关键词: 水泡性口炎病毒, 血管内皮屏障, 黏附连接, 低密度脂蛋白受体, Wnt/β-连环蛋白信号通路

Abstract:

Objective To discuss the damage effect of vesicular stomatitis virus （VSV） on the vascular endothelial （VE） barrier， and to clarify its mechanism. Methods The canine kidney cells were used to amplify VSV. The half tissue culture infective dose （TCID₅₀） of VSV was determined using mouse brain endothelial tumor bEnd.3 cells， and subsequent experiment was conducted using 300 times the TCID50. The bEnd.3 cells were divided into infection 0 h group， infection 4 h group， infection 8 h group， and infection 12 h group for VE barrier damage experiments due to VSV infection. The bEnd.3 cells were also divided into control group， infection group， and correction group for experiments to inhibit the VSV replication and restore the VE barrier. The bEnd.3 cells were inoculated into Transwell chambers to construct an in vitro VE barrier model. Cell voltage resistance meter was used to detect the transepithelial resistance （TER） in various groups after the bEnd.3 cells were infected with VSV at different time points；fluorescein isothiocyanate-dextran leakage assay was used to detect the permeability coefficients of the cells in various groups； immunofluorescence staining was used to observe the localization changes of VE-cadherin， β-catenin， and phosphorylated β-catenin （p-β-catenin） in cytoskeleton and adherens junctions （AJs） of the bEnd.3 cells after VSV infection； real-time fluorescence quantitative PCR （RT-qPCR） method was used to detect the expression levels of Wnt and β-catenin mRNA in the cells in various groups； Western blotting method was used to detect the expression levels of Wnt， β-catenin， and p-β-catenin proteins in the cells in various groups. Results The TCID₅₀ of VSV was 10^-4.5·100 μL^-1. The Transwell chamber experiment results showed that compared with infection 0 h group， the TERs in the cells in the other groups were significantly decreased （P<0.05）， and the permeability coefficients were significantly increased （P<0.05）. The immunofluorescence staining results showed that compared with control group， the cytoskeleton of the bEnd.3 cells in infection group was disordered， the cell gaps was increased， the linear index of AJs was significantly decreased （P<0.05）， and β-catenin and p-β-catenin translocated from the cell membrane to the perinuclear area. The RT-qPCR results showed that compared with infection 0 h group， the expression levels of Wnt mRNA in the cells in the other groups were significantly decreased （P<0.05）， while the expression levels of β-catenin mRNA showed no statistically significant difference （P>0.05）. The Western blotting results showed that compared with infection 0 h group， the expression levels of Wnt protein in the cells in the other groups were significantly decreased （P<0.05）， the expression levels of β-catenin showed no statistically significant differences （P>0.05）， and the expression levels of p-β-catenin were significantly increased （P<0.05）.After inhibiting the VSV replication and correcting the low density lipoprotein receptor （LDLR） abnormalities， the Transwell chamber experiment results showed that compared with infection group， the TER in the cells in correction group was significantly increased （P<0.05）， and the permeability coefficient was significantly decreased （P<0.05）.The immunofluorescence staining results showed that compared with infection group， the gaps in the cells in correction group were reduced， and the perinuclear aggregation of β-catenin and p-β-catenin in the cells was restrained. The RT-qPCR results showed that compared with infection group， the expression level of Wnt mRNA in the cells in correction group was significantly increased （P<0.05）. The Western blotting results showed that compared with infection group， the expression level of Wnt protein in the cells in correction group was significantly increased （P<0.05）， the expression level of β-catenin showed no statistically significant difference （P>0.05）， and the expression level of p-β-catenin was significantly decreased （P<0.05）. Conclusion VSV infection can cause the LDLR inactivation， reduce the expression level of Wnt protein， increase the phosphorylation level of β-catenin and cause its internalization， disrupt the stability of AJs， and ultimately lead to VE barrier damage.

Key words: Vesicular stomatitis virus, Vascular endothelial barrier, Adherent junction, Low density lipoprotein receptor, Wnt/β-catenin signaling pathway