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Zafirlukast protects blood-brain barrier integrity from ischemic brain injury
Journal Pre-proof Zafirlukast protects blood-brain barrier integrity from ischemic brain injury Chaosheng Zeng, Desheng Wang, Cong Chen, Lin Chen, Bocan Chen, Li Li, Min Chen, Huaijie Xing PII:
S0009-2797(19)31589-3
DOI:
https://doi.org/10.1016/j.cbi.2019.108915
Reference:
CBI 108915
To appear in:
Chemico-Biological Interactions
Received Date: 20 September 2019 Revised Date:
22 November 2019
Accepted Date: 5 December 2019
Please cite this article as: C. Zeng, D. Wang, C. Chen, L. Chen, B. Chen, L. Li, M. Chen, H. Xing, Zafirlukast protects blood-brain barrier integrity from ischemic brain injury, Chemico-Biological Interactions (2020), doi: https://doi.org/10.1016/j.cbi.2019.108915. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
Author contribution statement Chaosheng Zeng: Conceptualization, Methodology, Investigation, Software, Formal analysis, Writing-Review & Editing; Desheng Wang: Conceptualization, Methodology, Investigation, Software, Formal analysis, Writing-Review & Editing; Cong Chen: Supervision, Conceptualization, Writing-Original Draft, Writing-Review & Editing, Project administration, Data Curation; Lin Chen: Resources, Methodology, Investigation; Bocan Chen: Resources, Methodology, Investigation; Li Li: Resources, Methodology, Investigation; Min Chen: Resources, Methodology, Investigation; Huaijie Xing: Resources, Methodology, Investigation.
Title: Zafirlukast protects Blood-Brain Barrier Integrity from Ischemic Brain Injury Authors: Chaosheng Zeng#, Desheng Wang#, Cong Chen*, Lin Chen, Bocan Chen, Li Li, Min Chen, Huaijie Xing Affiliation Department of Neurology, The Second Affiliated Hospital of Hainan Medical University, Haikou city, Hainan Province, China. *, Corresponding author: Dr. Cong Chen Department of Neurology, The Second Affiliated Hospital of Hainan Medical University, No. 48, Baishuitang Road, Longhua District, Haikou city, 570311, Hainan Province, China. Tel: +86-0898-66809446; Fax: +86-0898-66809446; Email: [email protected] #
These authors contributed equally to this work.
Abstract Stroke has been considered the second leading cause of death worldwide, and ischemic stroke accounts for the vast majority of stroke cases. Some of the main features of ischemic stroke are increased brain permeability, ischemia/reperfusion injury, oxidative stress, and acute inflammation. Antagonism of cysLT1R has been shown to provide cardiovascular and neural benefits. In the present study, we investigated the effects of the cysLT1R antagonist zafirlukast both in vivo and in vitro using a middle cerebral artery occlusion (MCAO) mouse model and human brain microvascular endothelial cells (HBMVECs). In vivo, we found that zafirlukast pretreatment could reduce MCAO-induced increased brain permeability by rescuing the expression levels of the tight junction proteins occludin and ZO-1. In vitro, we found that zafirlukast could suppress the increase in endothelial monolayer permeability induced by OGD/R via rescue of occludin and ZO-1 expression; additionally, we found that zafirlukast prevented OGD/R-induced degradation of the extracellular matrix via inhibition of MMP-2 and MMP-9 expression. Finally, we found that zafirlukast could also inhibit OGD/R-induced activation of the critical proinflammatory regulator NF-κB by preventing phosphorylation and nuclear translocation of p65 protein. Together, our findings demonstrate a promising role for zafirlukast in preventing damage induced by ischemic stroke and reperfusion injury. Keywords: Zafirlukast; brain endothelial cells; brain permeability; NF-κB; tight junction; ZO-1
1. Introduction Stroke is the second leading cause of death around the globe, and in cases of survival, the patient is often left with significantly declined physical and/or cognitive function. According to the American Stroke Association, ischemic stroke accounts for 87% of all stroke cases. In ischemic stroke, the flow of blood to the brain is disrupted, usually due to blockage of a vessel, resulting in the tissue of the brain being starved of essential oxygen and nutrients. This is usually due to occlusion of the middle cerebral artery or its branches [1]. Thus, middle cerebral artery occlusion (MCAO) rodent models are a popular study model for stroke. Treatment for stroke is time-sensitive, and the window for treatment is only 4.5 hours after the occurrence of stroke [2]. Brain microvascular endothelial cells (BMVECs) along with other cell types comprise the blood-brain barrier (BBB), which plays a pivotal role in maintaining separation and homeostasis between the vasculature of the brain and the central nervous system (CNS). The BBB has selective permeability, allowing certain cell types, substances, and signaling molecules to travel freely between the circulatory system and the CNS. However, upon disruption of its function, the permeability of the BBB is altered, and a prolonged and robust inflammatory response is triggered [3;4]. Ischemia and subsequent reperfusion cause further injury to BMVECs, thereby initiating an increase in permeability and eventual breakdown of the BBB. This is referred to as oxygen-glucose deprivation and reoxygenation (OGD/R) injury. BMVECs also facilitate trans-membrane molecular transport through the BBB using tight-junction proteins. Tight junction proteins play a central role in regulating BBB permeability and maintaining the closeness of BMVECs. Of these, occludin regulates the fence function of the BBB while zonula occludens-1 (ZO-1) anchors occludin and other transmembrane proteins to the actin skeleton. Downregulation of occludin and ZO-1 is considered an indicator of BBB damage [5;6]. In addition, proteolytic enzymes such as matrix metalloproteinase-2 (MMP-2) and MMP-9 contribute to ischemia-induced breakdown of the BBB by degrading the components of the extracellular matrix and increasing the inflammatory response. However, the role of MMPs is complex. In their inactive form, MMPs confer protective effects on cells, but upon activation such as by ischemia, they cause damage to BMVECs and interrupt the function of the BBB [7]. MMP-2
has been shown to play a role in acute neuronal injury and delayed healing, while MMP-9 is implicated in increasing infarct volume and neurological decline [8]. Nuclear factor-κB (NF-κB) is regarded as a critical regulator of inflammation and has been considered a significant factor in neuronal damage following ischemic stroke. In normal conditions, NF-κB is sequestered in the cytoplasm, but upon cellular injury, the p65 protein component of NF-κB is translocated to the nucleus. This results in the activation of a wide array of pro-inflammatory cytokines [9]. Thus, inhibiting the activation of NF-κB signaling is an attractive therapeutic option for combating inflammatory diseases, including stroke. Zafirlukast is a cysteinyl-leukotriene type 1 receptor (CysLT1R) antagonist, which is commonly used for the treatment of asthma. Interestingly, adult-onset asthma has been associated with 2-fold increased risk of stroke and coronary heart disease in women [10]. CysLTs are recognized as potent inflammatory mediators that play a role in ischemia/reperfusion-induced injury. Zafirlukast has been shown to exert neuroprotective effects in a rat model of vascular dementia as well as cardioprotective effects [11;12]. Along with zafirlukast, other CysLT1R antagonists such as montelukast and pranlukast have also been shown to have potential protective effects against cerebral events, as per a recent review [13]. However, there is currently little research on the exact mechanisms of these potential effects. In the present study, we examined the effects of zafirlukast on ischemic stroke using three mouse models: a sham group, an MCAO + placebo group, and an MCAO + zafirlukast group. Also, using an in vitro BMVECs cell model, we tested the beneficial effects of zafirlukast against OGD/R-induced endothelial monolayer permeability and explored the underlying molecular mechanisms. 2. Materials and methods 2.1 Mouse MCAO model and drug administration C57/BL6 mice were purchased from Jackson Laboratory. All animal experiment protocols used in this study were approved by the animal care committee of Hainan Medical University. Briefly, occlusion of the middle cerebral artery was performed in anesthetized animals in the
MCAO + placebo and MCAO + zafirlukast groups by introducing a surgical filament and closing with sutures. Successful ischemia/reperfusion injury was confirmed by assessing neurological deficit in all mice once they had recovered from anesthesia. Infarct volume was assessed by TTC staining. For drug treatment, 10 mg/kg zafirlukast was injected intraperitoneally twice daily for 14 days, and during the MCAO experiment. 2.2 Neurological deficit scoring method To evaluate the severity of neurological deficit in mice [14], a five-point grading scale was used, wherein grade 0 = no visible sign of neurological deficit; grade 1 = failure to fully extend the contralateral forepaw when held by the tail; grade 2 = animals move in circles around the ipsilateral side; more severe, grade 3 = animal falls to the side contralateral to brain damage; grade 4 = animals stop moving and display minimal signs of consciousness. 2.3 Blood-brain barrier vascular leakage assay Evans blue dye staining was used to assess BBB permeability in vivo. Briefly, 2% Evans blue dye (EBD; 4 ml/kg) was injected into the tail vein immediately following stereotactic injection. As a negative control, sham mice with burr holes only were used. At the end of the experiment, mice in all groups were sacrificed and perfused with saline. Mice brains were collected as quickly as possible. Then, the brains were weighed and homogenized in 50% trichloroacetic acid (TCA). The resulting fluorescence intensity was measured with excitation at 620 nm and emission at 680 nm to index the concentration of EBD. 2.4 Tight junction immunostaining Next, we determined the integrity of the BBB by staining the tight junction proteins occludin and ZO-1 with corresponding antibodies. Briefly, the mice in each group were sacrificed and perfused using phosphate buffer saline (PBS). Whole-brain tissues were then sliced into 8 µm thick sections and embedded using OCT compound. Next, the sections were stained with anti-occludin and anti-ZO-1 primary antibodies, and Alexa Fluor 594-labeled anti-mouse secondary antibody. The images were visualized using a Zeiss fluorescent microscope.
2.5 Cell culture, OGD/R, and treatment HBMVECs used in all in vitro experiments were obtained from Cell Systems. Zafirlukast was purchased from Sigma-Aldrich and dissolved in dimethyl sulfoxide (DMSO). HBMVECs were maintained using a Cell Systems complete medium kit with 10% serum and CultureBoost-R™. For OGD, HBMVECs were incubated in an air-tight incubator with deoxygenated media for 6 h and then flushed with 1% O2, 5% CO2, and 94% NO. The cells were then reperfused in normal culture media under normoxic conditions (21% O2, 5% CO2) at 37 °C in the presence or absence of 2.5 and 5 µM zafirlukast. 2.6 Endothelial cell permeability in vitro assay To determine the permeability of the endothelial layer in vitro, we employed a 24-well receiver plate with 24 individual hanging cell culture inserts. Briefly, HBMVECs were seeded onto collagen-coated inserts. The resulting endothelial monolayer was exposed to OGD for 6 h followed by reperfusion media for 24 h in the presence or absence of 2.5 and 5 µM zafirlukast. FITC-Dextran was then applied on top of the cells, allowing the fluorescent molecules to pass through the endothelial cell monolayer. The rate at which the molecules passed through the monolayer was proportional to the permeability of the monolayer. To determine the extent of permeability, the fluorescent signal of the receiver plate well solution was measured. The fluorescence intensity was normalized to basal conditions and comparisons were made at multiple time points. 2.7 Real-time PCR analysis Total RNA was extracted from HBMVECs using an RNeasy Micro kit from Qiagen (Cat.74004). A Nanodrop spectrophotometer was used to quantify the RNA concentration. Then, 1 µg total RNA was used to synthesize cDNA with iScript™ reverse transcription Supermix for RT-qPCR (Invitrogen (Cat. 1708840)). SYBR-based real-time PCR was performed to detect the total mRNA transcripts of occludin, ZO-1, MMP-2, and MMP-9 on the ABI 7500 platform.
2.8 Western blot analysis HBMVECs under the different conditions were lysed using radio-immunoprecipitation assay (RIPA) buffer supplemented with protease inhibitor cocktail. Then, 20 µg total cell lysates were loaded into 4-20% precasted polyacrylamide gel electrophoresis (PAGE) gels in order to separate the proteins according to size. The separated protein mixture was then transferred onto polyvinylidene fluoride (PVDF) membranes, and the protein expression levels were determined by blotting with specific antibodies [15]. The following antibodies were used in this study: occludin, ZO-1, MMP-2, MMP-9, and β-actin. 2.9 Statistical analysis All experimental results are expressed as means ± standard deviation (SD). The SPSS statistical software package (v15.0) was used to assess the significance of differences between groups in this study. Statistical significance was assessed using analysis of variance (ANOVA). A P value of < 0.05 was considered to be statistically significant. 3. Results 3.1 Zafirlukast protects against MCAO-induced brain infarction and neurological deficit First, we employed an MCAO mouse model to determine the effects of zafirlukast in vivo. Infarct volume was calculated based on imaging results. As shown in Figure 1, MCAO + placebo mice had an infarct volume of 32.6% over baseline, which was only 15.4% in the MCAO + zafirlukast group, thereby indicating a reduction in infarct volume of over 50% after pretreatment with zafirlukast. Next, we measured the neurological deficit score of mice in the three groups. As shown in Figure 2, in the MCAO + placebo group, the neurological deficit was 3.3, which was reduced to only 1.8 in the MCAO + zafirlukast group, indicating a neuroprotective effect of cysLT1R antagonism by zafirlukast. 3.2 Zafirlukast protects against MCAO-induced increase in brain permeability and reduced expression of occludin and ZO-1
To determine the impact of pretreatment with zafirlukast on brain permeability, we employed the Evans blue staining method. As shown in Figure 3, the MCAO + placebo group had a 0.7-fold increase in brain permeability, while the MCAO + zafirlukast group had a 0.2-fold increase in brain permeability as compared to the sham control group. To determine whether this improvement in brain permeability was due to changes in the expression of tight junction proteins, we measured the expression levels of occludin and ZO-1 via real-time PCR and immunostaining. As shown in Figure 4, the mRNA expression of occludin was reduced by 54% in the MCAO + placebo group, but only 11% in the MCAO + zafirlukast group. The results of immunostaining show that the protein expression of occludin was reduced by 44% in the MCAO + placebo group, and only 4% in the MCAO + zafirlukast group, thereby demonstrating a significant protective effect of zafirlukast against MCAO-induced reduced occludin and ZO-1 expression. 3.3 Zafirlukast suppresses OGD/R-induced increase in endothelial monolayer permeability and reduction of occludin and ZO-1 Next, we performed a series of in vitro experiments using HBMVECs exposed to OGD/R in the presence or absence of 2.5 and 5 µM zafirlukast. Endothelial permeability was determined via FITC-dextran permeation. As shown in Figure 5, endothelial permeability was low at baseline, but significantly increased by OGD/R. However, treatment with the two doses of zafirlukast reduced endothelial permeability in a dose-dependent manner. As above, we then measured the expression levels of occludin and ZO-1 in the presence or absence of the two doses of zafirlukast. As shown in Figure 6A, OGD/R reduced the mRNA expression of occludin by 61%, which was rescued to reductions of only 35% and 9% by the two respective doses of zafirlukast. The mRNA expression of ZO-1 was reduced by 55%, but only 28% and 2% by 2.5 and 5 µM zafirlukast. At the protein level, we saw even more significant improvement. OGD/R reduced occludin protein expression by 55%, but only 31% and 2% after pretreatment with zafirlukast. The protein expression of ZO-1 was reduced by 45% after OGD/R, but only 19% after pretreatment with 2.5 µM zafirlukast and remarkably, 5 µM zafirlukast increased ZO-1 protein expression to 3% over baseline (Figure 6B). Thus, zafirlukast pretreatment has a remarkable positive effect on the expression of the tight
junction proteins occludin and ZO-1 after OGD/R. 3.4 Zafirlukast prevented OGD/R-induced MMP-2 and MMP-9 expression Real-time PCR and ELISA analyses were used to determine the effects of zafirlukast on OGD/R-induced expression of MMP-2 and MMP-9. As shown in Figure 7A, the mRNA expression of MMP-2 and MMP-9 was increased to 3.8- and 4.3-fold by OGD/R, while the dose of 2.5 µM reduced these values to 2.3- and 2.6-fold, respectively, and the 5 µM dose further reduced the mRNA expression of these two proteoglycans to only 1.5-fold. At the protein level, the concentration of MMP-2 was 366.8 pg/ml at baseline and 2678.9 pg/ml after OGD/R. For MMP-9, the protein concentration increased from 423.1 to 3822.9 pg/ml upon OGD/R. However, 2.5 and 5 µM zafirlukast mitigated the increase in MMP-2 expression to only 1883.5 and 1062.4 pg/ml, and that of MMP-9 to 2877.6 and 1536.7 pg/ml, respectively (Figure 7B). Thus, the reduction in brain permeability may be due in part to zafirlukast-mediated reduced expression of MMP-2 and MMP-9. 3.5 Zafirlukast inhibited OGD/R-induced activation of NF-κB Activation of the NF-κB pathway has been associated with the degradation of tight junction proteins. Finally, we investigated the effects of zafirlukast on OGD/R-induced activation of the NF-κB proinflammatory signaling pathway. Using lamin B as a control, we first measured the nuclear translocation of p65 protein. As shown in Figure 8A, OGD/R increased the nuclear translocation of p65 3.9-fold, which was reduced to only 2.7- and 1.7-fold by 2.5 and 5 µM zafirlukast. As shown in Figure 8B, the results of luciferase reporter assay show that OGD/R significantly increased NF-κB activation to 167.8-fold, which was remarkably reduced to only 67.4 and 32.8-fold by the two doses of zafirlukast. 4. Discussion Ischemic stroke is a major threat to human life throughout the world. However, treatment remains challenging, due in part to a lack of understanding of the mechanisms involved in ischemic neurovascular injury. In the present study, we investigated the role of the cysLT1R
antagonist zafirlukast in mitigating the effects of ischemic stroke both in vivo using a MCAO mouse model and in vitro using HBMVECs. Brain infarct volume is a significant independent determining factor of functional outcome after ischemic stroke and is often assessed in MCAO mouse models to indicate stroke severity [16;17]. Here, we found that pretreatment with zafirlukast at a concentration of 10 mg/kg twice daily for 14 days prior to and during MCAO led to a reduction in infarct volume of about half as compared to the placebo group, which is a significant improvement. Mitigating neurological deficit following stroke is a key goal of any stroke treatment. MCAO mouse model studies have found that pretreatment with montelukast, another cysLT1R antagonist, can reduce infarct volume and improve ischemia-induced neurological deficit, brain edema, and neuron density [13;18]. These findings are concordant with our own results showing that cysLT1R antagonism with zafirlukast significantly reduced infarct volume and neurological deficit score in MCAO mice. During our in vivo MCAO model experiments, we also found that pretreatment with zafirlukast significantly ameliorated MCAO-induced increased BBB permeability. A contemporary study, which also used both an MCAO mouse model and HBMVECs exposed to OGD/R, found similar effects of montelukast. Namely, montelukast pretreatment reduced ischemia-induced BBB permeability, which was attributed to decreased expression of MMP-2 and MMP-9, as well as rescued expression of occludin and ZO-1 [19]. Thus, antagonism of cysLT1R has a neuroprotective effect by reducing infarct volume, improving neurological deficit score, and reducing brain permeability via enhanced expression of tight junction proteins. These findings were confirmed both in vivo and in vitro. CysLTs have been shown to increase brain permeability and ischemic injury. Preventing the synthesis of cysLTs via inhibition of 5-lipoxygenase activating protein has been suggested as a potential treatment strategy against increased brain permeability and inflammation induced by traumatic brain injury [20; 21]. The role of proteoglycans in ischemic stroke has been thoroughly studied. Upon ischemic stroke, elevated expression of MMP-2 and MMP-9 leads to excessive breakdown of the BBB, thereby promoting increased infarct volume and inflammation. Additionally, MMP-2 has been associated with neuronal injury and impaired repair mechanisms, while MMP-9 is associated with increased infarct volume and
neurological deficit [22]. Here, we found that HBMVECs exposed to OGD/R injury expressed roughly 8-fold higher amounts of MMP-2 and MMP-9, while pretreatment with zafirlukast significantly reduced this increase to only around 2-fold. These findings are consistent with our in vivo findings that zafirlukast could reduce infarct volume and improve neurological deficit. The NF-κB cellular signaling pathway is widely regarded as a master regulator of the inflammatory response. Inflammation is a significant contributor to brain injury and declined neural function after ischemic stroke. Numerous studies have demonstrated that inhibition of NF-κB activation during ischemia/reperfusion can protect the brain from further damage and improve neurological deficit [23-25]. Here, we found that zafirlukast suppressed OGD/R-induced NF-κB activation through inhibition of the phosphorylation of p65 protein. Previous research has shown that montelukast can inhibit NF-κB activation by suppressing the nuclear translocation of p65 [26]. However, this is the first study to our knowledge identifying a similar effect of zafirlukast. Taken together, our findings demonstrate the potential of cysLT1R antagonism by zafirlukast as a preventative treatment against the deleterious effects of ischemic stroke by reducing infarct volume, improving neurological deficit, maintaining BBB stability, and attenuating NF-κB-mediated inflammatory response. Rescue of the expression of the tight junction proteins occludin and ZO-1, as well as inhibition of MMP-2 and MMP-9 expression, play a major role in facilitating the benefits of zafirlukast described above. Additional research is necessary to better understand the exact mechanisms through which the cysLT1R antagonist zafirlukast protects against ischemia-induced brain injury. References [1] Tressera-Rimbau A, Arranz S, Eder M, Vallverdú-Queralt A. Dietary Polyphenols in the Prevention of Stroke. Oxid Med Cell Longev. 2017:7467962. [2] Kassner A, Merali Z. Assessment of Blood-Brain Barrier Disruption in Stroke. Stroke. 2015; 46(11):3310-5. [3] Doll DN, Hu H, Sun J, Lewis SE, Simpkins JW, Ren X. Mitochondrial crisis in
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Fig. 1. Zafirlukast protects against middle cerebral artery occlusion (MCAO)-induced brain infarction in mice. Experimental mice were divided into three groups: non-treated sham mice, MCAO mice with placebo, and MCAO mice treated with zafirlukast (10 mg/kg body weight, twice a day) for 2 weeks before the MCAO experiment and during the MCAO experiment. Representative brain infarction images and quantification of infarction volume (****, P<0.0001 vs. sham group; ##, P<0.01 vs. MCAO group). Fig. 2. Zafirlukast protects against middle cerebral artery occlusion (MCAO)-induced neurological deficit in mice. Neurological deficit scores of mice were measured (****, P<0.0001 vs. sham group; ##, P<0.01 vs. MCAO group). Fig. 3. Zafirlukast protects against middle cerebral artery occlusion (MCAO)-induced increase in brain permeability in mice. Brain permeability of MCAO mice was measured by Evans blue staining (****, P<0.0001 vs. sham group; ##, P<0.01 vs. MCAO group). Fig. 4. Zafirlukast prevents middle cerebral artery occlusion (MCAO)-induced reduction in the expressions of tight junction proteins such as occludin and ZO-1. (A). mRNA levels of occludin and ZO-1; (B). Representative immunostaining of occludin and ZO-1 (***, P<0.001 vs. sham group; ###, P<0.001 vs. MCAO group). Fig. 5. Zafirlukast protects primary human brain microvascular endothelial cells (HBMVECs) against oxygen-glucose deprivation/reperfusion (OGD/R)-induced endothelial monolayer permeability. HBMVECs were exposed to OGD for 6 h, followed by exposure to reperfusion media for 24 h in the presence or absence of Zzafirlukast (2.5, 5 µM). Endothelial permeability was measured by FITC-dextran permeation (****, P<0.0001 vs. sham group; ###, P<0.001, ####, P<0.0001 vs. OGR/R group). Fig. 6. Zafirlukast restored OGD/R-induced reduction of occludin and ZO-1 in HBMVECs. HBMVECs were exposed to OGD for 6 h, followed by exposure to reperfusion media for 24 h in the presence or absence of Zafirlukast (2.5, 5 µM). (A). mRNA levels of occludin and ZO-1; (B). Protein levels of occludin and ZO-1 as measured by western blot analysis (***, P<0.001 vs. sham group; ##, P<0.01, ###, P<0.001 vs. OGR/R group).
Fig. 7. Zafirlukast prevented OGD/R-induced increase in MMP-2 and MMP-9 in HBMVECs. HBMVECs were exposed to OGD for 6 h, followed by exposure to reperfusion media for 24 h in the presence or absence of Zafirlukast (2.5, 5 µM). (A). mRNA levels of MMP-2 and MMP-9; (B). Protein levels of MMP-2 and MMP-9 as measured by ELISA (****, P<0.001 vs. sham group; ##, P<0.01, ###, P<0.001 vs. OGR/R group). Fig. 8. Zafirlukast inhibited OGD/R-induced activation of NF-κB in HBMVECs. HBMVECs were exposed to OGD for 6 h, followed by exposure to reperfusion media for 24 h in the presence or absence of Zafirlukast (2.5, 5 µM). (A). Nuclear levels of NF-κB p65 with Lamin B1 as an internal control; (B). Luciferase activity of NF-κB (****, P<0.0001 vs. sham group; ###, P<0.001, ####, P<0.0001 vs. OGR/R group).
Highlights 1.
Zafirlukast protected against MCAO-induced brain infarction and neurological
deficit; 2.
Zafirlukast protected against MCAO-induced increase in brain permeability and
reduction of occludin and ZO-1 3.
Zafirlukast suppressed OGD/R-induced endothelial monolayer permeability
4.
Zafirlukast prevented OGD/R-induced MMP-2 and MMP-9 expression and
NF-κB activation
Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
S0009-2797(19)31589-3
DOI:
https://doi.org/10.1016/j.cbi.2019.108915
Reference:
CBI 108915
To appear in:
Chemico-Biological Interactions
Received Date: 20 September 2019 Revised Date:
22 November 2019
Accepted Date: 5 December 2019
Please cite this article as: C. Zeng, D. Wang, C. Chen, L. Chen, B. Chen, L. Li, M. Chen, H. Xing, Zafirlukast protects blood-brain barrier integrity from ischemic brain injury, Chemico-Biological Interactions (2020), doi: https://doi.org/10.1016/j.cbi.2019.108915. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
Author contribution statement Chaosheng Zeng: Conceptualization, Methodology, Investigation, Software, Formal analysis, Writing-Review & Editing; Desheng Wang: Conceptualization, Methodology, Investigation, Software, Formal analysis, Writing-Review & Editing; Cong Chen: Supervision, Conceptualization, Writing-Original Draft, Writing-Review & Editing, Project administration, Data Curation; Lin Chen: Resources, Methodology, Investigation; Bocan Chen: Resources, Methodology, Investigation; Li Li: Resources, Methodology, Investigation; Min Chen: Resources, Methodology, Investigation; Huaijie Xing: Resources, Methodology, Investigation.
Title: Zafirlukast protects Blood-Brain Barrier Integrity from Ischemic Brain Injury Authors: Chaosheng Zeng#, Desheng Wang#, Cong Chen*, Lin Chen, Bocan Chen, Li Li, Min Chen, Huaijie Xing Affiliation Department of Neurology, The Second Affiliated Hospital of Hainan Medical University, Haikou city, Hainan Province, China. *, Corresponding author: Dr. Cong Chen Department of Neurology, The Second Affiliated Hospital of Hainan Medical University, No. 48, Baishuitang Road, Longhua District, Haikou city, 570311, Hainan Province, China. Tel: +86-0898-66809446; Fax: +86-0898-66809446; Email: [email protected] #
These authors contributed equally to this work.
Abstract Stroke has been considered the second leading cause of death worldwide, and ischemic stroke accounts for the vast majority of stroke cases. Some of the main features of ischemic stroke are increased brain permeability, ischemia/reperfusion injury, oxidative stress, and acute inflammation. Antagonism of cysLT1R has been shown to provide cardiovascular and neural benefits. In the present study, we investigated the effects of the cysLT1R antagonist zafirlukast both in vivo and in vitro using a middle cerebral artery occlusion (MCAO) mouse model and human brain microvascular endothelial cells (HBMVECs). In vivo, we found that zafirlukast pretreatment could reduce MCAO-induced increased brain permeability by rescuing the expression levels of the tight junction proteins occludin and ZO-1. In vitro, we found that zafirlukast could suppress the increase in endothelial monolayer permeability induced by OGD/R via rescue of occludin and ZO-1 expression; additionally, we found that zafirlukast prevented OGD/R-induced degradation of the extracellular matrix via inhibition of MMP-2 and MMP-9 expression. Finally, we found that zafirlukast could also inhibit OGD/R-induced activation of the critical proinflammatory regulator NF-κB by preventing phosphorylation and nuclear translocation of p65 protein. Together, our findings demonstrate a promising role for zafirlukast in preventing damage induced by ischemic stroke and reperfusion injury. Keywords: Zafirlukast; brain endothelial cells; brain permeability; NF-κB; tight junction; ZO-1
1. Introduction Stroke is the second leading cause of death around the globe, and in cases of survival, the patient is often left with significantly declined physical and/or cognitive function. According to the American Stroke Association, ischemic stroke accounts for 87% of all stroke cases. In ischemic stroke, the flow of blood to the brain is disrupted, usually due to blockage of a vessel, resulting in the tissue of the brain being starved of essential oxygen and nutrients. This is usually due to occlusion of the middle cerebral artery or its branches [1]. Thus, middle cerebral artery occlusion (MCAO) rodent models are a popular study model for stroke. Treatment for stroke is time-sensitive, and the window for treatment is only 4.5 hours after the occurrence of stroke [2]. Brain microvascular endothelial cells (BMVECs) along with other cell types comprise the blood-brain barrier (BBB), which plays a pivotal role in maintaining separation and homeostasis between the vasculature of the brain and the central nervous system (CNS). The BBB has selective permeability, allowing certain cell types, substances, and signaling molecules to travel freely between the circulatory system and the CNS. However, upon disruption of its function, the permeability of the BBB is altered, and a prolonged and robust inflammatory response is triggered [3;4]. Ischemia and subsequent reperfusion cause further injury to BMVECs, thereby initiating an increase in permeability and eventual breakdown of the BBB. This is referred to as oxygen-glucose deprivation and reoxygenation (OGD/R) injury. BMVECs also facilitate trans-membrane molecular transport through the BBB using tight-junction proteins. Tight junction proteins play a central role in regulating BBB permeability and maintaining the closeness of BMVECs. Of these, occludin regulates the fence function of the BBB while zonula occludens-1 (ZO-1) anchors occludin and other transmembrane proteins to the actin skeleton. Downregulation of occludin and ZO-1 is considered an indicator of BBB damage [5;6]. In addition, proteolytic enzymes such as matrix metalloproteinase-2 (MMP-2) and MMP-9 contribute to ischemia-induced breakdown of the BBB by degrading the components of the extracellular matrix and increasing the inflammatory response. However, the role of MMPs is complex. In their inactive form, MMPs confer protective effects on cells, but upon activation such as by ischemia, they cause damage to BMVECs and interrupt the function of the BBB [7]. MMP-2
has been shown to play a role in acute neuronal injury and delayed healing, while MMP-9 is implicated in increasing infarct volume and neurological decline [8]. Nuclear factor-κB (NF-κB) is regarded as a critical regulator of inflammation and has been considered a significant factor in neuronal damage following ischemic stroke. In normal conditions, NF-κB is sequestered in the cytoplasm, but upon cellular injury, the p65 protein component of NF-κB is translocated to the nucleus. This results in the activation of a wide array of pro-inflammatory cytokines [9]. Thus, inhibiting the activation of NF-κB signaling is an attractive therapeutic option for combating inflammatory diseases, including stroke. Zafirlukast is a cysteinyl-leukotriene type 1 receptor (CysLT1R) antagonist, which is commonly used for the treatment of asthma. Interestingly, adult-onset asthma has been associated with 2-fold increased risk of stroke and coronary heart disease in women [10]. CysLTs are recognized as potent inflammatory mediators that play a role in ischemia/reperfusion-induced injury. Zafirlukast has been shown to exert neuroprotective effects in a rat model of vascular dementia as well as cardioprotective effects [11;12]. Along with zafirlukast, other CysLT1R antagonists such as montelukast and pranlukast have also been shown to have potential protective effects against cerebral events, as per a recent review [13]. However, there is currently little research on the exact mechanisms of these potential effects. In the present study, we examined the effects of zafirlukast on ischemic stroke using three mouse models: a sham group, an MCAO + placebo group, and an MCAO + zafirlukast group. Also, using an in vitro BMVECs cell model, we tested the beneficial effects of zafirlukast against OGD/R-induced endothelial monolayer permeability and explored the underlying molecular mechanisms. 2. Materials and methods 2.1 Mouse MCAO model and drug administration C57/BL6 mice were purchased from Jackson Laboratory. All animal experiment protocols used in this study were approved by the animal care committee of Hainan Medical University. Briefly, occlusion of the middle cerebral artery was performed in anesthetized animals in the
MCAO + placebo and MCAO + zafirlukast groups by introducing a surgical filament and closing with sutures. Successful ischemia/reperfusion injury was confirmed by assessing neurological deficit in all mice once they had recovered from anesthesia. Infarct volume was assessed by TTC staining. For drug treatment, 10 mg/kg zafirlukast was injected intraperitoneally twice daily for 14 days, and during the MCAO experiment. 2.2 Neurological deficit scoring method To evaluate the severity of neurological deficit in mice [14], a five-point grading scale was used, wherein grade 0 = no visible sign of neurological deficit; grade 1 = failure to fully extend the contralateral forepaw when held by the tail; grade 2 = animals move in circles around the ipsilateral side; more severe, grade 3 = animal falls to the side contralateral to brain damage; grade 4 = animals stop moving and display minimal signs of consciousness. 2.3 Blood-brain barrier vascular leakage assay Evans blue dye staining was used to assess BBB permeability in vivo. Briefly, 2% Evans blue dye (EBD; 4 ml/kg) was injected into the tail vein immediately following stereotactic injection. As a negative control, sham mice with burr holes only were used. At the end of the experiment, mice in all groups were sacrificed and perfused with saline. Mice brains were collected as quickly as possible. Then, the brains were weighed and homogenized in 50% trichloroacetic acid (TCA). The resulting fluorescence intensity was measured with excitation at 620 nm and emission at 680 nm to index the concentration of EBD. 2.4 Tight junction immunostaining Next, we determined the integrity of the BBB by staining the tight junction proteins occludin and ZO-1 with corresponding antibodies. Briefly, the mice in each group were sacrificed and perfused using phosphate buffer saline (PBS). Whole-brain tissues were then sliced into 8 µm thick sections and embedded using OCT compound. Next, the sections were stained with anti-occludin and anti-ZO-1 primary antibodies, and Alexa Fluor 594-labeled anti-mouse secondary antibody. The images were visualized using a Zeiss fluorescent microscope.
2.5 Cell culture, OGD/R, and treatment HBMVECs used in all in vitro experiments were obtained from Cell Systems. Zafirlukast was purchased from Sigma-Aldrich and dissolved in dimethyl sulfoxide (DMSO). HBMVECs were maintained using a Cell Systems complete medium kit with 10% serum and CultureBoost-R™. For OGD, HBMVECs were incubated in an air-tight incubator with deoxygenated media for 6 h and then flushed with 1% O2, 5% CO2, and 94% NO. The cells were then reperfused in normal culture media under normoxic conditions (21% O2, 5% CO2) at 37 °C in the presence or absence of 2.5 and 5 µM zafirlukast. 2.6 Endothelial cell permeability in vitro assay To determine the permeability of the endothelial layer in vitro, we employed a 24-well receiver plate with 24 individual hanging cell culture inserts. Briefly, HBMVECs were seeded onto collagen-coated inserts. The resulting endothelial monolayer was exposed to OGD for 6 h followed by reperfusion media for 24 h in the presence or absence of 2.5 and 5 µM zafirlukast. FITC-Dextran was then applied on top of the cells, allowing the fluorescent molecules to pass through the endothelial cell monolayer. The rate at which the molecules passed through the monolayer was proportional to the permeability of the monolayer. To determine the extent of permeability, the fluorescent signal of the receiver plate well solution was measured. The fluorescence intensity was normalized to basal conditions and comparisons were made at multiple time points. 2.7 Real-time PCR analysis Total RNA was extracted from HBMVECs using an RNeasy Micro kit from Qiagen (Cat.74004). A Nanodrop spectrophotometer was used to quantify the RNA concentration. Then, 1 µg total RNA was used to synthesize cDNA with iScript™ reverse transcription Supermix for RT-qPCR (Invitrogen (Cat. 1708840)). SYBR-based real-time PCR was performed to detect the total mRNA transcripts of occludin, ZO-1, MMP-2, and MMP-9 on the ABI 7500 platform.
2.8 Western blot analysis HBMVECs under the different conditions were lysed using radio-immunoprecipitation assay (RIPA) buffer supplemented with protease inhibitor cocktail. Then, 20 µg total cell lysates were loaded into 4-20% precasted polyacrylamide gel electrophoresis (PAGE) gels in order to separate the proteins according to size. The separated protein mixture was then transferred onto polyvinylidene fluoride (PVDF) membranes, and the protein expression levels were determined by blotting with specific antibodies [15]. The following antibodies were used in this study: occludin, ZO-1, MMP-2, MMP-9, and β-actin. 2.9 Statistical analysis All experimental results are expressed as means ± standard deviation (SD). The SPSS statistical software package (v15.0) was used to assess the significance of differences between groups in this study. Statistical significance was assessed using analysis of variance (ANOVA). A P value of < 0.05 was considered to be statistically significant. 3. Results 3.1 Zafirlukast protects against MCAO-induced brain infarction and neurological deficit First, we employed an MCAO mouse model to determine the effects of zafirlukast in vivo. Infarct volume was calculated based on imaging results. As shown in Figure 1, MCAO + placebo mice had an infarct volume of 32.6% over baseline, which was only 15.4% in the MCAO + zafirlukast group, thereby indicating a reduction in infarct volume of over 50% after pretreatment with zafirlukast. Next, we measured the neurological deficit score of mice in the three groups. As shown in Figure 2, in the MCAO + placebo group, the neurological deficit was 3.3, which was reduced to only 1.8 in the MCAO + zafirlukast group, indicating a neuroprotective effect of cysLT1R antagonism by zafirlukast. 3.2 Zafirlukast protects against MCAO-induced increase in brain permeability and reduced expression of occludin and ZO-1
To determine the impact of pretreatment with zafirlukast on brain permeability, we employed the Evans blue staining method. As shown in Figure 3, the MCAO + placebo group had a 0.7-fold increase in brain permeability, while the MCAO + zafirlukast group had a 0.2-fold increase in brain permeability as compared to the sham control group. To determine whether this improvement in brain permeability was due to changes in the expression of tight junction proteins, we measured the expression levels of occludin and ZO-1 via real-time PCR and immunostaining. As shown in Figure 4, the mRNA expression of occludin was reduced by 54% in the MCAO + placebo group, but only 11% in the MCAO + zafirlukast group. The results of immunostaining show that the protein expression of occludin was reduced by 44% in the MCAO + placebo group, and only 4% in the MCAO + zafirlukast group, thereby demonstrating a significant protective effect of zafirlukast against MCAO-induced reduced occludin and ZO-1 expression. 3.3 Zafirlukast suppresses OGD/R-induced increase in endothelial monolayer permeability and reduction of occludin and ZO-1 Next, we performed a series of in vitro experiments using HBMVECs exposed to OGD/R in the presence or absence of 2.5 and 5 µM zafirlukast. Endothelial permeability was determined via FITC-dextran permeation. As shown in Figure 5, endothelial permeability was low at baseline, but significantly increased by OGD/R. However, treatment with the two doses of zafirlukast reduced endothelial permeability in a dose-dependent manner. As above, we then measured the expression levels of occludin and ZO-1 in the presence or absence of the two doses of zafirlukast. As shown in Figure 6A, OGD/R reduced the mRNA expression of occludin by 61%, which was rescued to reductions of only 35% and 9% by the two respective doses of zafirlukast. The mRNA expression of ZO-1 was reduced by 55%, but only 28% and 2% by 2.5 and 5 µM zafirlukast. At the protein level, we saw even more significant improvement. OGD/R reduced occludin protein expression by 55%, but only 31% and 2% after pretreatment with zafirlukast. The protein expression of ZO-1 was reduced by 45% after OGD/R, but only 19% after pretreatment with 2.5 µM zafirlukast and remarkably, 5 µM zafirlukast increased ZO-1 protein expression to 3% over baseline (Figure 6B). Thus, zafirlukast pretreatment has a remarkable positive effect on the expression of the tight
junction proteins occludin and ZO-1 after OGD/R. 3.4 Zafirlukast prevented OGD/R-induced MMP-2 and MMP-9 expression Real-time PCR and ELISA analyses were used to determine the effects of zafirlukast on OGD/R-induced expression of MMP-2 and MMP-9. As shown in Figure 7A, the mRNA expression of MMP-2 and MMP-9 was increased to 3.8- and 4.3-fold by OGD/R, while the dose of 2.5 µM reduced these values to 2.3- and 2.6-fold, respectively, and the 5 µM dose further reduced the mRNA expression of these two proteoglycans to only 1.5-fold. At the protein level, the concentration of MMP-2 was 366.8 pg/ml at baseline and 2678.9 pg/ml after OGD/R. For MMP-9, the protein concentration increased from 423.1 to 3822.9 pg/ml upon OGD/R. However, 2.5 and 5 µM zafirlukast mitigated the increase in MMP-2 expression to only 1883.5 and 1062.4 pg/ml, and that of MMP-9 to 2877.6 and 1536.7 pg/ml, respectively (Figure 7B). Thus, the reduction in brain permeability may be due in part to zafirlukast-mediated reduced expression of MMP-2 and MMP-9. 3.5 Zafirlukast inhibited OGD/R-induced activation of NF-κB Activation of the NF-κB pathway has been associated with the degradation of tight junction proteins. Finally, we investigated the effects of zafirlukast on OGD/R-induced activation of the NF-κB proinflammatory signaling pathway. Using lamin B as a control, we first measured the nuclear translocation of p65 protein. As shown in Figure 8A, OGD/R increased the nuclear translocation of p65 3.9-fold, which was reduced to only 2.7- and 1.7-fold by 2.5 and 5 µM zafirlukast. As shown in Figure 8B, the results of luciferase reporter assay show that OGD/R significantly increased NF-κB activation to 167.8-fold, which was remarkably reduced to only 67.4 and 32.8-fold by the two doses of zafirlukast. 4. Discussion Ischemic stroke is a major threat to human life throughout the world. However, treatment remains challenging, due in part to a lack of understanding of the mechanisms involved in ischemic neurovascular injury. In the present study, we investigated the role of the cysLT1R
antagonist zafirlukast in mitigating the effects of ischemic stroke both in vivo using a MCAO mouse model and in vitro using HBMVECs. Brain infarct volume is a significant independent determining factor of functional outcome after ischemic stroke and is often assessed in MCAO mouse models to indicate stroke severity [16;17]. Here, we found that pretreatment with zafirlukast at a concentration of 10 mg/kg twice daily for 14 days prior to and during MCAO led to a reduction in infarct volume of about half as compared to the placebo group, which is a significant improvement. Mitigating neurological deficit following stroke is a key goal of any stroke treatment. MCAO mouse model studies have found that pretreatment with montelukast, another cysLT1R antagonist, can reduce infarct volume and improve ischemia-induced neurological deficit, brain edema, and neuron density [13;18]. These findings are concordant with our own results showing that cysLT1R antagonism with zafirlukast significantly reduced infarct volume and neurological deficit score in MCAO mice. During our in vivo MCAO model experiments, we also found that pretreatment with zafirlukast significantly ameliorated MCAO-induced increased BBB permeability. A contemporary study, which also used both an MCAO mouse model and HBMVECs exposed to OGD/R, found similar effects of montelukast. Namely, montelukast pretreatment reduced ischemia-induced BBB permeability, which was attributed to decreased expression of MMP-2 and MMP-9, as well as rescued expression of occludin and ZO-1 [19]. Thus, antagonism of cysLT1R has a neuroprotective effect by reducing infarct volume, improving neurological deficit score, and reducing brain permeability via enhanced expression of tight junction proteins. These findings were confirmed both in vivo and in vitro. CysLTs have been shown to increase brain permeability and ischemic injury. Preventing the synthesis of cysLTs via inhibition of 5-lipoxygenase activating protein has been suggested as a potential treatment strategy against increased brain permeability and inflammation induced by traumatic brain injury [20; 21]. The role of proteoglycans in ischemic stroke has been thoroughly studied. Upon ischemic stroke, elevated expression of MMP-2 and MMP-9 leads to excessive breakdown of the BBB, thereby promoting increased infarct volume and inflammation. Additionally, MMP-2 has been associated with neuronal injury and impaired repair mechanisms, while MMP-9 is associated with increased infarct volume and
neurological deficit [22]. Here, we found that HBMVECs exposed to OGD/R injury expressed roughly 8-fold higher amounts of MMP-2 and MMP-9, while pretreatment with zafirlukast significantly reduced this increase to only around 2-fold. These findings are consistent with our in vivo findings that zafirlukast could reduce infarct volume and improve neurological deficit. The NF-κB cellular signaling pathway is widely regarded as a master regulator of the inflammatory response. Inflammation is a significant contributor to brain injury and declined neural function after ischemic stroke. Numerous studies have demonstrated that inhibition of NF-κB activation during ischemia/reperfusion can protect the brain from further damage and improve neurological deficit [23-25]. Here, we found that zafirlukast suppressed OGD/R-induced NF-κB activation through inhibition of the phosphorylation of p65 protein. Previous research has shown that montelukast can inhibit NF-κB activation by suppressing the nuclear translocation of p65 [26]. However, this is the first study to our knowledge identifying a similar effect of zafirlukast. Taken together, our findings demonstrate the potential of cysLT1R antagonism by zafirlukast as a preventative treatment against the deleterious effects of ischemic stroke by reducing infarct volume, improving neurological deficit, maintaining BBB stability, and attenuating NF-κB-mediated inflammatory response. Rescue of the expression of the tight junction proteins occludin and ZO-1, as well as inhibition of MMP-2 and MMP-9 expression, play a major role in facilitating the benefits of zafirlukast described above. Additional research is necessary to better understand the exact mechanisms through which the cysLT1R antagonist zafirlukast protects against ischemia-induced brain injury. References [1] Tressera-Rimbau A, Arranz S, Eder M, Vallverdú-Queralt A. Dietary Polyphenols in the Prevention of Stroke. Oxid Med Cell Longev. 2017:7467962. [2] Kassner A, Merali Z. Assessment of Blood-Brain Barrier Disruption in Stroke. Stroke. 2015; 46(11):3310-5. [3] Doll DN, Hu H, Sun J, Lewis SE, Simpkins JW, Ren X. Mitochondrial crisis in
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Fig. 1. Zafirlukast protects against middle cerebral artery occlusion (MCAO)-induced brain infarction in mice. Experimental mice were divided into three groups: non-treated sham mice, MCAO mice with placebo, and MCAO mice treated with zafirlukast (10 mg/kg body weight, twice a day) for 2 weeks before the MCAO experiment and during the MCAO experiment. Representative brain infarction images and quantification of infarction volume (****, P<0.0001 vs. sham group; ##, P<0.01 vs. MCAO group). Fig. 2. Zafirlukast protects against middle cerebral artery occlusion (MCAO)-induced neurological deficit in mice. Neurological deficit scores of mice were measured (****, P<0.0001 vs. sham group; ##, P<0.01 vs. MCAO group). Fig. 3. Zafirlukast protects against middle cerebral artery occlusion (MCAO)-induced increase in brain permeability in mice. Brain permeability of MCAO mice was measured by Evans blue staining (****, P<0.0001 vs. sham group; ##, P<0.01 vs. MCAO group). Fig. 4. Zafirlukast prevents middle cerebral artery occlusion (MCAO)-induced reduction in the expressions of tight junction proteins such as occludin and ZO-1. (A). mRNA levels of occludin and ZO-1; (B). Representative immunostaining of occludin and ZO-1 (***, P<0.001 vs. sham group; ###, P<0.001 vs. MCAO group). Fig. 5. Zafirlukast protects primary human brain microvascular endothelial cells (HBMVECs) against oxygen-glucose deprivation/reperfusion (OGD/R)-induced endothelial monolayer permeability. HBMVECs were exposed to OGD for 6 h, followed by exposure to reperfusion media for 24 h in the presence or absence of Zzafirlukast (2.5, 5 µM). Endothelial permeability was measured by FITC-dextran permeation (****, P<0.0001 vs. sham group; ###, P<0.001, ####, P<0.0001 vs. OGR/R group). Fig. 6. Zafirlukast restored OGD/R-induced reduction of occludin and ZO-1 in HBMVECs. HBMVECs were exposed to OGD for 6 h, followed by exposure to reperfusion media for 24 h in the presence or absence of Zafirlukast (2.5, 5 µM). (A). mRNA levels of occludin and ZO-1; (B). Protein levels of occludin and ZO-1 as measured by western blot analysis (***, P<0.001 vs. sham group; ##, P<0.01, ###, P<0.001 vs. OGR/R group).
Fig. 7. Zafirlukast prevented OGD/R-induced increase in MMP-2 and MMP-9 in HBMVECs. HBMVECs were exposed to OGD for 6 h, followed by exposure to reperfusion media for 24 h in the presence or absence of Zafirlukast (2.5, 5 µM). (A). mRNA levels of MMP-2 and MMP-9; (B). Protein levels of MMP-2 and MMP-9 as measured by ELISA (****, P<0.001 vs. sham group; ##, P<0.01, ###, P<0.001 vs. OGR/R group). Fig. 8. Zafirlukast inhibited OGD/R-induced activation of NF-κB in HBMVECs. HBMVECs were exposed to OGD for 6 h, followed by exposure to reperfusion media for 24 h in the presence or absence of Zafirlukast (2.5, 5 µM). (A). Nuclear levels of NF-κB p65 with Lamin B1 as an internal control; (B). Luciferase activity of NF-κB (****, P<0.0001 vs. sham group; ###, P<0.001, ####, P<0.0001 vs. OGR/R group).
Highlights 1.
Zafirlukast protected against MCAO-induced brain infarction and neurological
deficit; 2.
Zafirlukast protected against MCAO-induced increase in brain permeability and
reduction of occludin and ZO-1 3.
Zafirlukast suppressed OGD/R-induced endothelial monolayer permeability
4.
Zafirlukast prevented OGD/R-induced MMP-2 and MMP-9 expression and
NF-κB activation
Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: