| Objective:The mortality of ischemic stroke(Is)in China ranks the second in the world.In addition to thrombolytic therapy,there is no definite method to reduce neurological deficit caused by ischemic stroke.Especially in middle-aged people,the risk of stroke is increased and the recovery is slow.Therefore,from the perspective of basic research,improving the neurological function after ischemic stroke and promoting the neurogenesis of older rats are the focus of research.A large number of evidences have shown that Eph/ephrin pathway plays a regulatory role in cardiovascular system and embryonic vascular development,tumor growth,proliferation,metastasis and neurogenesis.Previous studies in our laboratory found that Morroniside can improve neurological function,reduce the volume of cerebral infarction,and promote the proliferation and differentiation of neural stem cells in the brain.According to the results of transcriptome sequencing and surface plasmon resonance,the interaction between receptor and ligand was analyzed,and EphB4 gene was screened.Then in vitro experiments found that the silencing of human embryonic stem cells EphB4 expression can increase astrocytes and inhibit neurogenesis.On the contrary,overexpression of EphB4 can maintain the proliferation and differentiation of neural stem cells,which is beneficial to functional prognosis.Therefore,it is inferred that it may be a target of neurogenesis,but it is still lack of in vivo experimental verification.In order to further clarify the mechanism of Morroniside promoting neurogenesis and determine its target,we designed conditional knockout EphB4 transgenic mice to continue to explore the effect of Morroniside on the neurogenesis of 9-month-old mice after stroke,so as to provide a new therapeutic target for the regeneration and repair of post-stroke nerves.Methods:(1)The neural progenitor cell specific EphB4 knockout(nestin creert2;EphB4-/-)transgenic mice model was established by injection of tamoxifen.(2)The model of middle cerebral artery occlusion(MCAO)was established in 25-30g weight knockout mice and control wild-type mice.After 30 minutes of ischemia,the thread plug was pulled out for reperfusion.After successful modeling,the mice were randomly divided into 8 groups:control sham operation group,control sham operation plus drug group,control model group,control model plus drug group,knockout sham operation group,knockout sham operation plus drug group,knockout model group and knockout model plus drug group.In sham operation,the blood vessels were separated without ligation.The sham operation group and model group were given Morroniside(270mg/kg)by gavage every day,while the sham operation group and model group were given the same amount of normal saline.After the model was established,the mice were asked to perform the behavioral tests of stick rotation,gait and new object recognition on the 1st,7th and 28th day after operation.(3)The neurogenesis of Brd U/EphB4/Nestin,Brd U/EphB4/SOX2 and Brd U/EphB4/DCX in SVZ were detected by immunofluorescence(IF)staining 7 days after cerebral infarction.Results:(1)Behavioral test results showed that:(1)rod rotation:compared with the model group 28 days after MCAO modeling,the time of rod rotation in the control group was significantly longer,but there was no difference between the EphB4 c KO group and the model group;(2)m NSS score:compared with the model group 28 days after MCAO modeling,the m NSS score in the control group was significantly lower,but there was no difference between the knockout group and the model group;(3)gait test:right Compared with the model group,catwalk gait parameters of mice in the irradiation group changed,which showed that the duration of mice passing the runway increased,the average speed of movement increased,the bos of the front and rear limbs decreased,and the step length of the front and rear limbs increased,while there was no difference between the knockout group and the model group.(2)After MCAO,the number of Brd U~+/Nestin~+and Brd U~+/DCX~+positive cells in the EphB4 c KO group was significantly lower than that in the wild group.Compared with the wild model group,the number of Brd U~+/Nestin~+/EphB4~+and the percentage of Nestin~+/EphB4~+,the number of Brd U~+/SOX2~+/EphB4~+cells and the percentage of SOX2~+/EphB4~+,the number of Brd U~+/DCX~+/EphB4~+cells and the percentage of DCX~+/EphB4~+were significantly increased after Morroniside administration.However,there was no statistical difference between the EphB4 c KO model group and the EphB4 c KO+Morroniside model group.Conclusion:Morroniside can promote the recovery of neural function and the proliferation and differentiation of neural stem cells in 9-month-old mice.After conditional knockout of EphB4,the recovery of neural function is not obvious,and the ability of promoting neurogenesis is weakened.This indicates that the mechanism of morroniside regulating neurogenesis may be related to EphB4 receptor,which provides a new target for stroke treatment. |
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