Effects Of Endogenous Neural Stem Cells Activated By AA/SVCT2 And Artesunate On The Neuroregeneration After Ischemic Stroke In Mice

Ischemic stroke is one of the important causes of disability and death worldwide,and 80%of patients are accompanied by neurological dysfunction.After stroke,the repair and reconstruction of the central nervous system（CNS）is crucial in restoring CNS architecture and function and has become the focus of current research.The studies have shown that neural stem/progenitor cells（NSPCs）are the key to cell replacement therapy for central nervous system injury.NSPCs can proliferate,migrate to the lesion sites and finally differentiate into neurons and glial cells,and play a therapeutic role in CNS diseases through supporting nutritients,regulating inflammatory response,replacing neurons by targeted differentiation,rebuilding neural circuits and functions,and paracring nerve growth factors,thereby promoting repair after injury.For ischemic stroke,due to the impaired blood-brain barrier,excitotoxicity and neuroinflammation obviously destroy the cell microenvironment.That is,in the damaged brain tissue,a clear pathological environment appears in the cell microenvironment,which is not conducive to the survival,neurogenesis and differentiation of endogenous NSPCs（eNSPCs）.The strategy of activating eNSPCs is to use the inherent characteristics of eNSPCs（expansionof the number of eNSPCs,migration to injury sites,and differentiation into mature cells to participate in neural network reconstruction）,and relies on the use of various exogenous"activation factors"（including growth factors and cytokines,etc.）to activate eNSPCs in the brain.Ascorbic acid（AA）is also known as vitamin C.Previous researches have shown that AA plays a significant role in embryonic cerebral development and in directing NSPC differentiation.Sodium-dependent vitamin C transporter 2（SVCT2）,which is mainly expressed in the CNS,is detected in NSPCs located in the inner and outer subventricular zone（SVZ）,suggesting the effect of AA on NSPCs might be due to modulating SVCT2.Most recently,studies have indicated that SVCT2 is expressed in NSPCs in SVZ,which is the discrete niche originating endogenous NSPCs for cell replacement remedy after ischemia.In addition,researches have demonstrated SVCT2 regulates NSPCs differentiation into neurons via amplifying vitamin C uptake.It has also been reported that NSPCs obviously proliferate in the SVZ,but only a few proliferated NSPCs can migrate towards the lesions after ischemia,indicating that promoting NSPCs migration toward the lesions is an evident issue for activating endogenous NSPCs after ischemia.However,whether SVCT2 is implicated in facilitating NSPCs migration and its underlying mechanism needs to be elucidated.Artesunate（ART）,a water-soluble derivative of artemisinin,penetrates the blood-brain barrier easily and exhibits anti-malarial functions with high-efficiency and low-toxicity.Thus,ART is widely used in clinical practice and is recommended by the World Health Organization for the treatment of malaria,especially for cerebral and falciparum malaria.ART has been intensively investigated due to its potential functions involved in anti-tumor,immune regulation,inhibition of inflammation and treatment of type I diabetes.An animal model of subarachnoid hemorrhage has been established in our previous study and the data revealed that ART could protect the blood-brain barrier by activating sphingosine 1 phosphate receptor1/phosphatidylinositol 3 kinase（S1pR1/PI3K）signaling pathway.To improve ischemic stroke-induced neuro-recovery,enhancing proliferation and differentiation of NSPCs could be a novel therapeutic approach.However,the effects of ART on the proliferation of NSPCs and ischemic stroke injury have not been elucidated.Part Ⅰ The effects and mechanism of AA and SVCT2 in reducing cerebral ischemiainjury by promoting NSPCs migrationObjective To explore the effects and mechanism of SVCT2 overexpression and AA intervention on migration of NSPCs and ischemia-reperfusion injury.Design and methods MCAO model was established in mice.Immunofluorescence and Western blotting（WB）were used to detect the expression levels of SVCT2 after MCAO.Overexpression of SVCT2 and combined application with AA（250mg/Kg）,then The rotarod test,corner test and beam walking were uased to detect the kinematics of MCAO model mice.TTC staining was used to observe the volume of cerebral infarction in MCAO model mice.Brdu injection was given while overexpressing SVCT2 to observe the state of NSPCs migrating along the corpus callosum and the number of Brdu positive neurons in the cortex and basal ganglia area around the cerebral infarction.Furthermore,the NSPCs oxygen glucose deprivation（OGD）model was constructed in vitro.The cells were divided into OGD,OGD+400μM AA,OGD+LV-SVCT2,OGD+400μM AA+LV-SVCT2 groups,and the migration of NSPCs was investigated.Immunofluorescence staining of tubulin and phalloidin was used to observe the changes of filamentous pseudopods of NSPCs.WB was used to detect the expression levels of SVCT2,CDC42 and F-actin.The CDC42-specific inhibitor ZCL278was used to evaluate the effect of CDC42 inhibition on the migration of NSPCs induced by SVCT2 overexpression.Results The in vivo experiment results revealed that 250 mg/Kg AA intervention significantly reduced the volume of cerebral infarction and mitigated motor dysfunction in MCAO mice.Immunofluorescence and WB experiments showed that the expression level of SVCT2 protein was significantly downregulated in mice at 1 day,3 days,7 days and 14 days after MCAO.Overexpression of SVCT2 by injection of lateral ventricle with recombinant AAV vector combined with the application of AA significantly decreased the volume of cerebral infarction and improved motor dysfunction in MCAO mice.Overexpression of SVCT2 and AA intervention could promote the migration and differentiation of NSPCs,but had no significant effect on the number of NSPCs.The in vitro experiment data showed that 200μM,400μM and1 mM AA significantly promoted the migration of NSPCs.Both SVCT2 overexpression and SVCT2 overexpression plus 400μM AA significantly promoted the migration of NSPCs,but SVCT2 shRNA intervention noticeably suppressed the migration of NSPCs.After OGD/R injury,the expression of SVCT2 on DCX-positive neuronal precursor cells was significantly reduced.The intervention of AA and SVCT2 after OGD/R injury could significantly promote the expression of CDC42/F-actin pathway related molecules.ZCL278 intervention significantly inhibited the formation of the filamentous pseudopods and the number of primary and secondary branches of NSPCs during migration,and reversed the effects of SVCT2 on the formation of filamentous pseudopodias.Conclusions The expression of SVCT2 is significantly down-regulated after cerebral ischemia,which may be the reason why AA is not effective in treating patients with cerebral ischemia.SVCT2 overexpression can play a role in promoting the migration of NSPCs through CDC42/F-actin.The combined use of SVCT2 overexpression and AA significantly relieves motor dysfunction and cerebral infarction volume in cerebral ischemic mice,which provides new therapeutic drugs and intervention targets for endogenous NSPCs in the treatment of stroke.Part Ⅱ Artesunate regulates neuroregeneration after ischemic stroke to reduceischemia-reperfusion injury in vivoObjective To explore the effects of ART in regulating neuroregeneration after ischemic stroke to reduce ischemia-reperfusion injury and investigate the role of PI3K/Akt/FOXO3a/p27^kip1ip1 signaling pathway in ischemic stroke.Design and methods Different doses of ART（50 mg/kg,150 mg/kg and 250 mg/kg）were used to evaluate the effects of ART on the recovery of motor function in MCAO mice.After intervening MCAO mice with 150 mg/kg ART and overexpression of FOXO3a,TTC staining analysis was used to detect cerebral infarct volume.Magnetic resonance diffusion tensor imaging（DTI）,transmission electron microscopy and Tuj1 immunofluorescence were used to determine white matter damage.Immunofluorescence staining was used to detect the expression levels of DCX,GFAP and Brdu in the SVZ and the cortex around the lesion.To further study the role of PI3K/Akt/FOXO3a/p27^kip1ip1 signaling pathway in the damage protection effects induced by ART in MCAO mice,MCAO mice were treated with 150 mg/kg ART and1 mg/kg wortmannin（PI3K inhibitor）.Functional behavior test was conducted to assess motor function at 1,3,7,and 14 days after MCAO.The volume of cerebral infarction was evaluated by TTC staining analysis.Three days after MCAO,the expressions of PI3K/Akt/FOXO3a/p27^kip1ip1 related molecules and Nestin（a marker of NSPCs）were examined by WB.Results 150 mg/kg ART significantly enhanced the evaluation results of nerve function scores,mine experiments and ladder experiments.MRI T2-weighted images and TTC staining showed that 150 mg/kg ART intervention reduced the volume of cerebral infarction in mice three days after MCAO injury.The results of DTI imaging,electron microscopy and Tuj1immunofluorescence staining showed that 150mg/kg ART also reduced the damage of white matter fiber bundles in the inner capsule area of??the injured side of mice after MCAO.By comparing the number of DCX+/Brdu+cells in the ipsilateral SVZ three days after MCAO and the number of DCX+/Brdu+cells in the cortex around the lesion,it was found that 150 mg/kg ART activated NSPCs to rapidly proliferate and differentiate into neurons.After overexpressing FOXO3a,the volume of cerebral infarction increased significantly.The motor nerve function defects and white matter damage in the inner capsule area of??the injured side increased,while the neurogenesis in the area around the cerebral infarction decreased.WB was used to detect the expression levels of FOXO3a/p27^kip1/Cyclin E/CDK2 signaling related molecules and Nestin and the results further confirmed the protective effect of ART on MCAO mice through the FOXO3a/p27^kip1ip1 signaling pathway.Additionally,the treatment with PI3K inhibitor wortmannin resulted in increased volume of cerebral infarction and decreased motor behavior.WB was used to detect the expression levels of PI3K/Akt/FOXO3a/p27^kip1ip1 signaling related molecules（p-AKT,p-FOXO3a,p27kip1）and Nestin,and the data confirmed that ART inhibited the phosphorylation of FOXO3a through PI3K/Akt signaling pathway,down-regulated P27^kip1,and protected mice from MCAO injury.Conclusions ART promotes neurogenesis through the PI3K/Akt/FOXO3a/p27kip1pathway,thereby saving penumbra damage,reducing white matter damage,and helping to restore function after ischemic stroke.Part Ⅲ Artesunate regulates the proliferation and differentiation of NSPCs in vitroObjective In order to explore the effects of ART on the proliferation and differentiation of NSPCs and confirm the promotion effct of ART on the proliferation of NSPCs by PI3K/Akt/FOXO3a/p27^Kip1ip1 signaling pathway after OGD/R injury in vitro.Design and methods NSPCs were treated with different concentrations of ART（0,0.4,0.8,1.6,3.2,6.4,12.8 and 25.6μmol/L）.The oxygen-glucose deprivation/reoxygenation（OGD/R）injured NSPCs were intervened with different concentrations of ART and 0.1μmol/L PI3K inhibitor wortmannin.CCK8 was used to detect the proliferation of NSPCs.WB and RT-qPCR were used to detect the expression of Nestin in NSPCs at 72 h after treatment with ART（0,0.4,0.8 and 1.6μmol/L）.Flow cytometry was used to detect the cell cycle of NSPCs at 72h after 0.8μmol/L ART treatment.Immunofluorescence staining of DCX and GFAP was used to determine the effects of 0.8μmol/L ART on the neuronal differentiation of NSPCs.After intervention of OGD/R injured NSPCs with 0.4μmol/L ART and 0.1μmol/L PI3K inhibitor wortmannin,CCK8 assay was used to examine the proliferation of NSPCs at 24 h and72 h,and WB was used to detect the expression of PI3K/Akt/FOXO3a/p27kip1 signal-related molecules and Nestin in NSPCs.Results 0.8μmol/L ART intervention promoted the proliferation of NSPCs,increased the percentage of DCX⁺cells,and decreased the percentage of GFAP⁺cells.The intervention with0.4μmol/L ART promoted the proliferation of NSPCs after OGD/R.Moreover,ART increased the expression levels of p-AKT and p-FOXO3a,and downregulated the expression level of p27^kip1.The intervention of PI3K inhibitor wortmannin reversed the effects of ART on the proliferation of NSPCs after OGD/R.Conclusions 0.8μmol/L ART intervention promotes the proliferation of NSPCs and neuronal differentiation of NSPCs.0.4μmol/L ART activates the PI3K/Akt/FOXO3a/p27kip1signaling pathway to promote the proliferation of NSPCs after OGD/R.