PHD2 Inhibition Protects Bone Marrow Mesenchymal Stem Cells From Oxidative Stress Damage

[Background]Transplantation of bone marrow mesenchymal stem cells（BMSC）has become a promising therapy in repairing damaged myocardial tissue caused by myocardial infarction（MI）.The oxidative stress,induced by ischemic and hypoxic microenvironment in the infarcted area,is not conducive to the survival of transplanted BMSCs,thus limiting the therapeutic effect of BMSCs.proline hydroxylase 2（PHD2）and hypoxia inducible factor 1α（HIF-1α）are important substances that regulate the survival of cells in hypoxic environment.PHD2 is a negative regulator of HIF-1αIn the normoxic state,PHD2 degrades HIF-1αthrough hydroxylation.Under the condition of hypoxia,PHD2 is inactivated and lose the control of HIF-1α,leading the accumulation of HIF-1α.HIF-1αcan be transported into the nucleus acting as transcription factor participating in the regulation of cell processes,such as proliferation,metabolism and so on.These fundamental researches suggest that silencing PHD2 may have protective effect on BMSCs in oxidative stress environment.[Objective]The aim of this study was to investigate the effect of silencing PHD2expression on BMSCs resistant to oxidative stress injury by modifying BMSCs with shRNA-PHD2.[Methods]The BMSC was extracted from SD rat via whole bone marrow adherence method.Lentivirus with shRAN-PHD2 target fragment was constructed and then transfect BMSCs,screening out the BMSCs which stably expresses PHD2-shRNA.The model of oxidative stress damage in vitro was constructed.The cells were divided into three groups:MSC group,MSC+H₂O₂ group and shPHD2-MSC+H₂O₂+group.To restrain the activity of HIF-1α,shPHD2-MSC+H₂O₂+YC-1 was added.The morphology and distribution of MSC were observed under optical microscope,and the surface specific CD molecule of MSC was detected by flow cytometry.oil red was performed after BMSC adipogenic differentiation.the BMSC was stained with alizarin red after osteogenic differentiation.The mRNA level of PHD2 and HIF-1αwas detected by RT-PCR.The protein expression level of PHD2 and HIF-1αwas detected by Western blotting.Apoptosis was detected by TUNEL staining and Annexin V-APC/7-AAD Apoptosis Detection.[Results]1.Under optical microscope,The long fusiform shape of BMSCs grew in vortex or parallel arrangement.2.The result of flow cytometry showed that the expression of CD markers on the surface of MSC was CD29（98.2%）,CD90（99.9%）,CD45（1.7%）and CD34（0.2%）.3.MSC can differentiate into adipocytes,the Lipid droplets in adipocytes can be stained by oil red O.BMSCs can differentiate into osteoblasts,calcified nodules and calcium deposits in osteoblasts can be stained by alizarin red.4.The results of RT-PCR showed that,compared with MSC group,The mRNA level of PHD2 decreased,while the mRNA level of HIF-1αincreased in shPHD2-MSC group（p<0.05）.The result of protein expression level in shPHD2-MSC group was consistent with that in RT-PCR.Compared with MSC group,the protein expression level of PHD2decreased while the protein level of HIF-1αincreased in shPHD2-MSC group（p<0.05）.5.According to TUNEL staining result,after being subjected to H₂O₂,the number of TUNEL positive cells significantly increased in MSC+H₂O₂ group,compared with MSC group（p<0.05）.However,the number of TUNEL positive cells in shPHD2-MSC+H₂O₂ group is much lesser than that in MSC+H₂O₂ group（p<0.05）.6.The results of Annexin V-APC/7-AAD Apoptosis Detection showed that the rate of apoptosis in shPHD2-MSC+H₂O₂ group was significantly lower than that in MSC+H₂O₂ group（p<0.05）.But,compared with shPHD2-MSC+H₂O₂ group,the apoptosis rate in shPHD2-MSC+H₂O₂+YC-1 group significantly increased（p<0.05）.[Conclusion]Silencing the expression of PHD2 obviously reduce the apoptosis rate of the MSC in the oxidative stress environment,besides of that,these protective effects is mainly dependent on the HIF-1 regulated by the PHD2.Our research provides the foundation for the following in vivo research and may offers new perspective for alleviating the poor retention rate of transplanted stem cells...