Study On The Role Of Mitophagy And Pyroptosis Induced By Nano-silver In Testicular Injury

Objective:To explore the role of mitochondrial autophagy and pyroptosis caused by silver nanoparticles(AgNPs)in testicular injury.Methods: 7-week-old ICR male mice were used after one week of adaptive feeding.40 mice were randomly divided into 4 groups,each group with 10 mice,and they were respectively infected with AgNPs 0 mg/kg(blank control group,C)and 25 mg/ kg(low dose group,L),50mg/kg(medium dose group,M),100 mg/kg(high dose group,H)for 60 days.The AgNPs solution was dissolved and mixed in PBS,and each mouse was given 2m L of AgNPs solution by intragastric administration every day.After the last exposure,the mice were fed for 24 hours,and the mice were sacrificed by orbital blood sampling.The obtained mouse blood was centrifuged at4°C within 2 hours,and the supernatant was taken to obtain mouse whole blood,which was stored in a refrigerator at 4°C for subsequent use.Take mouse testes and store them in a refrigerator at-80℃.Elisa kit was used to detect serum testosterone content and pyroptosis-related cytokines IL-1β and IL-18.HE staining to observe the changes in the morphology and structure of the testis.RT-q PCR was used to detect the m RNA expression levels of mitochondrial autophagy and pyroptosis-related proteins LC3,TOM20,Parkin,PINK1,Caspase-1 and NLRP3 in the testis.Western blot was used to detect the content of mitophagy and pyroptosis-related proteins LC3,TOM20,Parkin,PINK1,and pyroptosis-related proteins Caspase-1,NLRP3,GSDMD.Result:1.TEM observation shows that 95% of AgNPs particles have a particle size of 25.93nm±4.98 nm.HE staining showed that the testicular tissue morphology changed after AgNPs infection.Spermocytes were seen in the testes of mice in the low,medium and high dose groups.A small amount of eosinophilic substances exuded in the seminiferous tubules in the low-dose group.Abnormal changes occurred in the high-dose group,with obvious vacuolation of seminiferous tubules,thickened basement membrane,and disordered arrangement of spermatogenic cells.The number of sperms in the seminiferous tubules is significantly reduced.The epididymal tissue of the mice in group C and L was normal,the epididymal duct structure was complete,and the epithelial cells were rich in cytoplasm and arranged tightly.Interstitial edema and loose connective tissue were seen in the epididymis in group M and H.There was mild connective tissue hyperplasia around the epididymal duct in group H.2.Serum testosterone levels in groups L,M,and H were all higher than those in group C,and group M had the highest testosterone content(P<0.05).3.After 60 days of exposure to AgNPs,compared with group C,the mitochondrial autophagy-related protein PINK1 m RNA expression in group H was significantly increased(P<0.05),while the expression level of Parkin m RNA was not statistically significant(P>0.05).Compared with group C,the expression level of LC3A/LC3 B m RNA increased in a dose-dependent manner in groups L,M,and H(P<0.05);the expression level of mitochondrial membrane protein TOM20 m RNA increased in groups L,M,and H High,the increase in group H was the most obvious(P<0.05).4.Compared with group C,the expression of PINK1 protein in groups L,M,and H was higher than that in group C(P<0.05);the expression of mitochondrial autophagy-related protein Parkin in groups L,M,and H showed an increasing trend,but no Statistical significance(P>0.05);the expression of LC3 protein in the L,M,and H groups all increased significantly(P<0.05);the expression of TOM20 protein in the L,M,and H groups all increased in a dose-dependent manner(P< 0.05).5.Compared with the C group,the m RNA expression of the pyroptosis-related protein Caspase-1in the L,M,and H groups all increased,and the expression of the M and H groups increased the most significantly(P<0.05).The m RNA expression level of pyroptosis-related protein NLRP3 in the L,M,and H groups increased in a dose-dependent manner(P<0.05).6.Compared with the C group,the protein expression of the pyroptosis-related protein Caspase-1in the L,M,and H groups all increased,and the expression of the H group increased the most(P<0.05).The expression of pyroptosis-related protein NLRP3 in groups L,M,and H increased with the increase of AgNPs concentration,and the expression of group H was the most obvious(P<0.05).The expression of GSDMD protein showed an increasing trend,and the expression of M and H groups increased significantly(P<0.05).Compared with the C group,the serum pyroptosis-related cytokine IL-1β levels increased in the L,M,and H groups.Among them,the increase in group L and group H was the most obvious(P<0.05).The content of IL-18,a pyroptosis-related cytokine in serum,increased in groups L,M,and H compared with group C,and increased with the increase of AgNP concentration(P<0.05).Conclusion: 1.AgNPs can cause mitophagy and pyroptosis in mouse testes;2.The possible mechanism of testicular damage caused by AgNPs is: AgNPs exposure can activate the PINK1/Parkin signaling pathway in mouse testicular tissue cells,cause mitochondrial autophagy,induce the production of NLRP3 bodies,activate Caspase-1,and promote IL-1β and IL-18 is released,resulting in an increase in the expression of GSDMD protein,which leads to pyroptosis of testicular tissue cells.