| Background: Hypertension is the third most important risk factor for the global burden of disease in 2015.The morbidity rate of hypertension is about 20%,and there are about 1 billion hypertensive patients worldwide.In2010,9.4 million deaths were attributable to hypertension.In 2018,Circulation released the status of hypertension in China: according to the 2010 Chinese guidelines,the incidence rate of hypertension in people over 18 years old was 23.2%(about 245 million people),and that in prehypertension was 41.3%(about 435 million people).Long-term hypertension damages brain microvasculature,so cerebral hemorrhage is one of the most serious complications of hypertension.20%-30% of strokes are spontaneous cerebral hemorrhage,of which hypertensive cerebral hemorrhage is as high as 80%.Hypertension can also lead to cerebral arteriosclerosis,microvascular wall disease thickening,stenosis of the lumen,microvascular occlusion after thrombus formation,lacunar infarction,reduction of regional cerebral blood flow leading to brain atrophy and affect cognitive function.An increase of 10 mmHg in diastolic blood pressure can increase the degree of cognitive impairment,and the increase in diastolic blood pressure is also associated with anxiety and depressive symptoms.Preclinical studies have shown a linear relationship between blood pressure and alcohol intake,limiting alcohol consumption is an effective behavioral intervention for lowering blood pressure.Any alcohol consumption by men is associated with an increased risk of high blood pressure.Women who drink more than 2 drinks a day increase the risk of hypertension.In 2016,the female drinking rate in China rose to 16%,the male drinking rate rose to 48%,and the number of deaths caused by drinking was the highest in the world.The mechanism of alcoholic hypertensive brain damage remains unclear.The neurovascular unit(NVU)is composed of vascular cells(endothelial cells,pericytes,smooth muscle cells),glia(astrocytes,oligodendroglia,microglia),and neurons.NVU is critical to maintain the complex brain function of the central nervous system,the integrity of the blood-brain barrier(BBB)and the regulation of cerebral blood flow.At present,there have been no reports on alcoholic hypertensive brain damage from the perspective of NVU.This thesis not only uses behavioral tests,morphological analyses and biochemical techniques to study the characteristics of alcoholic hypertensive brain damage,but also used high-resolution mass spectrometry-based proteomics to detect differentially expressed proteins in NVU in alcoholic hypertensive(AH)rats.In data analysis,besides the conventional analysis strategy,we also used brain cell type proteomics database,vascular single cells database,angiogenesis factor and angiogenesis inhibitor PCR chip indexes,STRING database,VarElect database and AlzData database to obtain more comprehensive differentially expressed proteins information in AH rats.Matrix metalloproteinases(MMPs),members of the proteolytic enzyme family,play an important role in the process of vascular remodeling,cell migration,and processing of extracellular matrix(ECM)proteins and adhesion molecules.MMP2 is associated with hypertension and the level of MMP-2 was increased in the heart and blood in patients with hypertension.Increased MMP2 activity can hydrolyze a variety of ECM proteins leading to hypertensive endothelial dysfunction,and can also degrade endothelial nitric oxide synthase or cofactors to cause impaired endothelial function.Inhibition of MMP2 can reduce blood pressure and improve endothelial dysfunction in renal hypertension rats,insulin resistance rats and pregnancy-induced hypertension rats.The role of MMP2 in alcoholic hypertensive indued brain damage is unclear.Objective: 1.Use alcohol-preferring rat(P rat)establishs an AH rat model,and then uses behavioral tests,morphological analyses and biochemical techniques to study the characteristics of alcoholic hypertensive brain damage.2.To explore the role of MMP2 in hypertension and behavioral impairment in AH rats.Main research methods: 1.Model establishment and treatment 65 3-months old P rats were divided into 3 groups,Con group(n=18),5% EtOH group(n=20)and 30% EtOH group(n=27).The 30% EtOH group was intragastrically administered with 30% ethanol(EtOH,13 ml/kg/day),and the Con group and the 5% EtOH group were intragastrically administered with an equal volume of distilled water or 5% alcohol in the 30% EtOH group.To study the effect of inhibiting MMP2 on AH rats,3-months old P rats were divided into 2 groups,Con group(n=8)and 30% EtOH group(n=22).After 4 weeks of alcohol intragastric administration and behavioral testing,30% EtOH rats were randomly divided into 2 groups(30% EtOH+Veh and 30% EtOH+SB-3CT,n=8 and 14 respectively)treated with SB-3CT(MMP-2/9 inhibitor,intraperitoneally 25mg/kg/day)or vehicle for 1 week.2.Behavior detection blood pressure measurement 2.1 blood pressure measurement The systolic blood pressure(SBP),diastolic blood pressure(DBP)and mean blood pressure(MBP)were measured at 8:00-12:00 am by Tail-cuff method.2.2 Cognitive-related behavioral tests The spatial cognition of P rats was evaluated by Morris water maze test(MWMT),the escape latency reflects the spatial learning ability in the learning phase,and the number of crossings of the platform during the memory detection phase reflects the spatial memory ability.The cognitive memory of P mice was evaluated using recognition index(RI)in the novel object recognition test(NORT).2.3 Emotional related behavioral tests In open field test(OFT),we use the total distance and the central duration(s)to evaluate the spontaneous activity and anxiety of P rats.In elevated plus maze(EPM),we use the open-armed duration(%)and the open-armed entries to evaluate the anxiety of P rats.Depression was evaluated by OFT,Sucrose preference test(SPT),and Forced swimming test(FST).The decreased sucrose preference(%)reflected anhedonia,and the immobility time(s)in FST reflected hopelessness.3.Enrichment of NVU We enriched NVU with cold sucrose buffer based on a previously published methods.Morphological identification revealed that the isolated components were structurally intact and retained NVU interacting with adjacent cells.Meanwhile,marker proteins of NVU component cell types such as endothelial cells,pericytes,smooth muscle cells,astrocytes,oligodendrocytes,microglia and neurons were found in NVU proteomics,these results indicate that NVU is enriched by successful separation.4.Proteomics analysis Proteomics based on isobaric tags for relative and absolute quantification(iTRAQ)were used to detect differentially expressed proteins in NVU of Con and 30% EtOH group(n=3).The differentially expressed protein threshold was defined as: t-test statistics p<0.05,30% EtOH group was up-regulated compared to Con group fold changes greater than 1.2 fold,and less than 0.83 was down-regulated.Gene Ontology was used for general analysis from cell composition,molecular function,biological processes,and the KEGG pathway.For comprehensive and in-depth exploration of differentially expressed proteins information of NVU in AH rats,cell type analyses of differentially expressed proteins were based on brain cell type proteomics database and vascular single cells database,and differentially expressed proteins interactive network analysis,functional clustering and visualization were used Cytosacpe software.The functions of differentially expressed proteins were batch query from VarElect database.The differentially expressed proteins related to angiogenesis were analyzed by VarElect database and angiogenic factors and angiogenesis inhibitor PCR chips,and the differentially expressed proteins associated with Alzheimer's disease(AD)were analyzed using the AlzData database.5.Morphological tests Fluorescein isothiocyante(FITC)to assess BBB disruption,?-galactosidase staining,HE stain and Masson stain to detect pathological changes.In addition,immunofluorescence was used to detect the level of MMP2 in hippocampus,and immunohistochemistry was used to detect the cerebral microvessel density,number of neurons in the brain,the activation of microglia and astrocytes.6.Western blotting(WB)and the sandwich enzyme-linked immunosorbent assay(ELISA)Oxidative stress and proinflammatory cytokine levels were measured by ELISA,and hippocampal homogenate MMP2,MMP9,synapse-associated protein and tau were detected by WB.Results: 1.Chronic alcohol exposure Established an alcoholic hypertensive(AH)rat model During the 4 weeks of chronic ethanol exposure,the BEC of the 30% EtOH group was greater than 100mg/dl and increased in the first 3 weeks,reached 177.8±20mg/dl in the third week,while the BEC in the 5% EtOH group was less than 100mg/dl.When BEC is higher than 100mg/dl,it will have measurable effects on physiology and/or behavior,indicating that the exposure concentration of alcohol in the 30% EtOH group can affect the physiology or behaviors.The blood pressure was measured at the 7,14,21,and 28 days.Starting from the second week of chronic alcohol exposure,the systolic blood pressure(SBP,149.7±3.5mmHg),diastolic blood pressure(DBP,121.2±3.4mmHg)and mean arterial pressure(MBP,130.3±3.4mmHg)in the 30% EtOH was significantly increased compared with Con rats,and furtherly increased in the next two weeks.2.AH rats showed emotional and cognitive impairment Duration(%)in the open-arms of the 30% EtOH group in the EPM was 13±1.5%,which was significantly lower than the Con group(23±4.1%)and the 5% EtOH group(24±3.6%);And the number of entries in the open-arms of the 30% EtOH group(4.4±0.5times)was also significantly lower than Con rats(8.4±1.6 times).In SPT,the sucrose preference(%)of 30% EtOH rats in the SPT was declined by 74% compared with the Con rats,and declined by 66.7% compared to the 5% EtOH group.In OFT,the total distance of the 30% EtOH group(3160±114cm)decreased by 13.5% compared with the Con group(3654±170cm),and the central duration of the 30% EtOH group(25.57±4s)decreased by 37.6% compared with the Con group(41±4.7s).The first three days of learning in MWMT,the 30% EtOH rats had the longest latency to find the submerged platform.3.Brain damage and its proteome basis in AH rats 3.1 Classification and functional enrichment of differentially expressed proteins in the NVU of AH rats We isolated NVU from Con and 30% EtOH group(n=3)from brain tissue and identified.In the NVU proteomics results we found the specific marker proteins of endothelial cells,pericytes,smooth muscle cells,astrocytes,oligodendroglia,microglia,and neurons.In summary,the isolated cell population reflects the cellular composition of the NVU.Using proteomics analysis,5574 proteins were identified from the NVU,and 4755 proteins were quantified.When setting the quantification fold change?1.2 and p-value<0.05 for differentially regulated proteins,133 proteins up-regulated and 138 proteins down-regulated in 30% EtOH rats were obtained.To investigate the possible regulation network among the differentially regulated proteins,an interaction network analysis was performed by submitting the proteins to the STRING database(https://string-db.org/).The interaction network that comprised of 207 protein nodes was established for 30% EtOH group vs Con group.Further cluster analysis of differential expressed protein interaction networks revealed 13 different clusters,including gap junction,innate immune response,and extracellular matrix receptor interaction(ECM)-receptor interaction and oligodendrocyte differentiation et al.3.2 Cerebral microvessels injuries and proteome basis in AH rats Extravascular leakage of microvessels was assayed by FITC perfusion,which is a method of visualizing vasculature and extravascular leakage.The green fluorescence of FITC was observed outside of the microvessels in hippocampal fissure(HF),thalamus(TH),hypothalamus(HYP)and amygdala(AMY).AMY and HYP were the most obvious,and the TH part was extravascularly present,but no BBB injury was found in the cerebral cortex,hippocampal CA1,CA2,CA3 and DG.However,extravascular leakage of FITC was not observed in the brain parenchyma of Con rats.In 30% EtOH group of NVU,significantly decreased levels of gap junction proteins were observed,such as Gjb1,Tuba4 a,Tubb3,Tubb4 a,Tubb6,Grb2,Gjc3 and Tubb4b(18.5%,22.4%,22.4%,22%,28.8%,21.1%.20.9% and 17.4%,respectively).In addition,the CLDN11,ITGB3,MBP and FBN1 associated with the formation of BBB decreased by 18.6%,52.4%,22.5% and 17.2% in the 30% EtOH group,respectively,while CTNND2 and RAB13 were increased by 25.6% and 30.7%.By ?-galactosidase(SA-beta-Gal)staining detected senescent cells,we found 30% EtOH group showed positive staining in the cortex and hippocampus endothelial cells,while neurons and other cell types were negative staining.These results suggest that the primary senescence cell types in AH rats are cerebrovascular endothelial cells.Furthermore,the NVU senescence signal-related proteins in AH rats were studied.It was found that CDKN1 B,AGO1,MAP1LC3 A,DNAJB1,NUP54,PSMB10 and PSME1 were up-regulated by 114.7%,26.5%,32.1%,23.2%,20.8%,24.7%,and 20.3%,respectively.HIST2H2 AC,THBS1 and PRDX5 were down-regulated by 67.5%,61.5% and 21.5%,respectively.In NVU,STK39,ABAT,and DDAH2,which are negatively regulated in NVU,were down-regulated by 23.1%,24.7%,and 23.3%,respectively,which was consistent with the elevated blood pressure phenotype in AH rats.In addition,ENPEP with a blood pressure lowering effect was up-regulated by 25.3%.Compared with Con and 5% EtOH rats,the cerebral small vessel density(the percentage of area occupied by vessels in the analysis area),total blood vessel length,and branch index(the number of vessel junctions normalized per analysis area)in the 30% EtOH group were significantly increased in the prefrontal cortex(PFC)and the caudate putamen(CPu).However,there were no significant differences of vessel densities in the hippocampus,primary somatosensory cortex,corpus callosum and internal capsule among groups.The hippocampal MAG:PLP1 ratio of 5% EtOH and 30% EtOH group was significantly lower than that in Con group,suggesting pathological hypoperfusion in 5% EtOH and 30% EtOH.In NVU,a series of angiogenic factors and antiangiogenic factors were altered.9 differential expressed proteins promoted cerebral angiogenesis: the angiogenic factors STAB2,Grn,RAB13,and LGALS1 were significantly up-regulated by 34.9%,23.4%,30.7% and 22.1%,respectively;and antiangiogenic factors TBS1,PF4,ITGB3,SIRT2 were significantly down-regulated by 61.8%,67%,52.4%,21.4% and 20.9%,respectively.6 differential expressed proteins inhibited cerebral angiogenesis: the angiogenic factors TGFA and LOXL2 were down-regulated by 35% and 24.7%,and antiangiogenic factors BAIAP2,Prl,RNF213 and LGALS9 were up-regulated by 25.2%,26.2%,50.3% and 22.7%,respectively.The total fold changes of differentially expressed proteins related to angiogenesis and anti-angiogenesis were 15.38 fold and 8.11 fold,respectively.miR-126 is highly expressed in vascular endothelial cells.Compared with the Con rats,the levels of the two mature forms of miR-126,miR-126-5p and miR-126 b were significantly up-regulated.Targetscan predicted that 28 and 19 of the NVU differential proteins regulated by miR-126-5p and miR-126 b were down-regulated,respectively.3.3 The proteomic basis of cognitive impairment in AH rats The level of A?40 in the hippocampus of the 30% EtOH group was 17.3±1.3 pg/mg,which was significantly higher than that in the Con group(12.1±0.68 pg/mg tissue).In the 30% EtOH group,the levels of hippocampal tau phosphorylated at Thr205(pT205)and Thr231(pT231)were signifcantly increased.In addition,neuron numbers and synapse-related proteins were decreased in the hippocampus of the 30% EtOH rats.To investigate the underlying mechanisms of alcoholic hypertension induced cognitive impairment,we analyzed the correlation between NVU differentially expressed proteins and Alzheimer's disease(AD).The differential expressed proteins correlated with A? or tau were analyzed,199 proteins significant correlation with AD.64 differentially expressed proteins are correlated with ?-Amyloid(A?)pathological changes,40 proteins correlated with tau pathological changes and 30 overlap differential expressed proteins correlated with A? and tau.Among the 30 differentially expressed proteins correlated with A? and tau pathological changes,12 proteins positively correlated with A? and tau pathological changes were up-regulated,and 11 proteins negatively correlated with A? and tau pathological changes were down-regulated.In addition,there are 7 differentially expressed proteins that can ameliorate the pathological changes of A? and tau.77 differential expressed proteins in quantitative trait loci(eQTL)and early differentially expressed genes(DEGs),136 proteins showed the correlation with AD-risk in international genomics of Alzheimer's project(IGAP)data,and 42 overlap differential expressed proteins(17 up-regulated,25 down-regulated).These results suggest that the 42 differentially expressed proteins in the NVU are very important in cognitive impairment in AH rats.In addition,the axonal growth factors Kif5,Uchl1,PLP1 and Tubb3 in NVU were down-regulated by 18.1%,17.3%,17.7% and 22.4%,respectively.3.4 The proteomic basis of oxidative stress and inflammatory response signals in AH rats Morphological studies showed that hippocampal astrocytes increased in the hippocampal CA1 and CA3 regions of the 30% EtOH group compared with the Con group and the 5% EtOH group;the microglia cell area increased and the protrusion decreased,suggesting that these two types activation of glial cells.In NVU,we found a series of differentially expressed proteins were associated with oxidative stress and inflammatory response.For oxidative stress,CDC37,PRDX5,HIST2H2 AC,SIRT2 and APOD were down-regulated by 20.4%,21.%,67.5%,21.4% and 23.6%,respectively,and AGO1 was up-regulated by 26.5%.In this study,the inflammatory response-related Kng1,C4 B,RNF213,NR3C1,TAP1 and S100A9 in the NVU of AH rats were up-regulated by 24%,30.3%,50.3%,21.2%,33.6% and 37.7%,respectively.4.The important role of MMP2 in brain damage of AH rats.4.1 MMP2 activity is increased in AH rats Levels of hippocampal MMP2 and MMP9 were detected by western blotting,MMP9 showed no differences among groups.Compared with Con group,30% EtOH group had increases of 28.6% in MMP2 activity(active form/propeptide),and 26.7% increases of 26.7% compared to 5% EtOH rats.Hippocampi with MMP2-based immunofluorescence staining also found the level of MMP2 was increased in 30% EtOH group.Chronic ethanol exposure causes damage to various organs and systems,liver is a primary target for the detrimental effects of alcohol.HE stain and Masson showed significant inflammatory infiltration,lipid droplets and collagen deposition in the liver of AH rats.The expression abundance of Con MMP2 in different organs was lung>liver>heart>brain.Compared with the Con group,the level of MMP2 in 5% EtOH and 30% EtOH rats increases of 78.9% and 79.8% after chronic ethanol exposure,respectively.Compared with Con rats,the levels of MMP2 activity in the serum of 30% EtOH also up-regulated by 34.2%.4.2 Inhibition of MMP2 ameliorates hypertension and cognitive impairment in AH rats After 4 weeks of chronic ethanol exposure and 1 week of SB-3CT treatment,the blood pressure was measured at the 45 and 50 days.We found that 30% EtOH+SB-3CT group SBP,DBP and MBP were reduced to similar normal levels in the Con group,while 30% EtOH+Veh remained in hypertensive status.In NORT,recognition index(RI)of 30% EtOH+SB-3CT rats in the retention trial was significantly increased compared with the acquisition trial.While 30% EtOH+Veh rats could not distinguish between old and novel object.The movement tracks during the acquisition trial and retention trial in NORT also showed 30% EtOH+SB-3CT rats had more interest in the novel object as Con rats.These results suggest that SB-3CT can effectively reduce the blood pressure and improve cognitive impairment in AH rats.4.3 Inhibition of MMP2 maintains BBB integrity and reduces the inflammatory response in AH rats.SB-3CT treatment can effectively inhibit hippocampus MMP2.After FITC perfusion,a large amount of leaking FITC green fluorescence was observed in the hippocampus of the 30% EtOH+Veh group.While no FITC fluorescence leakage was observed in Con and 30% EtOH+SB-3CT group.Compared with the Con group,the hippocampus FITC fluorescence intensities in the 30% EtOH+Veh group were increased by 69.9%.After SB-3CT treatment,the hippocampus FITC fluorescence intensities in the 30% EtOH+SB-3CT group decreased by 44.9% compared to 30% EtOH+Veh group.Compared with Con group(IL-6: 22.7±1.0pg/ml;IL-1?:42.8±0.8pg/ml),the levels of serum IL-6(28.4±2pg/ml)and IL-1?(46.6±1.6pg/ml)were up-regulated by 24.8% and 8.9%,respectively.After 1 week of SB-3CT treatment,the serum pro-inflammatory cytokines IL-6 and IL-1? were decreased by 16.4% and 8%,respectively.Compared with Con group(IL-6: 676±18.8pg/g/tissue;IL-1?: 668.6±14.8pg/g/tissue),the levels of hippocampus IL-6(732.8±3.9pg/g/tissue)and IL-1?(723.4±4.1pg/g/tissue)in the 30% EtOH+Veh group were up-regulated by 8.4% and 8.2%,respectively.While 30% EtOH+SB-3CT group had a normal IL-6 and IL-1? levels compared to Con group.In addition,astrocytes activation in the hippocampus in EtOH+Veh rats were also inhibited by SB-3CT.By WB,we found that SB-3CT treatment eliminated hippocampal tau hyperphosphorylation and increased synapse-related proteins.Conclusion: In this research,we established an alcoholic hypertensive rat model,which showed hypertension phenotype,anxiety,depressive and cognitive impairments behaviors.Combining behavioral tests,morphological analyses and biochemical techniques to study the characteristics brain damage of AH rats,such as disruption of BBB,endothelial cell senescence,synaptic loss and tau hyperphosphorylation,ect.The activity of MMP2 increased in AH rats,and inhibition of MMP2 significantly improved cognitive impairment in AH rats,suggesting an important role of MMP2 in brain damage of AH rats. |
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