| Objective:To explore the repair effect of bone marrow mesenchymal stem cell（BMSCs）transplantation on injured kidneys induced by hexavalent chromium and its repair mechanism after rats were exposed to hexavalent chromium.Methods:The cells obtained from bone marrow of 5-7day Wistar rats were cultured in culture flasks,purified by adherence method and expanded.The third passage cultured cells were used to detect the cell cycle and BMSCs surface markers,including CD44,CD90 and CD45 by flow cytometry,and were induced to differentiate into adipocytes which were identified by oil red O staining.24 adult Wistar rats were randomly divided into the control group,model group in which rats renal injuries were induced by hexavalent chromium and cell therapy group.The rats in model and the therapy groups were intraperitoneally injected with the Cr（VI）solution at a concentration of 0.4 mg/kg of body weight（1.13 mg/kg body weight K2Cr2O7 solution）5 days a week for 1 month.The rats in the control group were injected with the same volume of normal saline At the end of the exposure,1×107BMSCs were transplanted into rats in the therapy group.The rats in the control and model groups were injected with an equal volume of phosphate buffered saline.The general state of the rats were observed and recorded every day.Two weeks after cell transplantation,serum creatinine（CR）and Urea（URE）content,the kidney weight and renal index,SOD activity,and MDA content were detected;the pathological changes of rats renal tissues were observed with HE staining;chromium content in rat kidney tissue were determinated by atomic absorption method;TUNEL method was used to detect cell apoptosis of renal tissue;immunohistochemical and western blotting methods were used to detect mitochondria pathway apoptosis-related proteins Bax,Bcl-2,Cyt c and Caspase 3,mitophagy-related proteins Beclin 1,P62,PINK1,Parkin,p-Parkin and LC3B;western blotting method was used to detect MAPK pathway proteins,which include p38,p-p38,JNK,p-JNK and ERK,p-ERK.Observation of the localization of CM-Dil-labeled stem cells in renal tissues was performed by laser confocal microscopy.Results:1.Isolation,culture,and identification of BMSCs:the flow cytometry results showed that in the third passage BMSCs,the expression of mesenchymal stem cell surface markers CD44 and CD90 was high,while that of CD45 was low,the surface marker of hematopoietic cell lines.And most derived-cells were in intermitotic period.The adipocytes differentiated from the cultured cells were stained by oil red O with red fat droplets.2.Repairing effects of BMSCs on renal injury of rats induced by hexavalent chromium:after exposure to hexachromium,the rats in the control group were in good condition;the rats in the model group decreased appetite,with dull yellow hair color compared to the control group.Compared to the model group,the rats in the therapy group were active and better appetite.Two weeks after cell transplantation,the renal weight in the model group was significantly lower than that in the control group（P<0.05）.The kidney index of rats in model group were significantly higher than that in control group and cell transplantation group（P<0.05）.The results of blood biochemical test showed that the content of CR and URE in the therapy group was significantly higher than that in control group,lower than that in the model group（P<0.05）.The results of oxidative stress-related indicators showed that,in model group,SOD activity was significantly lower,and MDA content was significantly higher than those the control and the therapy groups.There was no significant difference in chromium content in the kidney tissues between the model and therapy groups.HE staining showed that renal glomerular atrophy,renal tubular structure damage and epithelial cell damage were observed in the model group,and the pathohistological changes in the rats kidneys in the therapy group were obviously improved.3.The mechanism of repairing eff ects of BMSCs on rats renel injury caused by hexavalent chromium:CM-Dil-labeled BMSCs were found in injured kidneys in the therapy group by laser scanning confocal microscope.TUNEL assay showed that the percentage of apoptotic cells in rat renal tissues of the therapy group was significantly lower than that in the model group and higher than that in the control group（P<0.05）.The results of apoptosis-related proteins via the mitochondrial pathway were as follows:immunohistochemical results showed that the expression of Bax,Cyt c and Caspase 3 in the renal tissue of the therapy group was significantly higher than that in the control group and lower than that in the model group（P<0.05）;the expression of apoptosis suppression protein Bcl-2 was significantly lower than that in the control group and higher than that in the model group（P<0.05）;the results of western blot:results from total protein lysates showed that the expression of Bax in the model group was significantly higher than that in the control group,while the ratio of Bcl-2to Bax and Bcl-2 expression in the therapy group were significantly higher than that in the model group and lower than that in the model group（P<0.05）,the expression of Caspase 3 and Cyt c in the therapy group were significantly higher than those in the control group,and lower than that in the model group（P<0.05）;results from mitochondria protein lysate showed that the expression of Bax in the model group was significantly higher than that in the control group,and the expression of Bcl-2 and Cyt c were significantly lower than those in the therapy and control group.The mitochondria-free proteins lysates showed that in the therapy group,the expression of Bax and Cyt c was significantly lower than that in the model group and higher than that in the control group（P<0.05）.Detection results of mitochondria autophagy-related proteins by immunohistochemical and western blot showed that in the therapy group the expression of Beclin 1,PINK1,Parkin,p-Parkin and LC3B were significantly higher than that in the control group and lower than that in the model group and the expression of P62 was significantly higher than that in the model group and lower than that in the control group（P<0.05）.Detection results of MAPK signal pathway showed that the ratios of p-p38 to p38 and p-JNK to JNK in the therapy group were significantly higher than those in the control group,which were lower than those in the model group（P<0.05）,and the ratio of p-ERK to ERK was significantly lower than that in the control group,and higher than that in the model group（P<0.05）.Conclusions:The hexavalent chromium can accumulate in rat kidney,cause oxidative stress reaction,activate p38 and JNK phosphorylation,inhibit ERK phosphorylation,leading to activate mitochondria-mediated apoptosis and mitochondrial autophagy in renal tissues.BMSCs transplantation can repair renal injury induced by hexachromium,the possible mechanism may be related to inhibiting mitochondrial-mediated apoptosis and mitophagy mediated by MAPK pathway. |
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