Effect Of Autophagy On Cadmium-induced Damage Of Blood-testis Barrier In Mice

Cadmium（Cd）is a non-essential trace heavy metal element in the human body,and it is unequally distributed in the environment.Cadmium can accumulate in the body through direct exposure or occupational exposure.Cadmium has a long biological half-life in the human body,and its accumulation in organs such as the liver and testes can cause significant toxic effects.Spermatogenesis is an important process for maintaining and promoting male fertility,and the blood-testis barrier（BTB）,which is formed by tight junctions between Sertoli cells,plays a crucial role in the process of sperm production.At the same time,the BTB is also an important target of cadmium-induced male reproductive damage.Objective:To investigate the effect of cadmium-induced autophagy on the blood-testis barrier in adolescent mice and its regulatory mechanism.Methods:Twenty male C57BL/6 mice aged four weeks were randomly divided into control group[0 mg/（kg·d）],low-dose group[0.5 mg/（kg·d）],medium-dose group[1.0mg/（kg·d）],and high-dose group[2.0 mg/（kg·d）],and were injected intraperitoneally with cadmium chloride continuously for 28 days.The histological changes of testicular tissue were analyzed by HE staining,the integrity of the BTB was observed by a biological tracer,and the expression of BTB components ZO-1 and N-Cadherin protein was detected by Western blot.The TM4 cell line was treated with 0,2.5,5,and10μmol/L Cd Cl₂ for 24 h,and the localization and expression of microfilaments in the cells were observed by immunofluorescence probe.The changes of ZO-1 and N-Cadherin protein,as well as autophagy-related proteins LC3-II and p62,were detected by Western blot.Cells were treated with autophagy inhibitors3-methyladenine（3-MA）（60μmol/L）,chloroquine（CQ）（5μmol/L）,or autophagy activator rapamycin（RAPA）（50 nmol/L）in Cd Cl2（10μmol/L）-containing culture medium for 24 h.The localization and expression of microfilaments in the cells were observed by immunofluorescence probe,and the expression of LC3-II,p62,ZO-1,and N-Cadherin proteins was detected by Western blot.Results:Compared with the control group mice,the cadmium-treated group mice showed an increase in the interstitial space of the seminiferous tubules,formation of intracellular cavities in the germ cells,reduced cell hierarchy,and disordered arrangement,and the integrity of the blood-testis barrier（BTB）was disrupted.In the testicular tissue of mice treated with medium and high doses of Cd Cl₂,the expression levels of ZO-1 and N-Cadherin proteins were significantly down-regulated（P<0.05）.Compared with the control group cells,the TM4 cell cytoskeleton showed shrinkage and disordered arrangement.In cells treated with Cd Cl₂,the expression levels of ZO-1and N-Cadherin proteins were significantly down-regulated（P<0.001）,while the expression levels of LC3-II and p62 proteins were significantly up-regulated（P<0.05）.Compared with cells treated with 10μmol/L Cd Cl₂,cells co-treated with the autophagy inhibitor 3-MA and Cd Cl₂ showed significant up-regulation of ZO-1,N-Cadherin,LC3-II,and p62 protein expression（P<0.01）.Compared with cells treated with 10μmol/L Cd Cl₂,cells co-treated with the autophagy inhibitor CQ and Cd Cl₂ showed alleviated cytoskeletal damage,and significant up-regulation of ZO-1,N-Cadherin,LC3-II,and p62 protein expression（P<0.05）.Cells co-treated with the autophagy inducer RAPA and Cd Cl₂ showed enhanced cytoskeletal damage,and significant down-regulation of ZO-1,N-Cadherin,and p62 protein expression（P<0.05）,while LC3 protein expression was up-regulated（P<0.05）.Conclusion:Cadmium chloride can disrupt the integrity of the BTB in mice,and the mechanism may be related to the ability of cadmium to enhance autophagy levels in testicular supporting cells,regulating the expression levels of BTB proteins.