Changes Of IL-1β And IL-18 In Bradykinin-induced Blood-brain Barrier Opening In Combination With Papaverine

Cerebral glioma is a kind of malignant disease in central neural system,which is highest in mobidity and mortality rate.It accounts for 40%of all the cerebral tumors, but the cause for this disease remains elusive.BK nonselectively increases BBB permeability.The mechanism might involve in the following cascade of signal transduction reaction:BK combines with its BK₂ receptor,causing extacellular Ca²⁺ influx,intracellular Nitrixc Oxide and cyclic GMP increase,enhancing formation of transendothelial pinocytopic vehicles,or opening the tight junction,and finally opening the BTB.It has been found recently that intraarterial infusion of papaverine relieves cerebral vasospasms by inhibiting smooth musle contraction and it may simultaneously lead to a disruption of BBB.The exact molecular mechanism for papaverine is not well known,it is thought that this substance functions by inhibiting cAMP and cGMP phosphodiesterase activation in smooth muscle cells and by increasing the turnover of intracellular cAMP and CGMP.Some of the inflammatory agents discussed cause a reduction in arterial/arteriolar smooth muscle tone leading to vasodilatation or an increase in venular tone leading to a rise in intravascular hydrostatic pressure.Both could cause strentch of the tight junctional zone in capillaries/venules that could contribute to opening of paracellular pathway.It has been found that IL-1βinduces reversible BBB breakdown,which is mediated by neutrophils and could be eliminated in neutrophil-depleted animals.So the increase of BBB permeability may be due to the endogenous production of IL-1β. IL-1βinduced tissue cGMP concentration,and diputyril cGMP enhanced Ca²⁺release.IL-18 is the most recently identified member of IL-1 family and its role in the brain is unknown.In vitro IL-18 stimulates the production of IL-1βin primary astrocytes and of IL-1βand IL-8 via TNF-αin blood mononuclear cells.One hypothesis is that IL-18 may play a role in modulating IL-1βand thus indirectly affect the IL-1βdependent actions on the brain.The current study first established rat brain C6 tumor model and intracarotid infused BK/PA.We then measured the BTB permeability through Evans Blue staining, and observed the opening degree of tight junctions and the number of transendothelial pinocytopic vesicles by electron microscope,examined the experession of IL-1βand IL-18 to evaluate the relationship between the BTB permeability and content of IL-1βand IL-8,which would provide theoretic evidence to the mechanism underlying how IL-1βand IL-18 participate in the modulating of BBB permeability,and eventually provided new target and method to the clinical treatment of brain glioma.Materials1,Experimental animals:the adult female wistar rats(180-200g)were used, which were purchased from the center for Expeimental Animals of China Medical University.The total number was 160.All animal experiments were carried out in accordance with the National Institute of Health Guide for the Care and Use of Laborarory animals.2,Experimental cell line:the C6 glioma cell line was supplied by Department of Cytobiology in China Medical University.3,Experimental regents:rabbit polyclonal antibody anti-IL-1βand rabbit IL-18 polyclonal antibody(BosteCompany,Wuhan);bradykinin(BK)(Sigma-Aldrich,inc.); papaverine(PA)(shenyang first pharmacy);goat anti-habbit IgG conjugated with horseradish peroxidase and SABC immunohitochemistry kit(Boster Company,Wuhan); anti-β-actin antibody,goat polyclonal antibody anti-mouse IgG conjugated with horseradish peroxidase,and goat anti-rabbit IgG conjugated with FITC(Zhongshan Company,Beijing).4,Experimental instruments:CO₂ incubator,superclean bench,stereotaxic apparatus for rat brain,low temperature refrigeration centrifuge,ultroviolet spectro photometer,automatic running gel imaging system,GP electrophoresis apparatus, upright fluorescense microscope,-80℃deep freeze refrigerator,homeothermia freezing microtome,Motic Images Advanced 3.2 image analysis system. Methods1,Establishment of rat C6 brain tumor model.2,We pumped low dose of BK/PA into rat's brain with glioma via the carotid artery,and tested the different expressions of IL-1βand IL-18 protein in tumor district of each group(normal group,sham-operated group,PA group,BK group,PA+BK group, 1/2PA+BK group,PA+1/2BK group,1/2 PA+1/2BK group)with western blot.3,Measurement of BTB permeability.4,The opening of the BTB was observed by electron microscope.5,Western blot and Immunohistochemistry was used to detect the expression of IL-1βand IL-18.6,Image analysisThe datas obtained from Western blot were scanned with Chemi Imager 5500 V2.03 software;Integrated Density Value(IDV)of the IL-1βand IL-18 protein was calculated with Fluor Chen 2.0 software.The data of Immunochemistry were obtained and analysed semiquantitatively with Motic Images Advanced 3.2 image analysis system.The data of Immunolocalization of IL-1βand IL-18 protein were analysed with SPOT Advanced 4.0.2 software.7,Statistics analysisData were disposed with SPSS11.0 statistic software by One-Way ANOVA and they were expressed as mean±standard deviation,t test,P＜0.05 was considered statistic ally significant.Results1,Measurement of BTB permeabilityThe study has found that compared with normal group(0.244541±0.042274μg/g) and sham-operated group(0.345083±0.04982μg/g),the content of EB of other groups increased.The content of EB in PA+BK group was highest(0.960309±0.071750μg/g, P＜0.05),and the next was the 1/2PA+BK group(0.732120±0.067341μg/g,P＜0.05), BK group(0.663774±0.036010μg/g,P＜0.05),PA group(0.603132±0.34442μg/g, P＜0.05). 2,Changes of the opening of BTB by electron microscopeWe found no opening of BTB in normal group or sham-operated group by electron microscope.Slight opening could be detected in PA/BK group;obviously opening could be detected in PA+BK group,and only partial opening of BTB could be detected in 1/2PA+BK/PA+1/2BK group.NO:opening could be observed in 1/2PA+1/2BK group.3,Results of western blot analysisThe result is the IDV ratio of each group toβ-actin.The expression of IL-1βprotein was very low in normal group and sham operated group;after intracaroid infusion of BK/PA,the expression of IL-1βincreased.In PA group,the IDV ratio was 0.98570±0.04475(n=8,p＜0.05);while in BK group,the IDV ratio was 1.16340±0.08745(n=8,p＜0.05);the expression of IL-1βin PA+BK group increased to peak,the IDV ratio of which was 1.54100±0.08417(n=8,p＜0.05);and in 1/2PA+BK group and 1/2BK+PA group,the IDV ratio went down and had no statistical difference with PA/BK group;the expression of IL-1βin 1/2PA+1/2BK was low.The expression of IL-18 protein had the same tendency with IL-1β.The expression of IL-18 protein in normal group and sham-operated group was very low;after intracaroid infusion of BK/PA,the expression of IL-18 increased.In PA group,the IDV ratio was 0.97839±0.07478(n=8,p＜0.05);while in BK group,the IDV ratio was 0.99791±0.06956(n=8,p＜0.05);The expression of IL-18 in PA+BK group increased to peak,the IDV ratio of which was 1.61476±0.08623(n=8,p＜0.05);and in 1/2PA+BK group,the IDV ratio went down to 1.19166±0.07203(n=8,p＜0.05);in 1/2BK+PA group and 1/2PA+1/2BK group,the expression of IL-18 was low,only having statistical difference with normal group.4,Results of Immunochemistry analysisThere was no expression of IL-1βin normal group and sham-operated group.After infusion of PA,the expression of IL-1βon tumor capillaries was low.The IDV of IL-1βin PA group was 0.02580±0.00442(n=6,p＜0.05).And it was 0.02660±0.00423(n=6, p＜0.05)in BK group.The expression of IL-1βin PA+BK group reach the peak,the IDV of which was 0.03980±0.00425(n=6,p＜0.05);the expression of IL-1βin 1/2PA+BK group and PA+1/2BK group was low and had no statistical difference with PA/BK group.The expression of IL-18 on the tumor capillary had the identical tendency with IL-1βThe expression of IL-18 in normal group and sham-operated was low.In PA group,the IDV of IL-18 was 0.01960±0.00344(n=6,p＜0.05);while in BK group,it came to 0.02660±0.00216(n=6,p＜0.05);in PA+BK group,the expression was the highest,the IDV of which was 0.03600±0.00249(n=6,p＜0.05);in 1/2PA+BK group, and PA+1/2BK qroup,the expression of IL-18 was low,only having statistical difference with PA group;the expression of IL-18 in 1/2PA+1/2BK was very low.DiscussionThe current experiment showed:the combination of BK and PA had more significant effect on the opening of the BBB than single use of BK/PA.The expression of IL-1βand IL-18 increased accompanied with the increase of BBB permeability.It seemed that the combination of BK and PA could contribute to the opening of the BBB.The previous research had detected that the BK and PA could increase BTB permeability through the elevation of cGMP level.BK-activated downstream signaling targets,such as endogenous NO,sGC,or cGMP,could be independently modulated to elicit a selectively and enhanced BTB permeability response.And this is in accordance with our result that the combination of BK and PA can increase BTB permeability.The present results may indicate that the NO-dependent modulation of Ca²⁺is part of signaling cascade subserving some of the multiple functions of IL-1β.And NO/cGMP signaling is a part of the intracellular mechanism transducing IL-1β-mediated Ca²⁺release.It has been show that chronic expression of IL-1,in contrast to its acute delivery,can reversibly damage CNS integrity and implicates this cytokine or downstream components as major mediators of demyelination in chronic inflammatory and demyelinating diseases.Hu and Fraser showed that the cytokine interleukin IL-1βgreatly enhanced the response to BK under normal conditions.The expression of IL-1βhad some relationships with the BTB permeability.IL-1βmay be the downstream signaling targets of BK and PA inducing BBB opening.But it remains elusive how IL-1βlead the BBB opening through NO/cGMP signal system,and what roles of IL-1βis in this mechanism.IL-18 is the most recently identified member of IL-1 family.We identified that IL-18 expression level had the same tendency with that of IL-1β,and there might be some relationships between BBB permeability and expressions of these two cytokines, though the molecular mechanism is not clear.Take together,the current study demonstrate that the combination of BK and PA has synergetic effect to the opening of BTB.And single use of PA can also increase the permeability of the BBB.The expression of cytokine IL-1βand IL-18 has some relationships with the BBB permeability.We can develop a new approach to increase BTB permeability by combination of BK and PA,which could increase the delivery of drugs to brain tumors.Conclusion1,The current experiment firstly testified that the combination of BK and PA increased the permeability of BTB.2,The single use of PA increased the permeability of BTB.3,The combination of BK and PA increased the permeability of BTB and the expression of IL-1βand IL-18 in this process.