Study On Acrylamide-induced Apoptosis And Autophagy Of Mouse Spermatogonial Stem Cells C18-4

Objective:Acrylamide is a chemical material commonly used in industry,which is highly toxic and can enter the body throught the skin,the respiratory and digestive tract.Besides neurotoxicity,hepatotoxicity and embryonic development toxicity,acrylam-ide can also cause damage to the reproductive system.However,the potential mechanism of acrylamide in male reproductive system remains unclear.This study investigated explored the mechanism of acrylamide-induced apoptosis and autophagy of mouse spermatogonial stem cells(C18-4 cell).Methods:In order to investigate the effects of acrylamide on apoptosis and autophagy of mouse spermatogonial stem cells,the mouse spermatogonial stem cell line,C18-4cell was treated with different concentration of acrylamide(0,0.5,1,2 m M)for 24 h,and the cell activity was detected by CCK assay.The expression of apoptosis related proteins Cleaved Caspase-8,Cleaved Caspase-3,Bcl-2 and Bax and the number of Annexin V-FITC positive cells were detected by Western blot and Annexin V-FITC/PI double staining,respectively.Western blot and transmission electron microscopy were used to detect the expression of intracellular autophagy related proteins Atg5,Beclin1,LC3 and the number of intracellular autophagy vesicles.To determine whether oxidative stress is involved in acrylamide-induced apoptosis and autophagy of mouse C18-4 cells,the mouse C18-4 cells were treated with or without 5 m M N-acetyl-L-cysteine(NAC)and then treated with 2 m M acrylamide for 24 h.The cell activity,apoptosis and autophagy were detected by CCK assay,Annexin V-FITC/PI double staining,Western blot and transmission electron microscopy,respectively.The contents of MDA and GSH and the activities of SOD and GSH-PX in cells were detected by oxidative stress detection kit.In order to identify the role of autophagy in acrylamide-induced apoptosis of mouse C18-4 cells,the mouse C18-4 cells were pretreated with or without 3-methyladenine(3-MA)and then treated with acrylamide at 2 m M concentration for 24 h,cell viability and apoptosis were detected by CCK assay,Annexin V-FITC/PI double staining and Western blot,respectively.To further clarify whether oxidative stress-related genes are involved in the apoptosis and autophagy of mouse C18-4 cells induced by acrylamide,the mouse C18-4 cells were treated with acrylamide at 2 m M concentration for 24 h,and high-throughput screen-ing of differentially expressed genes was performed with RNA-seq and validatde with Real-time PCR.Results:The results of CCK assay showed that acrylamide inhibited the activity of mouse C18-4 cells in a concentration-dependent manner(p<0.05);Western blot results showed that acrylamide could significantly increase the protein expressions of Cleaved Caspase-8,Cleaved Caspase-3 and Bax in mouse C18-4 cells,while significantly inhibited the expression of Bcl-2 protein in the cells,suggesting that acrylamide can induce apoptosis of mouse C18-4 cells.Annexin V-FITC/PI double staining assay showed that acrylamide could significantly increase the number of Annexin V-FITC positive cells in mouse C18-4 cells,which further confirmed that acrylamide could induce apoptosis in mouse C18-4 cells.At the same time,acrylamide also significantly increased the expression levels of autophagy-related proteins Atg5,Beclin1 and LC3-II and the ratio of LC3-II to LC3-I in mouse C18-4cells,and the results of transmission electron microscopy showed that the intracellular autophagic vesicles in the acrylamide-treated group were significantly increased,these results suggested that acrylamide could induce autophagy in mouse C18-4 cells.The results of oxidative stress-related detection showed that the content of MDA in the acrylamide-treated mouse C18-4 cells acrylamide was significantly increased,while the content of GSH and the activities of GSH-PX and SOD were significantly decreased,suggesting that acrylamide could induce oxidative stress in mouse C18-4cells.After inhibiting oxidative stress in mouse C18-4 cells with NAC,acrylamide-induced apoptosis and autophagy levels were significantly reduced,suggesting that oxidative stress was involved in acrylamide-induced apoptosis and autophagy of mouse C18-4 cells.The autophagy inhibitor 3-Methyladenine(3-MA)could significantly reverse the inhibition of cell viability and apoptosis induced by acrylamide,suggesting that autophagy plays a cytotoxic role in acrylamide-induced apoptosis of mouse C18-4 cells.Meanwhile,RNA-seq and Real-time PCR showd that acrylamide could significantly increase the m RNA expression of oxidative stress-related genes Ldha,Atf4,Sod1,Sod3,Gpx7,Lonp1,Hsph1,Tra2 b in mouse C18-4cells,while decreased the m RNA level of Coll A1,which further indicated that acrylamideoxidative stress was involved in acrylamide induced apoptosis and autophagy of mouse C18-4 cells.Conclusions:Acrylamide could induce apoptosis and autophagy of mouse C18-4 cells.Acrylamide could induce oxidative stress of mouse C18-4 cells.Oxidative stress was involved in acrylamide-induced apoptosis and autophagy of mouse C18-4 cells.Autophagy played a cytotoxic role in acrylamide-induced apoptosis of mouse C18-4cells.