| Bilateral common carotid artery occlusion(2VO)-induced chronic cerebral hypoperfusion(CCH)in rats is the most widely used aminal model of vascular-related dementia at present,and neuroprotection in CCH model is becoming an important method to improve cognitive ability.Previous studies by our team and others have found that the dysfunction of neuroregulin 1(NRG1)and its main tyrosine kinase receptor ErbB4 signaling system could mediate direct neuronal damage(e.g.neuronal apoptosis);furthermore,high mobility group box-1(HMGB1)as an important pro-inflammatory molecular after activation has been reported to mediate indirect(delayed/secondary)neuronal damage induced by neuroinflammatory responses.The effect of the expression patterns and the regulation of these signaling systems on CCH model has not been systematically investigated.Besides,previous studies at home and abroad,including our own,have confirmed that mesenchymal stem cells(MSCs)and mesenchymal stem cell-derived exosomes(MSC-exo)have obvious immune-regulation and neuroprotective effects.Compared with cell therapy alone,MSC-Exo is easier in terms of penetrating BBB,avoiding the risk of tumorigenesis,facilitating freeze-thaw preservation(-20 C),all of which suggesting that MSC-Exo may be a better neuroprotective agent in clinical use.Based on these studies,we further explored the signaling mechanisms related to neuroprotection(NRG1/ErbB4 and HMGB1 signaling systems),and we discussed the general therapeutic effect of MSC-exo on CCH model and its regulatory role of the above signaling systems preliminarily.It can be divided into three parts as the following: Part 1: Direct neuronal damage mediated by NRG1/ErbB4 signaling system dysfunction and its regulation.Experiment 1: Correlation between hippocampal NRG1/ErbB4 expression and neuronal apoptosis in a rat model of chronic cerebral hypoperfusion Objective: To investigate the correlational relationship between the hippocampal NRG1/ErbB4 expression and neuronal apoptosis in rats with CCH.Methods: SD rats were treated with permanent 2VO to establish the animal model of CCH.Morris water maze(MWM)and NeuN staining were used to analyze cognitive impairments and neuronal loss,and ELISA,Western blotting and immunostaining were performed to see the expression of hippocampal NRG1/ErbB4 and the co-expression pattern of ErbB4 in 2VO rats.The immunostained cells of Terminal-deoxynucleoitidyl Transferase Mediated Nick End Labeling(TUNEL)and the protein expression of cleaved caspase 3/ caspase 3 tested by Westernblot in the hippocampal CA1 subarea were observed to see the neuronal apoptosis.Results: We found that 2VO could induce dramatic cognitive deficits as well as increased levels of neuronal apoptosis.Hippocampal CA1 NRG1(ELISA)and ErbB4(Immunostaining)were significantly reduced in the 2VO group compared to the sham group.Pearson’s correlation revealed that the expression of NRG1/ErbB4 directly correlated with neuronal apoptosis(NRG1: r =-0.869,P = 0.025 < 0.05;ErbB4: r =-0.859,P = 0.029 < 0.05).Conclusions: These findings indicate that hippocampal NRG1/ErbB4 signaling may be involved in the pathogenesis of CCH,especially neuronal apoptosis during CCH.Experiment 2: ErbB4 mediated NRG1’ role in neuroprotection and cognitive improvement during CCH.Objective: To further investigate the protective role of NRG1 in CCH and exploring the involvement of ErbB4 receptors in NRG1’s action using the AG1478,an ErbB4 inhibitor.Method: Permanent 2VO or sham operation was performed in SD rats.NRG1(100 μM)and AG1478(50 mM)was administered intraventricularly.Eight weeks post-surgery,cognitive impairment was analyzed using Morris water maze(MWM)and radial arm water maze(RAWM)tests,followed by histological assessment of the survival and apoptosis of hippocampal CA1 neurons using NeuN and TUNEL immunostaining respectively.Expression of apoptosis-related proteins and ErbB4 activation(pErbB4/ErbB4)was evaluated by Western blotting.Results: NRG1 significantly improved the performances in MWM(spatial learning and memory)and RAWM(spatial working and reference memory),attenuated hippocampal CA1 neuronal loss and apoptosis,upregulated the expression of pErbB4/ErbB4 and the anti-apoptotic protein Bcl-2,and downregulated the expression of pro-apoptotic proteins of Cleaved(Cl)-caspase3 and Bax.In addition,the protective effects of NRG1 could be partly abolished by AG1478.Conclusion: The current study suggested that NRG1 ameliorates cognitive impairment and neuronal apoptosis partly via ErbB4 receptors in rats with CCH.Part 2: Neuroinflammatory responses-related indirect neuronal damage mediated by HMGB1 and its regulation.Objective: To investigate the protective role of HMGB1 inhibition using anti-HMGB1 neutralizing antibody(Ab)against CCH in rats Method: SD rats underwent 2VO or sham operation.PBS,anti-HMGB1 Ab(1 mg/kg),or control IgG Ab(1 mg/kg)was intravenously administered post-operation.HMGB1 translocation,blood-brain barrier(BBB)permeability and glial activation were evaluated at 3 d,as well as the levels of inflammatory cytokines and oxidative stress.NeuN immunostaining and Morris Water Maze(MWM)were performed at 3 d,4 w and 12 w Results: Anti-HMGB1 neutralizing Ab inhibited HMGB1 translocation in hippocampal CA1 subarea and improved hippocampal HMGB1 level.Besides,anti-HMGB1 Ab preserved BBB integrity and reduced glial activation,in association with the related changes in oxidative stress and inflammatory cytokines at 3 d.Additionally,anti-HMGB1 neutralizing Ab improved hippocampal CA1 neuronal survival and behavioral outcomes in the chronic phase(4 w and 12 w).Conclusion: These findings suggest that HMGB1 neutralization suppresses hippocampal inflammatory responses and oxidative stress in the acute phase,and these changes exert long-lasting beneficial effects in the chronic phase of CCH.Part 3: The effect and related molecular mechanism of mesenchymal stem cell-derived exosomes on cognitive improvement and hippocampal CA1 neuronal protection in CCH model.Objective: To explore the neuroprotective effect and related molecular mechanism of MSC-exo on hippocampal CA1 region in CCH rat model.Methods: SD rats were randomly divided into three groups: sham group(PBS injection only),2VO group(bilateral common carotid artery ligation(2VO))and 2VO + MSC-exo group(MSC-exo lateral ventricular stereotactic injection 2 h after 2VO operation).The exomes separated from MSCs by differential centrifugation were identified in vitro(using CD81/CD63,transmission electron microscopy,nanoparticle tracking analysis,etc.).Morris water maze(MWM)analysis was performed 28 days after 2VO.At the meantime,immunohistochemical staining and Western blotting were used to analyze the effects of MSC-exo on neuronal protection,as well as the regulation of the ErbB4(ErbB4&NeuN expression level and pErbB4/ErbB4 expression level)and HMGB1 signaling system(HMGB1 translocation,TLR2,TLR4).Results: MSC-exo expressed CD81 and CD63 surface markers and showed typical rim of cup shape under electron microscopy,and nanoparticle tracking analysis showed that the diameter of the isolated materials were among 40-150 nm.After stereotactic injection in vivo,MSC-exo could significantly improve the spatial learning and memory performance of MWM and play a neuroprotective(improved NeuN-positive cells and decreased apoptotic index in TUNEL staining)and anti-neuroinflammatory role(regulation of the expression level of Iba1 and GFAP).Finally,MSC-exo could significantly up-regulate the number of ErbB4-NeuN positive cells and the expression of pErbB4/ErbB4,and down-regulate HMGB1 translocation and the expression of TLR2/4 down-regulated).Conclusion: The stereotactic injection of MSCs-exo can obviously ease the cognitive impairment and play a neuroprotective role in the rat model of CCH.These outcomes were probably achieved by regulating the expression of ErbB4 signaling and inhibiting the activation of HMGB1 signaling system. |
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