Effects Of VCAM-1 On Human Bone Marrow Mesenchymal Stem Cells In The Repair Of Cartilage

Objective:The migration,homing,osteogenic and chondrogenic differentiation of bone marrow mesenchymal stem cells play a key role in the repair of bone and cartilage injury.VCAM-1(vascular endothelial adhesion molecule-1)protein promotes leukocyte migration and mediates inflammation through cell adhesion,while injury,inflammation and degenerative diseases are closely related to ferroptosis.At present,there are few studies on VCAM-1 protein in human bone marrow mesenchymal stem cells(HBMSCs).In this study,the effects of VCAM-1 protein on the migration of HBMSCs and ferroptosis were studied in vitro.Methods:Firstly,human bone marrow mesenchymal stem cells(HBMSCs)were extracted,and their phenotypes were isolated,cultured and identified.The effect of different concentrations of VCAM-1 on the migration of HBMSCs was detected by Transwell migration experiment.HBMSCs was treated with recombinant VCAM-1 protein,ferroptosis inducer(RSL-3)and ferroptosis inhibitor(Fer-1).The subsequent experimental groups were divided into Control group,VCAM-1 group,RSL-3 group,VCAM-1+RSL-3 group and VCAM-1+RSL-3+Fer-1 group.The best concentration of iron death induced by RSL-3 was detected by CCK8,and the effect of different concentrations of VCAM-1 combined with RSL-3 on HBMSCs activity was detected.The expression of reactive oxygen species(ROS)in HBMSCs treated with VCAM-1 protein was detected by fluorescence probe,the content of lipid peroxide(LPO)was detected at the same time,and the change of mitochondrial membrane potential was detected by JC-1 fluorescence probe.Finally,the expression of iron death related genes(GPX4,SLC7A11,ACSL-4)was detected by RT-q PCR,and the expression of GPX4 protein was detected by cellular immunofluorescence.Results:The results of flow cytometry showed that the surface molecules CD73 、CD90、CD34 and HLA-DR expressed 96.42%,95.25%,0.07% and0.14%,respectively,of HBMSCs extracted and cultured,which accorded with the surface marker identification standard of HBMSCs.Transwell migration assay showed that VCAM-1 could promote HBMSCs migration.When the concentration was 200ng/ml,the cell migration ability was the strongest,the difference was statistically significant(P<0.01).RSL-3could induce ferroptosis and decrease the cell viability of HBMSCs(P<0.01).The half inhibitory concentration(IC50)for 4 hours was 0.308 u M.At the same time,the concentration of VCAM-1 at 200ng/ml-800ng/ml could increase the cytotoxicity of RSL-3 to HBMSCs(P<0.01).VCAM-1could increase the expression of ROS and LPO of HBMSCs(P<0.01),and promote the accumulation of LPO and the decrease of mitochondrial membrane potential induced by RSL-3(P < 0.05,RSL-3 group VS(VCAM-1+RSL-3)group).At the same time,Fer-1 could restore the expression of LPO and mitochondrial membrane potential to normal level(P > 0.05,(VCAM-1+RSL-3+Fer-1)group VS Control group).VCAM-1could inhibit the m RNA expression level of GPX4 and promote the m RNA expression level of ACSL4(P<0.05),which could be reversed by Fer-1,but did not inhibit the m RNA expression level of SLC7A11(P>0.05).Although VCAM-1 could not significantly inhibit the expression of GPX4 at protein level(P>0.05),it promoted the inhibition of GPX4 expression induced by RSL-3(P < 0.05,RSL-3 group VS(VCAM-1+RSL-3)group),and Fer-1protected the expression of GPX4 protein to some extent(P < 0.01,(VCAM-1+RSL-3+Fer-1)group VS(VCAM-1+RSL-3)group).Conclusions:VCAM-1 protein promotes the migration ability of HBMSCs,but also may promote ferroptosis induced by RSL-3 by increasing the accumulation of ROS and LPO and mitochondrial damage.