The Mechanism And Intervention Of Vascular Cognitive Impairment Induced By Chronic Cerebral Hypoperfusion Via Changing Of Differentially Expressed Proteins In Cortex And Hippocampus Of SD Rats

Background and purpose Chronic cerebral hypoperfusion(CCH)describes a state of chronic,sustained,moderate decrease in cerebral blood flow(CBF),which can lead to the occurrence and development of vascular cognitive impairment(VCI),including vascular dementia(VD);however,there is currently no definitively curative treatment.Reduction of cortical CBF and hippocampal neuron death are important pathological features of the initial and key stages of CCH-induced VCI.Therefore,improving cerebral perfusion and mitigating hippocampal neuron death appear to be the most reasonable approaches to improve cognitive impairment in CCH;however,the mechanisms involved and optimal intervention methods remain unclear.High-efficiency protein antibody microarray technology can identify diseases or drug-related biomarkers effectively.Dl-3-n-butylphthalide(NBP)is a racemic,lipid-soluble small molecule that can penetrate the blood brain barrier(BBB)and has multi-target effects.It has been demonstrated to be safe and effective in the treatment of a variety of neurological diseases.However,the specific molecular mechanism of NBP intervention in CCH remains unclear.Therefore,the specific molecular mechanism of CCH-induced VCI and potential therapeutic targets for NBP warrant further exploration.Methods Part I Forty-two rats were randomly divided into groups as follows: sham(n=10);and CCH(CCH 2-week [n=11];CCH 4-week [n=10];and CCH 8-week [n=11]).A bilateral common carotid artery occlusion(BCCAO)method was used to establish CCH rat models(i.e.,CCH 2-,4-,and 8-week [3 time points]).In addition,arterial spin labeling(ASL)and magnetic resonance angiography(MRA)of magnetic resonance imaging(MRI),were applied to dynamically determine changes in CBF and the diameters of the vertebral artery(VA).Diffusion tensor imaging(DTI)combined with luxol fast blue(LFB)and myelin basic protein(MBP)immunohistochemistry were used to evaluate white matter damage.The Morris water maze behavioral test was used to evaluate changes in cognitive function of learning and memory in CCH rats at different time points,and to validate the establishment of the CCH-induced VCI rats model.Furthermore,changes in hippocampal neuron morphology,the deposition of amyloid ?(A?1-42)and the expression of cortical blood-brain-barrier(BBB)-related proteins were assessed in CCH rats at different time points,and neuropathological changes in the brain tissue of CCH rats were evaluated.Part II Ninety-four rats were randomly divided into groups as follows: sham 2-week(n=11),sham 4-week(n=8),and sham 8-week(n=11);CCH 2-week(n=12),CCH 4-week(n=12),and CCH 8-week(n=12);and CCH+NBP treatment(NBP 2-week [n=9],NBP 4-week [n=9],and NBP 8-week [n=10]).First,antibody microarray technology was used to analyze differentially expressed cortical proteins in the CCH group at each time point(i.e.,CCH 2,4,and 8 weeks)compared with the sham operation group,and to analyze differentially expressed proteins in the time series.The Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)biological functions of these cortical differentially expressed proteins were analyzed.A protein-protein interaction network was constructed via STRING analysis on selected differentially expressed proteins in the cortex of CCH rats,and protein expression levels were verified by Western blot and immunostaining.After NBP intervention in the CCH rats,changes in the expression of selected cortex proteins were evaluated at the corresponding time points(i.e.,NBP 2,4,and 8 weeks).The expression of VEGF and CD34 positive cells were analyzed by immunofluorescence staining,and the correlation between Tie-2 and VEGF/CD34 positive cells was analyzed by Spearman correlation analysis,angiogenesis was marked by CD34+Ki67 immunofluorescence staining.Changes in CBF were determined using ASL of MRI,cerebral white matter fiber was assessed using DTI,and the diameters of the bilateral VAs were evaluated using MRA,to explore potential targets of NBP treatment in improving cortical CBF in CCH rats.Part III Forty-nine rats were randomly divided into groups as follows: sham 8-week(n=13);CCH 8-week(n=18);and CCH 8-week+NBP-treatment(NBP 8-week [n=18]).An antibody microarray technique was used to analyze the differentially expressed hippocampal proteins in CCH 8-week rats(i.e.,CCH-induced VCI)and compared with the sham group.Biological functions of GO and KEGG were analyzed for these differentially expressed proteins.Antibody microarray technology was again applied to analyze the differentially expressed hippocampal proteins in the sham operation,CCH 8-week,and CCH 8-week+NBP intervention(NBP)groups.Biological conservation was analyzed in the differentially expressed proteins,and the most interesting and conserved differentially expressed hippocampal protein was chosen.The protein-protein interaction network was analyzed using STRING,a Western blot method was used to verify protein expression levels,and immunofluorescence staining was used to label selected proteins in three types of nerve cells(Neu N for neurons,GFAP for astrocytes,and Iba1 for microglia).The Morris water maze behavior test was used to evaluate cognitive and functional changes in the three groups of rats.Hematoxylin and eosin,Nissl and terminal deoxynucleotidyl transferase d UTP nick-end labeling(TUNEL)staining,and transmission electron microscopy were used to analyze changes in hippocampal neurons.Finally,Western blotting was used to analyze the expressions of apoptosis-related proteins(Bcl-2,Bax,cleaved caspase-3),p-AKT and p-ERK1/2 signaling.A primary hippocampal neuron culture system was also established.OGD/R was used to mimic the CCH model in vitro,and the optimal concentration of NBP pretreatment and Ret inhibitor for hippocampal neuron intervention were selected.In six different conditions(Normal;Normal + OGD/R;Normal + OGD/R+NBP;Normal + Ret inhibitor;Normal + OGD/R + Ret inhibitor;and Normal + OGD/R + Ret inhibitor + NBP).Western blot and immunofluorescence staining were used to evaluate the expression of the proteins of interest and apoptosis proteins,and neuronal viability was assessed by CCK 8 method.To explore potential targets of NBP pretreatment to reduce hippocampal neuron apoptosis induced by CCH in rats.Results ?.According to the results of ASL,compared with the CBF before the BCCAO procedure,CBF in the cerebral cortex was still in a low perfusion state after the BCCAO procedure,and at 1,2,and 4 weeks after BCCAO,and still maintaining hypoperfusion at 8 weeks after the BCCAO operation.DTI combined with MBP and LFB staining revealed significant white matter damage at 4 and 8 weeks after BCCAO.The Morris water maze behavioral test revealed that at 2,4 and 8 weeks after BCCAO surgery,compared with the sham operation group,longer escape latency and fewer times of crossing the platform,indicating cognitive impairment in these rats.The above results revealed that the BCCAO procedure could establish a CCH-related VCI rat models(2 weeks after BCCAO [CCH 2 weeks];4 weeks after BCCAO [CCH 4 weeks];8 weeks after BCCAO [CCH 8 weeks]).In addition,A?1-42 intracellular deposition was observed and cortical BBB was impaired at 4 and 8 weeks after BCCAO.At 4 and 8 weeks after BCCAO,significantly delayed neuron loss and death(apoptosis)occurred in the hippocampus(CA1 and CA3 regions)of these rats.?.Compared with the sham group,there were 7 differentially expressed proteins(Tie-2,CNTFR IL-4,IL-10,integrin alpha-M beta 2,MDC,and TROY)in the cerebral cortex at 3 different time points in the CCH groups(i.e.,CCH 2-,4-,and 8-weeks).Time series analysis revealed that there were 10 differentially expressed proteins(TIMP-3,TAL1 A,IL-10,FADD,IL-6,LIX,integrin alpha-M beta 2,CCR4,Tie-2,and b FGF)in the cerebral cortex of the CCH groups compared with the sham group.Tie-2 protein was the only protein that was down-regulated at each time point in the CCH groups.Tie-2 is anticipated to be the target protein of interest in future studies.STRING analysis revealed that Ang-1 and Ang-2 proteins were highly correlated with Tie-2 protein,forming an Ang-1/Ang-2/Tie-2 signaling axis.Western blot and immunohistochemical staining revealed that Tie-2 protein expression was consistent with antibody microarray results in the CCH groups,compared with the sham group,and was significantly down-regulated in the entire CCH process(3 time points).In the earlier stages of CCH(CCH 2-and 4-weeks),the expression of Ang-1 was down-regulated and up-regulated in the later stage(i.e.,CCH 8-weeks),but the expression of Ang-2 was significantly up-regulated at all 3 CCH time points.After NBP treatment,the Ang-1/Ang-2/Tie-2 signal axis was altered: NBP treatment significantly upregulated Tie-2 expression;in the earlier stage of NBP treatment(NBP 2-and 4-weeks),Ang-1 expression was significantly up-regulated.In the later stage of NBP intervention(NBP 8-weeks),Ang-2 expression was significantly down-regulated.At the same time,the number of VEGF and CD34 positive cells/CD34+Ki67 microangiogenesis were significantly increased at the earlier stage of intervention(NBP 2-and 4-weeks)in the cortex of NBP-treated CCH rats.Compared with CCH 8-weeks,the diameter of the bilateral VAs in the NBP intervention group(NBP 8-weeks)was significantly increased.Compared with the CCH 8-week group,ASL revealed that cortical CBF in the NBP intervention group(NBP 8-week)had recovered to normal levels.DTI revealed increased white matter fiber density in the NBP intervention group.In Ang-1/Ang-2/Tie-2 signaling,Ang-1 and Tie-2 promoted angiogenesis,maturation,and stability.Ang-2 disrupts vascular stability,and increases vascular or BBB permeability.These results suggest that cerebral hypoperfusion in CCH rats is related to changes in the Ang-1/Ang-2/Tie-2 signal axis.NBP promoted angiogenesis and vascular remodeling in the cortex of CCH rats,and improved CBF by regulating the Ang-1/Ang-2/Tie-2 signaling axis.?.Compared with the sham group,six differentially expressed proteins(E-selectin,Fractalkine,GFR?1,GFR?2,IL-3,and Insulin)were found in the hippocampus of CCH 8-week rats(i.e.,CCH-induced VCI rats).After NBP treatment,antibody microarray re-analysis revealed that three proteins,TIMP-1,ACTH and GFR?1,were down-regulated in the hippocampus of CCH rats,while up-regulated in the hippocampus of the sham-and NBP-treated groups.GFR?1 protein has the best biological conservation,suggesting that it is the most similar to the human protein sequence.Therefore,GFR?1 is anticipated to be a target protein of interest in future studies.STRING analysis revealed that GDNF and Ret proteins were highly correlated with GFR?1 protein,forming the GDNF/GFR?1/Ret signaling axis.According to Western blot and immunostaining,GDNF/GFR?1/Ret signal expression was down-regulated in the hippocampus of the CCH 8-week group compared with the sham group.After NBP treatment,neuronal apoptosis in the CA1 and CA3 regions of the hippocampus were significantly reduced,the expression of GDNF/GFR?1/Ret signal was significantly up-regulated,and GDNF/GFR?1/Ret proteins were co-localized with hippocampal neurons,p-AKT and p-ERK1/2 were activated.Experiments were performed using primary hippocampal neurons to simulate the hypoxic environment of hippocampal neurons in vitro with OGD1h/R48h(OGD/R).OGD/R significantly down-regulated GDNF/GFR?1/Ret and p-AKT,p-ERK1/2 expression,and increased apoptosis of neurons(decreased neurons viability).Ret inhibitors could inhibit Ret expression,inhibit the expression of upstream GDNF/GFR?1 and downstream p-AKT and p-ERK1/2,and increase the apoptosis of neurons(reduced neuron viability).The decreased expressions of GDNF/GFR?1/Ret and p-AKT,p-ERK1/2,and the apoptosis of hippocampal neurons were more obvious under the combined inhibition of OGD/R and Ret.In addition,NBP pretreatment of hippocampal neurons resulted in up-regulated expression of GDNF/GFR?1/Ret signal and p-AKT and p-ERK1/2,and reduced apoptosis in hippocampal neurons(increased neuron viability)in the environment where OGD/R and/Ret were inhibited.This suggests that the down-regulation of GDNF/GFR?1/Ret signal caused by CCH in hippocampal neurons is the mechanism leading to apoptosis in hippocampal neurons.Moreover,intervention with NBP can activate GDNF/GFR?1/Ret signaling and downstream p-AKT and p-ERK1/2,to protect hippocampal neurons and improve cognitive function.Conclusions ?.The BCCAO method was used,lower CBF continuously in the cortex of rats and ultimately led to cognitive dysfunction,indicating that the VCI rat model induced by CCH was successfully established.In the established CCH rat model,exhibited white matter injury,intracellular deposition of A?1-42,BBB injury and delayed hippocampal neuron death(apoptosis)were found.II.Antibody microarray technology was used to identify differentially expressed proteins in the cerebral cortex and hippocampus of CCH rats.The biological functions of the differentially expressed proteins involved cytokines,growth factors,neuronal death and energy pathways.Tie-2 was an interesting target protein in the cortical tissue of CCH rats,while GFR?1 was an interesting target protein in the hippocampus of CCH rats;both will be investigated further in future studies.?.In the cortex of CCH rats,the state of low CBF was associated with decreased expression of Ang-1/Tie-2 and increased expression of Ang-2.By regulating changes in cortical Ang-1/Ang-2/Tie-2 signaling,NBP promoted angiogenesis and vascular remodeling,thus improving cortical CBF and reducing white matter damage in CCH rats.IV.In the hippocampal tissue of CCH rats,neuronal apoptosis was correlated with down-regulation of GDNF/GFR?1/Ret signaling.By up-regulating the expression of GDNF/GFR?1/Ret signaling in the hippocampus,NBP-treatment mitigated apoptosis of hippocampal neurons,promoted the viability of hippocampal neurons,and improved cognitive function in CCH-induced VCI rats.