Role And Mechanism Of Interferon-lambda In A Model Of Oxygen Glucose Deprivation/Reoxygenation-induced Blood-brain Barrier

Objective : Stroke is the second leading cause of death worldwide,and the overall prevalence of stroke in China is 2.6% in 2020.Ischemic stroke(IS)is the main type of stroke that can cause brain tissue death and focal neuronal damage.The size of the infarct and the severity of the neurological system after the attack depend on the time after the onset,the severity of ischemia,etc.It is a complex disease.Treatment of ischemic stroke depends on recanalization,however,restoration of blood flow in tissues previously subjected to ischemia leads to cerebral ischemia/reperfusion(I/R)injury,and improving blood-brain barrier(BBB)injury and protecting the integrity of the BBB can improve the damage caused by cerebral ischemia/reperfusion.injury,which is the focus of treatment.Oxygen glucose deprivation/reoxygenation(OGD/R)is a method to replicate ischemia/reperfusion injury in vitro.IFN-λ(interferon-lambda,IFN-λ)is a type III interferon,and the aim of this study was to investigate the effect and mechanism of IFN-λ on the BBB in a brain ischemia/reperfusion model simulated by OGD/R to provide a possible treatment for ischemic stroke.Methods:The BBB model was constructed by a Transwell co-culture model with b.End3 cells cultured in the upper chamber of the Transwell and astrocytes in the lower chamber.By oxygen glucose deprivation/reoxygenation(OGD/R)treatment of the BBB model and subsequent addition of IFN-λ intervention,the BBB model was divided into control group,OGD/R treatment group,and IFN-λintervention group,with three replicate wells in each group.Whether IFN-λ plays a role in the OGD/R model was examined by qRT-PCR assay;whether astrocytes and brain microvascular endothelial cells(b.End3),which constitute the Transwell co-culture model,can express the receptor for IFN-λ was determined by qRT-PCR assay;according to the Transwell permeability assay FITC-dextran-10000 permeation,the effect of IFN-λ treatment on the blood-brain barrier permeability of the OGD/R model was assessed;the mRNA and protein of the tight junction proteins(ZO-1,Occudin and Claudin-5)constituting the BBB were examined by qRT-PCR,Western blot and fluorescence confocal assay,expression and their integrity.Results:The qRT-PCR assay results showed that the expression of IFN-λ2 and IFN-λ3 was increased in the OGD/R-treated group compared with the control group(p<0.05).The qRT-PCR assay showed no significant difference in the expression of IFNLR1 and IL-10Rβ between the control and OGD/R-treated groups(p>0.05).The results of the Transwell permeability assay showed that the amount of FITC-dextran-10000 permeation was significantly lower in the IFN-λ intervention group compared to the OGD/R treatment group(p<0.05).The qRT-PCR assay showed that the expression of tight junction protein mRNA was increased in b.End3 cells in the IFN-λ intervention group compared with the OGD/R treatment group.Western blot assay showed increased expression of tight junction protein in b.End3 cells in the IFN-λ intervention group compared with the OGD/R-treated group(p<0.05).The results of immunofluorescence confocal assay showed that the mean fluorescence intensity of tight junction protein of b.End3 cells in the IFN-λintervention group was higher than that in the OGD/R-treated group(p<0.05).Conclusion : IFN-λ promotes tight protein integrity and connectivity and improves BBB permeability in the OGD/R model.