Effects Of Claudin-5 On Bradykinin-induced Blood-tumor Barrier Opening In Combination With Papaverine

ObjectiveGlioma is the most common brain tumor, the incidence rate has increased year by year and the morbidity is high. One of problems faced by the brain tumor patients is non-permeability of brain vasculature and the blood-brain barrier (BBB) restricts the hydrophilic chemotherapeutic agents getting into the tumor site. Therefore, how to open the brain-tumor barrier (BTB) selectively is the key factor to improve the chemotherapeutic effects of glioma patients.Bradykinin is the drug of selective opening BTB found by recent years. Studies demonstrated that it can open BTB selectively when bradykinin B₂ receptor agonist cereport combined with carboplation affecting on the brain tissue and it has been confirmed by clinical phaseâ…¡study. But the B₂ receptor system the cereport stimulates to modulate permeability of the BBB is highly sensitive and autoregulated. Cereport creates transient (15min) opening of the BBB which only allows entry of 1kDa-size molecules, which confined anti-tumor drugs application type and continuous action time. Recent research found intraarteria infusing PA can also open BTB selectively. Comparing with the action mechanism of bradykinin and papaverine, we found both of them could increase the second messenger cGMP in cells. Accordingly we speculated that BK and PA may increase the BTB permeability from certain same ways, combination of the two drugs has the synergistic effect.The study demonstrated tight junction (TJ) is the important factor to maintain BBB integrity. TJ constituted the barrier of ion and molecule passing membrane transport through the paracellular pathway. Claudin-5 is a major cell adhesion molecule of TJ in brain endothelial cells. The down-regulation of claudin-5 expression level can directly lead to disruption of TJ between brain microvascular endothelial cells. Claudin-5 was the necessary and sufficient condition in the formation of TJ.This study observed the changes of BTB permeability and examined the changes of claudin-5 expression level by the combination of BK and PA using the model of glioma rats, primarily understanding mechanism of the combination of BK and PA and provided the new treatment pathway for the central nervous system diseases.Materials and MethodsMaterials1.Experimental animals: The adult female Wister rats(180-200g) were used, which purchased from the Center for Experimental Animals of China Medical University. The total number was 230.2.Experimental cell line: rat C6 glioma cell strain provided by cytobiological laboratory of China Medical University.3.Experimental reagents: goat anti-rat claudin-5 polyclonal antibody (Santa Cruz Biotechnology, Inc.); Bradykinin (Sigma, U. S. A); rabbit ant-goat antibody conjugated with horseradish peroxidase and SABC immunohistochemistry kit (Boster Biological technology Company, Wuhan); Evance blue (Fluka separate packing); papaverine hydrochloride (Shenyang NO1 pharmaceutical Factory).4.CO₂ incubator; superclean bench; stereotaxic apparatus for rat brain; Perfusor Compact S micro-infusion pump; low temperature refrigeration centrifuge; ultraviolet spectro photometer; GP electrophoresis apparatus; -80℃deep freeze refrigerator; homeothermia freezing microtome; Motic Images Advanced 3.2 image analysis system.Methods1.Establishment of rat brain C6 tumor model.2.groups Combination of BK and PA on different timeCombination of BK and PA with different dose3.Measurement of BTB permeability.4.Investigation of tight junction protein claudin-5 expression by western blot analysis.5.Immunohistochemistry was used to detect the location and the expression of claudin-5.6.The data obtained from Western blot were scanned with Chemi Imager 5500 V2.03 software; Integrated Density Value (IDV) of the claudin-5 was calculated with Fluor Chen 2.0 software; the data of Immunohistochemistry were obtained and analyzed semiquantitatively with Motic Images Advanced 3.2 image analysis system.7.Statistics analysisData were disposed with SPSS11.0 statistic software by One-Way ANOVA and linear regression and they were expressed as mean±standard deviation, t test, P<0.05 was considered statistically significant.Results1. Measurement of BTB permeabilityIt is most effective when BK in combination with PA 60 minutes.when combination of BK and PA with different doses, compared with the content of EB in control group 0.266±0.036μg/g brain weight (n=10), EB contents in experimental groups were 0.497±0.040μg/g brain weight(PA, n=10, p<0.01), 0.563±0.058μg/g brain weight(BK, n=10, p<0.01), 1.004±0.023μg/g brain weight(PA+BK, n=10, p<0.01), 0.734±0.045μg/g brain weight (PA+1/2BK, n=10, p<0.01), 0.724±0.028μg/g brain weight (1/2PA+BK, n=10, p<0.01), 0.515±0.064μg/g brain weight (1/2PA+1/2BK, n=10, p<0.01); PA+BK, PA+1/2BK, 1/2PA+BK group vs. BK or PA group, p<0.01. 2. Results of Western blot analysisCompared with the integrated density value (IDV) ratio of claudin-5 to P-actin in control group was 0.397±0.009(n=8).The IDV ratios of claudin-5 to p-actin in experimental groups were 0.327±0.011(PA, n=8, p<0.01); 0.320±0.013(BK, n=8, p<0.01); 0.227±0.015(PA+BK,n=8, p<0.01); 0.262±0.019(1/2 PA+BK, n=8, p<0.01); 0.277±0.011(PA+1/2BK,n=8, p<0.01); 0.312±0.042(1/2PA+1/2BK, n=8, p<0.01); PA+BK,PA+1/2BK,1/2PA+BKgroup vs. BK or PA group, p<0.01.3. Results of Immunohistochemistry analysisIn control group, claudin-5 expressed mainly on tumor capillaries in brown blot or brown line. There was a significant decrease in claudin-5-like immunoreactivity in each experimental group(p<0.01).PA+BK> PA+1/2BK,1/2PA+BK group vs. BK or PA group, p<0.01. The mean optical density value in each group was 0.277±0.011 (control, n=8), 0.249±0.010(PA, n=8), 0.243±0.007(BK, n=8), 0.213±0.017(PA+BK, n=8), 0.218±0.014, (1/2PA+BKn=8), 0.224±0.017(PA+1/2BK, n=8), 0.252±0.013(1/2PA+1/2 BK, n=8) respectively.4. Regression analysis between BTB permeability and claudin-5 expression levelThe claudin-5 expression level decreased accompanying with the increasing of BTB permeability. There was a significant negative correlation between BTB permeability and claudin-5 expression level (y=0.4441-0.2293x, r=0.977, P<0.01).DiscussionThis study used the C6 glioma model to prove that the combination of BK and PA had synergistic effect on the opening of BTB, both of them may reduce the expression level of claudin-5, there was a significant negative-correlation between the claudin-5 expression level and BTB permeability.Bradykinin and its analogue cereport could open BTB selectively and reversibly. Previous study showed the mechanism of BK selective opening the BTB involved Ca²⁺ inflow, NO, cAMP and cGMP et al. Because BK could induce the tachyphylaxis which reduced its application on the clinical, PA was the familiar vasodilator agent, we can used its potent vasodilating effects to open the BBB. At present, it is thought that this substance acts by inhibiting cAMP and cGMP phosphodiesterase activation in smooth muscle cells and increasing the turnover of intracellular cAMP and cGMP. We found the same second messenger on the function approach of BK and PA, so that we speculated these two medicines may play an active role through the same approach and had the same synergistic effect. Through the combination of different dose of BK and PA, we found, the opening degree of BTB increase significantly comparing the combination with single administration of the two drugs.Study indicated TJ was the key factor to keep the integrity of BBB.TJ constituted the major barrier of ion and molecule passing membrane transport through the paracellular pathway. TJ was constituted by a group of molecular protein elements, such as claudins, occludin. Claudin-5 is the brain microvascular endothelial cell-specific protein in the TJ. Study found that claudin-5 was the necessary and sufficient condition in the constitution of TJ. The permeability of glioma patients had closely relation with the decreasing of claudin-5. In this study, BK and PA may reduce the expression level of claudin-5 on endothelial cells of tumor vasculature, combination made to reduce the expression level further. There is a linear regression between the claudin-5 expression level and opening of BTB, which indicated claudin-5 was the action target in the combination of two drugs, caused the TJ open and increase the BTB permeability, resulted in synergistic effect. Liu L B etc. demonstrated BK could increase the BTB permeability through reduced the expression level of occludin, claudin-5 and ZO-1, which is consistent with our study.PA increases intracellular cAMP and cGMP by inhibiting cAMP and cGMP phosphodiesterase activation. Ishizaki etc study showed that cAMP could promote phosphorlation of claudin-5 on threonine residues and increase expression of claudin-5 in porcine blood-brain barrier endothelial cells via a protein kinase A-dependent/independent pathway, and then promote TJ's function. Oak etc study found an increase of cGMP in the pericapillary area could increase BBB permeability. Shinji Fukui also found cGMP anague may significantly increase the permeability of BBB. Similarly, increase the level of cGMP can increase the permeability of tumor microvaculature. Evidently, the achievement of BK and PA down-regulated the claudin-5 expression level may through the increase of cGMP/ decrease of cAMP signal pathway.This study proved that combination of BK and PA existed the synergistic effect on the selective increasing the BTB permeability, and primarily proved this effect has relation with the decrease of claudin-5 expression level. The study result indicated that it may be considered to combine the BK and PA to further increase the concentration of drug which getting into the tumor tissue, and provided a new approach for the treatment of brain tumors.Conclusion1.The combination of BK and PA has synergistic effect on selective opening of BTB.2.Claudin-5 expression decrease is one of major elements of BK and PA increasing BTB permeability; there is a significant negative correlation between claudin-5 expression level and BTB opening degree by administering BK in combination with PA.