| Spinal cord injury（SCI）can cause severe sensory and motor function impairment,and even paralysis,leading to huge losses and burdens to the patient’s family and society.The development process of SCI includes primary injury and secondary injury.The secondary injury stage includes a series of complex pathological changes,such as ischemia,hypoxia,oxidative stress,inflammation,neuronal death and demyelination.Complicated secondary reactions can cause neuronal mitochondrial dysfunction and DNA damage,which leads to cell death.At the same time,changes in the microenvironment of the damaged site inhibit the repair and regeneration of neurons.These become the biggest obstacles to the repair and reconstruction of damaged tissues.Unfortunately,there is no effective clinical therapy that can completely cure SCI.Therefore,it is of great significance to explore the mechanism and methods of reducing neuron apoptosis and promoting neuron regeneration after SCI for the clinical therapy.Recently,stem cell has become a research hotspot in regenerative medicine and biological treatment that has been widely used in clinical practice.Mesenchymal stem cells（MSCs）are characterized as high availability,easy to culture,and low immunogenicity.Evidence has proved that MSCs could attenuate apoptosis and inflammatory response at the site of SCI and promote neuron regeneration,which made MSCs a potential biological tool for the repair and cure of SCI.As the energy center of cells,mitochondria can determine the fate of cells by their homeostasis and physiological activities.Our previous studies have found that mitochondrial transfer occurred during the transplantation of bone mesenchymal stem cells（BMSCs）to treat SCI,and the transfer of mitochondria has a more positive effect on damaged neurons.Therefore,in present study,we explore the mechanism of mitochondrial production and transfer,to provide a more feasible and effective way for the treatment of SCI.Firstly,the BMSCs were pre-stained with Mito Tracker Red CMXRos which could present mitochondria as red dots.After 24 hours of culture,the BMSCs conditioned-medium（BCM）was collected.Electron microscope observation and fluorescence-activated cell sorting confirmed that BMSCs could produce extracellular mitochondria.After pre-stained BMSCs and VSC4.1 spinal neuronoma cell line（VSC4.1 cells）were directly in a dish or indirectly co-cultured in a transwell system,red fluorescence signal was observed in the VSC4.1 cells,indicating that BMSCs-derived mitochondria were transported to VSC4.1 cells.In addition,when BMSCs were co-cultured with VSC4.1cells treated with oxygen-glucose deprivation（OGD）,the content of mitochondria internalized by VSC4.1 cells was significantly higher than that of normal VSC4.1 cells.However,do these BMSCs-derived mitochondria retain their original activity?Producing energy is the main function of mitochondria,so that ATP content detection can be one of the indicators of mitochondrial function.We collected the BMSC-conditioned medium（BCM）and filtered a part of the medium with a 0.22μm filter to delete the mitochondria as a control group（Md-BCM）.The ATP content measurement of the two groups showed that the ATP content in the normal medium is significantly higher than that in the control group,indicating that extracellular mitochondria still have the function of energy production.Meanwhile,the mitochondrial membrane potential assay（JC-1）showed that the mitochondrial membrane potential in the normal medium was higher than that in the control group,indicating that the extracellular mitochondria maintained normal membrane potential.Secondly,we found that when BMSCs were co-cultured with OGD-treated VSC4.1 cells,the expression of CD157 protein in BMSCs increased.Given that CD157is capable of signal transduction to regulate intracellular calcium ions,and intracellular calcium ions are closely related to the homeostasis of mitochondria,we suspected that CD157 in BMSCs could regulate the production and transfer of extracellular mitochondria.We constructed CD157 protein high-expressing adenovirus vector and sh RNA adenovirus vector,with the empty virus as a control.Transfected BMSCs were pre-stained with Mito Tracker Red CMXRos and were cultured for 24 hours.The culture medium of each group was collected for FACS and fluorescence intensity detection.The results showed that the positive rate of red fluorescence and fluorescence intensity in the medium of the CD157 high expression group（Over group）were significantly higher than those in the control group（Mock group）,and those in the sh RNA knockdown group（sh RNA group）were lower than the other two groups.These results indicated that the expression of CD157 might regulate the production of extracellular mitochondria,and the production of extracellular mitochondria increased with the increasing expression of CD157.When the transfected BMSCs were co-cultured with OGD-treated VSC4.1 cells,the fluorescence intensity statistics showed that the internalization of mitochondria in VSC4.1 cells in the Over group was significantly higher than that in the Mock group,while the sh RNA group was significantly lower than the Mock group.After being co-cultured with the transfected BMSCs,the axon length of VSC4.1 cells in the Mock group was significantly higher than that in the OGD group,the Over group was significantly higher than the Mock group,and the sh RNA group was significantly lower than the Mock group.The results indicated that the synaptic regeneration of damaged neurons might improved by the increase of extracellular mitochondria which were regulated by the over-expression of CD157.Thirdly,the transfected BMSCs were co-cultured with OGD-treated VSC4.1 cells.Immunofluorescence staining and western blotting were used to detect the expression of apoptosis-related proteins in VSC4.1 cells after co-culture.The results showed that the expression of pro-inflammatory factor Grp78 and the endonuclear NF-κB were both significantly downregulated in the Over group,while the expression of anti-apoptotic factor Bcl-xl protein increased significantly in the Over group.Meanwhile,we carried out the BMSCs transplantation in SCI rats,and the injured tissue sections were taken and sliced.The results of immunofluorescence staining were consistent with the cell experiments:The expression of Grp78 and NF-κB in the Over group were significantly downregulated,while the expression levels of p-Akt and GAP43 were significantly upregulated in the Over group.In addition,the BBB scores showed that after 21 days,the scores of rats in the Over group were significantly higher than that in the Mock group,and the scores of rats in the sh RNA group were significantly lower than that in the Mock group.In summary,the high level of CD157 in BMSCs may upregulate the production of extracellular mitochondria and promote the mitochondrial internalization of injured neurons,resulting in the inhibition of inflammatory response and apoptosis at the injured site,promoting the repairment and regeneration of neurons,and improving the motor function of SCI rats.Finally,we verified whether CD157 regulates mitochondria through the c ADPR-Ca2+signaling pathway at the cellular level.The results showed that CD157 did have cyclase activity.As c ADPR increased,the intracellular mitochondrial content,ATP content and calcium ion signal in BMSCs all increased.In consist with this,when the expression of CD157 changed,the calcium ion signal also changed,showing that the calcium ion signal increased with the increase of CD157 expression.All above indicated that CD157 in BMSCs might regulate BMSCs to produce and transfer extracellular mitochondria to injured neurons via the CD157/c ADPR/Ca2+signaling pathway.In conclusion,our study suggests that CD157 in BMSCs regulates the production and transfer of mitochondria to damaged neurons through the c ADPR-calcium ion signaling pathway and mediates inflammation and cell apoptosis at the injured site,promoting the plerosis of neuron and improving motor function of SCI rats. |
PDF Full Text Request