Cinnamophilin Inhibits Neutrophilic Respiratory Burst and Protects Against Ischemia-Reperfusion Brain Damage

Up to 40% of stroke patients did not recover their independence [1], which makes stroke a major public issue in the world. There are limited therapeutic treatments, available in clinic, because administration of tissue plasminogen activator (tPA) within 3 hours of symptom onset only benefits a small proportion of stroke patients [2-4], and also increases a risk of hemorrhagic transformation which may potentiate ischemic neuronal damage [5-9]. Thus, there is an urgent need for safe and effective neuroprotective agents that can beneficial for the majority of stroke patients.


Introduction
Up to 40% of stroke patients did not recover their independence [1], which makes stroke a major public issue in the world. There are limited therapeutic treatments, available in clinic, because administration of tissue plasminogen activator (tPA) within 3 hours of symptom onset only benefits a small proportion of stroke patients [2][3][4], and also increases a risk of hemorrhagic transformation which may potentiate ischemic neuronal damage [5][6][7][8][9]. Thus, there is an urgent need for safe and effective neuroprotective agents that can beneficial for the majority of stroke patients.
It is well known that neutrophils play an important role in the pathogenesis of ischemia reperfusion injury by releasing a variety of oxygen radicals including O 2 -., H 2 O 2 , and OH-. [10,11].
Following cerebral ischemia-reperfusion, neutrophils produce a variety of reactive oxygen species (ROS) when they are activated during inflammatory responses [12], a process which is known as the neutrophilic respiratory burst. NADPH oxidase which is located in neutrophils catalyzes the generation of O 2 -. from oxygen and NADPH [13][14][15] while O 2 -. is converted to H 2 O 2 by superoxide dismutase (SOD). Neutrophils utilize myeloperoxidase to further combine H 2 O 2 with chlorides to produce hypochlorite. Therefore, neutrophilic respiratory burst results in the release of O 2 -. and H 2 O 2 , which play an important mechanism of cerebral ischemia-reperfusion injury. It has been shown that neutrophil depletion or inhibition of neutrophil function exerted significant protective effects in ischemia reperfusioninduced brain injury [16][17][18][19]. One strategy to protect the brain against ischemic-reperfusion injury is to improve the endogenous antioxidant defense of the tissues at risk, to decrease oxidative damage by scavenging toxic free radicals [20][21][22] and to inhibit neutrophilic respiratory burst in activated neutrophils [23]. Cinnamophilin, (CINN;

Abstract
We have shown that administration of Cinnamophilin (CINN) effectively reduced oxidative damage, DNA lipid peroxidation, neutrophil infiltration, and ischemic brain damage by inhibiting oxidative stress and the resulting inflammation in experimental stroke. In this study the potential CINN to ameliorate neutrophilic respiratory burst and reduce neutrophil infiltration was investigated. Neutrophils pretreated or co-treated with CINN, were stimulated by phorbol 12-myristate 13-acetate (PMA) and the levels of superoxide radical (O 2 -.) and hydrogen peroxide (H 2 O 2 ) produced were determined by dihydroethidium (DHE) and dihydrorhodamine-123 (DHR) fluorescence assays, respectively, while myeloperoxidase activity (MPO) was measured by the guaiacol method. Our results showed that both pretreatment and co-treatment with CINN significantly inhibited H 2 O 2 production in PMA-stimulated neutrophils. Additionally, cotreatment, but not pretreatment, with CINN effectively inhibited O 2 -. production in the PMA-stimulated neutrophils. Both treatments did not effectively reduce the MPO activity in neutrophil. Finally, animals treated with CINN at reperfusion brain insults significantly reduced brain infarction and neutrophil infiltration, as well as improved neurobehavioral outcome following cerebral ischemic reperfusion. These results support pluripotent neuroprotection actions offered by CINN against cerebral ischemia-reperfusion.
[8R, 8S]-4, 4-dihydroxy-3, 3-dimethoxy-7-oxo-8, 8-neolignan), is a highly lipophilic compound isolated from Cinnamomum philippinense, which can readily penetrate the blood-brain barrier with a slow decay in the brain [24,25]. We have previously demonstrated that CINN not only reduced brain infarction and oxidative stress, but also improved behavioral outcome in a mouse model of transient middle cerebral artery (MCA) occlusion [26,27]. More recently, we have demonstrated that CINN exhibited anti-inflammatory actions against ischemic stroke [26]. In the present study, we further examined whether administration of CINN would reduce brain inflammation by inhibiting neutrophilic respiratory burst.

Materials and Methods
Animals C57BL/B6 mice, weighted 19-22 g, and Sprague-Dawley rats, weighed 250-280 g, were supplied by the University Laboratory Animal Center. All procedures performed were approved by the Subcommittee on Research Animal Care of the University Medical Center, and the standards meet the guidelines of the National Institutes of Health.

Chemicals and reagents
All chemicals were purchased from Sigma-Aldrich Co. (St Louis, MO) unless otherwise indicated. CINN was isolated from the root of Cinnamomum philippinense by the methods previously described [28] and was dissolved in 0.1% dimethylsulfoxide (DMSO, Sigma-Aldrich Co.) for in vitro studies.

Transient middle cerebral artery occlusion model
Adult Sprague-Dawley rats were anesthetized with 1% halothane in 70% N 2 O/30% O 2 . Animals were subjected to intra-arterial filament occlusion of the right proximal middle cerebral artery (MCA) for 90 minutes by the methods previously described [29]. During surgery, animals' rectal temperatures were maintained at 37.0 ± 1.0°C using a thermostatically controlled heating blanket (Harvard Apparatus, South Natick, MA, USA) and heating lamp. Effective MCA occlusion was confirmed by the measurements of a Laser-Doppler flowmetry (LaserFlo BMP2, Vasamedics, St. Paul, MN, USA). Sham-operated rat received similar surgical procedures excluding vascular opening and filament insertion.

Drug administration
Animals were randomly assigned to treatment protocol. The investigators were blinded to the treatment paradigm. CINN was dissolved in 45% aqueous hydroxypropyl-β-cyclodextrin (HPCD). An optimal dosage of CINN at 80 mg/kg was chosen based on its neuroprotective dose-response studies in a rodent model of stroke [27]. Animals were administered intravenously with CINN (80 mg/kg) or the same volume of HPCD at 2 hours after the onset of MCA occlusion.

Neurobehavioral testing
Body weight measurements and neurologic evaluations were assessed at 72 hours after the ischemia-reperfusion insult by one observer unaware of treatment protocol. Two neurologic grading systems were used: (i) a sensorimotor grading scale modified from previously published methods [30] with five categories (0-4) for forward and sideways visual placing tests of the affected forelimb and five categories (0-4) for motor outcome and (ii) a grading scale of 0-28 developed by Clark et al. [31].

Animal sacrifice and quantification of ischemic damage
Following 72 hours of survival, animal was sacrificed under anesthesia by transcardiac perfusion accomplished with 4% paraformaldehyde in 0.1 M phosphate-buffered saline. Brains were removed, stored in the same fixative for 24 hours, and sequentially immersed in 15 and 30% sucrose at 4°C for 48 hours. The brains were then embedded in Optimal Cutting Temperature compound (OCT, Miles Inc., Elkhart, IN, USA) and frozen in liquid nitrogen. The brains were sectioned coronally on a cryostat (HM-500O, Microm International GmbH, Walldorf, Germany). Serial sections of 40 µm at eight preselected coronal levels, with 1-mm intervals from the stereotaxic coordinates of the Bregma AP +2.22 to -4.78 mm, were mounted on poly-l-lysine-coated slides and dried at 37°C overnight. Brain infarction was determined by staining preselected brain slices with hematoxylin and eosin stain (HE). Under light microscopy, the areas of neuronal perikarya displaying typical morphological features of ischemic damage were delineated. Infarction volume was measured using a computerized image analyzer (MCID Elite, Imaging Research Inc., Ontario, Canada) and was expressed as a percentage of the contralateral hemisphere volume [29].

Detection of free radicals using dihydroethidium in situ
Brain sections were incubated with dihydroethidium (DHE) Molecular Probes, Eugene, OR, USA) in a humidified chamber, at 37°C for 30 minutes. The sections were rinsed in PBS for 15 minutes and incubated with DAPI in PBS for 15 seconds in a dark chamber, and then were rinsed in PBS and mounted with Aquamount. Fluorescence was assessed at excitation 365 nm and emission >420 nm for DAPI and was at excitation 510-550 nm and emission >580 nm for ethidium detection. The intensity of the oxidized HEt on the ischemic brain was observed and compared between the CINN-treated and vehicle-treated animals.

Immunohistochemistry for myeloperoxidase
Coronal brain sections were cut into 40 µm. A set of brain sections was processed for immunohistochemistry with polyclonal rabbit antimyeloperoxidase antibody (1:200, Thermo Fisher Scientific, Fremont, CA, USA) at 4°C overnight. After washes with PBS for 5 minutes three times, the bound antibody was visualized by labeling with goat antirabbit immunoglobulin G-fluorescein isothiocyanate (FITC) conjugate (Molecular Probes) as a fluorescent probe. Fluorescence was assessed at excitation 488 nm and emission 520 nm for FITC detection.

Neutrophils isolation
Mice were intraperitoneally injected with 5 mg/kg lipopolysaccharide (LPS) to induce neutrophils in blood. Cardiac blood collected 24 hours later was incubated with ACK buffer (0.15 M NH4Cl, 1 mM KHCO 3 , and 0.1 mM EDTA) for 6 minutes, followed by centrifugation at 800 g to remove red blood cells. Cells were then washed with Hank's balanced salt solution (HBSS, GIBCO, Grand Island, NY, USA) containing 0.05% bovine serum albumin (BSA).

Statistical analysis
Data were presented as the mean ± standard deviation of the mean (S. D.). The distribution of the date was verified by Levene tests before post hoc comparisons. Cytotoxic effects of CINN on the viability of neutrophil were analyzed by one-way analysis of variance (one-way ANOVA) with LSD protected least significant difference post hoc comparison. The data of respiratory burst of neutrophils were analyzed by Kruskal-Wallis/Mann-Whitney U tests because they were not found to follow normal distribution. The other data with normal/ (stock solution, 1 mM in dimethyl sulfoxide, diluted to 5 µm with PBS,

CINN inhibited superoxide production and neutrophil infiltration in the brain of animals subjected to transient MCA occlusion
Intracellular production of O 2 -. was measured by the staining of DHE. CINN administration significantly decreased DHE signals in the ischemic brain sections (Figure 2, P<0.05). Thus, CINN could attenuate the in situ accumulation of O 2 -. in the ischemic brain. In vehicle-treated controls, the ischemic reperfusion insult induced dramatically increased numbers of the MPO-expressing cells in the ischemic territory. CINNtreatment animals, however, significantly reduced the number of MPOimmunopositive cells, compared with controls (Figure 3, P<0.05).

Effect of CINN on cell viability of neutrophils
The cell viability was determined by the propidium iodine assay.
No significant cytoxicity to neutrophils was found with CINN at the concentrations of 1 to 100 µM ( Figure 4). CINN (300 µM), however, exhibited increased cytoxicity to neutrophils.  Cinnamophilin decreased superoxide radical production in the ischemic brain. In situ detection of intracellular superoxide radical production was measured by dihydroethidium (DHE) in the ischemic brain. The brain sections were double stained with DHE (red) and DAPI staining (blue). The CINN-treated rats had a significant decrease in the intensity of DHE signals, compared to the the vehicle-injected controls. Data are presented as mean ± standard deviation (S. D.), and n=6 per group. *P<0.05 compared with vehicletreated controls by using the unpaired Student's t test. Animal treated either with CINN or vehicle did not have altered local cortical blood perfusion or core temperature during a course of surgery (data not shown). Relative to controls, animals treated with CINN had significant reduced brain infarction and hemispheric edema. Treatment with CINN resulted in 22.9% reduction in infarction volumes (P<0.05; Figure 1A and 1B). However, CINN neither significantly reduced brain swelling nor effectively affected individual cortical and striatal infarcts (Figure 1 B and C; P>.05, respectively).

Discussion
Our results demonstrated that CINN could directly inhibit O 2 -. and H 2 O 2 produced by the PMA-induced respiratory burst in neutrophils. Although CINN did not directly suppress the MPO activity of neutrophils, the agent effectively reduced the MPO activity induced by cerebral ischemia-reperfusion injury observed in vivo ( Figure  3). Thus, CINN could protect the brain from ischemia-reperfusioninduced injury by inhibiting the respiratory burst actions in activated neutrophils.
We have demonstrated that CINN (80 mg/kg) effectively reduced brain damage induced by cerebral ischemic reperfusion insults in rats [27]. Additionally, the agent effectively improved the numbers of the surviving neurons in the penumbral parietal cortex and caudoputamen [27]. Our previous works have also demonstrated that CINN effectively scavenged free radicals and improved the endogenous antioxidant defense to the tissues at risk of brain infarction. Additionally, CINN had antioxidant effects by reducing the malondialdehyde levels in the ischemic brain tissues and the Fe 3+ -induced lipid peroxidation in rat brain homogenate [26]. Thus, treatment with CINN could result in a marked reduction in postischemic accumulations of ROS and, consequently, attenuated the extent of lipid peroxidation, oxidative  the PMA-stimulated neutrophils ( Figure 6B, P<0.05). The IC 50 for H 2 O 2 production was 25.4 µM and 13.9 µM in the pretreatment and cotreatment CINN protocols, respectively. We further investigated the effect of CINN on the MPO activity in the PMA-stimulated neutrophils, and have observed that CINN had no effect on the MPO activity in the PMA-stimulated neutrophils (data not shown). The findings with no effect of CINN against MPO activity in the PMA-activated neutrophils are inconsistent with the in vivo data of CINN observed in a rat model of cerebral ischemia-reperfusion. This, however, indicates that molecular mechanisms of neuroprotective effect could be influenced by neuroprotective action in the model of whole animals. Thus, it was very possible that the CINN-mediated reduction in the MPO activity observed in vivo might have simply reflected its neuroprotection and anti-inflammatory actions against ischemic stroke. Other mechanisms, in addition to the inhibitory action against the neurotrophilic respiratory burst observed with CINN in vitro, might also have contributed to the CINN's ability against cerebral ischemiareperfusion insults observed in vivo. Thus, the in vivo and in vitro studies were essentially independent and might not establish a causal relationship. In addition, the dose-response levels in the present studies actually varied greatly between the in vivo and the in vitro studies.
Our results confirmed that administration of CINN significantly inhibited the PMA-induced O 2 -. and H 2 O 2 in neutrophils. However, CINN has no effect to inhibit the MPO activity in neutrophils. Thus, CINN did not directly attenuate the MPO pathway. In previous studies, calphostin C, an inhibitor of protein kinase C (PKC) can obviously reduce the He-Ne laser-induced respiratory burst of neutrophils [32], and the PMA is a stimulator for PKC-mediated NADPH oxidase and nitric oxide (NO) synthase during respiratory burst of neutrophils [33]. Therefore, it was very possible that CINN could also be a PKC inhibitor, It should be emphasized that fluorescent dyes such as DHR (for H 2 O 2 ) and DHE (for superoxide radical) is not the best method of choice for radical detection, although it is more preferable in the cellular-based studies. The DHR and DHE actually react with many reactive oxygen species (and not only H 2 O 2 or superoxide radical), and this non-specific integrity may leads to the production of other interfering substances which could also fluoresce and interfere. Superoxide reacts with DHE and produces a specific reaction product, the 2-hydroxy-ethidium which should be isolated to estimate accurately superoxide radical concentration. This could partly explain the decrease in superoxide radical with the significant high S. D. bar, as seen in Figure 2.
Our previous works have shown that CINN could be served as an effective free radical scavenger and antioxidant, and has multifaceted neuroprotective properties acting against cerebral ischemia-reperfusion injury. In stroke animals, CINN significantly reduced the infarction volumes, proinflammatory cytokine levels, nitric oxide production, and improved neurobehavioral outcomes as well (Figure 7). Therefore, CINN could prevent neuronal damage by its direct antioxidant and antiinflammatory effects after ischemia-reperfusion. In the present study, we further demonstrated that CINN inhibited neutrophilic respiratory bursts by directly attenuating the PMA-induced O 2 -. and H 2 O 2 , but not the MPO activity, and thus reduced post-stroke neutrophilic infiltration in the ischemic brain. Further studies are needed to verify whether CINN would be a PKC inhibitor so as to reduce the NADPH oxidase activity and then to decrease the levels of O 2 -. More studies are also needed to demonstrate the neuroprotective mechanism of CINN in view of its potential role in the field of ischemic stroke. and, thus, leading to a decrease in the O 2 -production by reducing the NADPH oxidase activity, and this, however, needs further investigation.