Knockdown Of Macrophage Migration Inhibitory Factor(MIF)Reduces Parthanatos Caused By Oxidative Stress In Adipose-derived Stem Cells

Background:Traumatic central nervous system injury,such as spinal cord injury（SCI）,is a very common seen disease with a poor prognosis.Due to the non-renewable nature of nerve cells,stem cell transplantation has become the preferred method to treat such cases.In recent years,a large number of studies have proven the effectiveness of stem cell transplantation for SCI.Among them,adipose derived stem cells（ADSCs）are considered as suitable cases for tissue repair and to regeneration due to their rapid proliferation ability and multi-lineage potential.The most attractive aspects of ADSCs are their immunosuppressive properties,anti-apoptotic,anti-oxidant,and anti-inflammatory capabilities,coupled with their easy accessibility,which has gradually made them the most widely used transplant stem cell species in clinical.Nonetheless,the local adverse environment after SCI will decrease the survival rate of transplanted stem cells,which brings limitation to the therapeutic effect of stem cell transplantation.Oxidative stress is a key factor leading to this poor microenvironmental progress.Therefore,how to improve the survival rate after stem cell transplantation is an important issue to improve the therapeutic effect of stem cell transplantation.Studies have shown that oxidative stress causes stem cell DNA damage and induces PARP-1hyperactivation,which may be related to PARP-1-mediated cell death,parthanatos.Parthanatos is a PARP-1-dependent cell death first reported in 2007.It involves a wide range of diseases,including Parkinson’s disease,heart failure,ischemia-reperfusion injury,spinal cord injury,and ischemic and hypoxic brain damage.Purpose:This study was to explore whether the death of ADSCs exposed to oxidative stress is parthanatos,and if so,if knockdown of the key factor in the parthanatos process,macrophage migration inhibitory factor（MIF）may increase ADSCs survival.Methods:After exposed to oxidative stress,ADSCs death was measured using flow cytometry（annexin-V/PI）.To identify the difference between parthanatos and apoptosis,etoposide（ETO）was used to induce classical apoptosis as a comparison.Meanwhile,PJ-34 and Z-VAD-FMK were used as PARP-1 inhibitor and caspase inhibitor,respectively.Flow cytometry（annexin-V/PI）was performed again to detect the amount of cell death and the signal distribution.Immunofluorescence was performed to detect the PAR fluorescence signal to reflect PARP-1 activation.Western blot was used to detect the cleavage of PARP-1 and caspase-3 which are the typical features of apoptotic pathway.After parthanatos verified,knockdown of MIF was achieved by adenovirus transfection.Then ADSCs were exposed to H₂O₂ again,cell death was determined by flow cytometry assay,and alkaline and neutral comet assays also were used to identify the DNA strand breaks after H₂O₂ exposure when MIF is absent.Results:After exposure,ADSCs exhibited annexin-V/PI double positive cells,which could be blocked by PJ-34,while Z-VAD-FMK could not.In addition,strong PAR fluorescence signal was observed in H₂O₂ exposure group but not ETO exposure group,which also could be blocked by PJ-34.According to the western blot result,typical apoptotic features,PARP-1 and caspas-3 cleavage,could be seen in ETO exposure group but not H₂O₂ exposure group.After MIF knocked down,the cells death was significantly decreased after oxidative insults exposure.In addition,neutral and alkaline comet assays also showed significant decrease of DNA fragmentation.Conclusion:This study validates that MIF knockdown can reduce the death of ADSCs after oxidative insults exposure.It has been confirmed that MIF knockdown can inhibit parthanatos process.Therefore,similar to PARP-1 and AIF,MIF is expected to become a promising therapeutic target to improve ADSCs survival after transplantation under adverse microenvironment which may improve the therapeutic outcome of stem cells transplantation in central nervous system injuries.