Knockdown Of Macrophage Migration Inhibitory Factor(MIF),A Novel Target To Protect Neurons From Parthanatos Induced By Simulated Post-spinal Cord Injury Oxidative Stress

Spinal cord injury（SCI）is a very common seen disease of spine surgery department.Pathology process of spinal cord injury is divided into two phases:primary injury and secondary injury.The secondary injury that continues to damage within hours to months after the end of the primary injury is an important culprit that further causes neurological dysfunction after trauma.In the process of secondary injury,the continuous production of reactive oxygen species and nitrogen species and the mitochondrial dysfunction and DNA damage caused by these events are considered to be key events in the cell death signaling pathway.Poly（ADP-ribose）polymerase-1（PARP-1）,a DNA gap sensor enzyme,mediates the detection and repair of intracellular DNA damage,and responds to DNA damage induced by reactive oxygen/nitrogen substances.In general,PARP-1 is a ribozyme which will be hyperactivated by DNA damage,initiates DNA repair.PARylation,as one of the inner environment stabilization mechanisms,responds to slight oxidative damage and plays a role in the modulation of DNA repair.However,it can become a cell death promoting factor under continuous or excessive oxidative stress exposure.In 2007,Dawson’s team of Johns Hopkins University School discovered a new form of programmed brain cell death,in which the hyperactivation of poly PARP-1 causes caspase-independent cell death.They named this death“parthanatos”which compounded by the hub molecule“PARP”and the Greek word“thanatos”meaning“death”.The term cell death nomenclature committee also recognized this PARP-dependent cell death pathway in 2012,reporting that it is widely involved in many diseases,including Parkinson’s disease,heart attack,ischemia-reperfusion injury,spinal cord injury,and ischemic and hypoxic brain injury.In the following cascade,apoptosis-inducing factor（AIF）plays a key role.After PARP-1 activation,apoptosis-inducing factor rapidly responds to the cell death signal.Apoptosis-inducing factor was released from mitochondria since the membrane potential was disturbed by the PAR polymers.After translocated into the nucleus,apoptosis-inducing factor induces chromatin condensation and large-scale DNA fragmentation leading to cell death.It is known that parthanatos is highly related to DNA damage due to oxidative damage related to the pathology of spinal cord injury.However,the underlying mechanism of how apoptosis-inducing factor to cleave DNA strands was not clear until the nuclease activity of macrophage migration inhibitory factor was discovered in 2016.Many previous researches focused on reducing hyperactivation of PARP-1,or nuclear translocation of apoptosis-inducing factors,or hydrolysis of PAR polymers in parthanatos.However,PARP-1 has intrinsic physiological functions for DNA damage detection and repair.As well as apoptosis-inducing factor have been proven to be critical for mitochondrial respiration and function maintenance,and apoptosis-inducing factor knockout may cause neurodegeneration and abnormal development of the nervous system.Therefore,inhibition of PARP-1 and apoptosis-inducing factors may have inherent risks in the treatment of both acute and chronic central nervous system injuries.In consideration of this,it becomes worthwhile to identify the effectiveness of macrophage migration inhibitory factor knockdown in parthanatos process.In order to provide a novel therapeutic target similar to PARP-1 and apoptosis-inducing factor.On the other hand,it is reported that many in vitro research results related to neurons responding to oxidative stress have been based on the use of oxidative condition that have nothing to do with exact pathological extracellular environment.To avoid this limitation,the oxidative stress level in the present study was set according to a dynamically monitor result of intraspinal extracellular oxidant level in rat model after spinal cord contusion（150μM H₂O₂）.Of note,this oxidative stress level has been considered to be sublethal in certain cell types which may lead to growth arrest,cell senescence,apoptosis or parthanatos.Besides,it is also demonstrated that different types of cells have different tolerance and response to H₂O₂-induced oxidative stress.And increased oxidative stress can lead to caspase-3 inhibition and persistent PARP-1activation and lead to parthanatos.Therefore,it is controversial that identical oxidative stress will pro-apoptosis or pro-parthanatos in different cell types.Objective:The main purpose of the present study was to investigate if knockdown of macrophage migration inhibitory factor may protect neurons under this simulated post-spinal cord injury oxidative stress（150μM H₂O₂）from parthanatos.Methods:According to previous research,it was found that this simulated post-spinal cord injury oxidative stress（150μM H₂O₂）is sublethal in certain cell types.It has been reported different cell fates when cells in face of sublethal oxidative stress,including cell growth arrest,senescence,apoptosis and parthanatos.Therefore,it is first necessary to determine the response of neurons exposed to 150μM H₂O₂ for a long time,that is,whether this level of oxidative stress is a lethal concentration for neurons.After treatment,flow cytometry（annexin-V/PI）was performed to detect cell death.Considering the close relationship between apoptosis and parthanatos,the decision for cell to make to be pro-apoptosis or pro-parthanatos in face of adverse stimuli depends on multiple factors.Hence,if the lethality of this level of oxidative stress is verified,it still needs to further determine the cell death pathway is apoptosis or pathanatos.In this step,etoposide（ETO）was used to induce classical apoptosis（ETO group）compared with the oxidative stress exposure group（H₂O₂ group）,while using the PARP-1inhibitor PJ-34 and broad-spectrum caspase inhibitor Z-VAD-FMK to intervene both groups respectively.Flow cytometry（annexin-V/PI）assay was conducted to show cell death and the signal distribution of annexin-V/PI cell.Immunofluorescence was conducted to detect the fluorescence signal PAR polymers in the nucleus.Western blot was used to detect the classical apoptotic features,PARP-1 and caspase-3 cleavage.Immunofluorescence co-localization was performed to detect the downstream events after PARP-1 hyperactivation in parthanatos,namely the intracellular spatial changes of apoptosis-inducing factor and macrophage migration inhibitory factor.After parthanatos confirmed under this level of oxidative stress exposure,the macrophage migration inhibitory factor was knocked down by adenovirus transfection,and then re-exposed to the identical oxidative stress.Intracellular spatial changes of apoptosis-inducing factor and macrophage migration inhibitory factor was detected via immunofluorescence co-localization,changes in cell death was detected by flow cytometry（annexin-V/PI）.And then alkaline comet assay and neutral comet assay were performed to show DNA strand breaks after macrophage migration inhibitory factor knockdown.Results:First,the flow cytometry（annexin-V/PI）results after concentration gradient H₂O₂ treatment showed that 150μM H₂O₂ was lethal（annexin-V/PI double positive cells are 67.5%）.Secondly,cell death caused by H₂O₂ exposure can be blocked by PJ-34,but not Z-VAD-FMK.As well as apoptosis induced by etoposide can be blocked by Z-VAD-FMK but not PJ-34.In addition,there was no strong PAR fluorescence signal seen in etoposide group,while a significantly strong fluorescence signal appeared in H₂O₂ group,which also could be blocked by PJ-34.Next,the etoposide group showed classical PARP-1 and caspas-3 cleavage,but it was absent in H₂O₂exposure group.In the meantime,significant fluorescence signal of apoptosis-inducing factor and macrophage migration inhibitory factor were also observed in H₂O₂ exposure group which is absent in etoposide exposure group,either.After macrophage migration inhibitory factor was knocked down and re-exposed to H₂O₂,cell death was significantly decreased（13.6%,61.5%in control group,57.8%in negative control group,respectively）.Neutral comet assay and alkaline comet assay showed that both single and double strand breaks generation were significantly decrease after H₂O₂exposure after macrophage migration inhibitory factor knockdown.Interestingly,immunofluorescence co-localization showed that the nuclear translocation of macrophage migration inhibitory factor was significantly decreased after apoptosis-inducing factor knockdown,indicating that macrophage migration inhibitory factor nuclear recruitment may be apoptosis-inducing factor dependent,but not vice versa.Conclusion:This study is the first time 1)to use a simulated post-spinal cord injury oxidative stress（150μM H₂O₂）based on a rat spinal cord injury model intraspinal monitoring;2)and validated this oxidative stress concentration is lethal in nerve cells;3)and verified neurons under this level of oxidative stress died through parthanatos rather than apoptosis;4)demonstrated macrophage migration inhibitory factor knockdown can protect neurons from parthanatos after oxidative exposure.Macrophage migration inhibitory factor is expected to be a promising therapeutic target for the treatment of spinal cord injury,as well as other acute and chronic central nervous system injuries.