Tanshinone IIA protects the human blood–brain barrier model from leukocyte-associated hypoxia-reoxygenation injury

European Journal of Pharmacology 648 (2010) 146–152

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European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r

Cardiovascular Pharmacology

Tanshinone IIA protects the human blood–brain barrier model from leukocyte-associated hypoxia-reoxygenation injury Wen-jian Zhang a, Jie Feng a, Ran Zhou b, Li-ya Ye a, Hong-lin Liu a, Liang Peng a, Jin-ning Lou a, Cheng-hui Li b,⁎ a b

Institute of Clinical Medical Sciences, China-Japan Friendship Hospital, Beijing 100029, PR China Department of Anesthesiology, China-Japan Friendship Hospital, Beijing 100029, PR China

a r t i c l e

i n f o

Article history: Received 26 January 2010 Received in revised form 21 July 2010 Accepted 25 August 2010 Available online 15 September 2010 Keywords: Models Blood–brain barrier Permeability Reperfusion injury Drug

a b s t r a c t To investigate the in vitro effect of tanshinone IIA on leukocyte-associated hypoxia-reoxygenation injury of human brain–blood barrier (BBB), we established the BBB model by culturing purified primary human brain microvascular endothelial cells (HBMVEC) to confluence on cell culture insert. BBB was identified by tight junction, transendothelial electrical resistance (TEER) and the permeability of BBB to horseradish peroxidase (HRP). The effect of tanshinone IIA on the permeability of BBB was tested at 2 h after hypoxia and 1 h after reoxygenation with or without the supernatants of activated leukocytes. The effect of tanshinone IIA on leukocytes activation was analyzed by detection of MMP-9, cytokines and reactive oxygen species. The results showed that BBB formed by confluent HBMVECs had no cellular gap. Immunofluorescent staining for ZO-1 confirmed that the cells were connected by tight junction. Moreover, the BBB model had a higher TEER and a lower permeability for HRP than confluent HUVECs. The permeability of BBB for HRP was enhanced by hypoxia-reoxygenation and further greatly enhanced by adding the supernatants of activated leukocytes before reoxygenation. But such an effect was reversed by addition of tanshinone IIA before hypoxia. Moreover, tanshinone IIA could decrease the levels of MMP-9, TNF-α, IL-1α, IL-2, IFN-γ and reactive oxygen species in leukocytes. In conclusion, tanshinone IIA can protect BBB against leukocyte-associated hypoxia-reoxygenation injury by attenuating the activation of leukocytes and inhibiting the injury effects of leukocytic products. Tanshinone IIA may be a novel therapeutic agent for cerebral ischemia–reperfusion injury. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Ischemia–reperfusion injury, a serious problem for the therapy of ischemic brain diseases, is also a key process of influencing the development and outcome of ischemic cerebrovascular disease (Schaller and Graf, 2004). In addition, with the development of modern surgical techniques, it has been become necessary to transiently cease blood circulation for complicated procedures. Therefore developing a suitable drug for the treatment and prevention of ischemia–reperfusion injury is of great importance to neurologists, anesthesiologists and surgeons. Constructed mainly from brain microvascular endothelial cells, the blood–brain barrier (BBB) serves as a frontline defense for maintaining brain homeostasis with a low permeability. Ischemia–reperfusion has been known to damage the tight junctions of BBB and lead to permeability changes resulting in enhanced cerebral edema and neuron injury (Witt et al., 2003). The effect of leukocyte on the permeability of BBB is of key value for investigation in ischemia–reperfusion injury. It was reported that leukocytes aggregated in blood vessels and infiltrated

⁎ Corresponding author. Tel./fax: +86 10 84205819. E-mail address: [email protected] (C. Li). 0014-2999/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2010.08.040

into brain tissue during reperfusion (Buras and Reenstra, 2007). The activated leukocytes released proteinases, reactive oxygen species and cytokines to destroy tissues directly or indirectly (Vinten-Johansen et al., 2004; Jordan et al., 1999). Therefore the activation of leukocyte or inflammation contributed greatly to ischemia–reperfusion injury (Ritter et al., 2005; Buras and Reenstra, 2007). Furthermore, leukocytes might disrupt the BBB tight junctions and alter the BBB permeability (Kebir et al., 2007). However, we are still unclear about the effects of leukocytes on the permeability of BBB during ischemia–reperfusion. Therefore, it is important to understand the influence of leukocytes on the permeability of BBB during ischemia–reperfusion so as to develop protective drugs. As previously reported, Salvia miltiorrhiza could protect endothelial cells from ischemia–reperfusion injury (Han et al., 2008). Tanshinones are the major lipid-soluble pharmacological monomers of Danshen, the dried roots of S. miltiorrhiza Bunge (Labiatae), a well-known traditional Chinese medicine used for the treatment of cerebrovascular diseases including stroke (Lam et al., 2003). Tanshinone was shown to significantly reduce the infarct volume and improve the neurological deficits of ischemic injury by suppressing the oxidative stress and the radical-mediated inflammatory insults (Dong et al., 2009). In addition, tanshinone IIA might inhibit NADPH oxidase, scavenge peroxides and inhibit the expression of adhesion molecules in vascular endothelium and leukocytes. Tanshinone IIA also had an ameliorating effect on the

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microcirculatory disturbance and target organ injury after ischemia– reperfusion (Han et al., 2008). In our previous study, tanshinone IIA could offer obvious protection on the endothelial cells. But it has remained unclear about the action of tanshinone IIA on the permeability of BBB under ischemia–reperfusion. Therefore, in the present study, we tried to establish a BBB model of human brain MVEC (HBMVEC) to elucidate the in vitro effects of tanshinone IIA on the leukocytes-associated hypoxia-reoxygenation injury of human BBB model. It will provide significant experimental data for developing tanshinone IIA for the prevention and treatment of cerebral ischemia–reperfusion disorders. 2. Material and methods 2.1. Isolation, culture and identification of HBMVEC Tissue of human brain was obtained from an autopsy donor and its gray matter extracted within 3 h of death. The study protocol was approved by the Medical Ethics Committee of China-Japan Friendship Hospital. Microvascular endothelial cells were isolated from gray matter as described previously (J. Lou et al., 1998; J.N. Lou et al., 1998). The isolated human brain microvascular endothelial cells (HBMVEC) were re-suspended in endothelial cell culture medium (ECCM) (DMEM medium containing 20% fetal calf serum [FCS], 100 μg/ml penicillin/ streptomycin, 2 mmol/l L-glutamine, 40 U/ml heparin and 100 μg/ml endothelial growth supplement) and then inoculated into a 6-well culture plate coated with 2% gelatin. After incubating for 3–5 days, HBMVECs were purified by Dynabeads (Dynal A.S., Oslo, Norway) coated with Ulex europaeus agglutinin-1 (UEA-1) as previously described (Lou et al., 1999). Purified HBMVEC was cultured to confluence and then passaged by trypsin/EDTA treatment. HBMVEC within 3–5 passages was used in this study. The purified HBMVEC was identified by the uptake of Dil-Ac-LDL (Invitrogen, USA) examined under an IX71 fluorescence conversion microscope (OLYMPUS, Japan) and immunofluorescence for the expressions of classical marker of endothelial cell-CD31 (antibody from Sigma, USA) and tight junction protein-ZO-1(antibody from Invitrogen, USA) on the confluent HBMVEC. In addition, the purity of HBMVEC was analyzed by flow cytometry for the expression of von Willebrand factor (vWF). The details of the above experiments were summarized elsewhere (J. Lou et al., 1998; J.N. Lou et al., 1998). Human umbilical vein endothelial cells (HUVECs) were isolated and cultured from a fresh umbilical cord. Identified with vWF immunofluorescence staining, HUVEC cells of 3–5 passages were used for experimental controls. 2.2. Establishment and evaluation of BBB model 2.2.1. Establishment of BBB model HBMVEC was re-suspended in ECCM and seeded (5 × 104 cells/ well) onto a 2% gelatin-coated transparent and porous membrane of cell culture insert (Falcon, 8 μm pore size, 7.6 mm diameter, 0.45 cm2 surface area). After incubating in a CO2 incubator at 37 °C for 3–5 days, a confluent monolayer of HBMVEC was generated. Then the establishment of BBB was initially judged by a 4 h water-leaking test. Briefly, the additional ECCM was added into a donor chamber of cell culture insert until the liquid level inside rose 0.5 cm above that of acceptor chamber. Then the culture insert was cultured for over 4 h in a CO2 incubator at 37 °C. If the height difference of liquid level remained after 4 h, it indicated the water permeability encumbrance of cells, and the water leaking test records positive. A positive water leaking test means the basic formation of the BBB model. On the contrary, there was no difference or a limited difference between the medium levels of donor chamber and acceptor chamber results after incubation, which was designated as negative water-leaking test.

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2.2.2. Evaluation of the BBB model 2.2.2.1. Morphological identification of BBB. The confluent HBMVECs in cell culture insert positive for a water-leaking test were fixed with 2.5% glutaraldehyde at room temperature for 1 h. Rinsed three times with PBS, nylon membrane was then dehydrated and embedded to prepare the specimens for scanning and transmission electron microscopy (SEM and TEM) in accordance with the conventional methods. The cell specimens were observed under a JEOL 1010 TEM (JEOL, Tokyo, Japan) and JSM 6000F SEM (JEOL, Tokyo, Japan) respectively. 2.2.2.2. Determination of trans-endothelial electrical resistant (TEER) (Xie et al., 2005). Electrical resistance across BBB or confluent HUVEC cultured on cell insert was measured by EVOMTM volt ohmmeter (World Precision Instruments, USA) with 3 wells for each. TEER (Ω cm2) was calculated from the displayed electrical resistance on a readout screen by the subtraction of electrical resistance of a gelatincoated filter without cells and a correction for filter surface area. 2.2.2.3. Permeability of the BBB model. Confluent HBMVEC was selected as the BBB model to be judged primarily by water-leaking test and TEER. Confluent HUVEC was used as control. Horseradish peroxidase (HRP) (RZ = 3.0–3.5, enzyme activity N265 U/mg, MW = 44 kDa) was selected as an indicator to evaluate the permeability of BBB model. In brief, ECCM in an acceptor chamber was changed into 1140 μl experimental medium (ECCM without phenolsulfonphthalein and ECGS). ECCM in a donor chamber was substituted by 460 μl experimental medium containing 500 ng HRP so that the liquid inside and outside of cell insert were at the same level to avoid hydrostatic pressure. Samples of 50 μl were taken from the acceptor chamber at 0.5, 1.0, 2.0, 4.0, 8.0, and 24 h and 50 μl fresh experimental medium was added each time. At the end time-point, all the samples and diluted HRP standards were loaded in 96-well plates and reacted with 100 μl substrates (including 3,3′,5,5′-tetramethyl benzidine and H2O2). After stopping by H2SO4, OD of each sample was detected at a wavelength of 450 nm. The percentage of permeability was calculated by the following formula: P% =

Cacceptor ðng = mlÞ × Vacceptor ðng = mlÞ × 100% Cdonor ðng = mlÞ × Vdonor ðng = mlÞ

C is the concentration of HRP obtained from the standard curve, Vacceptor is 1.14 ml, Vdonor is 0.46 ml. 2.3. Hypoxia-reoxygenation of BBB To induce hypoxia, the experimental medium was used. BBBs with a positive water-leaking test were placed into an airtight chamber located inside an incubator at 37 °C and ventilated with a gas mixture consisting of 95% N2/5% CO2. After 2 h, BBBs were removed from hypoxia chamber and reoxygenated in a similar chamber ventilating continuously with a gas mixture of 95% O2 and 5% CO2 for another 1 h. For comparison, three BBBs were incubated under normoxic conditions for the same duration. 2.4. Influence of supernatants of activated leukocyte and tanshinone IIA on BBB model permeability under hypoxia-reoxygenation Leukocytes were isolated from peripheral blood of healthy volunteers by the standard method with Ficoll. After washing twice with DMEM, peripheral leukocytes were suspended in ECCM without phenolsulfonphthalein and seeded in a T25 flask at a density of 8 × 106 /ml. Then leukocytes were hypoxic for 2 h and reoxygenation for 1 h as described above. The supernatant was collected and used for the next step immediately.

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Tanshinone IIA was a kind gift of professor Liu Jie-wen, Tianjin Institute of Hematology, Chinese Academy of Medical Sciences. The formed BBB models were divided into six groups (n = 3 each): (1) normal control: BBBs normally cultured for 3 h; (2) hypoxic group: BBBs cultured at 37 °C, 95% N2 and 5% CO2 for 2 h; (3) hypoxicreoxygenation (H-R) group: BBBs cultured under hypoxic condition for 2 h and then reoxygenated under hyper-oxygen condition for another 1 h as described above; (4) leukocytic group: based on group H-R, the supernatants of leukocytes were added to each well of BBB before reoxygenation; (5) tanshinone IIA group: based on the leucocytic group, tanshinone IIA (a final concentration of 30 μg/ml) was added just before hypoxia; (6) BSA group: based on the leucocytic group, BSA 30 μg/ml instead of tanshinone IIA was added just before hypoxia. At the end of experiment, the permeabilities for HRP of BBBs were detected as described above.

3. Results 3.1. Purification and identification of HBMVEC HBMVEC was purified through UEA-1-conjugated magnetic Dynabeads. Upon incubation, some cells were selectively bound to the beads (Fig. 1A). After purification with a magnetic particle concentrator, only the cells bound with magnetic Dynabeads were collected (Fig. 1B). No bead was observed on HBMVEC after 5 passages (Fig. 1C). The purified cells uptake Dil-Ac-LDL (Fig. 1D) and express CD31. Immunofluorescence staining indicated that confluent cells expressed ZO-1 at the cell boundaries with continuous staining (Fig. 1F). Flow cytometric analysis indicated that over 95% of cells were positive for von Willebrand Factor (vWF) (Fig. 1G). 3.2. Evaluation of the BBB model

2.5. Action of tanshinone IIA on the functions of leukocyte under hypoxia-reoxygenation For analyzing the action of tanshinone IIA on functions of leukocyte, isolated leukocytes were suspended in DMEM with 10% FCS (for cytokine or reactive oxygen species detection) or DMEM without FCS (for MMP-9 detection) at a density of 1 × 106/ml, then were seeded in 24-well plate at a volume of 1 ml/well. The leukocytes were hypoxia-reoxygenated with different concentrations of tanshinone IIA (10, 30 and 100 μg/ml respectively) or without tanshinone IIA. The leukocytes cultured under normoxia were used as controls. At the end of reoxygenation, the supernatant of culture medium was collected and then the detections of MMP-9, cytokines and reactive oxygen species were performed as follows: 2.5.1. Zymography for MMP-9 The collected samples (without boiling) were fractionated by 10% SDS-PAGE with gelatin into a final concentration of 1.5 mg/ml (Sigma, St. Louis, USA). The standards of MMP-2 and MMP-9 were loaded as markers. The gel was soaked in 2.5% Triton-X100 twice with gentle shaking for 30 min at room temperature to remove SDS. Gel was washed with distilled water twice to remove the detergent and then incubated overnight at 37 °C in enzyme buffer (50 mM Tris, pH 7.5; 200 mM NaCl; 5 mM CaCl2 and 0.02% Brij-35). After incubation, gel was stained for at least 2 h with 0.5% Coomassie blue R-250 in 30% methanol and 10% acetic acid, de-stained with three washes of 30% methanol and 10% acetic acid. Proteolytic activity was visualized by clear, non-stained bands and was then photographed. 2.5.2. ELISA for cytokines The concentrations of TNF-α, IL-1α, IL-2 and IFN-γ in the supernatant of culture medium were detected by ELISA kits (R&D Company, USA). All the procedures followed the manufacturer's instructions. The concentrations of samples were calculated from a standard curve. 2.5.3. Detection of reactive oxygen species After the above-mentioned hypoxia-reoxygenation, the leukocytes were collected and reactive oxygen species was detected by kit (GENMED Company, USA) according to the manufacturer's protocol. Then the results were detected by microplate fluorescence reader (Molecular Devices Corp, CA, USA).

3.2.1. Morphological identification By SEM, the BBB model with positive water-leaking test was composed by monolayer confluent HBMVEC cells without cellular gap (Fig. 2A). Under TEM, it was observed that high electronic density tight junction existed between adjacent cells (Fig. 2B). 3.2.2. TEER of the BBB model As detected by EVOMTM, TEER of the BBB model formed by confluent HBMVEC was significantly higher than that of confluent HUVEC (Fig. 2C). The differences between the BBB model and confluent HUVEC were significant (P b 0.01). 3.2.3. Permeability of the BBB model The permeability of the BBB model and confluent HUVEC to HRP was detected. The results showed that the permeability of the BBB model was significantly lower than confluent HUVEC at all timepoints. Yet the leaked HRP increased with the lapse of time (Fig. 2D). 3.3. Increased permeability of the BBB model under hypoxia-reoxygenation and enhancement by supernatants of activated leukocytes Compared with the BBB model cultured under normoxia, the HRP permeability of BBB model increased after hypoxia for 2 h and increased markedly under reoxygenation time-dependently. The permeability of the BBB model increased significantly at 1 h reoxygenation (Fig. 3A). Therefore, the condition of hypoxia 2 h after a 1 h reoxygenation was used thereafter. Furthermore, the supernatants of activated leukocytes were found to enhance the effect of hypoxia-reoxygenation (Fig. 3B). There were statistically significant differences between the control and HR groups and between HR and leukocytic groups. 3.4. Tanshinone IIA protecting BBB from leukocyte-associated hypoxia-reoxygenation injury To evaluate the effect of tanshinone IIA on the permeability of the BBB model, tanshinone IIA was added just before hypoxia-reoxygenation. The result showed that the HRP permeability of the BBB model in tanshinone IIA group was significantly lower than that of group HR with the supernatants of activated leukocytes (Fig. 4) (P b 0.01). It indicated that tanshinone IIA protected the BBB model from leukocyte-associated HR injury. 3.5. Inhibition of leucocytic activities by tanshinone IIA

2.6. Statistical analysis Software SPSS 11.5 was used for statistical analysis. The results were expressed as mean ± S.D. and the Mann–Whitney U-test was used to evaluate the differences between two groups. The values of P b 0.05 and P b 0.01 were taken to denote statistical significance.

To investigate the protective mechanisms of tanshinone IIA, we detected the effects of tanshinone IIA on the functions of leukocytes under hypoxia-reoxygenation (HR). The result of zymography indicated that leukocytes released a great mount of MMP-9 after HR, but the amount of MMP-9 significantly decreased when incubated with

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120 160 200 80

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Fig. 1. Purification and identification of HBMVEC. A–C: Purification of HBMVEC by Ulex europaeus-conjugated magnetic beads (×200) UEA-1-conjugated magnetic beads selectively bound to HBMVEC (A), then only beads-bound HBMVECs were collected with the magnetic particle concentrator (B) and beads lost after 5 passages(C). D: Uptake of Dil-Ac-LDL (red fluorescence) (×400). E: Immunofluorescence staining for CD31 on confluent HBMVEC (×400). F: Immunofluorescence staining for ZO-1 on confluent HBMVEC (×400). G: Flow cytometric analysis for vWF.

glial cells from harmful insults. The permeability of BBB increased in some ischemia–reperfusion disorders in which leukocytes act as a key insulting factor. Therefore the drugs capable of inhibiting leukocytes may be effective for ischemia–reperfusion. In the present study, a human BBB model of confluent HBMVEC was established. It was shown that S. miltiorrhiza monomer tanshinone IIA could protect BBB from leukocyte-associated hypoxia-reoxygenation injury. BBB consists of brain microvascular endothelial cells surrounded by pericytes and end-feet astrocytes. At present, there are several in vitro types of BBB models: established by immortalized cell lines (Terasaki and Hosoya, 2001), co-cultured MVEC with astrocyte (Hurst and Fritz,

tanshinone IIA during HR (Fig. 5A). Moreover, it showed a significant dose–effect relationship. The results of ELISA for cytokines showed that the production of TNF-α, IL-1α, IL-2 and IFN-γ was inhibited by tanshinone IIA (Fig. 5B). Reactive oxygen species, another source of important injury factors of leukocytes, were also inhibited by tanshinone IIA. A good dose–effect relationship was observed as well (Fig. 5C). 4. Discussions The blood–brain barrier constructed from brain microvascular endothelial cells serves as a frontline defense to protect neurons and

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Fig. 2. Evaluation of in vitro BBB model. A: SEM showed no gap between adjacent cells (shown as arrows) (×3000). B: TEM for tight junctions (shown as arrows) (×20,000). C: Detection of transendothelial electrical resistance. D: Comparison of HRP permeability in different endothelial barriers. In panels C and D, the results were expressed as means± S.D. of 3 independent experiments. *P b 0.05, **P b 0.01, BBB model vs. confluent HUVEC.

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Permeability for HRP (%)

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Fig. 3. Effect of hypoxia-reoxygenation on the permeability of BBB. A: Permeability of BBB under normoxia or hypoxia-reoxygenation condition (HR: a 2 h hypoxia followed by 1 h reoxygenation). B: Permeability of BBB after hypoxia-reoxygenation. N: BBB cultured under normal condition for 3 h. H: BBB cultured under hypoxic condition for 2 h. HR: BBB cultured under hypoxic condition for 2 h followed by 1 h reoxygenation HR+ WBC: BBB cultured under hypoxic condition for 2 h followed by 1 h reoxygenation with the supernatants of activated leukocytes. The triplicate results were expressed as means± S.D. **P b 0.01, H group vs. control group, HR group vs. control group, and HR + WBC group vs. HR group.

1996; Kondo et al., 1996) or monolayer of MVEC (Franke et al., 2000). BBB established by immortalized cells could not form tight junctions and the modeling method was too complicated (Terasaki and Hosoya, 2001). BBB of co-cultured MVEC with astrocyte had a higher TEER but a similar permeability as compared with BBB of monolayer MVEC (Kondo et al., 1996). Therefore we established BBB of monolayer MVEC by culturing purified HBMVEC on cell culture insert. To judge the formation of BBB, a 4 h water-leaking test was conducted. Then the morphology and function of BBB were evaluated

Fig. 4. Protection of BBB from hypoxia-reoxygenation injury by tanshinone IIA. N, H, HR and HR+ WBC represented the same means as in Fig. 3. Tanshinone IIA: BBB cultured under HR condition in the presence of tanshinone IIA. BSA: BBB cultured under HR condition in the presence of bovine serum albumin. The triplicate results were expressed as means ± S.D. **P b 0.01, H group and HR group vs. control group, and HR + WBC group vs. HR group. ##P b 0.01, tanshinone IIA group vs. HR + WBC group.

by electron microscopy, TEER and permeability for HRP. The tight junction of inter-endothelial cells is the basic structure of BBB. Only capillary-derived MVEC has the capacity of developing intercellular tight junction. In the present study, the BBB model constructed by purified capillary origin HBMVEC cells had no intercellular gap between MVEC under SEM. And tight junctions were confirmed by TEM and immunofluorescence staining for ZO-1. After barrier formation, TEER of BBB was 6.1 times that of confluent HUVEC. The 4 h permeability of macromolecule HRP (MW 40,000) was less than 1%. These results indicated that this kind of BBB model had barrier functions as well as structural features similar to its in vivo counterpart. There are three significant advantages with this BBB model: (1) Simple preparation. With a high rate of barrier formation (N90 percent), barrier formation is easy to judge by water leaking test. (2) Quick barrier formation. Barriers usually could be formed within 5–7 days after HBMVEC was inoculated into insert. (3) Long-term barrier sustaining. The formed barriers are generally maintained for 5–7 days. By investigating the BBB model, it was found that the permeability of BBB was greatly boosted by hypoxia-reoxygenation and further enhanced by the supernatants of activated leukocytes. But tanshinone IIA offered significant protection for BBB. This result was consistent with previous reports (Matsuo et al., 1995) showing that ischemia– reperfusion injury was reduced by neutrophilic depletion. In vivo, a large number of leukocytes were infused into ischemic area and sequentially caused respiratory activation in the process of reperfusion. The products released by activated leukocytes could inflict tissue injuries (Welbourn et al., 1991; Hartl et al., 1996). Tanshinone IIA might affect the leukocytes by inhibiting its activation or reducing the injury by products of activated leukocytes. The supernatants of leukocytes contained various products of activated leukocyte to enhance the hypoxia-reoxygenation injury of BBB. When tanshinone IIA was added before reoxygenation, the injury became attenuated. It indicated that tanshinone IIA could inhibit the effects of activated leukocytic products. Thus it was feasible to employ tanshinone IIA as a therapeutic drug for ischemia–reperfusion injury. However, if tanshinone IIA was used for the prevention of ischemia–reperfusion injury, the inhibition of leukocyte activation might be equally important. It has been found that three types of products were involved in leukocyte-mediated injury (Welbourn et al., 1991; Weiss, 1989): proteinase, cytokines and reactive oxygen species. We analyzed the action of tanshinone IIA on the activation of leukocytes as well. The result of zymography indicated an increase in MMP-9 activity in leukocytes suffering from hypoxia-reoxygenation. MMP-9 might digest matrix membrane underlining MVEC, destroy the integrity of BBB, enhance the permeability of BBB and facilitate the in vivo infiltration of more inflammatory cells into ischemic tissue. Tanshinone IIA could effectively attenuate MMP-9 release in a dose-effect dependent way. Cytokines released by activated leukocyte participated in the process of ischemia–reperfusion injury and it worsened the damage of BBB (Krizanac-Bengez et al., 2006). We found that there were elevated levels of TNF-α, IL-1α, IL-2 and IFN-γ of leukocytes after hypoxia-reoxygenation. Moreover, such an effect might be reversed by tanshinone IIA. The gene of TNF-α was up-regulated in ischemic tissue (Zhang et al., 2005). When TNF-α was injected into cerebral tissue, microvessels became injured(Farkas et al., 2006). Other cytokines, such as IFN-γ, could up-regulate the TNF receptors and then enhance the action of TNF-α. IL-2 could promote the proliferation of T cells and then contribute to the ischemia–reperfusion injury (Zwacka et al., 1997). Furthermore IL-1 and TNF-α could up-regulate the expression of MMP-9. On the contrary, pro-TNF-α might be activated by MMP-9(Vecil et al., 2000). Therefore the injury roles of cytokines released by leukocytes could be mediated in many ways. Our data show that tanshinone IIA is effective in reducing reactive oxygen species level, which is in accordance with a previous report that S. miltiorrhiza is effective for anti-oxidative stress (Ding et al.,

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Fig. 5. Effect of tanshinone IIA on the activation of leukocytes by hypoxia-reoxygenation. A: Effect of tanshinone IIA on the MMP-9 activity of leukocytes activated by hypoxiareoxygenation. B: The effect of tanshinone IIA on cytokines of leukocytes activated by hypoxia-reoxygenation. The triplicate results were expressed as means ± S.D. *P b 0.05, **P b 0.01, HR group vs. N group; #P b 0.05, ##P b 0.01, tanshinone IIA group vs. HR group. C: Effect of tanshinone IIA on reactive oxygen species of leukocytes activated by hypoxiareoxygenation. The triplicate results were expressed as means ± S.D. **P b 0.01, HR group vs. N group; #P b 0.05, ##P b 0.01, tanshinone IIA group vs. HR group. N: Leukocytes cultured under normal condition. H: Leukocytes cultured under hypoxic condition. HR: Leukocytes cultured under hypoxic condition followed by reoxygenation. tanshinone IIA: leukocytes cultured under HR condition in the presence of different doses of tanshinone IIA.

2006). Oxidative stress occurred during reperfusion at ischemic tissue and the production of reactive oxygen species was induced by leukocyte. In addition, reactive oxygen species could deactivate proteinase inhibitor around leukocyte and increase leucocytic proteinase. With a potent effect of anti-oxidative stress, tanshinone IIA played a protective role for ischemia reperfusion injury (Wang et al., 2006). Therefore ischemia–reperfusion injury could be reduced through the inhibition of leukocytic activity (Liou et al., 2003).

5. Conclusion The monolayer of HBMVEC cultured in cell culture insert provides a relevant in vitro model for studying the BBB level. With this model, the permeability of BBB is enhanced by hypoxia-reoxygenation and such an effect was enhanced by leukocyte. But these phenomena may be abolished by the treatment of tanshinone IIA. Furthermore, tanshinone IIA protects BBB by inhibiting the activation of leukocyte

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and injury through the products of activated leukocytes. Therefore tanshinone IIA is a potential drug candidate for the treatment or prevention of ischemia–reperfusion injury.

Acknowledgments This study was supported by National Nature Science Foundation of China (No. 30571795 to Cheng-hui Li).

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