| The high mortality rate of cerebral ischemic diseases is a serious threat to human health and poses a serious burden on patients'families and society.It is generally believed that the molecular mechanisms involved in ischemic brain injury include excitatory amino acid release,abnormal brain energy metabolism,intracellular Ca2+overload,increased oxygen free radical production,increased release of inflammatory cytokines and adhesion molecules,and activation of apoptotic genes.In recent decades,a large number of studies have been carried out on the treatment of cerebral ischemic diseases,mainly focusing on recanalization,free radical scavenging,neuroprotection,etc.,but sometimes the prognosis is still poor,and it is impossible to fundamentally compensate for cerebral ischemic injury.The area lost a lot of nerve cells.Therefore,how to actively and effectively promote the recovery of neurological function during cerebral ischemia recovery is still the focus of current ischemic stroke research.Since the 1990s,neural stem cell transplantation has made progress in the treatment of neurological diseases such as Parkinson's disease,ischemic cerebrovascular disease,senile dementia,and spinal cord injury.It has become one of the research hotspots in the field of neuroscience at home and abroad.Neural stem cells?neural stem cells,NSCs?have the potential of self-renewal and multi-directional differentiation,and can differentiate into neurons,astrocytes and oligodendrocytes under the induction of specific microenvironment.Their good fusion ability and low immunogenicity make neural stem cells Long-term survival after transplantation.The Notch signaling pathway was found to be mutated in Drosophila and is one of the major signal transduction pathways known to be involved in nerve growth and development after cerebral ischemia.The Notch signaling pathway is involved in the neural development of the central nervous system.After cerebral ischemic injury,this signaling pathway is involved in the repair of the nervous system.Through the regulation of the Notch-Hes signal transduction system,the Notch signaling pathway can inhibit the differentiation of neural stem cells into neurons or glial cells,thereby indirectly maintaining the undifferentiated state and self-renewal of neural stem cells.Hes gene belongs to the basic helix-loop-helix?bHLH?gene family,in which Hes1 and Hes5 are highly expressed in NSCs,which are important effector molecules downstream of Notch signaling pathway and have important effects on proliferation and differentiation of neural stem cells.?-secretase is a key enzyme in Notch signaling,and various studies have shown that?-secretase inhibitor{N-[N-?3,5-difluorophenacetyl-L-ala nyl?]-S-phenyl-glycine T-butyl ester,DAPT}is a Notch signaling pathway inhibitor that specifically blocks?-secretase activity.DAPT preempts the Notch receptor hydrolysis site in presenilin?PS?and blocks Notch receptor intracellular?NICD?hydrolyzes,thereby affecting a series of life activities of nerve cells.In the early stage of our group,primary neural stem cell transplantation was performed in rats with cerebral ischemia-reperfusion.It was confirmed that neural stem cell transplantation can alleviate ischemic brain damage in rats and has neuroprotective effects,but its mechanism has not been elucidated.This study focused on the effect of Notch signaling pathway on the transplantation of NSCs in cerebral ischemia rats,and established a model of focal cerebral ischemia in the middle cerebral artery occlusion?MCAO?of rats,and performed NSCs transplantation after modeling.DAPT pretreatment was used to investigate the Notch signaling pathway of neural regeneration after neural stem cell transplantation in cerebral ischemia rats.Objective:Establish a rat model of focal cerebral ischemia in middle cerebral artery occlusion?MCAO?,and transplant NSCs into cerebral ischemia rats.To investigate whether neuroprotective effects of Notch signaling pathway inhibitor?-secretase inhibitor on neural stem cells after cerebral ischemia in rats and observe the changes of protein in Notch signaling pathway,and to explore the nerve regeneration after transplantation of NSCs in cerebral ischemia rats Notch signaling pathway mechanism.Methods:63 SD rats,after successful cerebral ischemia model,were randomly divided into 3 groups:model group,neural stem cell transplantation group?transplantation group?,DAPT+transplantation group.There are 21 rats in the sham operation group,only the exposed blood vessels were separated under anesthesia.The neurobehavioral scores of rats in each group were observed 7 days after NSCs transplantation.The volume of cerebral infarction was observed by TTC staining.The pathological changes of brain tissue were observed by Nissl staining and HE staining.The expression of Brdu/NeuN positive cells was detected by immunofluorescence double labeling.The expressions of Notch1,Hes1 and Hes5 in the brain tissue of each group were detected by immunohistochemistry and Western blotting.Results:1.In the sham operation group,the neurological function score was zero,no cerebral infarction,Nissl body was abundant,cell morphology was intact,and Brdu/NeuN positive cells were negative.Compared with the sham operation group,the neurological score of the model group was increased,with obvious neurological defects,obvious cerebral infarction,disordered arrangement of nerve cells,and few nissl bodies.Neurons are severely damaged,with nuclear pyknosis and karyolysis.The number of surviving neurons was significantly reduced?P<0.05?,and a small number of Brdu/NeuN positive cells were expressed?P<0.05?.Compared with the model group,the transplantation group and the DAPT+transplantation group had different degrees of neurological function score reduction,the cerebral infarction volume decreased,and the number of nerve cell survival increased?P<0.05?,the expression of Brdu/NeuN positive cells increased?P<0.05?,and the indexes of DAPT+transplantation group recovered most obviously.2.Compared with the sham operation group,the positive expression of Notch1,Hes1 and Hes5 in the model group was significantly increased,and the protein content was significantly up-regulated?P<0.05?.Compared with the model group,the expression of Notch1,Hes1 and Hes5 in the transplantation group and DAPT+transplantation group were partially expressed,and the protein content was decreased?P<0.05?.Compared with the transplanted group,the positive expression of Notch1,Hes1 and Hes5 in the DAPT+transplantation group was further reduced,and the protein content was further reduced?P<0.05?.Conclusion:Inhibition of Notch signaling pathway can significantly reduce the neurological function score after neural stem cell transplantation in rats with cerebral ischemia,significantly reduce the volume of cerebral infarction,reduce the degree of nerve cell damage,and promote the differentiation of transplanted NSCs into neurons.The use of Notch signaling pathway inhibitors has neuroprotective effects on cerebral ischemia rats after neural stem cell transplantation and promote nerve regeneration.The mechanism is mainly related to down-regulation of the expression of Notch1,Hes1 and Hes5. |
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