| BackgroundAbout 87%of patients with stroke are ischemic stroke.Currently,thrombolysis and thrombectomy are effective methods for the treatment of acute ischemic stroke.However,due to strict time window(within 4.5 h)restrictions and adverse effects such as increased bleeding risk,this method can only be applied to a small number of patients.Neuroprotective agents are still an effective method for the treatment of acute ischemic stroke,that is,inhibit the death of cells such as neurons in the ischemic penumbra.So far,this strategy of simply taking neuroprotective agents has not been effective in actual clinical treatment,we must try our best to promote the repair ability of the central nervous system.Notoginsenoside R1(NGR1)is a unique saponins isolated from panax notoginosides.The previous research of the research group found that the preventive administration of NGR1 has obvious neuroprotective effect on the acute stage of ischemic stroke injury,but whether the therapeutic administration of NGR1 can repair neurological function by promoting angiogenesis and neurogenesis has not yet been studied.ObjectivesTo explores the targets of NGR1 promoting neurorestoration in ischemic stroke from the two-key links of angiogenesis and neurogenesis,and systematically clarifies the role of NGR1 in promoting neurorestoration in ischemic stroke by digging deeply into the pathways and key targets of NGR1.The mechanism of action provides experimental basis for further research on the treatment of ischemic stroke with administration of Panax notoginseng,and lays a theoretical foundation for the clinical application of Panax notoginseng.MethodsEstablish middle cerebral artery occlusion/reperfusion(MCAO/R)-induced rat cerebral ischemia model and oxygen glucose deprivation/reperfusion(OGD/R)-induced cells model.NGR1 was injected intraperitoneally immediately after MCAO surgery for 28 consecutive days.?Using TTC staining method to detect the effect of different doses of NGR1 on cerebral infarction volume in rats.?Using immunological methods(Nissl staining methods)to evaluate the effects of different doses of NGR1 on the morphology and activity of rat cortex and hippocampal neurons.?Using Doppler imaging,immunofluorescence double labeling,two-photon imaging,transmission electron microscope,scratch experiment,EdU proliferation and tube experiments investigate the promoting effect of NGR1 on angiogenesis.?Using ELISA,matrix-assisted laser desorption/ionization mass spectrometry imaging(MALDI-MSI),scratch experiment,EdU proliferation experiment,western blot method to analyze the mechanism of NGR1 promoting angiogenesis,and use the nicotinamide phosphoribosyltransferase(NAMPT)inhibitor FK866 and SIRT1 inhibitor Ex-527 for further verify the molecular mechanism of NGR1 on angiogenesis after ischemic stroke.?Using behavioral experiment,immunofluorescence double labeling,western blot,ELISA,MALDI-MSI,scratch experiment and EdU proliferation experiment.?Using immunofluorescence double labeling,scratch experiment,EdU proliferation experiment,western blot method to analyze the mechanism of NGR1 promoting neurogenesis,and use TrkB inhibitor ANA-12 and PI3K inhibitor LY294002 to further verify the molecular mechanism of NGR1 on neurogenesis after ischemic stroke.Results1.The results showed that the therapeutic administration of NGR1 for 7 days can significantly improve the volume of local cerebral infarction in rats,and the high dose(40 mg·kg-1)and middle dose(20 mg·kg-1)are more obvious.Nissl staining results showed that compared with the model group,the loss of Nissl corpuscles in the cortex and hippocampus CA1 area of rats was significantly improved in the NGR1 high dose(40 mg·kg-1)and middle dose(20 mg·kg-1)group,the activity of neurons was significantly improved,and no obvious vacuole structure was found.In addition,the middle dose of NGR1(20 mg·kg-1)improved the loss of Nissl corpuscles in the CA1 area of the hippocampus and increased neuronal activity more significantly.2.The results showed that NGR1(20 mg·kg-1)treatment for 28 days can significantly restore cerebral blood flow in cerebral ischemic rats,promote the proliferation of new neurons CD31+/EdU+,increase blood vessel density and the number of vascular bifurcations,increase total blood vessel length,and improve cerebral microvascular endothelial cells structure.NGR1(20 mg·kg-1)treatment for 28 days can significantly increase the expression of angiogenic factors VEGF,TGF-?,b FGF2 and PGNF in cortical tissues and serum.NGR1(20 mg·kg-1)treatment for 7 days can significantly improve the expression levels of energy metabolism factors NAD,ATP,ATPase,NADPH,ADP,AMP and GMP.In vitro,12.5-50 ?M NGR1 incubation treatment can significantly improve the migration,proliferation and tube formation of HBMEC brain microvascular endothelial cells.The effect of NGR1 on the migration and proliferation of HBMEC brain microvascular endothelial cells was inhibited by the NAMPT inhibitor FK866 and SIRT1 inhibitor Ex-527.Through in vivo and in vitro experiments,it was further found that NGR1 treatment can significantly inhibit the degradation of NAMPT caused by temperature and protease,increase the expression level of NAMPT,and up-regulate the expression of SIRT1.The up-regulation of SIRT1 can deacetylate the internal domain of Notch.This inhibits DLL4-Notch signal transduction,leading to the up-regulation of VEGFR-2 in endothelial progenitor cells,thereby enhancing EPC angiogenesis.The angiogenesis function of NGR1 is blocked by FK866 and Ex-527.On the other hand,NGR1(20 mg·kg-1)treatment can significantly increase the expression level of NAMPT,up-regulate NAD+,increase the expression of SIRT1/2/3,increase mitochondrial protection,and improve energy metabolism,while the effect of NGR1 on improving energy metabolism is blocked by FK866.3.The results showed that NGR1(20 mg·kg-1)treatment for 28 days can significantly improve the behavioral indicators of cerebral ischemia rats,promote the proliferation of neonatal neurons DCX+/EdU+,Nestin+/EdU+,NeuN+/EdU+.NGR1(20 mg·kg-1)treatment for 28 days promote the proliferation of oligodendrocyte APC+/EdU+.The increases the expression of neurotrophic factors BDNF,NGF and NT-4 in cortical tissue and serum,increases the expression levels of synaptic proteins SYN,PSD95,MAP-2 and Tau-1 after ischemia/reperfusion injury,and promotes release of the neurotransmitters,such as glutamate,N-acetylaspartate and K+.In vitro,12.5?100 ?M NGR1 incubation treatment can significantly increase the migration and proliferation of PC12 neuronal cells.The effect of NGR1 on PC 12 neuronal cell migration and proliferation was inhibited by TrkB inhibitor ANA-12 and PI3K inhibitor LY294002.Through in vivo and in vitro experiments,it was further found that NGR1 treatment can significantly increase the expression level of BDNF,specifically activate the expression of its receptor TrkB,and up-regulate the expression of CREB and Akt,the downstream effectors of BDNF signaling.The activation of BDNF/Akt/CREB signal pathway by NGR1 was inhibited by ANA-12 and LY294002.ConclusionsNGR1(20 mg·kg-1)treatment for 28 days can significantly promote neurorestoration and angiogenesis in rats with ischemic stroke.Its mechanism of action is related to the regulation of NAMPT/Notch/VEGFR-2 signaling pathway.In addition,therapeutic administration of NGR1 can significantly improve neurological damage caused by ischemia,and promote neurogenesis.Its mechanism of action is related to the activation of the BDNF/Akt/CREB signaling pathway. |
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