Study On The Damage To Testicular Sertoli Cells By Hexavalent Chromium Through Ferritinophagy-mediated Ferroptosis In Mice

Objective:Hexavalent chromium [Cr(Ⅵ)] is a heavy metal pollutant,affects male reproductive function and causes testicular damage,but the detailed mechanism has not been fully clarified.Sertoli cells are one of the targets that Cr(Ⅵ)causes damage to the testes.This study aims to establish a model of mouse testicular sertoli cells(TM4 cell line)injury induced by Cr(Ⅵ),observe the toxic effect of Cr(Ⅵ)on TM4 cells,and further explore the role and mechanism of ferritinophagy mediated ferroptosis in TM4 cells injury caused by Cr(Ⅵ).Methods:TM4 mouse testicular Sertoli cell line was routinely cultured and subcultured,and CCK-8 assay was used to detect the cell viability after exposure to 1.25-40 μM Cr(Ⅵ)at 12,24 and 48 h.Ultrastructural changes were observed by transmission electron microscopy.DCFH-DA probe was used to detect the intracellular ROS levels.GSH and MDA contents and total iron content were measured by the corresponding kits.Western blot method was used to detect the expressions of blood-testis barrier-related proteins including CX43 and ZO-1,ferroptosis-related proteins including FPN1,Tf R1,SLC7A11 and GPX4,autophagy-related proteins including LC3 B,P62 and BECLIN1,and ferritinophagy-related proteins including NCOA4 and FTH1.After the ferroptosis inhibitor Ferrostatin-1 and 5 μM Cr(Ⅵ)were administered to TM4 cells simultaneously,the cell viability,ROS levels and ferroptosis-related proteins were detected.After the autophagy inhibitor 3-MA and 5 μM Cr(Ⅵ)were administered to TM4 cells simultaneously,the cell viability,ROS levels,autophagy-related,ferritinophagy-related and ferroptosis-related proteins were detected.The NCOA4 gene was silenced using siRNA technology,the efficiency of siRNA silencing was tested,and after TM4 cells were exposed to 5 μM Cr(Ⅵ),ROS levels,ferroptosis and ferritinophagy-related proteins expressions were detected.Results:1.The damage effect of Cr(Ⅵ)on TM4 cells: The CCK-8 results showed that Cr(Ⅵ)can induce the death of TM4 cells in a dose-dependent and time-dependent manner.The results of electron microscope showed that cell structure of the control group was normal,Cr(Ⅵ)treatment increased the number of autophagosomes and autolysosomes,increased mitochondrial membrane density,decreased mitochondrial cristae,and normal nuclear morphology were observed in the chromium exposed group.Western blot showed that the levels of blood-testis barrier proteins ZO-1 and CX43 decreased with the increase of exposure dose,and the levels of the two proteins in the exposure groups significantly lower than those in the control group(P<0.05).2.Cr(Ⅵ)induced ferroptosis in TM4 cells: The kits results showed that the GSH content decreased with the increase of Cr(Ⅵ)exposure dose,while the MDA content,ROS level,and iron content increased with the increase of Cr(Ⅵ)exposure dose in TM4 cells,and there was significantly different in the above indexes between 5 μM,10 μM and the control groups(P<0.05).Iron homeostasis and ferroptosis-related protein Tf R1 increased with increasing exposure dose,the levels of them were significantly higher than those in the control group(P<0.05),while the expression of FPN1 and GPX4 proteins decreased with increasing exposure dose,the levels of the proteins were significantly lower than those in the control group(P<0.05);the expression of SLC7A11 protein in 10 μM exposure group significantly decreased compared with that in the control group(P<0.05)2.Inhibition of ferroptosis saves the Cr(Ⅵ)-induced damage in TM4 cells:Inhibition of autophagy prevents the Cr(Ⅵ)-induced TM4 cell death: After treated with ferroptosis inhibitor Fer-1,cell viability significantly increased(P<0.05),ROS levels significantly decreased(P<0.05),GPX4 protein expression significantly increased(P<0.05),and Tf R1 protein expression significantly decreased(P<0.05)in inhibitor Fer-1 combined with Cr(Ⅵ)group(P<0.05).3.The role of ferritinophagy in the damage of TM4 cells caused by Cr(Ⅵ): The expression of autophagy-related proteins LC3 B II/I and BECLIN1 increased with the increase of exposure dose,the levels of them were significantly higher than that in the control group;P62 protein expression decreased with the increase of exposure dose,that in the 5 μM and 10 μM groups significantly lower than that in the control group(P<0.05).and the expression of degradation of ferritin proteins NCOA4 increased with the increase of exposure dose,that in the 5 μM and 10 μM groups significantly higher than that in the control group(P<0.05).FTH1 protein expression decreased with the increase of exposure dose,that in the 5 μM and 10 μM groups significantly lower than that in the control group(P<0.05).4.Inhibition of autophagy prevents the Cr(Ⅵ)-induced damage in TM4 cells: After treated with autophagy inhibitor 3-MA,cell viability significantly increased,ROS levels significantly decreased,LC3 B II/I and NCOA4 protein expression significantly decreased,while the expression of P62,GPX4 and FTH1 proteins significantly increased compared with the Cr(Ⅵ)expsoure group(P<0.05).5.The role of NCOA4-mediated ferritinophagy in the ferroptosis of TM4 cells caused by Cr(Ⅵ): After NCOA4 gene silencing,the level of ROS decreased,the expression of NCOA4 and Tf R1 significantly decreased and the expression of FTH1 and GPX4 significantly increased compared with the Cr(Ⅵ)group(P<0.05).Conclusions:1.Cr(Ⅵ)can cause damage to TM4 cell.2.Cr(Ⅵ)can cause TM4 cells damage through ferroptosis;after treatment with ferroptosis inhibitor Fer-1 can reduce the occurrence of ferroptosis in TM4 cells induced by Cr(Ⅵ).3.Cr(Ⅵ)can cause damage to TM4 cells through ferritinophagy-mediated ferroptosis.Autophagy inhibitor 3-MA as well as siRNA silencing of the NCOA4 gene can alleviate the occurrence of ferritinophagy-mediated ferroptosis in TM4 cells induced by Cr(Ⅵ).