reperfusion-induced blood-brain barrier impairment by increasing tight junction protein expression and decreasing inflammation, oxidative stress, and apoptosis in an in vitro system

European Journal of Pharmacology 854 (2019) 224–231

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Geniposide protects against hypoxia/reperfusion-induced blood-brain barrier impairment by increasing tight junction protein expression and decreasing inflammation, oxidative stress, and apoptosis in an in vitro system

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Changxiang Li, Xueqian Wang, Fafeng Cheng, Xin Du, Juntang Yan, Changming Zhai, Jie Mu, Qingguo Wang∗ School of Traditional Chinese Medicine Department, Beijing University of Chinese Medicine, Beijing, 100029, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Geniposide In vitro blood-brain barrier Oxygen glucose deprivation and reoxygenation Tight junction Anti-inflammatory Anti-apoptotic

The blood-brain barrier (BBB) is involved in the pathogeneses of ischemic stroke (IS). Geniposide (GEN), an iridoid glycoside isolated from Gardenia jasminoides Ellis, has been used for the treatment of IS. However, the effects of GEN on the BBB are poorly understood. In vitro disease models of the BBB could be very helpful for the elucidation of underlying mechanisms and the development of novel therapeutic strategies. Therefore, we established an in vitro BBB model composed of primary cultures of brain microvascular endothelial cells and astrocytes. We then used this in vitro model to investigate the effect of GEN on the function of the BBB. Oxygen glucose deprivation and reoxygenation (OGD/R) significantly increased permeability and cell apoptosis in this in vitro BBB model. Notably, GEN pretreatment effectively improved the BBB function by decreasing the permeability of the BBB, promoting expression of tight junction proteins (zonula occludens-1, claudin-5, and occludin) and gamma-glutamyl transpeptidase, increasing transendothelial electrical resistance, mitigating oxidative stress damage and the release of inflammatory cytokines, downregulating the expression levels of matrix metallopeptidases-9 (MMP-9) and MMP-2, and increasing the release of brain derived neurotrophic factor and glial cell derived neurotrophic factor. Therefore, GEN can ameliorate the BBB dysfunction induced by OGD/R conditions through multiple protective mechanisms. The findings suggest that GEN may be an appropriate drug for restoring the barrier function of the BBB.

1. Introduction The blood-brain barrier (BBB) is a regulatory interface that separates the circulation from the central nervous system (CNS) (Banerjee et al., 2016). The BBB consists of continuous highly specialised vascular endothelial cells, pericytes, perivascular astrocyte end-feet, and noncellular basement membranes, and the astrocytic foot process covers 99% of the capillary basement membrane (Hawkins et al., 2015). Significant features of the mature BBB include the presence of tight junction (TJ) complexes that markedly limit paracellular permeability, specific efflux transport systems, and characteristic enzymatic properties (Tóth et al., 2011). TJ proteins are a hallmark for the integrity of the BBB that composed of transmembrane proteins (claudin and occludin family proteins) and cytoplasmic proteins (zonula-occludens [ZO]-1, ZO-2, ZO-3, and cingulin), which link transmembrane proteins to the actin cytoskeleton (Bradbury, 1984). TJ proteins are responsible

for the high electrical resistance and the low paracellular permeability of the BBB (Ruck et al., 2014). Destruction of the BBB is a critical event in the development and progression of ischemic stroke (IS). The pathology caused by IS increases BBB permeability, thus leading to the swelling of the brain (Saraiva et al., 2016). Previous studies have shown that hypoxia stress can induce the breakdown of TJ proteins (Colin L et al., 2010), and disruption of TJ proteins severely impairs the integrity and functionality of the BBB and is thus involved in the progression of IS (Lv et al., 2018). The attenuation of BBB dysfunction has been demonstrated to be the valuable therapy for neuronal damage (Abbott et al., 2006), and accumulating evidence indicates that maintaining BBB function is crucial for reducing IS injury. Therefore, most of the current treatments are designed to improve the BBB. In particular, improving the impaired BBB TJ in neurological diseases is a rapidly evolving research field, and the BBB has become a promising drug target.

∗ Corresponding author. School of Traditional Chinese Medicine Department, Beijing University of Chinese Medicine, 11 Beisanhuandong Road, Chao yang District, Beijing, 100029, China. E-mail address: [email protected] (Q. Wang).

https://doi.org/10.1016/j.ejphar.2019.04.021 Received 19 March 2019; Received in revised form 5 April 2019; Accepted 8 April 2019 Available online 14 April 2019 0014-2999/ © 2019 Elsevier B.V. All rights reserved.

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Fig. 1. Outline of this study. GEN: Geniposide; EBSS: Earle's balanced salt solution; TEER: transendothelial electrical resistance; BMECs: brain microvascular endothelial cells; OGD: oxygen-glucose deprivation; BBB: Blood-brain barrier.

2.3. Immunofluorescent staining

Geniposide (GEN) is an iridoid glycoside compound purified from the fruit of Gardenia jasminoides Ellis (Shan et al., 2017). Previous studies have demonstrated that GEN protects the CNS against hypoxia/ reoxygenation-induced injury (Zhang et al., 2017). Our recent studies indicate that GEN combined with different bile acids reduces oxygenglucose deprivation and reoxygenation (OGD/R) damage (Cheng et al., 2018; Chongyang et al., 2018). Because protecting BBB function is critical for reducing IS injury, our current study focused on investigating the protection of GEN against BBB breakdown after OGD/R and the underlying mechanisms. In the current study, we established an in vitro BBB model and analysed the BBB functions. We further evaluated the anti-oxidative, anti-inflammatory, and anti-apoptotic effects of GEN using an OGD (1 h)/R (24 h) model in the in vitro BBB system.

After fixing with 4% paraformaldehyde, cells were blocked and permeabilised for 1 h using a mixture of 0.3% (v/v) Triton X-100 (Fisher Scientific, Pittsburgh, PA, USA) and 10% (v/v) goat serum (Sigma). When ZO-1 was detected, all cells were first fixed, and astrocytes were then scraped off from the proximal side of the insert. Cells were incubated with one of the following primary antibodies overnight at 4 °C: anti-glial fibrillary acidic protein (anti-GFAP, 1:200 dilution; ab10062, Abcam), anti-factor VIII (anti-VIII, 1:100 dilution; ab6994, Abcam) (Liu et al., 2014), and anti-zonula occludens-1 (anti-ZO-1, 1:100 dilution; 21773-1-APP, Proteintech) antibodies. Subsequently, cells were incubated with the secondary antibody (goat anti-mouse IgG or FITC, 1:200 dilution, ab6785, Abcam; donkey anti-rabbit IgG or Alexa Fluor 555, 1:200 dilution, ab150074, Abcam; or goat anti-rabbit IgG or FITC, 1:200 dilution, ab6717, Abcam), followed by counterstaining with 4′, 6-diamidino-2-phenylindole (C0065, Solarbio). Digital images were captured using a fluorescence microscope (Olympus Inc., Tokyo, Japan).

2. Materials and methods 2.1. Animals Newborn Sprague-Dawley rats were purchased from Beijing Weitong Lihua Experimental Animal Technology (Beijing, China). Animal experimental procedures were carried out in accordance with China's Guidelines for Care and Use of Laboratory Animals and were approved by the Ethics Committee of Experimental Animals of Beijing University of Chinese Medicine (BUCM-3-2016040201-2003).

2.4. Construction of the in vitro BBB model The most frequently used filters include Transwell™ polycarbonate inserts (3460, 0.4 μm, Corning), on which cells develop a tight cell monolayer (DM and KH, 2012). First, astrocytes (2 × 105 cells/cm2) were seeded under the insert membrane in a Petri dish. After 4 h, the Transwell insert was placed upside down in plates. At 24 h, BMECs were seeded on the inner side of the inserts coated with gelatin at the density of 3 × 105 cells/cm2. The experiments started at 144 h. The procedure for establishing the model is shown in Fig. 1. The system in which these two cells were cultured together was classified as the co-culture group, and the BBB model with only BMECs was classified as the mono-culture group. This study compared the barrier function between the monoculture and co-culture groups.

2.2. Primary culture of brain microvascular endothelial cells (BMECs) and astrocytes BMECs and astrocytes were respectively separated from SpragueDawley rats aged 2–3 weeks and 24 h, as described previously (Park et al., 2017; Xue et al., 2013). BMECs: Cortical tissues were minced and suspended in an equal volume of 25% BSA (g/v), and the cell pellet was washed by centrifuging 4 times at 1600×g for 5 min. The obtained microvessels were incubated with collagenase type II (1.0 mg/ml, Invitrogen, Carlsbad, CA, USA) and DNase (1.5 mg/ml) at 37 °C for 1 h. The cell pellet was cultured in DMEM/F12 with 20% FBS(v/v), basic fibroblast growth factor (1.0 ng/ml), heparin (100.0 mg/ml), penicillin (100.0 U/ml), and streptomycin (100.0 mg/ml). Astrocytes: The cerebral cortex was digested with 0.5 mg/ml EDTAtrypsin and DNase I at 37 °C for 10 min. The cell suspension was filtered through a nylon mesh with a pore size of 70 μ m and was then centrifuged at 1000 rpm for 3 min. The pellet was resuspended with the aforementioned medium. Cells (2.5 × 104 cells/cm2) were plated on poly-Lysine-coated 75 cm2 flasks in a CO2 incubator under 5% CO2 atmosphere at 37 °C.

2.5. Transmission electron microscopy The membranes of the Transwell inserts in the co-culture group were removed and fixed in 2% glutaral solution and 4% paraformaldehyde (Sigma-Aldrich) overnight at 4 °C (Yamamizu et al., 2017) and were then washed with cacodylate buffer. The cells were immersed in 0.5% OsO4 solution at 4 °C for 20 min. Samples were dehydrated in a series of graded ethanol and propylene oxide, stained with uranyl acetate and lead citrate, and then embedded in Luveak 812 (Nacalai Tesque). The intercellular TJ was observed using a Hitachi 7100 transmission electron microscope (Olympus, Tokyo, Japan). 2.6. OGD/R insult and drug administration GEN was prepared at a concentration of 25 μg/ml or 6.25 μg/ml. 225

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2.11. Western blot analysis

The BBB models were randomly divided into the following four groups: (1) control group; (2) Model group; (3) GEN-H group (GEN, 25 μg/ml); (4) GEN-L group (GEN, 6.25 μg/ml). As shown in Fig. 1, the medium was replaced with deoxygenated Earle's balanced salt solution (EBSS, cc0044, Leagene Biotech Co. Beijing, China) without glucose at 168 h to mimic ischemic-like conditions in vitro. Subsequently, the BBB models were placed into the sealed Anaero container with an Anaero Pack (Mitsubishi, Tokyo, Japan) for 1 h to initiate the OGD insult (Liao et al., 2016). OGD was stopped by replacing EBSS with the complete medium with 20% FBS, 1% penicillin, and streptomycin, and the cocultures were cultured under normoxic condition at 37 °C for 24 h to mimic reoxygenation (R). The cocultures with the above treatment were defined as the model group. The cocultures in the control group were incubated in DMEM/ F12 with 20% FBS, 1% penicillin, and streptomycin without the above OGD/R treatment. The cocultures in the GEN-H and GEN-L groups were treated with GEN for 24 h before OGD/R and throughout the OGD/R process.

Astrocytes were scraped off from the proximal side of the insert before protein extraction of BMECs. Protein extracts were obtained using Minute™ Plasma Membrane Protein Isolation and Cell Fractionation Kit (Invent Biotechnologies) according to the manufacturer's instruction. Protein samples were separated by SDS-PAG and were then transferred to a PVDF membrane. After blocking for 1 h in 10% nonfat milk, the membrane was incubated with anti-occludin, antiZO-1, anti-Claudin5, anti-matrix metallopeptidases-9 (anti-MMP-9), anti-MMP-2, or anti-β-actin antibodies overnight at 4 °C. The membranes were incubated with anti-rabbit IgG (1:2000–1:4000) for 1 h at 25 °C. The immunolabeling was detected using enhanced chemiluminescence reagents (PerkinElmer, Waltham, MA). 2.12. Statistical analysis All values are presented as mean ± S.D. All experiments were performed at least three times. The data were analysed with the SPSS 20 statistical package. Comparison among different groups was carried out with Student's t-test and one-way analysis of variance followed by the least significant difference (LSD) test. A P-value of < 0.05 was considered statistically significant.

2.7. Measurement of transendothelial electrical resistance (TEER) TEER was examined using an epithelial-volt-ohm resistance meter (ERS-2, Millipore, Germany) in each group. The cultures were allowed to equilibrate for 20 min. TEER of the inserts was calculated by subtracting the resistance of the blank inserts from that of the inserts without BMECs and multiplying the subtracted values by the area of the insert (Costa et al., 2014; Zhu et al., 2012).

3. Results 3.1. Morphological and functional features of the in vitro BBB model

2.8. Sodium fluorescein (SF) permeability measurements

The purities of astrocytes and BMECs were > 98% and > 99%, respectively (Fig. 2A–B). The in vitro BBB model including BMECs and astrocytes must be firstly validated and compared with the monoculture model to ensure its accuracy. As shown in Fig. 2D, ZO-1 was successfully observed in the co-culture group using the immunofluorescence analysis. Detailed ultrastructural examination by electron microscopy clearly identified the adjacent endothelial cells linked by TJs in BMECs cocultured with astrocytes (Fig. 2C, arrow). In addition, the characteristics of the in vitro BBB models were compared between the monoculture and co-culture groups. The TEER differed greatly between the mono-culture and co-culture groups. A maximum TEER of 224.99 ± 5.51 Ω cm2 was reached at 5 d in the co-culture group, but a maximum TEER of 149.28 ± 4.59 Ω cm2 was found at 6d in the monoculture group (Fig. 2E). Therefore, these results provide further evidence that coculture of BMECs with astrocytes largely favours the formation of tight endothelial monolayers. Under normal conditions, astrocytes are essential for maintaining BBB permeability. We further investigated the effect of astrocytes on BBB after OGD (1 h)/R (24 h). The TEER values were significantly lower in the monoculture group (71.26 ± 4.45 Ω.cm2) than in the co-culture group (142.08 ± 6.81 Ω.cm2) after OGD/R (P < 0.01) (Fig. 3A). The expression levels of ZO-1, claudin-5, and occludin, the best-known integral membrane protein of TJ, significantly increased in the presence of astrocytes on normoxic condition. The expression levels of TJ protein were significantly higher in the presence of astrocytes than in the absence of astrocytes (monoculture) after OGD/R (Fig. 3B). The permeability coefficient of SF was lower in the co-culture group (4.39 ± 0.11 × 10-6 cm/s) than in the mono-culture group (6.48 ± 0.45 × 10-6 cm/s) on normoxic condition. After OGD/R, the highest paracellular Papp of SF was 52.33 ± 4.45 × 10-6 cm/s in the mono-culture group and 42.67 ± 3.89 × 10-6 cm/s in the co-culture group (Fig. 3C). The extracellular matrix connects endothelial cells with neighbouring neuroglial cells including astrocytes and pericytes to sustain the properties and function of mature BBB. The involvement of MMPs in the BBB disruption in stroke has been demonstrated in numerous studies (Ronaldson and Davis, 2012). This present study revealed significantly increased MMP-9 secretion in the co-culture of BMECs with astrocytes compared with the monoculture of BCECs after

The original culture media in the upper chamber were aspirated and replaced with the permeability test culture media with sodium fluorescein (100 μg/ml) (Liu et al., 2014; Shaik et al., 2013). Aliquots (100 μl) were collected from the lower chamber and were replaced with the pre-equilibrated blank culture media (100 μl) at 15, 30, 45, and 60 min. Apparent permeability (Papp) in each group was calculated as follows: Papp = (dM/dt)/(A × C). 2.9. Cell apoptosis assay Apoptosis of BMECs was determined by annexin V-FITC/PI double staining assay kit (KGA105-KGA108, Kegen, Nanjing, Jiangsu Province, China) according to the manufacturer's protocol. 2.10. Inflammation, neurotrophic factor, and oxidative stress measurements The supernatant was collected. Cells were washed three times in cold PBS, collected gently using a cell scraper (Corning). Cells in each well were lysed with 100 μl cell lysate on ice for 30 min, and stored in −20°C. If necessary, the samples were diluted in the appropriate diluent and analysed. Interleukin -1β (IL-1β; LH-E10099RA, Wuhan Liu he Biotechnology Co., Wuhan, China), Interleukin-6 (IL-6; LHE10103RA, Wuhan Liu he Biotechnology Co., Wuhan, China), tumour necrosis factor-α (TNF-α; KE20001, proteintech, USA), brain-derived neurotrophic factor (BDNF; LH-E10031RA, Wuhan Liu he Biotechnology Co., Wuhan, China), and glial cell line-derived neurotrophic factor (GDNF; ELR-GDNF-1, raybioptech, USA) were assessed using ELISA kits according to the manufacturer's instructions. The optical density was measured at the wavelength of 450 nm using a microplate reader (Biotek, ELX800, USA). Malondialdehyde (MDA; A003-1, Jiancheng, Nanjing, China), superoxide dismutase (SOD; A001-3, Jiancheng, Nanjing, China), lactate dehydrogenase (LDH; A020-2, Jiancheng, Nanjing, China), and gammaglutamyl transpeptidase (γ-GT; c017-2, Jiancheng, Nanjing, China) were measured using commercial kits according to the manufacturer's instructions. 226

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Fig. 2. Morphological and functional features of the in vitro BBB model. A. GFAP-positive astrocytes (green). B. vWF-positive BMECs (red). C. Transmission electron microscopy of tight junction proteins (arrow). D. Immunostaining for ZO-1 in the in vitro BBB model (red, arrow). E. Comparison of the TEER between the monoculture and co-culture groups. TEER was continuously tested in 7 days (left panel). *P < 0.05, **P < 0.01. GFAP: glial fibrillary acidic protein; ZO: zonula occludens-1. vWF: Von Willebrand factor.

fluorescein permeability were found in the BBB model with astrocytes after OGD/R; however, treatment with GEN significantly increased TEER and decreased sodium fluorescein permeability. In addition, GEN treatment significantly increased γ-GT levels after OGD/R (Fig. 4E). Notably, 25 μg/ml GEN showed greater treatment effects than did 6.25 μg/ml GEN.

OGD/R (Fig. 3D). The γ-GT activity, a reliable marker for the BBB, was higher in the co-culture group than in the mono-culture group on normoxic condition. However, the γ-GT activity was significantly lower in the mono-culture group (14.01 ± 2.07 U/L) than in the co-culture group (20.84 ± 1.81 U/L) after OGD/R (Fig. 3E). γ-GT is enzyme abundantly present on the apical surface of endothelial cells and reliable markers for the BBB(Liu et al., 2011). Previous studies also suggested astrocytes and neurons could increase γ-GT activity of vascular endothelial cells (Tian et al., 2016; Xiang et al., 2015; Xue et al., 2013). Flow cytometry was used to detect apoptosis of BMECs in the experimental groups (Fig. 3F). The results showed that OGD/R induced obvious apoptosis of BMECs, and the cell apoptosis was greatly reduced in the co-culture group (31.57 ± 1.98%) compared with the mono-culture group (39.5 ± 1.67%). In summary, the function of BBB was higher in co-culture group than in mono-culture group. The findings indicate that the in vitro BBB model including BMECs and astrocytes is a suitable model for understanding the functions of the BBB.

3.3. Effects of GEN on oxidative damage and inflammation after OGD/R in the BBB model The BBB damage is caused by increased oxidative stress and inflammation [28]. As shown in Fig. 5A–F, OGD/R significantly increased the expression levels of inflammatory cytokines (IL-6, IL-1β, and TNFα) and enhanced oxidative stress (MDA, SOD, and LDH) compared with the vehicle control; however, GEN treatment (25 μg/ml or 6.25 μg/ml) can dramatically attenuate the effects in a dose-dependent manner.

3.2. GEN improves in vitro BBB dysfunction induced by OGD/R injury

3.4. Effects of GEN on apoptosis and the release of neurotrophic factors after OGD/R in the BBB model

The protective effect of GEN on the BBB in an OGD/R environment was further investigated in this study. As shown in Fig. 4A, the expression levels of ZO-1, occluding, and claudin-5 were significantly lower in the model group than in the control group; however, the expression levels of these proteins were significantly higher in the GEN group than in the model group. MMP-9 and MMP-2 were higher in the model group than in the control group, but they were significantly lower in the GEN group than in the model group (Fig. 4B). As shown in Fig. 4C and D, significantly decreased TEER and increased sodium

Flow cytometry was used to detect apoptosis of BMECs in each group (Fig. 6A). Apoptosis of BMECs was greatly increased in the BBB model after OGD/R. Significantly increased apoptosis was observed in the model group compared with other groups, and GEN treatment significantly inhibited OGD/R-induced neuronal cell apoptosis in a dose-dependent manner. Moreover, significantly decreased levels of BDNF (Fig. 6B) and GDNF (Fig. 6C) were observed in the model group compared with the control group, and GEN treatment attenuated the effects in a dose-dependent manner. 227

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Fig. 3. Functional features of the in vitro BBB model on normoxic and OGD/R condition. A. Comparison of the TEER between the mono-culture and co-culture groups on normoxic and OGD/R condition. B. Comparison of protein expression levels of ZO-1, occluding and claudin-5 on normoxic and OGD/R condition. C. Comparison of the Papp between the mono-culture and co-culture groups on normoxic and OGD/R condition. D. Comparison of protein expression levels of matrix metallopeptidases (MMP)2 and MMP9 between the mono-culture and co-culture groups on normoxic and OGD/R condition. E. Comparison of the γ-GT between the monoculture and co-culture groups on normoxic and OGD/R condition. F. Comparison of apoptosis after OGD/R between the mono-culture and co-culture groups. Quantified results were normalised to β-actin expression levels. *P < 0.05, **P < 0.01. Papp: apparent permeability; γ-GT: gamma-glutamyl transpeptidase.

4. Discussion

protective effects on BBB permeability and TJ protein expression. Moreover, GEN significantly inhibited oxidative stress, inflammation, and apoptosis and increased BDNF and GDNF levels in a dose-dependent manner. The findings indicate that the coculture model might be a very useful in vitro model to evaluate the effect of vasculoprotective agents on the BBB. Studies have demonstrated that the disruption of the BBB causes brain oedema. Hypoxia, a significant stress factor, induces significant BBB disruption. Notably, direct and indirect contributions of astrocytes are of critical importance for hypoxia-mediated BBB permeability and

In this study, we successfully established an in vitro BBB system consisting of astrocytes and BMECs. Higher TEER, lower Papp, and higher expression levels of TJ proteins were observed in the co-culture system, in which astrocytes were seeded in a contact orientation allowing astrocytic end-feet to pass through the pores and interact with BMECs. The in vitro BBB model was then used to investigate the effects of GEN treatment on the changes in BBB integrity and the mechanisms underlying the BBB breakdown after OGD/R. GEN showed significant 228

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Fig. 4. GEN improves the in vitro BBB impairment induced by OGD/R injury. A. Increased ZO-1, occluding, and claudin-5 expression levels after GEN treatment (25 μg/ml or 6.25 μg/ml) B. Reduced MMP-2 and MMP-9 expression levels after GEN treatment C. Effects of GEN treatment on the TEER. D. Effects of GEN treatment on the Papp. E. Effects of GEN treatment on the γ-GT activity. Quantified results were normalised to β-actin expression levels. *P < 0.05, **P < 0.01.

Fig. 5. GEN treatment attenuates oxidative damage and inflammation induced by OGD/R. Effects of GEN treatment (25 μg/ml and 6.25 μg/ml) on IL-6 (A), IL-1β (B), TNF-α (C), MDA (D), SOD (E), and LDH (F). *P < 0.05, **P < 0.01. IL-6: Interleukin-6; IL-1β: Interleukin beta; TNF-α: tumour necrosis factor alpha; MDA: malondialdehyde; SOD: superoxide dismutase; LDH: lactate dehydrogenase. 229

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Fig. 6. GEN treatment attenuates apoptosis and increases the release of neurotrophic factors after OGD/R in the BBB model. Effects of GEN on apoptosis of BMECs (A) and the secretion of BDNF (B) and GDNF (C) in the BBB model. *P < 0.05, **P < 0.01. BDNF: brain-derived neurotrophic factor; GDNF: glial cell line-derived neurotrophic factor.

therapeutic drug for ischemic stroke.

maintenance of BBB properties. Astrocytes are resistant to hypoxia and ischemia because of the high capacity to maintain ATP levels. In addition, astrocytes are activated after hypoxic/ischemic injury and secrete a large number of factors, including basic fibroblast growth factor, TGF-β1, GDNF, and BDNF, which modulate the BBB function (Bathina and Das, 2015; Engelhardt et al., 2014). For instance, GDNF and BDNF trigger multiple mechanisms to exert anti-inflammation, anti-apoptosis, and anti-oxidation effects (Chen et al., 2017; Géral et al., 2013; Smith and Cass, 2007). As demonstrated in this study, the protective effects of GEN treatment were related to the upregulation of BDNF and GDNF. Pathophysiological mechanisms of cerebral ischemia-reperfusion include the release of excitotoxic neurotransmitters, intracellular Ca2+ accumulation, oxidative damage, neuronal apoptosis, and neuronal inflammation (Kalogeris et al., 2016). The interaction between inflammatory cytokines (IL-6, TNF-α, and IL-1β) and BMECs initiates inflammation and leads to the increased permeability of BBB (Jin et al., 2013). Moreover, multiple free radical mediators, including MDA, LDH, and SOD, play essential roles in the development of IS (Sun et al., 2018). Previous studies have demonstrated that GEN has neuroprotective effects in in vitro and in vivo models of neurophysiological diseases by suppressing apoptosis and inflammation and enhancing antioxidation properties (Huang et al., 2018). In this study, GEN decreased the expression levels of IL-6, MDA, LDH, TNF-α, and IL-1β and increased the expression levels of SOD in the OGD/R-induced BBB impairment model. The protective effects of GEN will contribute to the attenuation of the OGD/R-induced BBB impairment. The effect of GEN on the CNS is lower than expected, and BBB integrity and the release of neurotrophic factors should thus be focused. Pre-incubation of BBB with GEN protected the cells from the damage caused by OGD/R. The underlying mechanisms might include downregulation of ischemia-induced inflammation, oxidation, and apoptosis, maintenance of the BBB integrity, and upregulation of BDNF and GDNF. The present findings indicate that GEN might be a useful and novel

5. Conclusions In the present study, we successfully established an in vitro BBB model with BMECs and astrocytes. This in vitro BBB model had the basic morphological structure and physiological and pathological function of the in vivo BBB. This model was then employed to investigate the protective effects of GEN on the BBB after hypoxia/reperfusion injury. Notably, GEN pretreatment exerted multiple protective effects against OGD/R injury. Therefore, GEN could be a promising agent for the treatment of hypoxia-ischemia brain injury associated with BBB disruption. Funding This work was supported by the National Natural Science Foundation of China (Grant No.81430102). Acknowledgements All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. References Abbott, N.J., Rönnbäck, L., Hansson, E., 2006. Astrocyte-endothelial interactions at the blood-brain barrier. Nat. Rev. Neurosci. 7, 41–53. Banerjee, J., Shi, Y., Azevedo, H.S., 2016. In vitro blood-brain barrier models for drug research: state-of-the-art and new perspectives on reconstituting these models on artificial basement membrane platforms. Drug Discov. Today 21, 1367–1386. Bathina, S., Das, U.N., 2015. Brain-derived neurotrophic factor and its clinical implications. Arch. Med. Sci. 11, 1164–1178. Bradbury, M.W., 1984. The structure and function of the blood-brain barrier. Fed. Proc. 43, 186–190.

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