Bone Marrow Mesenchymal Stem Cells Repair Testicular Injury Caused By Hexavalent Chromium By Regulating Autophagy And Ferroptosis

Objective:Chromium compounds are widely used in production,among which hexavalent chromium compounds have a strong toxic effect.As one of the target organs of heavy metal toxicants,testis is vulnerable to being damaged by hexavalent chromium,which affects male reproductive health.At present,there is no ideal treatment for testicular injury caused by hexavalent chromium.Previous results of our researches demonstrated that exposure to cadmium could cause testicular tissue damage in rats,and bone marrow mesenchymal stem cells（BMSCs）transplantation could repair cadmium-induced testicular injury.Therefore,we hypothesize BMSCs can also repair the testes injury caused by hexavalent chromium.In this paper,the BMSCs were transplanted into the injured rats testes caused by hexavalent chromium to observe the repairing effect of BMSCs transplantation on testicular injury caused by hexavalent chromium and further to explore the potential mechanism of BMSCs repairing testicular injury caused by hexavalent chromium.Method:Isolation,culture,identification and labeling of BMSCs:bone marrows cells were collected from Wistar sucking rats aged under one week and amplified by adherent purification method.Cell surface markers including CD45 and CD90 and cell cycle were determined by flow cytometry.Cultured cells were induced to differentiate into adipocytes and osteoblasts.CM-Dil labelled BMSCs and the percentage of labelled BMSCs was calculated.The repairing effect of BMSCs transplantation on the rat testes injury caused by hexavalent chromium:24 male Wistar rats were randomly divided into control group,model group and treatment group,with 8 rats in each group.The rats in model group and treatment group were injected with potassium dichromate solution at a dose of0.4mg/kg·bw intraperitoneally.The rats in control group were injected with the same volume of saline.Potassium dichromate was injected 5 days a week for 30 days.24 h and 48 h after chromium was not given to rats any longer,5×10⁶CM-Dil labelled BMSCs were transplanted into the rats in the treatment group via retro-orbital injection,respectively,and the same volume of PBS was injected into the control group and the model group.The general state of diet and activity of rats were observed.Two weeks after cell transplantation,the rats were sacrificed.Chromium content in testicular tissue of rats was determined by atomic absorption spectrometry.The histological changes of testes in each group were observed by HE staining,and the pathological score was performed according to Johnsen score standard.Lillie Fe²⁺staining was used to detect the change of iron content in testis of each group.The location of BMSCs in damaged testes of the treated group was observed by laser confocal microscope.The expressions of autophagy-related proteins LC3B and Beclin1,proteins in AKT/m TOR signal pathway including AKT,p-AKT and m TOR in rat testis were detected by Western blot（WB）.The expressions of LC3B,Beclin1,AKT,p-AKT,m TOR,p-m TOR,4-HNE,GPX4,SLC7A11 and Tf R1 proteins were detected by immunohistochemistry.Result:Isolation,culture,identification and labeling of BMSCs:most of the third-generation BMSCs were in G1 phase,with almost no expression of surface marker CD45 and high expression of CD90 by flow cytometry.The cultured cells had the ability to differentiate osteoblasts and adipocytes because the differentiated cells were positive by Oil red O and alizarin red staining.BMSCs labelled by CM-Dil were observed under laser confocal microscope,percentage of labelled BMSCs was93.56%±2.87%.The above confirmed the cultured cells were BMSCs.The repairing effect of BMSCs transplantation on the rat testes injury caused by hexavalent chromium:after exposure,the activity and appetite of rats from model group and treatment group were decreased.After the cell transplantation,the above parameters of rats from the treatment group were improved.The testes of rats in the control group showed normal cell arrangement in the seminiferous tubules and normal spermatogenic function.In model group,there were reduced mature sperms in seminiferous tubules of testis,and the arrangement of Sertoli cells and spermatogonia cells was loose,and the spermatogenic function was impaired.Compared with the model group,the testis of rats in the treatment group had more mature sperm,and the arrangement of Sertoli cells,spermatogonia cells and spermatocytes arranged orderly.Johnsen score evaluation results showed that the scores of model group and treatment group were significantly lower than those of control group（P<0.05）,and the scores of treatment group were significantly higher than that of model group（P<0.05）.BMSCs labelled by CM-Dil were observed in the testis of rats in the treatment group by laser confocal microscope.Atomic absorption spectrometry showed that the chromium content in testes of model and treatment group was significantly higher than that in control group（P<0.05）,and there was no statistical difference between model and treatment group.Lillie staining results showed that the content of Fe²⁺in testis of rats in model group increased while that in treatment group decreased.Immunohistochemistry and WB results showed that the expressions of autophagy-related proteins Beclin1 and LC3B in the model group and the treatment group were significantly higher than those in the control group（P<0.05）,and the expressions of Beclin1 and LC3B in the treatment group were lower than those in the model group（P<0.05）.Immunohistochemical results of ferrpotosis-related proteins showed that the expression of 4-HNE and Tf R1 proteins in model group and treatment group were significantly higher than those in control group（P<0.05）,and the expression of 4-HNE and Tf R1 proteins in treatment group were significantly lower than those in model group（P<0.05）.The expression of GPX4 and SLC7A11 proteins in model group and treatment group was significantly lower than that in control group（P<0.05）,and the expression of GPX4 and SLC7A11 proteins in treatment group was significantly higher than that in model group（P<0.05）.Immunohistochemistry results showed that p-AKT and p-m TOR protein expression in the model group and the treatment group was significantly lower than that in the control group（P<0.05）.The expression of p-AKT and p-m TOR protein in the treatment group was significantly higher than that in the model group（P<0.05）.The m TOR protein expression in the model group was significantly lower than that in the control group（P<0.05）,and the m TOR protein expression in the treatment group was significantly higher than that in the model group（P<0.05）.There was no significant difference in the expression of AKT protein among all groups.The WB results of AKT,p-AKT and m TOR proteins were consistent with the trend of immunohistochemical results.Conclusion:BMSCs can repair the testes injury caused by hexavalent chromium after transplantation.BMSCs may repair testicular injury induced by hexavalent chromium in rats by regulating autophagy and ferroptosis.BMSCs may regulate autophagy and ferroptosis by AKT/m TOR pathway.