Cadmium-induced Damage To The Rat Testicular Sertoli Cells And Its Repairing By Bone Marrow Mesenchymal Stem Cells Conditioned Medium

Objective:Cadmium is a heavy metal element with strong bioaccumulation and long-term toxicity.It is easy to accumulate in the organism,which can cause damage to the male reproductive system,result in sperm deformity,infertility and other diseases.The testis is the most sensitive to cadmium,so further exploration of the mechanism of cadmium damage to testicular Sertoli cells is of great significance for the treatment of cadmium-induced damage to testicular tissue.In addition to,there was no ideal therapy for cadmium-induced damage to testes.Previous studies of our research group have found that the bone marrow mesenchymal stem cells（BMSCs）transplantation could repair the cadmide-induced testicular injury in rats,but its repairing mechanism is not clear.This study aimed to explore the damage to testicular Sertoli cells induced by cadmium chloride and the mechanism underlying apoptosis in testicular injury by establishing the in vitro model of testicular Sertoli cells induced by cadmium chloride,and subsequently,the repairing effect of BMSCs-conditioned medium（BMSCs-CM）on the testicular Sertoli cells injury induced by cadmium chloride and its mechanism were discussed after testicular Sertoli cells induced by cadmium and BMSCs-conditioned medium were co-cultured.Method:Cadmium-induced damage to the rat Sertoli cells:Rat testicular Sertoli cells were separated and cultured in vitro,identified by HE,SudanⅣand Fuergen staining.Cytotoxicity of Sertoli cells caused by cadmium chloride was determined with CCK-8 assay,then cadmium chloride concentrations of 0,20,40 and 80μmol/L were used for the subesequent experiments.Then Sertoli cells were exposed to the above concentrations for 24 h,the Sertoli cells in the 0μmol/L CdCl₂ group were given an equal volume of PBS.The ultrastructural changes of Sertoli cells were observed by transmission electron microscopy.The apoptosis rate of Sertoli cells exposed to cadmium was determined by the method of AnnexinⅤ-FITC/PI.The expression of Bax and Bcl-2 proteins was detected by double immunofluorescence staining.The expression levels of the proteins associated with mitochondrial apoptosis,Bax,Bcl-2,XIAP,Smac,Cleaved Caspase-3,Caspase-3 and Caspase-7were detected by Western Blot.The repairing effect of BMSCs conditioned medium on Sertoli cells injury caused by cadmium:the BMSCs of Wistar rats born 5-7 days were cultured,purified and expanded.The cell cycle and surface markers,CD45 and CD90 of the third generation BMSCs were detected by flow cytometry.And BMSCs were induced to differentiate into adipocytes and osteoblast,which were identified by oil red O staining and alizarin red staining,respectively.The 3rd to 5th generations of BMSCs conditioned medium were collected and concentrated 15 times by ultrafiltration.Sertoli cells were divided into a 0μmol/L cadmium chloride group,40μmol/L cadmium chloride group,40μmol/L cadmium chloride with V-ZAD-FMK group（the apotosis inhibitor V-ZAD-FMK was added to the medium after Sertoli cells were exposed to 40μmol/L cadmium chloride）,and 40μmol/L cadmium chloride with 15 times BMSCs conditioned medium group（Sertoli cells exposed to cadmium and BMSCs-conditioned medium were co-cultured）.The expression of Bax and Bcl-2 proteins was detected by double immunofluorescence staining.The expression of Smac and XIAP proteins of Sertoli cells in different groups was detected by immunofluorescence.The expression levels of mitochondrial apoptosis-related proteins Cleaved Caspase-3 and Caspase-3 proteins were detected by Western Blot.Result:Cadmium-induced damage to the rat Sertoli cells:After the Sertoli cells primarily cultured were passaged and expanded,the cells showed irregular shapes with processes by HE staining,orange lipid droplets by Sudan IV staining,and bipolar corpuscula in nucleus by Feulgen staining.CCK-8 results showed that the cell viability decreased with the increase of time and the dose of cadmium chloride exposure.The results of electron microscopy showed that the structure of mitochondria was clear and the nuclear membrane was complete in 0μmol/L CdCl₂group.The number of organelles decreased,mitochondria swelled and the mitochondrial ridge structure broke down in the Sertoli cells exposed to cadmium.Chromatin concentration edge aggregation appeared in the 80μmol/L CdCl₂ group.The results of AnnexinⅤ-FITC/PI showed that the number of apoptotic cells increased with the increase of CdCl₂ concentration（P<0.05）.The results of double immunofluorescence staining showed that the ratio of Bax/Bcl-2 and the expression of Bax protein significantly increased as the dose of CdCl₂ increased（P<0.05）,the expression of Bcl-2 protein significantly decreased as the dose of CdCl₂ increased（P<0.05）,there was significantly different among all the groups（P<0.05）.Western Blot results showed that the ratio of Bax/Bcl-2 and the expression of Bax,cleaved caspase-3,Smac and Caspase-7 proteins increased with the increase of cadmium exposure dose（P<0.05）,and the expression of Bcl-2、XIAP and Caspase-3 proteins decreased significantly,and there was a singnificant difference amgong the groups（P<0.05）.The repairing effect of BMSCs conditioned medium on cadmium-induced the damage to Sertoli cells of rat testis:After BMSCs were purified by adherent method,passaged and expanded,cells mophorlogy was long spindle.The results of flow cytometry detection showed that most of the third generation BMSCs were in the G1phase,and in the cells there was nearly no expression of CD45,but expression of CD90 was high in the cells.BMSCs were induced to differentiate into adipocyte and osteocyte.CCK-8 results showed that compared with 40μmol/L CdCl₂ group,cytotoxicity in 40μmol/L CdCl₂+Z-VAD-FMK and 40μM CdCl₂+15×BMSCs-CM group was significantly reduced（P<0.05）.The observation results under a light microscope show that the cells in the 40μmol/L CdCl₂ group shrank and shed,and the number of cells decreased.In the 40μmol/L CdCl₂+Z-VAD-FMK group and the40μmol/L CdCl₂+15×BMSCs-CM group,the number of cells increased and the cell morphology was relatively complete.The results of double immunofluorescence staining showed that copared with 0μmol/L CdCl₂ group,the ratio of Bax/Bcl-2 and the expression of Bax protein were significantly increased,the expression of Bcl-2protein were significantly decreased in the 40μmol/L CdCl₂ group（P<0.05）,and that in the 40μmol/L CdCl₂+Z-VAD-FMK and 40μmol/L CdCl₂+15×BMSCs-CM group the ratio of Bax/Bcl-2 and the expression of Bax protein were significantly lower and the expression of Bcl-2 protein were significantly higher than that of the40μmol/L CdCl₂ group（P<0.05）.The results of fluorescence detection showed that the expression of Smac protein was the highest in the 40μmol/L CdCl₂ group,but that in 40μmol/L CdCl₂+Z-VAD-FMK and 40μmol/L CdCl₂+15×BMSCs-CM group was significantly lower than that in 40μmol/L CdCl₂ group（P<0.05）,whereas the expression of XIAP in 40μmol/L CdCl₂ group was the lowest,but that in 40μmol/L CdCl₂+Z-VAD-FMK and 40μmol/L CdCl₂+15×BMSCs-CM group was significantly higher than that in 40μmol/L CdCl₂ group（P<0.05）.Western Blot results showed that the expression of Cleaved Caspase-3 protein in the 40μmol/L CdCl₂ group was significantly higher than that in the other groups（P<0.05）,the expression of Caspase-3 protein was significantly lower than that in the other gourps（P<0.05）.Conclusion:1.Cadmium chloride can cause the damage to rat testis Sertoli cells.The mitochondria-pathway apoptisis mediated by Smac-XIAP pathway may be the one of the mechanism underlying cadmium-induced damage to the rat Sertoli cells.2.The conditioned medium of BMSCs can repair Sertoli cells damage caused by CdCl₂.It can be the one of the mechanisms underlying the repairing effect of BMSCs on the cadmium-induced damage to Sertoli cells that the mitochondria apoptosis mediated by Smac-XIAP pathway was inhibited.