RC,RL and RM groups was lower than that in C,L and M groups.The difference was statistically significant?p<0.05?,there was no significant difference in other groups?P>0.05?;Compared with C,L,M and H groups,the expression of SOD mRNA in RC,RL,RM and RH groups was lower than that in C,L,M and H groups.the difference was statistically significant?p<0.05?.there was no significant difference between other groups?P>0.05?The expression of GSTpi mRNA was lower in RL,RM,and RH groups than in L,M,and H groups.The expression of GSTpi mRNA was lower in the RL,RM,and RH groups than in the L,M,and H groups.The difference was statistically significant?p<0.05?.Compared with groups M and H,the expression of GSTpi mRNA in TM and TH groups was higher than that in M and H groups in TM and TH groups?p<0.05?.Conclusion:Activation of Nrf2 signaling pathway may be one of the important mechanisms that antagonize oxidative damage induced by manganese in TM3 cells.