BMSCs Repair Hexavalent Chromium-induced Liver Injury By Reducing Endoplasmic Reticulum Stress-mediated Apoptosis Via SIRT1/HIF-1? Signaling Pathway In Rats

Objective:To observe the repairing effects of bone marrow mesenchymal stem cells(BMSCs)transplanted by the vein on liver injury induced by hexavalent chromium[Cr(?)]in rats and explore whether its possible repairing mechanism is reducing endoplasmic reticulum stress-mediated apoptosis via SIRT1/HIF-1?pathway.Methods:Separation,purification and culture of BMSCs were performed by whole bone marrow adherence method.Flow cytometry assay was used to detect the cell cycle and surface markers CD45 and CD90.The adipogenic induction medium was used to induce BMSCs to differentiate into adipocytes which were identified by oil red O staining.24 Wistar rats were randomly divided into control,model and cell therapy group,8 rats in each group.Wistar rats were treated with the potassium dichromate solution containing 0,0.4 and 0.4 mg/kg·bw hexavalent chromium ion by intraperitoneal injection,5 times a week,continuous exposure for 30 days.24 h and48 h after potassium dichromate solution was no longer administered to rats,rats in the cell therapy group were treated with chloromethyl-benzamidodialkylcarbocyanine(CM-Dil)-labeled BMSCs(0.1 ml of cell suspension containing 5×10~6 cells),respectively.The rats in the control and model groups were given the same volume of phosphate buffered saline.The food consumption,drinking water and mental state of the rats were observed.2 weeks later,the rats were sacrificed.The blood was taken to determine the levels of ALT and AST.Rats liver were taken to perform HE staining for observating the liver pathological changes.Flame atomic absorption spectrometry was used to detect the chromium content in liver tissue.Laser confocal microscopy was used to observe the location of BMSCs in rat livers.The MDA content and SOD activity were detected;TUNEL method was used to detect the apoptosis rate of liver cells.The expressions of SIRT1,HIF-1?,endoplasmic reticulum stress-mediated apoptosis-related proteins Grp78,ATF6,Cleaved-caspase12,CHOP,Bcl-2 and Bax were determined by Western blot analysis.The expression of SIRT1,HIF-1?,Grp78,ATF6,Cleaved-caspase12 and CHOP proteins was detected by immunohistochemical method.Results:The third-passage BMSCs were fibroblast-like and arranged in an orderly manner.The flow cytometry assay results showed 84.40%of the cells were in the G1phase,and CD90 was highly expressed,while CD45 was hardly expressed.Oil red O staining was positive after 21 days of adipogenic induction.2 weeks after cell treatment,the mental state of the rats in the cell therapy group was improved,and the food consumption and drinking water status were improved.The gross observation of the liver showed that the liver in the model group was hard with obvious round edge,but the livers changes in the cell therapy group were improved with slightly round edges.Automated blood biochemical analyzer showed that AST and ALT levels in the cell therapy group were significantly higher than those in the control group and lower than those in the model group(P<0.05).HE staining showed that the rat liver tissue structure in the control group was regular,and the liver cells were arranged in a strip shape.In the model group,liver tissues appeared a large number of inflammatory cells infiltration,vacuolization,cell necrosis and hepatic sinusoidal dilatation.The liver tissues in the therapy group were improved significantly compared with the model group.The results of the TUNEL assay showed that the apoptosis rate of liver cells in the model and cell treatment groups was significantly higher than that in the control group(P<0.05),while the apoptosis rate of the liver cells in the treatment group was lower than that in the model group(P<0.05).The MDA content in the model and cell therapy group was significantly higher than the control group,and the MDA content in the cell therapy group was lower than model group(P<0.05).The activity of SOD in the model group and cell therapy group was lower than that in the control group,and the activity of SOD in the cell therapy group was higher than the model group(P<0.05).Immunohistochemistry and Western blot assays showed that the expression of SIRT1 in the cell therapy group was higher than the other two groups,HIF-1?and the endoplasmic reticulum stress-mediated apoptosis-related proteins,including Grp78,ATF6,CHOP,Cleaved-caspase12 and Bax were lower than those in the model group,the expression of Bcl-2 was higher than the model group and lower than the control group(P<0.05).Laser confocal microscopy observation revealed that CM-Dil-labeled BMSC_S were localized in the rats livers of the cell therapy group.Conclusions:Cr(?)can cause liver damage in rats.BMSCs can localize in the rat liver and reduce the rat liver injury.The possible mechanism may be related to the mechanisms of BMSCs reducing endoplasmic reticulum stress-mediated hepatocyte apoptosis via the SIRT1/HIF-1?signaling pathway.