The Role And Mechanism Of Oxidative Stress-Mediated ATM/p53 Pathway In MC-LR-induced Autophagy In Male Germ Cells

ObjectivesMicrocystin-LR(MC-LR)is a human carcinogen produced by cyanobacteria blooms,threatening the health of the male reproductive system.This study aimed to explore the effect of MC-LR on DNA damage and autophagy and its regulatory mechanism in male germ cells.By analyzing the relationship between oxidative stress and DNA damage and exploring the role of ATM/p53 pathway in inducing autophagy,it provided a new molecular mechanism for MC-LR to induce DNA damage and autophagy in male germ cells.It also provided a reliable theoretical basis for further research on the damage of the human male reproductive system induced by MC-LR.Methods1.Assess the degree of pathological damage by MC-LR to the testis of C57BL/6N miceForty-eight C57BL/6N mice were randomly divided into 8 groups,and injected intraperitoneally for 14 days.The weight change and final testis tissue weight of the mice were recorded.Western Blot and microcystin detection kit were used to detect the level of MC-LR in testis tissue and serum.H&E staining was used to detect pathological damage in testis tissue.2.Determine the concentration of MC-LR and the intervention dose of antioxidant NAC in GC-1 cellsGC-1 cells exposed to different concentrations of MC-LR or NAC for 24 hours.The CCK-8 kit was used to detect the cell viability to determine the subsequent experimental concentrations of MC-LR and NAC.Western Blot was used to detect the expression level of MC-LR in GC-1 cells.3.Detect the effect of MC-LR on DNA damage and oxidative stress related indicators in mouse testis tissue andGC-1 cellsWestern Blot,immunofluorescence and hydroxyl radical(hydroxyl radical,·OH)detection kit combined with microplate reader were used to detect the expressions of?-H2AX,8-hydroxy-deoxyguanosine(8-hydroxy-2'-deoxyguanosine,8-OHdG)and·OH in mouse testis tissue andGC-1 cells.Comet assay detects the degree of DNA damage in GC-1 cells.4.Detect the effect of MC-LR on the expression level of ATM and its downstream related proteins and the level of autophagy in mouse testis tissue andGC-1 cellsWestern Blot was used to detect the expression level of ATM and its downstream related proteins and autophagy-related proteins in mouse testis tissue andGC-1 cells.Immunofluorescence was used to detect the expression level of LC3 protein in mouse testis tissue andGC-1 cells.MDC staining was used to detect the change of autophagosome content in GC-1 cells.Results1.MC-LR accumulated in moouse testicular tissue and caused histopathological damageAfter 14 days of intraperitoneal injection of C57BL/6N mice,it was found that MC-LR could enter the mouse testis through the blood circulation,resulting in a significant decrease in the weight and gonadal index of the mice(P<0.05),and inducing a deepening of the pathological damage of the testis.2.Oxidative stress induced DNA damage in mouse testis after MC-LR exposureMC-LR increased the ability to produce·OH,and the expression levels of?-H2AX and DNA oxidative damage marker protein 8-OHdG(P<0.05)in mouse testis.Antioxidant NAC pretreatment significantly reduced the production capacity of·OH,and the expression levels of?-H2AX and 8-OHdG.3.The cell viability first increased and then decreased after GC-1 cells are exposed to different concentrations of MC-LR and NACAfter GC-1 cells were exposed to different concentrations of MC-LR for 24 h,the IC₅₀ of MC-LR to GC-1 cells was calculated to be 24?g/mL.The subsequent experimental concentrations in GC-1 cells were determined to be 3,6 and 12?g/mL.After exposure to different concentrations of NAC,it was found that when the NAC concentration was 5 mM,the cell activity was the highest.So the subsequent experiment concentration of NAC was determined to be 5 mM.Western Blot results showed that MC-LR can enter GC-1 cells 24 h after exposure.4.MC-LR exposure induced DNA damage in GC-1 cells through oxidative stressMC-LR significantly increased the Olive tail moment(OTM)and DNA content of comet tail in trailing cells(Tail DNA%)in GC-1 cells,led the expression level of?-H2AX significantly increased(P<0.05).MC-LR also increased the·OH production capacity in GC-1 cells and induced the high expression of 8-OHdG.NAC pretreatment significantly reduced the production capacity of·OH and the expression level of 8-OHdG,resulting in a decrease in the expression levels of OTM,Tail DNA%and?-H2AX in GC-1 cells.5.DNA damage-induced ATM/p53 pathway is involved in autophagy induced by MC-LR in mouse testis andGC-1 cellsMC-LR induced the increase of the phosphorylation level of ATM,CHK2 and p53 protein in mouse testis tissue andGC-1 cells,as well as the increase of the expression of autophagy-related protein Beclin1,ATG5,ATG12,ATG5-ATG12 and LC3?/LC3?(P<0.05).KU55933 pretreatment significantly reduced the protein phosphorylation levels of ATM,CHK2 and p53,and at the same time reduce the autophagy level of cells.Conclusions1.MC-LR can cause DNA damage in male germ cells of mice through oxidative stress.2.The ATM/p53 pathway induced by DNA damage plays an important role in the autophagy induced by MC-LR.