The Role Of Swiprosin-1 In The Regulation Of The Permeability Of Blood-brain Barrier

Blood-brain barrier(BBB) refers to a vascular structure of a variety of cells and non-cellular components, locating between the central nervous system(CNS) and the peripheral blood circulation. The BBB consists of brain microvascular endothelial cells(BMEC) which surrounded by other cells(such as astrocytes, pericytes, microglia and neurons, etc.), forming the function together. The mainly function of the BBB is to limit substances access to the CNS, maintaining the stability of the internal environment of the CNS. Damaging the function of BBB has occurred in almost all CNS disorders. In this situation, large number of components in vessels crosses the BBB targeting to the CNS. This could further exacerbate the deterioration of the disease. At the same time, many drugs treating CNS disorders could hardly enter the CNS because of the limitation of BBB. How to control and regulate the permeability of BBB is important to develop the drugs treating the CNS disorders. Therefore, finding novel proteins to regulate the permeability of the BBB is significant for the treatment in the CNS.AimsOur group found a novel protein named swiprosin-1 in the cerebral cortex. The protein mainly expresses in brain microvascular endothelial cells. Swiprosin-1 is a small molecular weight protein which was first reported in 2004. Current reports focused its function on the regulation of immuce cells such as B lymphocytes and T lymphocytes. Little is known about the other aspects of the role of swiprosin-1, especially in the CNS. We found that overexpression swiprosin-1 in vitro could downregulate the expression level of tight junction protein ZO-1. As BMEC is the basic structural of the BBB, we speculate that swiprosin-1 may play a role in the regulation of the permeability of the BBB. This paper aims to find the role of swiprosin-1 in regulation of the BBB permeability, and further clarify its mechanism under the phenomenon. We expect to find a novel potential target for the regulation of the BBB.Methods1. The localization of swiprosin-1 in the cerebral cortex and the expression differences in animal models with hyperpermeability BBB.(1) Swiprosin-1 expression was observed in different tissues and organs in normal mice using immunoblotting.(2) Swiprosin-1 expression and distribution was detected in the brain using immunohistochemy.(3) Rat brain microvascular endothelial cells(BMEC) were cultured in vitro and of which swiprosin-1 expression was detected by the use of immunocytochemistry.(4) Three animal models with hyperpermeability BBB were established. They are middle cerebral artery occlusion(MCAO), LPS treatment induced systemic immune response, cold injury model. The damaging of the BBB was confirmed by intravenous injection of Evan's blue, and then detecting the fluorescence.(5) Detect the expression of claudin-5, VE-cadherin and caveolin-1 in the three animal models with hypermeability BBB, compared with normal conditions.(6) Detect the expression of swiprosin-1 in the different animal models with hypermeability BBB;(7) Detect the expression of swiprosin-1 in the BMEC with oxygen glucose deprivation(OGD) treatment.2. Swiprosin-1 expression level influences the permeability of the BBB.(1) Swiprosin-1 knockout mice(Swp-/- mice) were engineered. Detect the permeability of Evan's blue, comparing with wild-type mice(Swp +/+).(2) Swiprosin-1 overexpression mice(Swp OE mice) were engineered. Detect the permeability of Evan's blue, comparing with wild-type mice(Swp WT). Compare the fluorescence images and the quantitative level between the two groups.(3) Compare the fluorescence images and the quantitative level between the Swp-/-mice and Swp +/+ mice after the treatment of MCAO for 24 h.3. The mechanism under swiprosin-1 regulates the permeability of the BBB.(1) Detect the distribution of the tight junction proteins, occludin and ZO-1, in the cerebral cortex of Swp OE mice compared with Swp WT mice in normal conditions using immunofluorescence.(2) Detect the protein expression level of the tight junction proteins, occludin and ZO-1, in the cerebral cortex of Swp OE mice compared with Swp WT mice in normal conditions using western blot.(3) Detect the distribution of the tight junction proteins, occludin and ZO-1, in the infarcted and non-infarcted area of cerebral cortex of Swp-/- mice compared with Swp +/+ mice after MCAO using immunofluorescence.(4) Detect the protein level of the tight junction proteins, occludin and ZO-1, in the infarcted and non-infarcted area of cerebral cortex of Swp-/- mice compared with Swp+/+ mice after MCAO using western blot.(5) Detect the protein level of ROCK-1 in the cerebral cortex of Swp OE compared with Swp WT using western blot.(6) Detect the protein level of ZO-1in BMEC overexpressed swiprosin-1 by western blot, and after the treatment of ROCK-1 inhibitor Y27632.(7) Detect the protein level of ROCK-1, in the infarcted and non-infarcted area of cerebral cortex of Swp-/- mice compared with Swp +/+ mice after MCAO.(8) Detect the protein level of ZO-1 in the BMEC with swiprosin-1 knockdown compared with the treatment of OGD 6h.(9) Detect the protein level of ZO-1 and occludin after the treatment of OGD 6h, with or without the treatment of Y27632.(10) The study of the protein-protein interaction between swiprosin-1 and ZO-1 in cerebral cortex.4. The role of swiprosin-1 on the regulation of tight junction proteins and cytoskeletal protein.(1) Detect the distribution of occludin and ZO-1, in the BMEC overexpressed swiprosin-1 with or without the treatment of Y27632.(2) Detect the distribution of claudin-5, in the BMEC overexpressed swiprosin-1 with or without the treatment of Y27632.(3) Detect the distribution of F-actin, in the BMEC overexpressed swiprosin-1 with or without the treatment of Y27632.(4) Detect the distribution of ZO-1, after the treatment of OGD 6h in BMEC, with or without the treatment of Y27632.(5) Detect the distribution of F-actin, after the treatment of OGD 6h in BMEC, with or without the treatment of Y27632.5. The function of swiprosin-1 effects on other endothelial cells functions in vitro.(1) Cell morphology was observed after OGD treated for 6h in BMEC after down regulation with swiprosin-1.(2) Detect the cell morphology and migration activity in BMEC with swiprosin-1 overexpression.(3) Detect the tube formation ability in BMEC with swiprosin-1 overexpression.Results1. Three animal models with dysfunctional BBB were successfully established, and swiprosin-1 expression was detected.(1) Swiprosin-1 was expressed in multi-tissues. Swiprosin-1 was expressed in the cerebrum, cerebellum, medulla oblongata in the CNS.(2) Swiprosin-1 is mainly distributed in the brain blood vessels of cerebral cortex.(3) The immunofluorescence results indicated swiprosin-1 was mainly expressed in the cytoplasm of the primary BMEC.(4) Three animal models of increasing BBB permeability were successfully established, and swiprosin-1 expression level was detected. After intravenous injection of Evan's blue, blue overall observation showed obvious infiltration, slice red fluorescence could be detected after section.(5) The expression level of claudin-5, VE-cadherin were down-regulated, and caveolin-1 up-regulated in three animal models.(6) The expression level of swiprosin-1was upregulated when the BBB permeability increased in vivo.(7) The expression level of swiprosin-1was upregulated in vitro with the treatment of OGD, reaching the highest level at 4h.2. Swiprosin-1 affects the permeability of the BBB in vivo.(1) BBB permeability has no significant differences between Swp-/- mice and Swp+/+ under physiological conditions.(2) BBB permeability increased significantly in Swp mice compared with Swp mice under physiological conditions;(3) Swp-/- mice had lower Evan's blue exudative effusion in cerebral cortex than Swp+/+ mice after MCAO 24 h.3. Swiprosin-1 regulated the BBB permeability through tight junction proteins.(1) Occludin and ZO-1 distributed uncontinuous in the cerebral cortex of Swpmice.(2) The expression level of occludin and ZO-1 decreased in the cerebral cortex of SwpOE mice, with claudin-5 unchanged.(3) The distribution of occludin and ZO-1 remained more continuous in the infarct area of Swp-/- mice after MCAO, with Evan's Blue concentrated into the cerebral vessels.(4) The expression level of occludin and ZO-1 decreased less in the infarct area of cerebral cortex in Swp-/- mice after MCAO 24 h.(5) The expression level of ROCK-1 in the cerebral cortex of SwpOE mice was significantly higher than in the SwpWT mice.(6) The expression level of ZO-1 was significantly decreased in BMEC after overexpression swiprosin-1, and could be inhibited by Y27632.(7) The expression level of ROCK-1 increased significantly in the infarct area of cerebral cortex in Swp+/+ mice after MCAO; while maintained unchanged in Swp-/-mice.(8) The expression level of ZO-1 was significantly higher in BMEC deleted swiprosin-1 after the treatment of OGD for 6h.(9) The expression level of occludin and ZO-1 was significantly down-regulated in BMEC after the treatment of OGD for 6h,and could be inhibited by Y27632.(10) There exist an protein-protein interaction between swiprosin-1 and ZO-1 in the cerebral cortex of wild-type mice.4. Swiprosin-1 affects tight junction proteins and cytoskeletal protein distribution of BMEC cells.(1) Occludin and ZO-1 distributed uncontinuous in BMEC overexpressed swiprosin-1, and could be inhibited by Y27632.(2) Claudin-5 distribution remained unchanged in BMEC overexpressed swiprosin-1;(3) F-actin tends to distribute towards membrane in BMEC overexpressed swiprosin-1, and could be inhibited by Y27632.(4) ZO-1 distributed uncontinuous in BMEC after OGD treatment, and could be inhibited by Y27632.(5) F-actin tends to distribute towards membrane in BMEC after OGD treatment, and could be inhibited by Y27632.5. Swiprosin-1 regulated other functions of endothelial cell.(1) The morphology of BMEC changes significantly under the treatment of OGD, and could be inhibited by Y27632.(2) The morphology of BMEC changes significantly under the treatment of OGD, and could be inhibited by swiprosin-1 deletion in vitro.(3) Pseudopodia stretched out from the cell body in BMEC overexpressed swiprosin-1, suggesting migration capacity enhancement.(4) The cell migration ability enhanced in BMEC overexpressed swiprosin-1.(5) The tube-formation ability had no significant change in BMEC overexpressed swiprosin-1.Conclusions1. Swiprosin-1 is an important protein involved in the regulation of BBB permeability.2. Swiprosin-1 regulated BBB permeability mainly by affecting the expression and distribution of occludin, ZO-1, together with affecting the distribution of F-actin.3. ROCK-1 involved in swiprosin-1 regulating BBB permeability.