The Role Of IRE1 Signaling Pathway In Cadmium-induced Apoptosis Of Mouse Spermatogonia

Background Cadmium is one of the main occupational and environmental toxicants,posing a huge threat to human health.Cadmium exposure can cause damage to many tissues and organs of the body,and can lead to the decline of male sperm quality and even male infertility.The previous in vivo studies of our research group found that cadmium can induce apoptosis in mouse testicular germ cells,which may be the cause of the damage of cadmium to the male reproductive system.However,the molecular mechanism of cadmium-induced apoptosis of male germ cells still needs further research.Therefore,we established a model of GC-1 cells in vitro to explore the role of IRE1signaling pathway in cadmium induced apoptosis of mouse spermatogonia.Objective In this study,GC-1 cells cultured in vitro were used as a model,and GC-1cells were treated with Cd Cl₂,endoplasmic reticulum stress inhibitor 4-PBA,and IRE1αspecific inhibitor STF-083010,respectively.To explore the role of endoplasmic reticulum stress IRE1 signaling pathway in cadmium-induced apoptosis of spermatogonia in mouse testes.Methods（1）In order to explore the toxic effect of cadmium on GC-1 cells and the effect of endoplasmic reticulum stress,different doses of Cd Cl₂（1μM,5μM,10μM,20μM）were used to treat GC-1 cells for different times（4h,8h,12h,24h）.CCK8 method was used to detect cell viability;TUNEL and Annexin V-FITC methods were used to detect cell apoptosis;RT-PCR and western blot methods were used to determine the expression of cell ERS-related indicators.（2）In order to explore the effect of endoplasmic reticulum stress on the apoptosis of GC-1 cells induced by cadmium,GC-1 cells were pretreated with different doses of 4-PBA（20μM,40μM,80μM,100μM）for 2h,and then incubated with 20μM Cd Cl₂ for 24h,use CCK8 method to detect cell viability.GC-1 cells were pretreated with 100μM 4-PBA for2h,and then incubated with 20μM Cd Cl₂ for 24h.RT-PCR and western blot were used to detect the expression of ERS-related indicators;TUNEL and Annexin V-FITC were used to detect cell apoptosis.（3）In order to explore the effect of IRE1 signaling pathway in cadmium-induced apoptosis of GC-1 cells,GC-1 cells were pretreated with different doses of STF-083010（20μM,40μM,80μM,100μM）for 2h,and then incubated with 20μM Cd Cl₂ for 24h,use CCK8 method to detect cell viability.GC-1 cells were pretreated with 80μM STF-083010for 2h,and then incubated with 20μM Cd Cl₂ for 24h.Western blot was used to detect the expression of IRE1 signaling pathway related indicators;TUNEL and Annexin V-FITC were used to detect cell apoptosis.Results（1）Compared with the control group,the cell viability of the Cd Cl₂ group was significantly reduced,and the percentage of apoptotic cells was significantly increased,and both had a time-effect relationship（P<0.05）.The expression of ERS-related m RNAs such as ATF6,CHOP,GRP78 and XBP-1 in the Cd Cl₂ group increased（P<0.05）,and the expression of ERS-related proteins such as P-e IF2α、CHOP、GRP78、ATF6α、P-IRE1α、XBP-1 and P-JNK also increased（P<0.05）.（2）After treating GC-1 cells with 4-PBA and Cd Cl₂,compared with the control group,the cell viability,the percentage of apoptotic cells,and the expression of ERS-related m RNA and protein in the 4-PBA group did not change significantly.Compared with the Cd Cl₂ group,the cell viability of the 4-PBA+Cd Cl₂ group was significantly increased,and the percentage of apoptotic cells was significantly decreased（P<0.05）.In addition,the expression of ERS-related m RNAs such as ATF6,CHOP,GRP78 and XBP-1 in the4-PBA+Cd Cl₂ group was significantly decreased（P<0.05）,and the expression of ERS-related proteins such as P-e IF2α,CHOP,GRP78,ATF6α,P-IRE1α,XBP-1and P-JNK was significant decrease（P<0.05）.（3）After treating GC-1 cells with STF-083010 and Cd Cl₂,compared with the control group,the cell viability,the percentage of apoptotic cells,and the expression of IRE1signal-related proteins in the STF-083010 group did not change significantly.Compared with the Cd Cl₂ group,the cell viability of the STF-083010+Cd Cl₂ group was significantly increased,and the percentage of apoptotic cells was significantly decreased（P<0.05）.In addition,the expression of ERS-related proteins such as P-IRE1α,XBP-1 and P-JNK in the STF-083010+Cd Cl₂ group was significantly decreased（P<0.05）.Conclusion（1）Cadmium activates endoplasmic reticulum stress in mouse spermatogonia and can induce apoptosis.4-PBA has an inhibitory effect on cadmium-activated spermatogonia endoplasmic reticulum stress,and it has a protective effect on cadmium-induced spermatogonia apoptosis.It suggests that endoplasmic reticulum stress may play an important role in cadmium-induced spermatogonia apoptosis.（2）Cadmium activates the IRE1 signaling pathway in mouse spermatogonia.The IRE1specific inhibitor STF-083010 can inhibit the expression of IRE1 signaling in spermatogonia and has a protective effect on spermatogonia apoptosis.It is suggested that the IRE1 signal pathway of spermatogonia activated by cadmium plays an important regulatory role in apoptosis.