The Mechanism Of Cathepsin D Regulating The Permeability Of Brain Microvascular Endothelial Cells By Up-regulating Cavin2

Objective: The increased permeability of the blood-brain barrier(BBB)is an important pathological basis of inflammation after cerebrovascular diseases.The increased permeability of BBB causes leukocyte-endothelial cell interaction and transendothelial cell migration into the brain,thus aggravating inflammatory injury of the central nervous system and leading to the destruction of neurons.In this study,Mouse brain microvascular endothelial cells(b End.3)was used to simulate BBB model in vitro to observe the effect of cathepsind(CTSD)on permeability changes of b End.3 cells and to explore its mechanism.Methods: 1.High-sugar medium containing 10% fetal bovine serum was cultured in incubator at 37 ℃ and 5%CO2.Cells were passed once for 2-3d,and cells at logarithmic growth stage were selected for the experiment.2.Research Methods:Firstly,MTT colorimetric assay was used to detect the effect of CTSD concentration at0ng/ml,50 ng/ml,100 ng/ml,200ng/ml,400ng/ml and 800ng/ml on cell proliferation activity.Then,b End.3 cells were used to establish an in vitro BBB model.The permeability changes of b End.3 cells in mice at the above CTSD concentrations were measured by Transwell in the Fluorescein isothiocyanate labeled dextrance osmosis assay.Cavin-2 was detected by western blot(WB)and immunofluorescence staining when CTSD concentrations were 0ng/ml,200ng/ml,400ng/ml and 800ng/ml Expression levels of response,Cavin-2,Zonula Occludens-1(ZO-1)and VE-cadherin in b End.3cells.The localization of Cavin2 protein on the surface of the cell membrane between the control group and 800ng/ml CTSD was investigated by Total Interal Reflection fluorescence Microscopy(Tirf),and the changes in the number of vesicles in the cell membrane were detected by transmission electron microscopy.Immunofluorescence staining was used to determine whether nuclear factor-κ-gene binding(NFκB)signaling pathway and Cavin2 expression of b End.3 cells were determined by WB.Pepstatin A(Pep A)and Pyrrdidine dithiocarbamate(PDTC)were used as inhibitors to observe whether they could inhibit the effect of CTSD on the permeability of b End.3.Results: 1.MTT assay results of cerebral microvascular endothelial cells at different concentrations of CTSD showed no significant difference in cell activity.2.With the increase of CTSD concentration,FITC-dextrance through the Transwell chamber increased significantly,and CTSD concentration was positively correlated with the permeability of cerebral microvascular endothelial cells.3.The immunofluorescence results showed that the fluorescence intensity of Cavin-2protein increased with the increase of CTSD concentration,while the fluorescence intensity of ZO-1 and VE-cadherin protein did not change.4.WB results showed that with the increase of CTSD concentration,the expression level of Cavin-2 protein increased,while the expression levels of ZO-1 and VE-cadherin did not change.5.Tirf microscope results showed that Cavin2 protein was mainly located on the endothelial cell membrane of cerebral microvessels,and the expression of Cavin2 protein in 800ng/ml group was higher than that in the control group.6.Electron microscope results showed that the number of vesicles near the cell membrane of cerebral microvascular endothelium in 800ng/ml group was higher than that in the control group.7.Compared with the control group,the NFκB signaling pathway was activated into the nucleus in the higher CTSD concentration group,and this phenomenon could be inhibited by PDTC.8.WB and immunofluorescence results showed that Cavin-2 protein expression was increased in the group with high CTSD concentration,which could be inhibited by PDTC but not by Pep A.9.The detection of FITC-dextrance concentration in Transwell showed that PDTC could block the increase of cerebral microvascular endothelial permeability caused by CTSD.Conclusion: 1.CTSD can increase the permeability of cerebral microvascular endothelial cells in a dose-dependent manner.2.CTSD can lead to the up-regulation of cavin2 expression in cerebral microvascular endothelial cells,but has no significant effect on the tight junction structure.3.The up-regulation of Cavin2 protein by CTSD is realized through activation of NFκB signaling pathway in cerebral microvascular endothelial cells.