Effects And Mechanisms Of Morroniside On The Proliferation And Differentiation Of Neural Stem Cells After Ischemic Stroke

With the characteristics of high morbility, mortality and disability rate, focal cerebral ischemia is becoming the second cause of death and first disability in adult in our country which causing a great burden to society and family. Stroke can stimulate the proliferation of transition on endogenous neural stem cells in short time to improve self repair, which providing insights into stroke treatment. But brain damage-induced neurogenesis is too short to repair the function in the chronic phase of stroke. So looking for endogenous drug or method to further enhance the proliferation and differentiation of neural stem cells, eventually achieving the nerve repair process is particularly important. In the present study,based on the hypothesis of “neurovascular homeostasis rehabilitation”proposed by Prof Wen Wang of Xuanwu Hospital in 2007, we tested the effects and mechanisms of morroniside on the proliferation and differentiation of neural stem cells after ischemic stroke.Middle cerebral artery occlusion(MCAO) model were performed on Sprague-Dawley male rats weighting 260-280 g. After Zea-Longa’s method was used to examine neurological function, the rats were randomly divided into sham-operated group, model group and morroniside-low(30mg/kg), morroniside-middle group(90mg/kg),morroniside-high group(270 mg/kg). Immunofluorescence staining as well as western-blot method were used for analysis the neurogenesis after stroke. The changes in expression of proteins related ephrin-B2/EphB4 signaling pathway were also observed.The results showed that :(1) 7 and 14 days after ischemia, Type B neural stem cells(NSCs)、type C amplifying precursors cells and type A neuroblasts were increased significantly compared to sham-operated groups, at 14 days, type B NSCs 、 type C amplifying precursors cells gradually decreased, while type A neuroblasts increased significantly, at28 days, type B、type C and type A cells were recovered to the level of sham-operated groups in the SVZ; at 7 and 14 days following MCAO,Type B neural stem cells(NSCs)、type C amplifying precursors cells and type A neuroblasts were increased significantly compared to shamoperated groups, at 14 days, type B NSCs、type C amplifying precursors cells and type A neuroblasts gradually increased, at 28 days, type B、type C and type A cells were no difference compared to sham-operated groups in the pri-infact cortex, which showed that endogenous neural stem cell proliferation reached the peak at 7 days, then differentiation into neuroblasts cells migrating to the ipsilateral cortext, at 28 days, there were also no new neurons in the pri-infact cortex, probably the damaged cortex cannot provide the microenvironment required for the differentiation of neural stem cells. Treatment with morroniside 7days and 14 days futher enhanced this increase in the number of type B cells and type C cells in the SVZ and in the cortex, at 28 days, the type A neuroblasts cells and mature neurons were raised significantly by treatment with morroniiside in the peri-infarct cortex, suggesting morroniside could promote NSCs proliferation and differentiation and directional differentiation into neurons in the cortex after MCAO.(2) EphB4 and ephrin-B2 appeared to be expressed in Radial glia-like neural stem cells(type B cells) and persisted as these cells became neuronal precursors(type C cells), neuroblasts(type A cells) and eventually mature neurons in the pri-infact cortex, indicating the potential relationship between the ephrin-B2/EphB4 signaling pathway and neurogenesis.(3) 7 days after MCAO, the Immunofluorescence shows that the expressions of ephrin-B2、EphB4 were increased significantly, suggesting that the ephrin-B2/Eph B4 signaling pathway was activated following MCAO, treatment with morroniside can regulate the expression of these proteins effectively and promoting the activation of proteins CyclinD1、CDK4、CDK6 and Abl downstream of the ephrin-B2/EphB4 signaling.The number of these double-positive cells BrdU+/EphB4+ and Nestin+/EphB4+ were significantly increased 7 days after MCAO,treatment with morroniside at a dose of 270 mg/kg/day further enhanced the increase in the number of these cells. Overall, these data indicate that administration of morroniside may promote NSC proliferation after focal cerebral ischemia via ephrin-B2/EphB4 signaling.(4) Double-label immunofluorescence showed that EphB4+/ SOX2+and EphB4+/DCX+ cells was significantly increased 7 days after MCAO.The number of EphB4+/SOX2+ and EphB4+/DCX+ cells were significantly increased in the morroniside-treated group compared to MCAO groups, at 28 days, EphB4+/NeuN+ cells were significantly increased in morroniside groups compared to MCAO group, suggesting that morroniside can promote NSCs directional differentiation into neurons in the cortex after MCAO via ephrin-B2/EphB4 sigalling pathway.In conclusion, morroniside could promote the proliferation and differentiation of NSCs in the SVZ after MCAO, then migrating into the damaged cortex and differentiate into mature neurons to replace the nerve cells of death to promote the recovery of the neurological function.Otherwise, the ephrinB2/EphB4 signaling pathway may be involved in promoting the proliferation and differentiation of endogenous NSCs of morroniside., suggesting the specific targets of morroniside on promoting neurovascular repair which provided a potentially new strategy for stroke treatment.