Aim of the study
Tight junctions (TJs) are major components of the blood-brain barrier (BBB) that physically restrict the paracellular diffusion of blood-borne substances between endothelial cells into the CNS. TJ proteins are associated with cholesterol-enriched regions of plasma membrane known as lipid rafts, which are critical for the trafficking, positioning and function of TJ proteins. In this study, we investigated the effect of SMI on the expression and trafficking of the key TJ-associated protein, occludin, in lipid rafts. Materials and
Conclusions
SMI helps maintain the proper assembly of the TJ multiprotein complex in lipid rafts, thereby helping to preserve BBB functional integrity during focal cerebral ischemic insult. Our findings enhance our understanding of the mechanisms underlying the neuroprotective effect of SMI in cerebral ischemia.
Methods
Using a neutral pH, rat cerebral microvessels were subjected to detergent-free density-gradient fractionation to isolate lipid rafts containing occludin. Transmission electron microscopy (TEM) was performed to study the effects of drug administration on ultrastructural changes to TJs. Western blotting (WB), immunofluorescence (IF), and co-immunoprecipitation (COIP) were used to observe the localization and function of TJ-associated proteins.
Results
We successfully isolated cerebral microvessels and separated lipid rafts from plasma membranes. With SMI treatment, extravasation of FITC-albumin decreased around the cerebral vessels by IF, the tight junctions were found to still be intact and the basement membrane appeared to be of uniform thickness in TEM. Compared with the untreated group, the co-expression of flotillin-1 and occludin in microvascular endothelial cells was increased and distributed continuously in SMI treatment as shown in double label IF. SMI significantly increased the translocation of occludin to lipid raft fractions by WB and COIP. Conclusions: SMI helps maintain the proper assembly of the TJ multiprotein complex in lipid rafts, thereby helping to preserve BBB functional integrity during focal cerebral ischemic insult. Our findings enhance our understanding of the mechanisms underlying the neuroprotective effect of SMI in cerebral ischemia.