The Experimental Study Of Erythropoietin Signaling Pathway On The Proliferation And Differentiation Of Endogenous Neural Stem Cells In Adult Spinal Cord

Erythropoietin(Epo)is a hematopoietic growth factor secreted by the kidneys and fetal liver,a member of the type I cytokine superfamily,which has previously been a key role in stimulating the proliferation and differentiation of hematopoietic stem cells.Clinically widely used in the treatment of various causes of anemia.Erythropoietin receptor(Epo R)is a specific receptor of Epo,which belongs to the type I family of single-transmembrane cytokine receptors,and Epo reacts with Epo R to activate downstream signaling pathways to exert various biological effects.In recent years,the non-hematopoietic effects of Epo signaling have been reported,particularly in the various types of central nervous system injuries,which have received extensive attention.Epo and EpoR are widely expressed in glial cells,neurons and endothelial cells in the central nervous system(CNS).Studies have demonstrated that Epo shows the effects of neuroprotection and promoting nerve regeneration in stroke,multiple sclerosis,autoimmune encephalomyelitis,schizophrenia,brain injury and spinal cord injury(SCI)and other central nervous system diseases.SCI is a costly,and common pathological condition which always causes disability,brought huge pain and loss to the family and society and there is no effective treatment till now.The pathology of SCI divided into two phase: the primary physical damage and secondary chemical damage.Due to the unpredictability of the primary injury,the current treatment research mainly focuses on the control of various acute ischemic necrosis and various inflammatory reactions during the secondary injury.The study found that Epo can significantly promote the recovery of neurological function after SCI,its role may be through anti-inflammatory,anti-apoptosis and promote vascular regeneration and other mechanisms to complete,and now the initial report of Epo in clinical trial application has the positive outcome,which making Epo be one of the most promising candidates for the treatment of SCI.Epo signaling pathway promotes the neurological recovery following brain injury and stimulating endogenous neural stem cells(NSCs)differentiating into neurons is an important mechanism,which is also a key issue in the application of NSCs to repair SCI.Adult spinal cord belongs to the "non-neurogenic" area and no new neuron born under normal circumstances.A large number of endogenous NSCs proliferation after SCI,however,because the damage to the internal environment of spinal cord most of them differentiated into astrocytes eventually which form scars that actually hinder nerve reconnection.Since stem cell differentiation is directly related to the microenvironment in which it is located,the neuroprotective effect of Epo signaling pathway may improve the internal environment after SCI,which provides opportunities for neurogenesis of endogenous NSCs in the adult spinal cord.Epo promotes functional recovery following SCI has been widely confirmed,a variety of mechanisms of action has been found,but its effect of proliferation and differentiation of spinal cord endogenous NSCs and its role as a part of its repairing mechanism had not been reported in the literature.This study is divided into three parts to investigate the effect of Epo signaling pathway on the proliferation and differentiation of endogenous NSCs in adult rats spinal cord.The first part is the effect of Epo on the proliferation and differentiation of endogenous NSCs following SCI in rats.After preparation of rat SCI model by modified Allen's method,the experimental group was injected intraperitoneally(i.p.)with Epo(5000 U/kg)×7 days and the control group was treated with i.p.saline at the same volume for 7 days.Immunofluorescence staining of endogenous NSCs number,neurons,astrocytes and oligodendrocytes was performed at 2 days,8 days and 14 days after surgery and the expression of Epo and Epo R were detected by Western blot.The results showed that the proportion of neurons(P<0.01)and oligodendrocyte differentiation(P<0.05)were significantly higher in the experimental group,the proportion of astrocytes(P<0.05)was significantly decreased,but the number of endogenous NSCs was no significant difference between the two groups.Epo and Epo R were increased by Epo injection,but there was no significant difference between the two groups at 2 days and 8 days.The experimental group was significantly stronger than the control group(P<0.01)in 14 days.The results indicated that Epo intraperitoneal injection can significantly prolong the action time of Epo signaling pathway,promote the neurogenesis and oligodendrogenesis,reduce the differentiation of astrocytes,but have no effect on the proliferation of endogenous NSCs following SCI.The second part is the effect of Epo on the proliferation and differentiation of endogenous NSCs in normal rats.The rats in the experimental group were treated with i.p.Epo(5000 U/kg)×7 days.The control group was treated with i.p.saline at the same volume for 7 days.After 2 days,8 days and 14 days,NSCs,neurons,astrocytes and oligodendrocytes were stained by immunofluorescence,and the expression of Epo and Epo R was detected by Western blot.The results showed that Epo intraperitoneal injection at 2 days and 8 days can significantly increase the expression of Epo(P<0.01),but the expression of Epo at 14 days has no difference with the control group.The expression of Epo R was significantly higher than that of the control group at three time points(P<0.01).There is no effect on the number and differentiation of endogenous NSCs.The third part is the effect of Epo on the proliferation and differentiation of endogenous NSCs in vitro.The direct effect of Epo signaling pathway on endogenous NSCs was confirmed.Isolated and cultured the endogenous NSCs from adult spinal cord after SCI and the cells were divided into three groups: Epo(10U/ml),vehicle and Epo + inhibitor. After 7 days of in vitro culture,the neurospheres counting,the mean diameter measurement and flow cytometry were performed.The expression of Epo and Epo R was detected by Western blot.Neurons,astrocytes and oligodendrocytes were stained by immunofluorescence.The results showed that the expression of Epo and Epo R was significantly increased,the ratio of neurons(P<0.01)and oligodendrocyte differentiation(P<0.05)in the experimental group was significantly higher,the proportion of astrocytes(P<0.05)was significantly reduced in Epo group.The inhibitor can eliminate the effect of Epo,the number of endogenous NSCs between the two groups was no significant difference.In summary,SCI and in vitro experiments have shown that exogenous Epo can can prolong or enhance the role of Epo signaling pathway,thereby promoting neurogenesis and oligodendrogenesis of endogenous NSCs,reducing the differentiation of astrocytes,which has no effect on proliferation.For normal rats,exogenous Epo can activate the signal pathway but has no effect on proliferation and differentiation of endogenous NSCs.