The Effects And Mechanisms Of Insulin-Like Growth Factor 1 And Dental Pulp Stem Cell Transplantation In Cerebral Ischemic Stroke

BackgroundCerebral ischemic stroke induces severe brain damage,increases adult morbidity and mortality,and has become a global healthcare problem that brings a huge burden to the society.Specific treatments are so limited that only a few patients can be treated effectively.Therefore,it is urgent to clarify the pathological mechanism of ischemic stroke,so as to find new therapeutic targets and obtain effective treatment methods.As an important bioactive substance,insulin-like growth factor-1(IGF-1)not only plays a vital role in the development and formation of central nervous system,but also controls the excitability of neurons,regulates the metabolism of nerve cells and promotes the proliferation and survival of nerve cells in adult brain tissue,suggesting its important roles in regulating the structure and function of the nervous system.Recently,we found that there was a complex relationship between IGF-1 and the disease progression and prognosis of ischemic stroke.IGF-1 was closely linked with outcomes of patients that the higher level of circulating IGF-1 was accompanied with the improved neurological outcomes.When an ischemic stroke attacked rats suffered diabetes,the rats obtained IGF-1 therapy exhibited a small infarct volume,however,the specific mechanism was unknown.Phosphoinositide 3-kinase(PI3K)/Protein kinase B(AKT)signaling pathway is the main downstream molecular mechanism how IGF-1 exerts biological functions.The activation of PI3K/AKT signaling pathway has been proved to alleviate ischemic injury by multiple mechanisms such as inhibition on inflammatory response.Moreover,it is reported that PI3K/AKT signaling pathway can be activated by mechanical stress,leading to enhancement of YAP/TAZ production,as a result of promotion in cell proliferation and tissue growth in Drosophila.Our previous study has confirmed that the enhanced expression of YAP/TAZ induces improved neurological function and promoted neurological recovery of rats underwent ischemic stroke.Therefore,it is reasonable to detect whether IGF-1 regulates the Hippo/YAP signaling pathway by activating PI3K/AKT cascade to exert its neuroprotective effects following ischemic stroke.Then,with the support of lots of in-depth research and rapid development of life sciences,the stem cell-based therapy has the huge potential to be an effective treatment for ischemic stroke.Dental pulp stem cells(DPSCs),a subtype of mesenchymal stem cell(MSCs)that originate from embryonic neural crest,DPSCs exhibit a superior potential for neurogenic differentiation,which renders DPSCs as a promising candidate for stem cell transplantation therapy following ischemic stroke.In addition,IGF-1 plays an important role in promoting the proliferation and differentiation of DPSCs,besides,the effects of both IGF-1 and DPSCs involve the promotion in proliferation and differentiation of nerve cells.There is a question that whether DPSCs transplanted into the brain exert neuroprotection by interacting with IGF-1 and enhancing the biological function of IGF-1?Part Ⅰ Expressive changes and neuroprotective effects of IGF-1 in cerebral ischemic strokeObjectivesTo clarify whether IGF-1 is involved in the pathological process of ischemic stroke and the protective effects of IGF-1 on ischemic-damaged cells and brain tissue.MethodsIn the in vitro study,we exposed cultured PC12 cells,SH-5YSY cells,and cortical primary neurons,to oxygen-glucose deprivation(OGD).In the in vivo study,SpragueDawley rats were subjected to middle cerebral artery occlusion(MCAO).Proper IGF-1 treatment was performed to ischemic-damaged cells and rats suffered MCAO.Immunofluorescence staining and western blotting were used to detect the expressive changes of IGF-1 in brain tissue with ischemic injury.In vitro,CCK8 kit and western blotting were used to detect the cellular survival and apoptosis;In vivo,the neurological function,infarct volume,brain edema and neuronal apoptosis of rats were detected by neurological functional score,TTC staining,brain water content and western blotting.Results The expression level of IGF-1 was upregulated in brain tissue of rats due to ischemic injury.IGF-1 therapy increased the expression of IGF-1 in injured brain tissue;IGF-1 increased the cell viability of cells with ischemic injury and inhibited the apoptosis;IGF-1 improved the neurological function,reduced the infarct volume,reduced brain edema and inhibited neuronal apoptosis of rats with cerebral ischemia.Conclusions IGF-1 expression was increased in the ischemia injured brain tissue and IGF-1 was involved in the physiological process of ischemic stroke.Part Ⅱ IGF-1 exerts neuroprotective effects against ischemic stroke through PI3K/AKT-Hippo/YAP molecular cascadeObjectives To detect whether IGF-1 alleviates ischemic injury through PI3K/AKTHippo/YAP molecular cascade.Methods In the in vitro study,we exposed cultured PC12 cells,SH-5YSY cells,and cortical primary neurons,to OGD.In the in vivo study,Sprague-Dawley rats were subjected to MCAO.Cells and rats underwent ischemic injury were treated with IGF-1 and/or LY294002(inhibitor of PI3K/AKT signaling pathway).In vitro,CCK8 kit and western blotting were used to detect the survival and apoptosis of cells and the expression levels of PI3 K and YAP;In vivo,the neurological function,infarct volume,brain edema,neuronal apoptosis and the expression levels of PI3K/AKT and YAP/TAZ were detected by neurological function score,TTC staining,brain water content,immunofluorescence staining,immunohistochemical staining,TUNEL staining and western blotting.Results LY294002 treatment reduced the cell viability and promoted apoptosis of cells underwent ischemic injury.LY294002 not only downregulated the expression of PI3 K,but also reduced YAP production.LY294002 weakened the effects of IGF-1 on apoptosis and expression of PI3 K and YAP in cells with ischemic injury;LY294002 aggravated the neurological dysfunction,increased the infarct volume,aggravated cerebral edema,downregulated the expression of PI3K/AKT and YAP/TAZ and promoted neuronal apoptosis of rats with ischemic damage.LY294002 weakened the protective effects of IGF-1 on rats suffered ischemic stroke.Conclusions PI3K/AKT-Hippo/YAP molecular cascade existed and participated in the pathological process of ischemic stroke.PI3K/AKT signaling pathway could exert neuroprotective effects by regulating Hippo/YAP signaling pathway;IGF-1 could regulate PI3K/AKT-Hippo/YAP molecular cascade and the enhancement of IGF-1-PI3K/AKT-Hippo/YAP molecular cascade effectively alleviated ischemic injury of rats.Part Ⅲ Dental pulp stem cell transplantation facilitates neuroprotection following cerebral ischemic stroke via enhancing IGF-1-PI3K/AKT-Hippo/YAP molecular cascadeObjectives To identity whether DPSC transplantation exerts neuroprotective effects against ischemic stroke through IGF-1-PI3K/AKT-Hippo/YAP molecular cascade.Methods Intracerebral transplantation of DPSCs was performed in rats subjected to MCAO.The neurological function,infarct volume,brain edema,TUNEL staining,Neu N immunofluorescence staining,Neu N immunohistochemical staining and western blotting were used to detect the neurological function,infarct volume,brain edema,apoptosis of nerve cells(especially neurons),and the expression levels of IGF-1,pAKT and YAP.Results Intracerebral transplantation of DPSCs reduced the neurological dysfunction and brain edema,decreased the infarct volume,inhibited the apoptosis and loss of nerve cells,especially neurons,and up-regulated the expression of IGF-1,p-AKT and YAP.Conclusions Intracerebral injection of DPSCs exerted protective effects against ischemic stroke through enhancing IGF-1-PI3K/AKT-Hippo/YAP molecular cascade.