Basic And Clinical Research On The Correlation Between Blood-brain Barrier Related Proteins Cx43?AQP-4 With Stroke Prognosis

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Effect of Inhibiting Cx43 Hemichannel in Blood-Brain Barrier on Cerebral Ischemia/Reperfusion Injury and Its Related MechanismsObjective: Neurovascular unit(NVU)is the structural and functional unit of the brain,and its core structure is the blood-brain barrier(BBB).Intercellular communication between NVU cells is mainly through cell junctions,the most important gap junction protein is connexin 43(Cx43),which constitutes gap junctions and hemichannels.We chose Cx43 as a therapeutic target and select Cx43 hemichannel specific blocker-Gap19,to conduct basic animal research on mouse Middle Cerebral Artery Occlusion(MCAO)and Oxygen-Glucose Deprivation(OGD)model to explore its effect on cerebral ischemia/reperfusion injury and related mechanisms.Materials and Methods: 1.In vivo experiment: Used adult male ICR mice as experimental animals,which were randomly divided in to sham group,sham+Gap19 group,I/R group,I/R+Gap26 and I/R+Gap19.And constructed mice MCAO model to simulate the cerebral ischemia/reperfusion injury.Injected Gap19/Gap26(10 ?g)into the lateral ventricle of different group mice at 1h after ischemia.After 24 h reperfusion,the mice were scored for neurological deficits.The volume of cerebral infarct ion was measured by TTC staining.The white matter damage was detected by Luxol Fast Blue(LFB)staining.Western blot was used to detect the expression of Cx43,Toll like receptor 4(TLR4)and its downstream channel protein in infarcted tissues.2.In vivo experiments: Extracted primary astrocytes,brain microvascular endothelial cells(BMECs)and neurons from different age mice for primary culture,identified cell types by immunofluorescence,co-culture to construct a NVU system and constructed Oxygen-Glucose Deprivation(OGD)model.The optimal concentration of Gap19 was determined by MTT assay.was detected by ethidium bromide(Et Br)assay.The expressions of Cx43,TLR4 and its downstream channel protein after OGD were detected by Western blot.TNF-? and IL-1? level were detected by enzyme-linked immunosorbent assay(ELISA).Liopolysaccharide(LPS)was used to further validate the mechanism of action of Gap19.Results: In vivo experiment: Compared with the I/R group,the neurological deficit was alleviated in I/R+Gap19 group,and the volume of cerebral infarction was significantly reduced,which was superior than Gap26(P<0.05).At the same time,results showed that LFB-positive myelin density of the injured striatum in the Gap19 group was significantly increased(P<0.05),which could reduce white matter damage.Western Blot showed that the expression of Cx43 and TLR4 increased after ischemia injury(P<0.05),and Cx43 expression decreased after Gap19 intervention(P<0.05).Compared with the I/R group,the expression levels of TLR4 pathway-related proteins My D88,NF-?B p-p65,TNF-? and IL-1? were significantly decreased in the I/R+Gap19 group(P<0.05).In vitro experiment: Gap19 intervention can significantly increase the cell viability after OGD,and the best concentration was 100?M(P<0.05).Through Et Br experiment,compared with control group,large numbers of hemichannels were opened in the astrocytes after OGD(P<0.05),while the dye uptake in the brain vascular endothelial cells and neurons was not significantly changed(P<0.05).Gap19 also reduced the expression of Cx43,TLR4 pathway proteins and inflammatory cytokines in vitro.Immunofluorescence staining showed that Cx43 and TLR4 were co-expressed in astrocytes after OGD.Meanwhile,Gap19 inhibited LPS-induced TLR4 pathway activation and decreases TNF-? and IL-1? levels.Conclusions: The Cx43 hemichannel-specific inhibitor Gap19 exerts neuroprotective effects on cerebral ischemic injury in mice by inhibiting TLR4-mediated signaling pathway.Part ? Relationship between Blood-Brain Barrier Associated Protein AQP-4 Level and Progressive StrokeObjective: Aquaporin 4(AQP-4)is the most abundant aquaporin in the brain,which exists in the astrocytes' foot processes and participates in the formation of BBB and regulates water molecule transport.AQP-4 expression is up-regulated after ischemia,increasing the permeability of BBB and aggravating brain edema.The level of AQP-4 was detected in peripheral blood of patients with acute ischemic stroke(AIS)and its correlation with progressive stroke was analyzed.Materials and Methods: AIS patients within 24 hours after the onset in neurology of the North Jiangsu People's Hospital from September 2018 to February 2019 were enrolled and confirmed by CT or MRI.Fasting venous blood was collected within 24 hours of admission,and serum AQP-4 levels were determined by ELISA.Collect general clinical data from patients with AIS.The NIHSS score increased by 4 points or more after admission was judged to be progressive stroke.The clinical data and AQP-4 levels were compared between the patients with progressive stroke and non-progressive stroke,and the risk factors for progressive stroke were analyzed by a two-class logistic regression model.Results: A total of 104 AIS patients who met the inclusion criteria were included in the study,among which 39(37.5%)were progressive stroke patients and 65(62.5%)were non-progressive stroke patients.1.There was a significant difference in low-density lipoprotein levels between the two groups(P<0.05).2.The serum AQP-4 level in the progressive stroke group(774.94±163.32pg/ml)was higher than that in the non-progressive stroke group(408.32±133.25pg/ml),and the difference was statistically significant(P<0.05);3.Logistic regression analysis showed that the elevated AQP-4 levels were independent risk factors for progressive stroke.Conclusions: Elevated AQP-4 may lead to aggravation of the blood-brain barrier permeability,leading to exacerbation of brain injury,which may be an independent risk factor for progressive stroke.