BHD Promotes Proliferation And Differentiation Of Neuroal Stem Cells Via Upregulating Autophagy Following Oxygen-glucose Deprivation/Reoxygenation Injury

ObjectiveThe decrease in cerebral blood flow after a stroke and the consequent hypoxia trigger a complex cascade of reactions,leading to cell death and neurological deficits.Current treatment strategies are mainly neuroprotection and thrombolytic therapy,whose purpose is to reduce neuronal death and protect damaged cells,but neither therapy can regenerate new neurons and repair damaged brains.The activation of neurogenesis after brain injury is of great significance to regenerative medicine after cerebral ischemia.More and more researches are focusing on drugs that can stimulate adult neurogenesis in different stages.Buyang huanwu decoction(BHD)is a classic prescription for the treatment of "qi deficiency and blood stasis " type of stroke.Previous studies have found that Buyang Huanwu Decoction can protect and improve many aspects such as cerebral ischemic injury neurons,blood vessels,glial cells,and brain microenvironment by regulating calcium signals,excitotoxicity,inflammation and other mechanisms.effect.In addition to the above mechanisms,Buyang Huanwu Decoction can also stimulate the proliferation of neural progenitor cells in the hippocampus and subventricular zone after ischemia,and promote axon regeneration after ischemia by regulating axon growth-related proteins and improving synaptic plasticity.Studies have found that autophagy is also closely related to the survival of nerve cells and the differentiation of neural stem cells(NSCs).There is evidence that Buyang Huanwu Decoction can regulate autophagy and protect neuron survival after oxygen glucose deprivation/reoxygenation(OGD/R).The clinical efficacy of Buyang Huanwu Decoction has been widely recognized,but its role in repairing nerve damage and how to stimulate nerve regeneration is still unclear.Therefore,we hypothesized that an effective dose of Buyang Huanwu Decoction-containing serum can restore neural stem cell damage after glucose and oxygen deprivation and promote neurogenesis.Autophagy may be involved in the process of Buyang Huanwu Decoction regulating NSCs.The study uses an in vitro glucose and oxygen deprivation/reoxygenation model to simulate the hypoxic environment,and explores the possible mechanism from two parts:cell experiment and molecular experiment.Methods1.The neuroprotective effect of Buyang Huanwu Decoction-containing serum against hypoxia modelThe primary neural stem cells were isolated and cultured from the hippocampus of SD rats,and the cells wore randomly divided into 5 groups:normoxia group,model group,Buyang Huanwu Decoction low-dose group(5%medicated serum),Buyang Huanwu Decoction Dose group(10%medicated serum),Buyang Huanwu Decoction high-dose group(20%medicated serum);observe.the cell morphology by optical microscope,use immunofluorescence to identify NSCs and differentiate into neurons and astrocytes Potential;establish a glucose and oxygen deprivation model;CCK-8 and LDH to detect the best dose-effect relationship and protective effect of Buyang Huanwu Decoction.2.The role and mechanism of autophagy in Buyang Huanwu Decoction in promoting the neurogenesis of neural stem cellsIsolation and culture of primary neural stem cells from the hippocampus of SD rats were randomly divided into 5 groups:normoxia group,model group,Buyang Huanwu decoction group,rapamycin group(Rapa),autophagy inhibitor 3-Methyladenine(3-MA)combined with Buyang Huanwu Decoction group.Detect the expression of autophagy-related proteins P62,Beclin-1,LC3B by Western-blot technology;mark the autophagy protein LC3B on the confocal microscope with the ad-mCherry-GFP-LC3B adenovirus tri-label,and detect autophagy flow;5-Acetylene-2-deoxyuridine(EdU)labeling method to detect the proliferation of NSCs;immunofluorescence method to detect brain-derived neurotrophic factor(BDNF),β-tubulin-Ⅲ(βtubulin Ⅲ)and glial fibrillary acidic protein(GFAP).Result:1.The neuroprotective effects of Buyang Huanwu Decoction against the damage of neural stem cells from glucose and oxygen deprivation:After the first generation of God inoculated the stem cells for 12 hours,a large number of cell aggregates and evenly scattered cells were observed under a light microscope.After culturing for 4 d,the suspended cells grew in a spherical shape,with different sizes,good refractive index,and smooth edges.Neural stem cells of the third generation were labeled with Neural Stem Cell Nestin Antibody,which showed a positive reaction.The third-generation neural stems induced by differentiation medium for 7 days were immunofluorescently stained with neuron marker β tubulin Ⅲ and glial cell marker GFAP,showing a positive reaction.Compared with the normoxic group,the survival rate of neural stem cells was not significantly reduced after 2 hours of modeling(P<0.05);the survival rate of cells was significantly reduced at 4 hours of modeling(P<0.05);the survival rate of cells was significantly reduced at 6 hours of modeling,The distribution of large broken cells(P<0.01).The dose-effect relationship of Buyang Huanwu Decoction showed that:compared with the normoxia group,the neural stem cell viability of the model group was significantly reduced(P<0.05);compared with the model group,the 5%volume fraction of medicated serum did not significantly improve Protective effect;10%medicated serum compared with the model group cell viability increased slightly(P>0.05);20%medicated serum significantly improved cell survival rate(P<0.01).The results of lactate dehydrogenase leakage rate showed that:compared with the normoxia group,the leakage rate of the model group increased significantly(P<0.01);compared with the model group,the leakage rate of 5%drug-containing serum was not significantly reduced(P>0.05);The leakage rate of 10%medicated serum decreased significantly and played a certain protective effect(P<0.05);the leakage rate of 20%medicated serum decreased significantly(P<0.05).2.Buyang Huanwu Decoction in the treatment of ischemic stroke promotes nerve regeneration and the mechanism of autophagy in it:WB results showed that LC3II,Beclinl and P62 had no statistical significance after glucose and oxygen deprivation compared with the normoxic group;compared with the model group,LC3II and Beclinl were significantly up-regulated in the BHD group(P<0.05,P<0.01),and the expression of P62 was down-regulated(P<0.01).Autophagy activity is up-regulated.With the addition of autophagy agonists and inhibitors,compared with the model group,LC3II and Beclinl in the Rapa group were significantly up-regulated,which was consistent with the trend in the administration group(P<0.01,P<0.01),and P62 expression was inhibited(P<0.01);3-In the MA+BHD group,LC3II and Beclinl were down-regulated(P<0.01,P<0.01),and the expression of P62 was up-regulated(P<0.01).Ad-mCherry-GFP-LC3B further verified the zishi flux.The green fluorescence of the BHD group was quenched in an acidic environment and turned red.The Rapa group showed a similar trend,and 3-MA partially blocked the autophagy activity.Compared with normal oxygen group,β-catenin protein expression was not significantly different after glucose oxygen deprivation.Compared with model group,β-catenin protein was up-regulated in BHD group,and up-regulated in RAPA group(P<0.01,P<0.01),but there was no difference in 3-MA group.Compared with normal oxygen group,p-PI3K was down-regulated and p-AKT was not significantly changed after glucose oxygen deprivation(P<0.05,P>0.05).Compared with model group,p-PI3K and p-AKT were down-regulated in BHD group,indicating that the pathway was inhibited(P<0.05,P<0.01).The autophagy flux was further verified by AD-mCherry-GFP-LC3B,and the green fluorescence in the BHD group was red when quenched in acidic environment,while that in the RAPA group showed a similar trend,and the autophagy activity was partially blocked by 3-MA.The results of Edu proliferation showed that:compared with the normoxia group,the number of Edu staining of cells in the model group was significantly reduced;compared with the model group,the number of Edu-positive cells in the BHD and Rapa groups was significantly increased;there was no significant change in the 3-Ma+BHD group.Immunofluorescence results showed that the number of β tubulin Ⅲ,GFAP and BDNF positive cells in the model group decreased compared with the normoxia group;compared with the model group,the number of positive cells in the BHD group increased,and the number of positive cells in the Rapa group increased,3-MA+BHD The number of positive cells in the group decreased.Conclusion1.Glucose oxygen deprivation/reoxygenation can significantly reduce the cell viability of rat NSCs.Compared with the model group,BHD significantly improved the survival of rat NSCs.2.BHD can induce the production of autophagosomes in NSCs after OGD,increase the formation and transport of LC3II,increase the expression of Beclin 1 but decrease the expression of p62,and increase the number of positive cells for Edu,β3tubulin,GFAP and BDNF significantly.The autophagy agonist Rapa played the same protective and promoting effect,and the autophagy inhibitor 3-MA can partially block the neuroprotective and differentiation ability of BHD.3.BHD may affect autophagy activity by down-regulating PI3K-Akt pathway,and the crosstalk between autophagy and β-catenin may be the reason why Bhd protects NSCs and promotes proliferation and differentiation.4.After glucose and oxygen deprivation,BHD may participate in autophagy in the baohu and neurogenesis of NSCs.In summary,BHD pre-protection can reduce the damage of rat NSCs caused by glucose and oxygen deprivation by up-regulating autophagy,and promote proliferation and differentiation.