brain research 1589 (2014) 126–139

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Research Report

Neuroprotective effects of gallic acid against hypoxia/reoxygenation-induced mitochondrial dysfunctions in vitro and cerebral ischemia/reperfusion injury in vivo Jing Sun, Yun-zi Li, Yin-hui Ding, Jin Wang, Ji Geng, Huan Yang, Jie Ren, Jin-yan Tang, Jing Gaon Neurobiology Laboratory, School of Pharmacy, Jiangsu University, No. 301 Xuefu Road, Zhenjiang 212013, P R China

ar t ic l e in f o

abs tra ct

Article history:

Oxidative stress and mitochondrial dysfunction are frequently implicated in the pathology

Accepted 15 September 2014

of secondary neuronal damage following cerebral ischemia/reperfusion. Recent evidence

Available online 22 September 2014

suggests that gallic acid (GA) reverses oxidative stress in rat model of streptozotocininduced dementia, but the roles and mechanisms of GA on cerebral ischemia/reperfusion

Keywords:

injury remain unknown. Here we investigated the potential roles and mechanisms of GA in

Gallic acid

hypoxia/reoxygenation induced by sodium hydrosulfite (Na2S2O4) in vitro and cerebral

Cerebral ischemia/reperfusion

ischemia/reperfusion induced by middle cerebral artery occlusion (MCAO) in vivo. 3-(4, 5-

injury

dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, 5, 50 , 6, 60 -tetrachloro-

Hypoxia/reoxygenation injury

1, 10 , 3, 30 -tetraethylbenzimidazol carbocyanine iodide (JC-1), Dichlorofluorescin diacetate

Mitochondrial dysfunction

(DCF-DA) and MitoSOX fluorescent assay, Clark-type oxygen electrode, firefly luciferase

Mitochondrial permeability

assay, and calcium-induced mitochondrial swelling were conducted to detect cell death,

transition pore

mitochondrial membrane potential (MMP), intracellular and mitochondrial reactive oxygen species (ROS), oxygen consumption, ATP level, and mitochondrial permeability transition pore (MPTP) viability. We firstly find that modulation of the mitochondrial dysfunction is an important mechanism by GA attenuating hypoxia/reoxygenation insult. To further

Abbreviations: ANT, C,

medium; DMSO, GA,

adenine nucleotide translocator; BCA,

bicinchoninic acid; CsA,

cyclosporin A; CypD,

Cytochrome C; DAPI, 4', 6-diamidino-2-phenylindole; DCF-DA, dichlorofluorescin diacetate; DMEM, dimethylsulfoxide; EB, ethidium bromide; EBSS,

gallic acid; H & E,

MCAO,

haematoxylin and eosin; JC-1,

middle cerebral artery occlusion; MDA,

permeability transition pore; MTT, dinucleotide phosphate; NOX,

Earle's balanced salt solution; FBS,

fetal bovine serum;

5, 50 , 6, 60 -tetrachloro-1, 10 , 3, 30 -tetraethylbenzimidazol carbocyanine iodide;

malondialdehyde; MMP,

mitochondrial membrane potential; MPTP,

3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide; NADPH,

NADPH oxidases; ROS, reactive oxidative species; rt-PA,

TTC, 2, 3, 5-triphenyl-tetrazolium chloride; TUNEL, n Corresponding author. Fax: þ86 511 85038451 806. E-mail address: [email protected] (J. Gao). http://dx.doi.org/10.1016/j.brainres.2014.09.039 0006-8993/& 2014 Elsevier B.V. All rights reserved.

Cyclophilin D; Cyt

dulbecco’s modified Eagle's

dUTP nick-end labeling; VDAC,

mitochondrial

nicotinamide adenine

recombinant tissue plasminogen activator;

voltage-dependent anion channel.

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assess the effects of GA on cerebral ischemia/reperfusion injury, 2, 3, 5-triphenyltetrazolium chloride (TTC) staining, dUTP nick-end labeling (TUNEL) assay, and Cytochrome C (Cyt C) release were performed in MCAO rats. The results support that GA is useful against cerebral ischemia/reperfusion injury as a potential protective agent. & 2014 Elsevier B.V. All rights reserved.

1.

Introduction

Ischemic stroke is an acute cerebrovascular event associated with brain tissue damage due to significant deprivation of oxygen and glucose caused by a reduction or complete blockade of artery supplying blood to the brain (Chen et al., 2011). Already five minutes after the onset of ischemia neurons begin to die (Radermacher et al., 2013). Therefore, early restoration of cerebral blood flow by using recombinant tissue plasminogen activator (rt-PA) is crucial for sustaining neuronal viability (Radermacher et al., 2013). Unfortunately, reperfusion is believed to contribute to delayed secondary brain injury because the freshly arriving oxygen will serve as a substrate for excessive reactive oxidative species (ROS) production (Chen et al., 2011; Radermacher et al., 2013; Schaller and Graf, 2004). Mitochondria have long been known to play a critical role in the pathogenesis of cerebral ischemia/reperfusion injury, via ROS generation, mitochondrial dysfunction, and mitochondrial (type II) apoptosis (Christophe and Nicolas, 2006; Turrens, 2003). Mitochondria are abundant in cerebral tissue, and mitochondrial complex I is a major source of cerebral intracellular ROS (Turrens, 2003). Cerebral ischemia/reperfusion injury promotes secondary mitochondrial dysfunction, which is characterized by reduction of mitochondrial membrane potential (MMP), depletion of ATP synthesis, and inducing a sudden increase in permeability of the mitochondrial permeability transition pore (MPTP). Finally, excessive amounts of ROS are released (Heo et al., 2005; Sanderson et al., 2013; Siesjo et al., 1999). Imbalance between generation and degradation of ROS collectively leads to oxidative stress (Radermacher et al., 2013). Much evidence suggests that oxidative stress is a fundamental mechanism of cerebral ischemia/reperfusion injury (Chen et al., 2011; Schaller and Graf, 2004). Therefore, improving mitochondrial dysfunction and inhibiting oxidative stress are beneficial in the treatment of cerebral ischemia/reperfusion injury.

MPTP is considered to be one of the most important targets for modulating mitochondrial dysfunction following cerebral ischemic injury (Schinzel et al., 2005; Tsujimoto et al., 2006; Vaseva et al., 2012). MPTP is formed at contact sites between the inner and outer mitochondrial membranes and consists of the voltage-dependent anion channel (VDAC), the adenine nucleotide translocator (ANT), and Cyclophilin D (CypD), the mitochondrial isoform of the peptidylprolyl cistrans isomerase cyclophilin chaperone family located on the inner membrane of mitochondria (Elrod and Molkentin, 2013). According to Halestrap, MPTP is closed during the ischemic period and opening has been shown to occur during reperfusion (Halestrap, 2009). In response to opening of MPTP, MMP is dissipated and the death effector proteins such as Cytochrome C (Cyt C) are released from the inter-membrane space, which initiate mitochondrial (type II) apoptosis (Gómez-Crisóstomo et al., 2013). In addition, the neuroprotective effect of ischemic post-conditioning or treatment with cyclosporin A (CsA) in cerebral ischemia/reperfusion injury contributes to inhibition of MPTP opening (Cho et al., 2013; Siesjo et al., 1999; Sun et al., 2012). However, as an immunosuppressant drug, the serious adverse reaction of the immune system limits its clinical application (Rezzani, 2006). Gallic acid (GA, 3, 4, 5-trihydroxybenzoic acid, Fig. 1) is one of the most important plant polyphenolic compounds, which can be abundantly found in natural plants, tea, and red wines (Shahrzad et al., 2001). Recent research find that GA attenuates streptozotocin-induced memory deficits by inhibiting oxidative stress and activating related enzyme-dependent signaling systems (Kade and Rocha, 2013; Mansouri et al., 2013). In addition, as the impact of GA on the regulation of systems energy metabolism in rat, the relationship between GA and mitochondria has become an active topic of exploration (Shi et al., 2013). However, no study has tested the effects of GA on cerebral ischemia/reperfusion injury, and the mechanisms are also barely known. In the current study, we have investigated the potential roles and mechanisms of GA in hypoxia/reoxygenation induced by sodium hydrosulfite (Na2S2O4) in vitro and cerebral ischemia/reperfusion induced by middle cerebral artery occlusion (MCAO) in vivo.

2.

Results

2.1. Protective effects of GA against hypoxia/ reoxygenation-induced cytotoxicity in SH-SY5Y cells

Fig. 1 – Chemical structure of Gallic acid (GA), 3, 4, 5-trihydroxybenzoic acid.

It is generally accepted that Na2S2O4-induced hypoxia/reoxygenation elicited remarkable neurons injury (Wei et al., 2013; Zhang and Eyzaguirre, 1999). The MTT assay revealed that

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Fig. 2 – Protective effects of GA against hypoxia/reoxygenation-induced cytotoxicity in SH-SY5Y cells. (A) Concentrationdependent effect of Na2S2O4 induced-hypoxia/reoxygenation injury on cell viability in SH-SY5Y neuroblastoma cells. Cells were exposed to different concentrations of Na2S2O4 for 2 h, then replacing the normal medium for additional 2 h. The cell viability was assessed by an MTT assay. (B) Inhibition of Na2S2O4-induced hypoxia/reoxygenation injury decrease in cell viability by GA in SH-SY5Y cells. Cells were exposed to GA at the concentrations of 0.1–10 μM for 48 h or were pretreated with GA (0.1–10 μM) for 24 h prior to incubation with Na2S2O4 for 2 h, then replacing the normal medium for additional 2 h. The cell viability was assessed by an MTT assay. Data are reported as the means7S.D. (n¼ 5). Non-treated cells served as controls. ## Po0.01, #Po0.05 versus control group, **Po0.01, *Po0.05 versus model group.

Na2S2O4-induced hypoxia/reoxygenation decreased cell viability in a concentration-dependent manner. As shown in Fig. 2A, the viability of the cells exposed to Na2S2O4 at concentrations of 1–5 mM for 2 h and replacing the normal medium for an additional 2 h decreased to 98.3276.58%, 98.0372.59%, 89.3076.08%, 70.7178.78%, and 69.2178.48% of the control value, respectively. Compared to the control group, Na2S2O4 significantly decreased the cell viability (% of control) at 4 mM (Fig. 2A, n¼ 5, Po0.01). Based on this result,

a treatment of 4 mM Na2S2O4 for 2 h and replacing the normal medium for an additional 2 h was used to induce SH-SY5Y cell hypoxia/reoxygenation injury in subsequent experiments. Compared to the model group, pretreatment with GA at 0.1 and 1 μM did not attenuate hypoxia/reoxygenationinduced cytotoxicity (Fig. 2B, n ¼5, P40.05). We therefore increased the amount of GA to 10 μM. Compared to the model group, the cell viability significantly increased to 92.9872.79% of the control value at 10 μM (Fig. 2B, n¼ 5, Po0.05). These

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Fig. 3 – GA reverses hypoxia/reoxygenation-induced oxidative stress. Cells were pretreated with 0.1–10 μM GA for 24 h before incubation with Na2S2O4 for 2 h, then replacing the normal medium for additional 2 h. Then these treated cells were loaded with 30 μM DCF-DA for 30 min. (A) Intracellular ROS levels were determined based on DCF-fluorescence by fluorescent inverted microscope. A-a: control; A-b: model; A-c: 0.1 μM GA; A-d: 1 μM GA; A-e: 10 μM GA. (B) DCF fluorescence was measured by flow cytometry. Histogram showing the intracellular fluorescence intensity of DCF in different treatment cells (n ¼3). (C) Histogram showing cellular MDA levels in different treatment cells (n ¼ 5). Data are reported as the means7S.D. Non-treated cells served as controls. Scale bars: 50 μm ##Po0.01 versus control group, **Po0.01 versus model group.

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data demonstrate that GA protects SH-SY5Y cells against hypoxia/reoxygenation-induced cytotoxicity in a concentrationdependent manner. In another experiment, 48 h of treatment with various concentrations (0.1–10 μM) of GA alone did not lead to any apparent increase in the viability of cells (% of control) (Fig. 2B, n¼ 5, P40.05). Consequently, 0.1, 1 and 10 μM of GA were chosen as drug treatment concentration in subsequent experiments.

2.2. GA reverses hypoxia/reoxygenation-induced oxidative stress Oxidative stress is known to cause secondary neuronal damage in hypoxia/reoxygenation injury, which is the result of an imbalance between the generation and degradation of ROS (Radermacher et al., 2013). In this study, a fluorescent probe, Dichlorofluorescin diacetate (DCF-DA), was used as a specific marker for quantitative intracellular ROS formation. In comparison to the control group, the intracellular DCF-fluorescence intensity in the model group significantly increased (Fig. 3A-a, b; B, n¼ 3, Po0.01). Compared to the model group, pretreatment

with either 1 or 10 μM GA for 24 h before Na2S2O4 exposure dramatically decreased the intracellular DCF-fluorescence intensity (Fig. 3A-b, d, e; B, n¼ 3, Po0.01); moreover, the intracellular DCF-fluorescence intensity was not significantly changed in the GA 0.1 μM group (Fig. 3A-b, c; B, n¼ 3, P40.05). Lipid peroxidation product is also widely used as a marker of cell membrane damage induced by oxidative stress (Sies, 1997). In the present study, we also tested the level of malondialdehyde (MDA) after pretreatment with GA. As shown in Fig. 3C, the levels of intracellular MDA significantly increased in the control group (0.658570.2538 nmol/mg protein) compared with the model group (8.021971.2785 nmol/ mg protein) (Fig. 3C, n¼ 5, Po0.01). Compared to the model group, pretreatment with either 1 or 10 μM GA for 24 h before Na2S2O4 exposure significantly decreased the levels of intracellular MDA to 0.880670.3887 and 0.325970.1142 nmol/mg protein, respectively (Fig. 3C, n ¼5, Po0.01). There was no significant charge between the model group and the 0.1 μM GA group (Fig. 3C, n¼ 5, P40.05). Taken together, these findings suggest a role for GA in promoting SH-SY5Y cell survival partially through inhibiting oxidative stress.

Fig. 4 – GA inhibits hypoxia/reoxygenation-induced mitochondrial ROS accumulation. Cells were treated as described in Fig. 3 and then incubated with the MitoSOX Red. (A) Intracellular red fluorescence of MitoSOX Red was determined by fluorescent inverted microscope. A-a: control; A-b: model; A-c: 0.1 μM GA; A-d: 1 μM GA; A-e: 10 μM GA. (B) The intracellular red fluorescence of MitoSOX Red was measured by flow cytometry. Histogram showing the intracellular fluorescence intensity of MitoSOX Red in different treatment cells (n ¼3). Data are reported as the means7S.D. Non-treated cells served as controls. Scale bars: 50 μm ##Po0.01, #Po0.05 versus control group, **Po0.01, *Po0.05 versus model group.

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2.3. GA inhibits hypoxia/reoxygenation-induced mitochondrial ROS accumulation During reoxygenation after hypoxia, increased oxygen supply results in overproduction of ROS in mitochondria (Christophe and Nicolas, 2006; Heo et al., 2005; Siesjo et al., 1999). In the current study, a fluorescent probe, MitoSOX Red, was used as a specific marker for quantitative mitochondrial ROS accumulation. Compared to the control group, the MitoSOXfluorescence intensity in the model group was significantly increased (Fig. 4A-a, b; B, n ¼3, Po0.01). Pretreatment with GA 10 μM effectively suppressed the level of mitochondrial ROS by significantly decreasing the MitoSOX-fluorescence intensity (Fig. 4A-b, e; B, n¼3, Po0.05). There was no significant charge between model group and 0.1 or 1 μM GA group (Fig. 4A-b, c, d; B, n¼ 3, P40.05). These data indicate that GA

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inhibits hypoxia/reoxygenation-induced oxidative stress partially through suppressing the accumulation of mitochondrial ROS.

2.4. GA reverses hypoxia/reoxygenation-induced mitochondrial dysfunctions To establish whether GA attenuated hypoxia/reoxygenationinduced mitochondrial ROS accumulation by modulating mitochondrial dysfunctions, the level of MMP, oxygen consumption, and ATP synthesis was evaluated. The lipophilic cationic probe 5, 50 , 6, 60 -tetrachloro-1, 10 , 3, 30 -tetraethylbenzimidazol carbocyanine iodide (JC-1) was used to evaluate MMP. As shown in Fig. 5A-a, b; B, the control cells clearly appeared red. However, hypoxia/reoxygenation injury rapidly caused MMP dissipation, as shown by the increase in green

Fig. 5 – GA reverses hypoxia/reoxygenation-induced mitochondrial dysfunctions. Cells were treated as described in Fig. 3 and then incubated with the membrane potential indicator JC-1. (A) Intracellular red and green fluorescence of JC-1 was determined by fluorescent inverted microscope. A-a: control; A-b: model; A-c: 0.1 μM GA; A-d: 1 μM GA; A-e: 10 μM GA. (B) The intracellular red and green fluorescence of JC-1 was measured by flow cytometry. Histogram showing the red/green fluorescence intensity ratio in different treatment cells (n¼ 3). (C) Histogram showing the average oxygen consumption rates measured by Clark Oxygen Electrode in different treatment cells (n ¼ 5). (D) ATP content was measured by firefly luciferase assay. Histogram showing cellular ATP content in different treatment cells (n¼ 3). Data are reported as the means7S.D. Nontreated cells served as controls. Scale bars: 50 μm. ##Po0.01, #Po0.05 versus control group, **Po0.01, *Po0.05 versus model group.

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fluorescence and the concomitant disappearance of red fluorescence, which significantly decreased the red/green fluorescence intensity ratio (% of control) at 43.4877.22% (Fig. 5A-a, b; B, n¼ 3, Po0.01). Compared with the model group, pretreatment with either 1 or 10 μM GA significantly attenuated the changes in MMP as indicated by the repression of green fluorescence and restoration of red fluorescence. The red/green fluorescence intensity ratio was increased to 71.7177.54% and 85.7973.95% of control, respectively (Fig. 5A-b, d, e; B, n ¼3, Po0.01). Moreover there was no significant charge between model group and 0.1 μM GA group (Fig. 5A-b, c; B, n¼ 3, P40.05). Clark electrode was used for determining the level of oxygen consumption. Compared with the control group, the level of oxygen consumption (nmol/min/104 cells) significantly decreased from 0.090370.0125 to 0.062270.0019 (Fig. 5C, n¼5, Po0.01). Compared with the model group, the level of oxygen consumption (nmol/min/104 cells) in the 10 μM GA group was increased to 0.075170.0055 (Fig. 5C, n¼5, Po0.05). Moreover there was no significant charge between model group and 0.1 or 1 μM GA group (Fig. 5C, n¼5, P40.05). Consistent with the oxygen consumption data, the ATP content was significantly decreased in model group (27.1870.01 nmol/mg protein) in comparison with control group (58.3172.94 nmol/mg protein). (Fig. 5D, n ¼3, Po0.01). It was observed that pretreatment with either 1 or 10 μM GA showed a protective response in terms of significantly increased ATP content to 47.6770.67 and 51.3273.31 nmol/ mg protein, respectively, when compared with model group (Fig. 5D, n¼ 3, Po0.01). These data suggest that GA exerts antioxidative activity partially by modulating hypoxia/reoxygenation-induced mitochondrial dysfunctions.

2.5. GA prevents calcium-induced MPTP opening in isolated mitochondria The MPTP opening leads to dissipation of the MMP and a sudden decrease in ATP levels, which is considered to be one of the most important signs of mitochondrial dysfunction following cerebral ischemic injury (Schinzel et al., 2005; Vaseva et al., 2012). To investigate the effect of GA on MPTP viability, mitochondria isolated from rat livers were subjected to be treated with 100 μM CaCl2 to induce MPTP opening. The ΔA540 value in the model group was significantly higher than control groups, while ΔA540 value in the CsA group was significantly lower than model groups (Fig. 6B, n¼5, Po0.01). This observation confirmed in our system that mitochondrial swelling resulted from calcium-induced opening of the MPTP. Mitochondria pretreatment with either 1 or 10 μM GA for 5 min before CaCl2 exposure failed to swell under identical conditions. The ΔA540 value in the GA 1 and 10 μM group was significantly lower than model groups (Fig. 6B, n¼5, Po0.01). These data suggest that MPTP is one of the key targets of GA regulation of mitochondrial dysfunctions.

2.6. GA protects rats against transient focal cerebral ischemia/reperfusion injury To further assess the neuroprotective effects of GA on cerebral ischemia/reperfusion injury, a model of transient

focal ischemia produced by MCAO was employed. According to the doses of treatment of streptozotocin-induced cerebral oxidative stress (Mansouri et al., 2013), we designed the doses of three groups in preliminary experiments as 50 mg/kg, 37.5 mg/kg and 25 mg/kg respectively. Compared with the MCAO group, the infarct volume (% of the contralateral hemisphere) in the GA (50 mg/kg) group was significantly smaller than that of the MCAO group (Supplementary Fig. 2, n ¼5, Po0.01). However, there was no significant difference in the infarct volume between GA (37.5 or 25 mg/kg) and MCAO groups (Supplementary Fig. 2, n ¼5, P40.05). Consequently, 50 and 25 mg/kg of GA were chosen as drug treatment dose in the formal experiment. After MCAO for 2 h, followed by a 24 h reperfusion, the ischemic infarctions appear white and regularly include the neocortex and basal ganglia as confirmed by TTC staining (Fig. 7A). The mean arterial blood pressure, glucose and heart rate of all animals were all monitored and maintained within normal ranges in each of the experimental animal (Supplementary Table 1, n¼ 26). No neurological deficits were observed in the sham-treated rats, whereas rats subjected to MCAO were scored 2, 3 or 4 in the neurological assessment following 2 h of reperfusion (Supplementary Fig. 3A, n ¼16, P40.05). After 24 h of reperfusion, compared with the MCAO group, the neurological deficits score in the GA 50 mg/kg group was significantly reduced (Supplementary Fig. 3B, n ¼16, Po0.01), while the GA 25 mg/kg group and CsA 10 mg/kg were not (Supplementary Fig. 3B, n¼ 16, P40.05). Compared with the sham group, the total infarct volume (% of contralateral hemisphere) in the MCAO group was significantly increased to 42.0373.22% (Fig. 7A; B, n¼ 8, Po0.01). Furthermore, compared to the MCAO group, an intravenous injection of GA 50 mg/kg significantly reduced the total infarct volume (% of contralateral hemisphere) to 27.2872.52% (Fig. 7A; B, n¼ 8, Po0.01). There was no significant difference between the MCAO group and the GA 25 mg/kg group (Fig. 7A; B, n¼8, P40.05). Of note CsA is thought to be an inhibitor of the MPTP, and pretreatment with CsA 10 mg/kg decreases infarct volume after cerebral ischemia/reperfusion injury (Cho et al., 2013; Muramatsu et al., 2007). In the present study, compared to the MCAO group, an intravenous injection of CsA 10 mg/kg significantly reduced the total infarct volume (% of contralateral hemisphere) to 26.4273.03% (Fig. 7A; B, n¼ 8, Po0.01). We also examined the anti-apoptotic effect of GA using the dUTP nick-end labeling (TUNEL) assay in various groups. Consistent with the infarct volume data, the number of TUNEL-positive cells (% of DAPIþ cells) significantly increased in the per-infarct region in the MCAO group (27.6570.61%), whereas TUNEL staining was negative in sham-operated rats (Fig. 7C-a, b; D, n¼4, Po0.01). Compared with the MCAO group, pretreatment with GA (50 or 25 mg/kg) significantly reduced the number of TUNEL-positive cells (% of DAPIþ cells) in the per-infarct region to 13.6472.01% and 18.977 2.51%, respectively (Fig. 7C-b, c, d; D, n¼4, Po0.05). In comparison to the MCAO group, pretreatment with CsA 10 mg/kg significantly reduced the number of TUNELpositive cells (% of DAPIþ cells) to 17.6573.61% (Fig. 7C-b, e; D, n¼ 4, Po0.05). It is generally accepted that Cyt C released from mitochondria is dependent on opening of the MPTP (Gómez-Crisóstomo

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Fig. 6 – GA prevents calcium-induced MPTP opening in isolated mitochondria. (A) The curve is a typical sample of mitochondrial swelling induced by 100 μM CaCl2. Mitochondrial swelling was measured by monitoring mitochondrial size at an absorbance of 540 nm. GA (1 μM or 10 μM final concentration), CsA (1 μM final concentration), and buffer were added to the mitochondrial suspension 5 min before CaCl2 exposure in each groups. A change of absorbance within 10 min with a spectrophotometer indicated mitochondrial swelling. (B) The difference (ΔA540) between the maximum A540 reading (immediately after CaCl2 addition) and the minimum A540 reading (10 min after CaCl2 addition) was used for statistical analysis. Histogram comparing ΔA540 values (A540 max  A540 min) among groups (n ¼ 5). Data are reported as the means7S.D. Non-treated cells served as controls. ##Po0.01, #Po0.05 versus control group, **Po0.01 versus model group.

et al., 2013; Naranmandura et al., 2012). The data showed that the expression of Cyt C in mitochondria was significantly reduced in the MCAO group compared to that in sham groups (Fig. 7E; F, n¼4, Po0.01). In contrast, its cytosolic expression was significantly enhanced in the MCAO group compared with

sham groups (Fig. 7E; G, n¼4, Po0.01). Compared with the MCAO group, pretreatment with GA 50 mg/kg or CsA 10 mg/kg significantly increased the expression of Cyt C in mitochondria and reduced the expression of Cyt C in cytosolic (Fig. 7E; F; G, n¼4, Po0.01). There was no significant difference between the

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Fig. 7 – GA protects rats against transient focal cerebral ischemia/reperfusion injury. (A) GA attenuated transient MCAOinduced infarction assessed by TTC staining. Following 24 h of reperfusion after 2 h of MCAO in Sprague-Dawley rats, five consecutive TTC-stained coronal brain slices arranged in cranial to caudal order are shown. The white brain area represents infarcted tissue. (B) Histogram showing the infarct volume (% of contralateral hemisphere) in TTC-stained brain sections (n¼ 8). (C) Photographs of H & E and TUNEL staining in the per-infarct area form ipsilateral cerebral cortex. C-a: sham group; C-b: MCAO group; C-c: MCAOþGA 50 mg/kg group; C-d: MCAOþGA 25 mg/kg group; C-e: MCAOþCsA 10 mg/kg group. (D) Histogram showing the percentage of TUNEL-positive cells which described as the number of TUNEL-positive cells/total number of cells (n ¼4). (E) Protein expression of mitochondrial and cytosolic Cytochrome C (Cyt C) in the per-infarct area forming ipsilateral cerebral cortex was analyzed by western blot. (F, G) Histogram showing the relative intensities of the bands in each sample was semi-quantified by quantity software (n ¼4). Data are reported as the means 7S.D. Scale bars: 50 μm ## Po0.01 versus control group, **Po0.01, *Po0.05 versus model group.

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MCAO group and the GA 25 mg/kg group (Fig. 7E; F; G, n¼ 4, P40.05). The cerebral ischemia and reperfusion injury did not induce any appreciable alteration of the protein levels of COX Iv and β-actin (Fig. 7E). These data further assessed that GA exerted neuroprotective effect partially via modulating MPTP opening. Altogether, these findings suggest that GA provides neuroprotection on focal cerebral ischemia/reperfusion injury in rats.

3.

Discussion

In this study, we have investigated the role of GA in cerebral ischemia/reperfusion injury both in vitro and in vivo, and highlighted for the first time that modulating of the mitochondrial dysfunction was an important mechanism by GA attenuating oxidative stress following hypoxia/reoxygenation insult. In addition, using a model of Ca2þ-induced mitochondrial swelling, we identified that MPTP probably was one of the key targets of GA regulation of mitochondrial dysfunctions mentioned above. Although pathology-associated ROS production emanates from various different sources, several studies support the assertion that mitochondrial dysfunctions and nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOX) related to oxidative damage are the major contributors to neurodegenerative diseases (Christophe and Nicolas, 2006; Sorce and Krause, 2009). Our results are partially consistent with this phenomenon, which showed that the level of mitochondrial ROS in the model group was more increased than the control group. GA effectively decreased the level of the mitochondrial ROS and reversed the mitochondrial respiratory dysfunction, indicating that GA exerts antioxidative activity partially by modulating hypoxia/reoxygenation-induced mitochondrial dysfunctions. We will further elaborate the antioxidant effect of GA under NOX inhibited conditions, and the model of ethidium bromide (EB)-induced mitochondrial DNA depletion will also be used to investigate the mechanisms of antioxidative effects of GA (Nacarelli et al., 2014). MPTP is considered to be one of the most important targets of mitochondrial dysfunction following cerebral ischemic injury (Schinzel et al., 2005; Tsujimoto et al., 2006; Vaseva et al., 2012). In response to opening of MPTP, swollen mitochondria exhibit an increase in volume, and MMP is dissipated in response to the arrest of the function of the respiratory complexes (I–V), which contributes to an inhibition of ATP biosynthesis (Martel et al., 2012). Although the above data strongly suggested that GA reversed hypoxia/ reoxygenation-induced mitochondrial dysfunctions by inhibiting the MPTP opening, direct proof was necessary to substantiate its local action of mitochondria. Mitochondrial swelling was often considered an artifact until seminal studies by Douglas Hunter and Robert Haworth in the late seventies who established the MPTP as a tightly regulated and reversible phenomenon, and a pore formation was suggested (Haworth and Hunter, 1979). As known, mitochondria are important in regulating the Ca2þ homeostasis, and Ca2þ is believed to trigger a conformational change of the responsible proteins in MPTP formation (Eliseev et al., 2009;

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Rizzuto et al., 2012). Therefore, this process has been investigated in association with Ca2þ-induced mitochondrial swelling. Moreover, there seems to be differences in Ca2þ sensitivity for comparing brain and liver mitochondria. Grancara et al. found that not brain mitochondria but rat liver mitochondria could amplify the mitochondria swelling induced by calcium, and it was concluded that brain mitochondria are insensitive to MPTP induction and swelling by calcium (Grancara et al., 2012). Furthermore, several studies have used Ca2þ-induced liver mitochondrial swelling to evaluate the status of MPTP in cerebral ischemia/reperfusion injury (Schinzel et al., 2005; Vaseva et al., 2012). To investigate whether the protective effect of GA against mitochondrial dysfunction was associated with inhibition of MPTP opening, in this study mitochondria isolated from rat livers were subjected to be treated with 100 μM CaCl2 to induce MPTP opening. CsA is a specific MPTP inhibitor, which is thought to limit reperfusion injury by antagonizing MPTP opening and inhibiting mitochondrial (type II) apoptosis (Cho et al., 2013; Muramatsu et al., 2007). In the current study, we did not find any significant charge in ΔA540 value between the CsA pretreatment group and the control group. This observation confirmed in our system that mitochondrial swelling resulted from calcium-induced opening of the MPTP. Mitochondria pretreatment with either 1 or 10 μM GA for 5 min before CaCl2 exposure failed to swell under identical conditions, suggesting that GA effectively inhibited the Ca2þinduced MPTP opening. Moreover, it remains controversial whether mitochondria swelling is associated with MPTP opening. Of note, transient opening of MPTP could be involved in recombinant Bax-induced proton dissipation without any identifiable mitochondria matrix swelling (Jürgensmeier et al., 1998). In any case, a mere model of mitochondrial swelling for MPTP formation is inadequate. The MPTP has been suggested as one of the mechanisms of release of Cyt C from mitochondria during apoptosis (Gómez-Crisóstomo et al., 2013; Naranmandura et al., 2012). Consistent with the mitochondrial swelling data, western blot results demonstrated that the cytosolic concentration of Cyt C was dramatically reduced in the GA pretreatment group. Taken together, these data indicate that GA protects against mitochondrial dysfunction partially through inhibiting the MPTP opening. To establish whether GA protected against cerebral ischemia/reperfusion in vivo, we analyzed the infarction volume and cell apoptosis using the model of MCAO. The results of our in vivo studies first showed that GA effectively attenuated ischemia injury by decreasing the infarct volume and the number of TUNEL-positive cells in MCAO rats. According to the references, the rats were administered CsA (10 mg/kg, intravenous injection) 20 min before ischemia onset (Cho et al., 2013; Muramatsu et al., 2007). The results clearly showed the infarct volume and the number of TUNELpositive cells in the CsA treatment group were significantly less than the MCAO group. However, as an immunosuppressor drug, no clinical trial evaluated the impact of CsA in ischemic stroke (Rezzani, 2006). GA protects against hypoxia/ reoxygenation-induced mitochondrial dysfunction and MCAO-induced cerebral ischemia/reperfusion injury, indicating that GA is useful as a potential mitochondrial-targeted protective agent against ischemic stroke.

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In conclusion, we highlighted for the first time that GA provides neuroprotection on focal cerebral ischemia/reperfusion injury in MCAO rats, and protects against hypoxia/ reoxygenation insult partially through regulation of the mitochondrial dysfunction. Furthermore, the molecular mechanisms of GA inhibition of MPTP opening and the relationship with the effects against mitochondrial dysfunction will be studied in the further experimental investigation.

was discarded, and DMSO was added to solubilize the formazan reaction product with shaking for 5 min. The optical density (OD) was spectrophotometrically measured at 570 nm using a microplate reader (Molecular Device, Spectra Max 190, USA) with DMSO as the blank. Cell viability of the control group was defined as 100%. Other groups' cell viabilities were expressed as a percentage of the control.

4.3.4.

4.

Experimental procedure

4.1.

Chemicals and reagents

Gallic acid (GA) and cyclosporin A (CsA) were purchased from Cayman Chemical (Shanghai, China). Dimethylsulfoxide (DMSO), sodium hydrosulfite (Na2S2O4), 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), and 2, 3, 5-triphenyltetrazolium chloride (TTC) were purchased from Sigma Aldrich (Beijing, China). All other reagents were of analytical grade and commercially available.

4.2.

Experimental design

This study was composed of three parts. The first part was designed to determine the neuroprotective effects of GA on hypoxia/reoxygenation injury in vitro. The second part aimed to assess the effects of GA on hypoxia/reoxygenation-induced mitochondrial dysfunctions and calcium-induced MPTP opening in vitro. The third part was designed to determine the neuroprotective effects of GA protection against cerebral ischemia in vivo.

4.3. Part 1 – The neuroprotective effects of GA on hypoxia/reoxygenation injury in vitro 4.3.1.

Cell cultures

Human SH-SY5Y neuroblastoma cells were cultured in Dulbecco's modified Eagle's medium (DMEM) and 10% fetal bovine serum (FBS). All cultures were maintained in 10 cm tissue culture dishes to approximately 90% confluence in a humidified atmosphere of 5% CO2–95% air at 37 1C.

4.3.2.

Hypoxia/reoxygenation model and GA pre-treatment

Hypoxia/reoxygenation model was measured by Na2S2O4 as described by Wei et al. (2013) with some modifications . The cells were treated with Na2S2O4 at a concentration of 4 mM in the glucose-free Earle's balanced salt solution (EBSS, pH 7.4) medium for 2 h (hypoxia). Hypoxia/reoxygenation procedure was terminated by replacing the anoxic medium for an additional 2 h (reoxygenation). Various concentrations of GA (0.1, 1, 10 μM) were added into the cultures 24 h before the onset of Na2S2O4-inducing hypoxia/reoxygenation. Control cultures were treated in an identical way without inducing hypoxic injury.

4.3.3.

Assessment of cell viability

Cell viability was measured by MTT assay. Briefly, MTT was added to the culture medium at a final concentration of 0.5 mg/ml and incubated at 37 1C for 4 h. Then the medium

Measurement of intracellular ROS

DCF-DA (Molecular Probes, Beijing, China) was used to estimate intracellular ROS. Briefly, cells were loaded with 30 μM DCF-DA at 37 1C for 30 min. The results were visualized using fluorescent inverted microscope (Nikon, Ti-E Live Cell Imaging System, Japan). Alternatively, fluorescence intensity was monitored using flow cytometry (BD Immunocytometry Systems, USA) at excitation/emission maxima of 488/525 nm.

4.3.5.

Determination of malondialdehyde (MDA) levels

The MDA level was measured with the respective commercial kits (Beyotime, Haimen, Jiangsu, China). MDA levels were measured by a method based on a reaction with thiobarbituric acid. The optical density at 532 nm was measured with a microplate reader (Molecular Device, Spectra Max 190, USA).

4.3.6.

Determination of mitochondrial ROS levels

MitoSOX Red (Molecular Probes, Beijing, China) was used to monitor mitochondrial ROS state as recommended by the manufacturer. Briefly, cells were incubated with 5 μM MitoSOX Red in culture medium for 10 min at 37 1C. Finally, the results were visualized using a fluorescent inverted microscope (Nikon, Ti-E Live Cell Imaging System, Japan). Alternatively, fluorescence intensity was monitored using flow cytometry (BD Immunocytometry Systems, USA) at excitation/emission maxima of 510/580 nm.

4.4. Part 2 – The effects of GA on hypoxia/reoxygenationinduced mitochondrial dysfunctions and calcium-induced MPTP opening in vitro The grouping and administration methods of GA were identical to those described in Part 1.

4.4.1. Assessment of mitochondrial membrane potential (MMP) A cationic dye, JC-1 (Molecular Probes, Beijing, China), was used to monitor the MMP. Briefly, cells were treated with JC-1 10 μM for 30 min incubation at 37 1C. The results were visualized using a fluorescent inverted microscope (Nikon, Ti-E Live Cell Imaging System, Japan). Alternatively, fluorescence intensity was monitored using flow cytometry (BD Immunocytometry Systems, USA) at excitation/emission maxima of 525/590 nm.

4.4.2.

Oxygen consumption measurements

To estimate oxygen consumption, the Clark-type Oxygen Electrode System was carried out using methods previously published by Papandreou et al. (2006). The data are exported to a computerized chart recorder (Oxygraph 1.01, Hansatech, Norfolk, UK), which calculates the rate of oxygen consumption.

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A small stir bar maintains the cells in suspension, and a peltier heating block maintains the temperature at 37 1C.

4.4.3.

Firefly luciferase ATP assay

ATP quantification was measured with the respective commercial kits (Promega, Beijing, China). In brief, the process consisted of transferring 50 μl to a white 96-well plate and adding 50 μl of Cell Titre-GloW reagent. The plate was briefly agitated and then incubated in dark for 10 min before measuring luminescence in the fluorescence microplate reader (Molecular Device, Spectra MaxGemini, USA).

4.4.4.

intravenous injection) or CsA (10 mg/kg, intravenous injection) 20 min before ischemia onset. The dosage, administration route and timing of treatment of CsA and GA were based on previous literature (Cho et al., 2013; Muramatsu et al., 2007) and our preliminary experiment. Focal cerebral ischemia was induced by MCAO (see later). The mean arterial blood pressure, glucose and heart rate were measured at 30 min before the insertion of the suture, insertion of the suture, and in the initial 30 min of reperfusion. After model assessment (Dirnagl, 2010), 40 male SD rats were used in the experiment (n¼ 8). All animals were humanely sacrificed 24 h following reperfusion under anesthesia.

Isolation of mitochondria

Mitochondria were isolated from the livers from rat according to the method of Schinzel et al. (2005). In brief, mitochondria were isolated from the livers of mice by standard differential centrifugation and resuspended in isolation buffer (0.2 M sucrose/10 mM Tris-Mops, pH 7.4/0.1 mM EGTA–Tris) and stored on ice. Mitochondrial protein content was quantified by using the bicinchoninic acid (BCA) protein assay (Beyotime, Haimen, Jiangsu, China).

4.4.5. Measurement of mitochondrial permeability transition pore (MPTP) activity To estimate MPTP activity, the calcium-induced mitochondrial swelling was carried out using methods previously published by Schinzel et al. (2005). The isolated mitochondria were diluted in swelling buffer (120 mmol/L KCl, 10 mmol/L Tris–HCl, 20 mmol/L MOPS, 5 mmol/L KH2PO4, pH 7.4) to a final concentration of 100 mg protein/ml at 251C. The swelling was induced by adding 100 μM CaCl2 and measured with a spectrophotometer (Molecular Device, Spectra Max 190, USA) as a decrease in light scattering at 540 nm for 10 min. GA (1 μM or 10 μM final concentration) and CsA (1 μM final concentration) were added to the mitochondrial suspension 5 min before CaCl2 exposure in the treatment groups. The difference (ΔA540) between the maximum A540 reading (immediately after CaCl2 addition) and the minimum A540 reading (10 min after CaCl2 addition) was used for statistical analysis.

4.5. Part 3 – The neuroprotective effects of GA protection against cerebral ischemia in vivo 4.5.1.

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Animal preparation and experimental groups

Adult male Sprague-Dawley rats weighing 250–300 g were purchased from Jiangsu University Laboratory Animals Center, Zhenjiang, China, and kept under standard housing conditions at a temperature between 20 1C and 23 1C, with a 12 h light– dark cycle and a relative humidity of 50%. All animal tests and experimental procedures were approved by the Administration Committee of Experimental Animals in Jiangsu Province and the Ethics Committee of Jiangsu University. The design for part 3 is shown in Supplementary Fig. 1.

4.5.1.1. Experiment I: Evaluation of infarct volume. A total of 60 male SD rats, weighing 250–300 g, were randomly assigned to five groups (n¼ 12): sham group, MCAO group, GA (50 mg/kg) group, GA (25 mg/kg) group, and CsA (10 mg/kg) group. The GA and CsA-treated group was administered GA (25 and 50 mg/kg,

4.5.1.2. Experiment II: Anti-apoptotic effect of GA in MCAO rats. The grouping and administration methods of the drugs were identical to Experiment I. 70 male Sprague-Dawley rats, weight 250–300 g, were randomly assigned to five groups (n¼ 14). After model assessment (Dirnagl, 2010), 40 male Sprague-Dawley rats were used in the experiment (n ¼8). Four rats in each group were used for TUNEL assay, and the remaining four rats in each group were used to determine the expression of Cyt C.

4.5.2.

Induction of focal cerebral ischemia

Focal cerebral ischemia was induced by MCAO according to the method of Longa et al. (1989). To block the origin of the middle cerebral artery, the nylon suture (diameter of approximately 0.26 mm) was introduced through the external carotid artery into the internal carotid artery and advanced approximately 18–20 mm intracranially from the common carotid artery bifurcation. After 2 h of MCAO, the suture was removed to restore blood flow. The rats in the sham operation group underwent vessel exposure without MCAO. Body temperature was regulated at 37.070.5 1C with a heating pad and lamp when necessary. The following exclusion criteria were applied during the experiment (Dirnagl, 2010): – Mortality of animals. – No stroke (The rat with neurological deficits score of 0 point will be excluded). The method of neurological deficits evaluation is according to Longa et al. (1989). Score 0: No apparent neurological deficits; Score 1: Contralateral forelimb flexion; Score 2: Decreased resistance to lateral push; Score 3: Spontaneous movement in all directions and contralateral circling when pulled by tail; Score 4: did not walk spontaneously and had depressed levels of consciousness. – Problems during induction of MCAO (excessive bleeding, prolonged operation time Z15 min, thread placement).

4.5.3.

Assessment of cerebral infarct volume

To analyze the cerebral infarction, TTC staining was carried out using methods previously published by Leker et al. (2003). The sections were stained with 2% TTC (Sigma Aldrich, Beijing, China) in saline for 30 min at 37 1C and photographed. Then photographs of the five sections were analyzed for infarct volume by a blinded observer using image analysis software (Image-Pro Plus 6.0). Brain Infarct volumes were calculated according to the following formula: (contralateral volume undamaged ipsilateral)/contralateral volume (%).

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4.5.4.

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Histopathological analysis

Rat brains were removed, fixed in 4% paraformaldehyde in phosphate-buffered saline for 72 h at room temperature, dehydrated through a graded ethanol series, and embedded in paraffin. Sections that were 4–5 μm thick were cut in the coronal plane and stained with haematoxylin and eosin (H & E) staining, which was performed as previously described (Fischer et al., 2008).

Appendix A.

Supporting information

Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.brainres. 2014.09.039.

r e f e r e n c e s 4.5.5. nuclei

dUTP nick-end labeling (TUNEL) assay for apoptotic

TUNEL staining was measured with the respective commercial kits (Vazyme Biotech Co., Ltd, Shanghai, China). In brief, the sections were treated with protease K (20 mg/ml) for 15 min at room temperature. Reaction buffer containing terminal deoxynucleotidyl transferase was directly applied to tissue sections and incubated for 1 h at 37 1C. After being washed with PBS, sections were incubated with 40 , 6-diamidino-2-phenylindole (DAPI) (Molecular Probes, Beijing, China) for 10 min at room temperature to detect nuclei. Data are expressed as the ratio of TUNEL-positive cells to total cells.

4.5.6. Sample preparation and Western blot analysis for Cytochrome C (Cyt C) The protocol of isolating brain samples from the penumbra has been published previously by Kramer et al. (2010). Tissues homogenate was prepared to isolation of mitochondria and cytosolic protein by a Mitochondria Isolation Kit according to the manufacturer's guidelines (Beyotime, Haimen, Jiangsu, China). Thirty-microgram protein samples were sizefractionated by 8% SDS-PAGE and immunoblotted with a monoclonal rabbit anti-Cyt C antibody (1:1000, Cell Signal Technology, Shanghai, China). β-Actin detected by mouse anti-β-actin monoclonal antibody (1:2000, Cell Signal Technology, Shanghai, China), and COX IV detected by mouse anti-COX IV monoclonal antibody (1:1000, Cell Signal Technology, Shanghai, China) served as control for the amount of protein loaded.

4.6.

Statistical analysis

Except neurological deficit scores, all values are expressed as the mean7S.D., and the statistical significance of difference between groups was determined by a two-way analysis of variance (ANOVA) test. Neurological deficit scores were expressed as the median, and the statistical significance of difference between groups was determined by a nonparametric Mann–Whitney test. A P-valueo0.05 was considered to indicate statistical significance.

Acknowledgments This research was supported by Fund for Natural Science Foundation of Jiangsu Province (BK20140574), Senior Talent Cultivation Program of Jiangsu University (13JDG063), A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institution (PAPD), and in part by the National Natural Science Foundation of China (81373400).

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