Research Of The Mechanism Of Nogo-A/NgR Promoting Nerve Regeneration In BMSCs During Recovery Cerebral Ischemia Through Notch1/HES1 Pathway

Research Background:Stroke,also known as "stroke",has a high mortality and disability rate worldwide.Stroke can be divided into ischemic stroke and hemorrhagic stroke.During ischemic stroke,the blood flow is temporarily or permanently interrupted,and the oxygen and glucose transport is blocked,resulting in the dysfunction of local brain tissue function and metabolism,and even partial death.In the past 30 years,the crude mortality rate of stroke in China has continued to rise,and the rising rate is higher than that of other countries in the world,contrary to the decline trend in developed countries.According to the data of the Global Burden of Disease(GBD),the incidence rate of ischemic stroke in China increased from 117/100000 to 145/100000 in the 15 years from 2005 to 2019,which brought huge burden to the medical and health system.The current situation of delayed treatment of stroke patients in China is relatively serious.The proportion of patients who miss the treatment opportunity in the acute phase is very high,up to 70%.Therefore,it is necessary to explore effective treatment methods for ischemic stroke in the recovery period,including the recovery of nervous system structure and function.At present,neurovascular regeneration is the main research direction of neural function repair after cerebral ischemia.The transplantation of bone marrow mesenchymal stem cells(BMSCs)has therapeutic effects on the injury of the nervous system.It can secrete various neurotrophic factors under the damaged microenvironment and promote the regeneration of nerve vessels at the damaged site.BMSCs transplantation can down-regulate the expression of the inhibitory factor Nogo-A/NgR pathway,thus promoting the nerve regeneration and functional repair of cerebral ischemia in the recovery period;Notchl inhibitor can inhibit the expression of HES1 gene and promote neuronal differentiation.In the treatment of subarachnoid hemorrhage with BMSCs,it was found that BMSCs could inhibit the expression of HES-1 in Notchl pathway.Therefore,this study is mainly to explore whether BMSCs transplantation therapy can inhibit Notch1/HES1 pathway by down-regulating the expression of Nogo-A/NGR,thereby regulating and controlling the growth of neuronal axons and promoting the recovery of neural function after cerebral infarction.Objective:To investigate the effect of bone marrow mesenchymal stem cells(BMSCs)on the function and repair of the nervous system of rats with cerebral ischemia in the recovery period,and the effect on Notchl/HES1 pathway and Nogo-A expression.Method:(1)Extracting BMSCs through whole bone marrow adhesion method and identifying BMSCs using flow cytometry;(2)Several male SD rats were randomly divided into Sham group,MCAO group,MCAO+PBS group,MCAO+BMSCs group,MCAO+DMSO group,and MCAO+DAPT group.SD rat middle cerebral artery ischemia-reperfusion(MCAO)model was prepared using an improved Zea Longa suture method.On the 14th day after surgery,PBS,BMSCs,DMSO,and DAPT were injected into the ventricles through stereotactic injection;(3)Seven days after stereotactic injection into the ventricles,mNSS scores,TTC staining,and MRI examinations were performed on each rat in each group;(4)On the 7th day after ventricular stereotactic injection,each rat in each group was perfused and the brain was taken out to prepare frozen sections of rat brain tissue.HE staining was used to observe the morphological changes of histopathology in the infarcted area of the brain,and immunofluorescence was used to detect the positive expression and expression level of Nogo-A,Notchl,HES1 in the infarcted area and surrounding areas of each rat’s brain tissue in each group.Result:At 7 days after treatment,there was a certain degree of decrease in mNSS scores in the MCAO+BMSCs group compared to the MCAO group and MCAO+PBS group;Compared with the MCAO group and the MCAO+DMSO group,the mNSS score of the MCAO+DAPT group showed a certain degree of decrease;The magnetic resonance imaging examination of the rat head suggests that at 7 days after treatment,compared with the MCAO group and MCAO+PBS group,the volume of cerebral ischemic lesions in the MCAO+BMSCs group decreased to a certain extent;Compared with the MCAO group and the MCAO+DMSO group,the volume of cerebral ischemic lesions in the MCAO+DAPT group decreased to a certain extent;Observing the TTC staining results of rat brain slices in each group,it can be seen that compared with the MCAO group and MCAO+PBS group,the MCAO+BMSCs group has a certain reduction in the ischemic area of the rat brain;Compared with the MCAO group and MCAO+DMSO group,the cerebral ischemic area of the MCAO+DAPT group rats decreased to a certain extent;By comparing the HE staining results of rat brain slices in each group,it can be seen that compared with the MCAO group and the MCAO+PBS group,the MCAO+BMSCs group has a relatively better brain tissue structure;Compared with the MCAO group and the MCAO+DMSO group,the brain tissue structure of the MCAO+DAPT group is relatively better;At 7 days after stereotactic injection into the ventricles,compared with the MCAO group and MCAO+PBS group,the NogoA,Notch1,Hes1 immunofluorescence intensity of the MCAO+BMSCs group decreased to a certain extent;Compared with the MCAO group and the MCAO+DMSO group,the NogoA,Notchl,and Hes1 immunofluorescence intensity of the MCAO+DAPT group also decreased to a certain extent.Conclusion:BMSCs transplantation has a positive effect on the repair of nervous system function in rats after middle cerebral artery ischemia and reperfusion,which is mainly manifested in promoting the regeneration of nerves and vessels,and its therapeutic effect may be produced by down-regulating the expression of Nogo-A/NGR and inhibiting Notch1/HES1 pathway.