Salviaolate Protects Rat Brain from Ischemia-Reperfusion Injury through Inhibition of NADPH Oxidase

• Introduction
• Results
• Discussion
• Materials and Methods
• Reagents
• Animal experiment
• Assessment of neurological deficit score and measurement of infarct volume
• Cell experiment
• Measurement of cellular apoptosis
• Western blot analysis for NOX2 and NOX4 protein expression
• Measurement of activities of NOX and caspase-3
• Measurement of H2O2 content
• Lactate dehydrogenase release assay
• Statistical analysis
• Supporting information
• Acknowledgements
• References

Abstract

Salviaolate is a group of depside salts isolated from Danshen (a traditional Chinese herbal medicine), with ≥ 85 % of magnesium lithospermate B. This study aims to investigate whether salviaolate is able to protect the rat brain from ischemia/reperfusion injury and the underlying mechanisms. Rats were subjected to 2 h of cerebral ischemia and 24 h of reperfusion to establish an ischemia/reperfusion injury model. The neuroprotective effects of salviaolate at different dosages were evaluated. A dosage (25 mg/kg) was chosen to explore the neuroprotective mechanisms of salviaolate. Neurological function, infarct volume, cellular apoptosis, nicotinamide adenine dinucleotide phosphate-oxidase activity, and H₂O₂ content were measured. In a nerve cell model of hypoxia/reoxygenation injury, magnesium lithospermate B was applied. Cellular apoptosis, lactate dehydrogenase, nicotinamide adenine dinucleotide phosphate-oxidase activity, and H₂O₂ content were examined. Ischemia/reperfusion treatment significantly increased the neurological deficit score, infarct volume, and cellular apoptosis accompanied by the elevated nicotinamide adenine dinucleotide phosphate-oxidase activity and H₂O₂ content in the rat brains. Administration of salviaolate reduced ischemia/reperfusion-induced cerebral injury in a dose-dependent manner concomitant with a decrease in nicotinamide adenine dinucleotide phosphate-oxidase activity and H₂O₂ production. Magnesium lithospermate B (20 mg/kg) and edaravone (6 mg/kg, the positive control) achieved the same beneficial effects as salviaolate did. In the cell experiments, the injury (indicated by apoptosis ratio and lactate dehydrogenase release), nicotinamide adenine dinucleotide phosphate-oxidase activity and H₂O₂ content were dramatically increased following hypoxia/reoxygenation, which were attenuated in the presence of magnesium lithospermate B (10⁻⁵ M), VAS2870 (nicotinamide adenine dinucleotide phosphate-oxidase inhibitor), or edaravone (10⁻⁵ M). The results suggest that salviaolate is able to protect the brain from ischemia/reperfusion oxidative injury, which is related to the inhibition of nicotinamide adenine dinucleotide phosphate-oxidase and a reduction of reactive oxygen species production.

Introduction

Danshen, the dried root of Salvia miltiorrhiza Bunge (Lamiaceae), has been widely used as a traditional Chinese herbal medicine for a long time to improve body functions, such as promoting blood circulation, removing blood stasis, and regulating meridians and collaterals [1], [2], [3]. To date, a number of pharmaceutical dosage forms of Danshen, like capsules, granules, oral liquids, injectables, dripping pills, and sprays, are commercially available in China [1]. Among them, a dosage form of injection, named Danshen salviaolate injection, is commonly used in clinics for adjunctive therapy of ischemic heart disease [4]. Salviaolate is a group of depside salts containing magnesium lithospermate B (MLB, ≥ 90.0 %), rosmarinic acid (≥ 5.1 %), and lithospermic acid (≥ 1.9 %) ([Fig. 1]), which are biologically active ingredients isolated from the aqueous extract of Danshen [5].

Since MLB is the major component of salviaolate, its pharmacological actions have been extensively studied. MLB has been shown to possess multiple pharmacological activities, such as anti-inflammation, antioxidation, and anti-apoptosis [6], [7], [8]. Among them, the antioxidative property of MLB is particularly impressive because its antioxidative effect is reported to be greater than that of α-lipoic acid [9], a super antioxidant available as a dietary supplement or pharmaceutical drug in many countries. MLB was able to reduce the size of myocardial infarction, suppress ischemia/reperfusion (I/R)-induced cardiomyocyte apoptosis, and improve myocardial microperfusion [10], which were related to, at least in part, its antioxidative activity [11]. In addition to the beneficial effects on the ischemic heart, MLB was also able to exert neuroprotective effects against ischemic strokes [12]. Although the antioxidative action of MLB is well recognized, the underlying mechanisms remain largely unknown.

NADPH oxidases (NOX) are considered a major source of reactive oxygen species (ROS) production in the cardiovascular and central nervous systems [13]. Numerous studies have demonstrated that NOX is activated following myocardial or cerebral I/R [14], [15], [16], accompanied by an increase in ROS generation and myocardial or cerebral oxidative injury, suggesting that a reduction of NOX-derived ROS may have a potential value in the prevention of myocardial or cerebral I/R injury. Based on previous reports, we hypothesize that the antioxidative activity of MLB is related to the inhibition of NOX.

To date, most studies regarding the aqueous extract of Danshen focus on MLB. Actually, salviaolate is the major clinical formula for depside salts from Danshen, which contains more than 85 % of MLB and other analogues [17]. The main purpose of this study was to explore the effect of salviaolate on cerebral I/R injury and the mechanisms responsible for its antioxidative activity. By using a rat model of focal cerebral I/R injury, we first evaluated the inhibitory effect of salviaolate on NOX-mediated cerebral oxidative injury in vivo. To confirm the findings of the in vivo study, we established a nerve cell model of hypoxia/reoxygenation (H/R) injury in vitro to mimic the conditions of I/R injury in vivo. Combining MLB with the specific inhibitor of NOX, VAS2870, we verified that the neuroprotective effect of MLB was related to the inhibition of NOX.

Results

A 5-point rating scale of a neurological deficit score is commonly used for the evaluation of neurological functions in an MCAO rat model. In the first set of animal experiments, there was no significant change in neurological function in the sham group compared to that in the normal control group. Two-hour cerebral ischemia and 24-h reperfusion caused a significant increase in the neurological deficit score. Salviaolate treatment significantly decreased the neurological deficit score caused by I/R in a dose-dependent manner ([Fig. 2 A]). There was no infarct in both the control and sham groups. I/R caused a significant increase in infarct volume, which was attenuated by salviaolate in a dose-dependent manner ([Fig. 2 B, C]). The vehicle of salviaolate did not display such an effect. The dose-effect studies showed that salviaolate achieved almost the maximal beneficial effect on cerebral I/R injury at the dosage of 25 mg/kg, which was chosen for the following studies.

In the second set of animal experiments, the protective effects of salviaolate on the I/R-induced neurological deficit and cerebral infarction were confirmed ([Fig. 3]). MLB (the major component of salviaolate) or edaravone (the positive control) treatment could also significantly reduce I/R-induced injuries, including neurological deficit and cerebral infarction. The vehicle of salviaolate had no such effect.

As displayed in [Fig. 4], it is hard to see terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells in both the control and sham groups. I/R treatment significantly increased the number of TUNEL-positive cells per cm²; this increase was attenuated by salviaolate. In agreement with the TUNEL data, I/R treatment significantly increased caspase-3 activity, another index for apoptosis, which was downregulated by salviaolate. MLB or edaravone treatment also reduced I/R-induced apoptosis. The vehicle of salviaolate did not show such an effect as that of salviaolate on apoptosis and caspase-3 activity.

Compared to the normal control group, there was no significant change in NOX (NOX2 and NOX4) protein levels and NOX activity in the sham group. Cerebral I/R caused a significant increase in NOX (NOX2 and NOX4) protein levels and NOX activity, which was downregulated in the presence of salviaolate ([Fig. 5 A–C]). In agreement with the changes in NOX activity, H₂O₂ content, a NOX-derived product, in the I/R group was significantly increased. The I/R-induced H₂O₂ production was blocked by salviaolate ([Fig. 5 D]). MLB or edaravone treatment also exerted the inhibitory effect on NOX activity and H₂O₂ production. The vehicle of salviaolate did not show such an effect as that of salviaolate on NOX activity and H₂O₂ production.

As displayed in Fig. S1 C, Supporting Information, I/R treatment dramatically decreased anti-O₂ ⁻ formation activity, which was reversed by salviaolate, MLB, or edaravone. The vehicle of salviaolate did not show such an effect on anti-O₂ ⁻ formation activity. I/R treatment also significantly decreased the activities of superoxide dismutase (SOD) and catalase, which were not affected by salviaolate, MLB, or edaravone (Fig. S1 A, B, Supporting Information).

To further verify the results regarding salviaolate in the rat model of cerebral I/R, a nerve cell (NG108–15) model of H/R was chosen to mimic the study in vivo. MLB, the major component of salviaolate, was used in the cell experiments. To evaluate H/R-induced NG108–15 cell injury, cellular apoptosis (Hoechst staining) and necrosis [lactate dehydrogenase (LDH) release] were analyzed. The results of Hoechst staining showed that the percentage of apoptotic cells in the H/R group was significantly elevated compared with the control group, and the elevated apoptosis was abolished by treating the cells with MLB, VAS2870 (a specific inhibitor of NOX), or edaravone ([Fig. 6 A]). The vehicle of MLB had no such effect.

H/R not only induced NG108–15 cell apoptosis but also caused NG108–15 cell necrosis. As displayed in [Fig. 6 B], H/R significantly increased LDH release versus the control group, which was attenuated in the presence of MLB, VAS2870, or edaravone. Like MLB, salviaolate could also exert similar effects on H/R-induced cell apoptosis and LDH release (Fig. S2, Supporting Information). The vehicle of MLB did not show such effect.

Compared to the normal control group, there was no significant change in NOX activity in the control group. H/R treatment caused a significant increase in NOX activity, which was downregulated in the presence of MLB, VAS2870, or edaravone ([Fig. 7 A]). In agreement with the changes in NOX activity, H₂O₂ content in the H/R group was significantly increased. The H/R-induced H₂O₂ production was blocked by MLB, VAS2870, or edaravone ([Fig. 7 B]). The vehicle of MLB did not show such an effect as that of MLB on NOX activity and H₂O₂ production.

Discussion

In this study, we evaluated the protective effect of salviaolate on the ischemic brain and the underlying mechanisms by using a rat model of cerebral I/R injury. Our results clearly showed that I/R treatment significantly increased neurological dysfunction, brain tissue, or nerve cell injury (such as necrosis and apoptosis) concomitant with the elevated NOX (NOX2 and NOX4) expression, NOX activity, and H₂O₂ production, and these effects were significantly attenuated by salviaolate. The beneficial effects of salviaolate were further confirmed by a nerve cell model of H/R injury, which mimics the condition of I/R in vivo. To the best of our knowledge, this was the first study to provide evidence that the neuroprotective effect of salviaolate was related to the inhibition of NOX and the reduction of ROS production.

The mechanisms responsible for cerebral I/R injury are multi-facial. Among them, oxidative stress is believed to play a key role in brain cell injury following I/R [18], [19]. In this study, the ROS (H₂O₂) production in the brains or NG108–15 cells was significantly increased following I/R or H/R treatment, supporting the contribution of oxidative stress to I/R or H/R injury. In clinics, although therapy with general antioxidants displays some positive effects on the reduction of cerebral I/R injury, the curative effects are not good enough and the clinical applications are still limited [20]. As a popular traditional Chinese herbal medicine, Danshen possesses the functions of antioxidation, anti-inflammation, and anti-apoptosis [1], [11], which are believed to be helpful for the prevention of I/R injury. Thus, identification of ingredients from Danshen with a potential in treating ischemic heart or brain diseases has attracted researchers in many countries, particularly in the Asiatic countries.

Early studies mainly focused on the lipophilic compounds from Danshen, while recent studies have focused more on hydrophilic compounds. To date, more than 50 chemical constituents have been isolated and identified from the aqueous extracts of Danshen [1]. Among them, MLB is a major aqueous extract ingredient of Danshen and preserves the most pharmacological activities of Danshen [5], [6], [7], [11], [21]. In theory, MLB is an ideal drug for therapy of ischemic heart or brain diseases because it is not only helpful for blood flow restoration but also beneficial to the reduction of I/R injury. However, it is a time-consuming and high-cost process to isolate pure MLB from Danshen. Most of time, MLB is just used as a research tool to represent the active component of the water-soluble fraction of Danshen. Compared to MLB, depside salts, named salviaolate, from Danshen are much easier to obtain and to spread in the clinic.

Recently, an injection form of salviaolate has been approved by the Chinese government for therapy of ischemic heart diseases [22]. But ischemic stroke has not been listed for the indication for salviaolate yet. In the present study, we provided evidence for the first time that salviaolate could protect the rat brain from I/R injury. The neuroprotective effect of salviaolate was apparently related to its function of antioxidation because salviaolate treatment could significantly decrease the production of ROS in the rat brain. The results also showed that salviaolate could achieve the same neuroprotective effect as MLB, suggesting that salviaolate could represent MLB for clinical purposes. Since previous studies from others and ours demonstrated that NOX was a major source for ROS in the brain and NOX (particularly the NOX2 and NOX4 subunits) was activated during cerebral ischemia [23], [24], we therefore examined the correlation between salviaolate and NOX. We have found that both salviaolate and MLB could significantly block the I/R-induced NOX activation in the brain concomitant with the decreased H₂O₂ content, suggesting that the neuroprotective effect of salviaolate or MLB is due to, at least partially, the inhibition of NOX. We have also found that the activities of SOD and catalase in rat brains were decreased following I/R. However, salviaolate treatment did not affect the activities of SOD and catalase, supporting that salviaolate might be a NOX inhibitor. It is well known that the immediate product of NOX is O₂ ⁻ , which dismutates into H₂O₂ either spontaneously or through the catalysis of SOD. It was reasonable to see that anti-O₂ ⁻ formation activity was increased while H₂O₂ production was reduced in the ischemic brain once NOX was inhibited by salviaolate. Based on these results, nevertheless, we still could not rule out the direct effect of salviaolate on O₂ ⁻ and H₂O₂ generation as a ROS scavenger.

To further verify the findings in the in vivo experiments, we performed the cell experiments with MLB or salviaolate. The neuroprotective activity of salviaolate was verified in the NG108–15 cell model of H/R injury. In addition, VAS2870, a specific inhibitor of NOX [25], [26], was also included in the cell experiments. Like the NOX inhibitor VAS2870, MLB treatment was able to attenuate H/R-induced NG108–15 cell injury (apoptosis and necrosis) accompanied by decreased NOX activity and H₂O₂ content, confirming our findings in the in vivo study.

In the present study, we chose edaravone as a positive control because it acts as a potent antioxidant to protect the brain from oxidative injury [27], [28]. Our results showed that edaravone was able to exert a neuroprotective effect in vivo or in vitro, which is related to the inhibition of NOX activation. To the best of our knowledge, this is the first report that the antioxidative function of edaravone is involved in, at least in part, NOX inhibition. Edaravone was initially marketed in Japan in 2001 [28], but to date its clinical applications are still limited to a few countries (such as Japan and India) [29], indicating that edaravone might not be an ideal drug for ischemic brain diseases due to some possible reasons like unacceptable side effects or unclear pharmacological mechanisms [30]. Different from synthetic edaravone, salviaolate is isolated from the natural herb Danshen, a traditional Chinese medicine, which is frequently used for the treatment of cardiovascular diseases such as coronary heart disease and cerebrovascular disease in China [5]. Randomized clinical trials and clinical experience in China indicated that Danshen-derived products are safe, with a low side-effect profile [1], [5]. Based on reports regarding edaravone and Danshen, it is reasonable to speculate that salviaolate may have clinical advantages over edaravone in the reduction of cerebral I/R oxidative injury. It is worth to point out that salviaolate is a mixture of depside salts, which contain MLB and its analogues, such as lithospermic acid and rosmarinic acid. Although MLB (≥ 90 %) is the major ingredient of salviaolate, we could not rule out the possible role of MLB analogues in the protection of the brain against I/R injury.

In summary, the results presented in this study demonstrate for the first time that salviaolate is able to protect the brain from I/R oxidative injury, which is related to the inhibition of NOX and a reduction of ROS production.

Materials and Methods

Reagents

Salviaolate (depside salts from Danshen), which contains MLB (≥ 90.0 %), rosmarinic acid (≥ 5.1 %), and lithospermic acid (≥ 1.9 %), was purchased from Greenvalley Pharmaceutical Company. MLB, with a purity ≥ 99.4 %, was provided by the Shanghai Institute of Materia Medica. Edaravone (purity ≥ 99.5 %) was purchased from the National Institute of Metrology of China. VAS2870 was offered by Sigma. Kits for Hoechst staining, measurements of LDH release, caspase-3, SOD, and catalase activity, superoxide, and H₂O₂ contents were obtained from the Beyotime Institute of Biotechnology, while kits for the TUNEL assay and NOX activity measurement were purchased from Roche and GENMED SCIENTIFICS INC, respectively. The antibodies against NOX2, NOX4, and β-actin were provided by Abcam, Santa Cruz, and Beyotime Institute of Biotechnology, respectively.

Animal experiment

Male Sprague-Dawley rats weighing 250–300 g (8 ~ 9 weeks old) were obtained from the Laboratory Animal Center, Xiang-Ya School of Medicine, Central South University, China. The animals were fasted for 24 h before the experiments, with free access to tap water. The study was performed following the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health (NIH Publication, 8th edition, 2011) and the ARRIVE guidelines (Animal Research: Reporting In Vivo Experiments). The experiments were approved by the Central South University Veterinary Medicine Animal Care and Use Committee (No. 2013–0015, 2013).

To establish the rat model of I/R injury, the rats were subjected to middle cerebral artery occlusion (MCAO) as we described previously [31]. Detailed information is available as Supporting Information. The first set of experiments was designed to examine the dose-effect relationship between salviaolate and cerebral I/R injury. The animals were randomly allocated to eight groups (n = 8 per group): (1) the control group (without surgery), (2) the sham group (underwent surgical procedures but without ischemic insult), (3) the I/R group (subjected to 2 h of ischemia followed by 24 h of reperfusion), (4–7) the salviaolate groups [treated with salviaolate (2, 10, 25, or 50 mg/kg, respectively, intravenous [i. v.], dissolved in normal saline) 30 min after the ischemia surgery], and (8) the vehicle group [treated with an equal volume of normal saline, (i. v.) 30 min after the ischemia surgery]. At the end of reperfusion, the neurological deficit score was assessed first, and then the brain tissues were collected for infarct volume measurements.

The second set of experiments was performed to explore the mechanisms underlying the neuroprotective effect of salviaolate. The animals were randomly allocated to seven groups (n = 18 per group): (1) the control group, (2) the sham group, and (3) the I/R group (treated with the same procedures as before), (4) the salviaolate group [treated with salviaolate (25 mg/kg, i. v.) 30 min after the ischemia surgery], (5) the MLB group [treated with MLB (20 mg/kg, dissolved in normal saline) 30 min after the ischemia surgery], (6) the edaravone group [treated with edaravone (6 mg/kg, i. v.) 30 min after the ischemia surgery, as a positive control here], and (7) the vehicle group (treated with an equal volume of normal saline, i. v., 30 min after the ischemia surgery). At the end of reperfusion, the neurological deficit score was assessed first, and then the brain tissues of the nine rats from each group were saved for infarct volume measurements, whereas the brain tissues (dissected from the ischemic boundary area) of the remaining nine rats from each group were collected for the TUNEL assay or other measurements (activities of caspase-3 and NOX, and H₂O₂ content).

Assessment of neurological deficit score and measurement of infarct volume

Twenty-four hours after reperfusion, the functional consequences of brain injury were evaluated by an investigator blinded to the experimental groups according to a five-point neurological deficit score (0 = no deficit, 1 = failure to extend the left forepaw, 2 = decreased grip strength of left forepaw, 3 = circling to the left by pulling the tail, 4 = spontaneous circling).

The infarct volume was evaluated by 2,3,5-triphenyltetrazolium chloride (TTC) staining. After a neurological function evaluation, the rats were sacrificed under the condition of anesthesia. The brains were rapidly removed and were sliced into 2-mm thick coronal sections with the aid of a brain matrix and were used for TTC staining (detailed information available as Supporting Information).

Cell experiment

NG108–15 neural cells were seeded at 1 × 10⁴ cells/cm² and cultured in DMEM, supplemented with 10 % fetal bovine serum, penicillin (100 IU × mL⁻¹), streptomycin (100 mg ×mL⁻¹), and L-glutamine (2 mmol × L⁻¹). The cultures were maintained at 37 °C in 95 % air/5 % CO₂ in a humidified incubator for two days, and then washed with PBS and rendered quiescent in serum-free DMEM for 24 h before the experiments. To establish the H/R model, NG108–15 cells were subjected to 5 h of hypoxia (O₂/N₂/CO₂, 1 : 94 : 5) in preconditioned hypoxic medium followed by 20 h of reoxygenation, where hypoxic medium was replaced with fresh medium upon switching to reoxygenation.

NG108–15 cells were randomly divided into four groups (eight individual experiments per group): the control group, without any treatment; the H/R group, cells were subjected to 5 h of hypoxia followed by 20 h of reoxygenation; the MLB plus H/R group, MLB (10⁻⁵ M) was added to the culture medium before the cells were subjected to H/R; the edaravone plus H/R group, edaravone (10⁻⁵ M) was added to the culture medium before the cells were subjected to H/R; the VAS2870 plus H/R group, VAS2870 (10⁻⁵ M), a specific inhibitor of NOX, was added to the culture medium before the cells were subjected to H/R; the vehicle plus H/R group, an equal volume of DMSO (0.5 %, final concentration) was added to the culture medium before the cells were subjected to H/R. At the end of the experiments, culture mediums or cells were collected for analysis of cellular apoptosis, LDH release, NOX activity, and H₂O₂ content.

Measurement of cellular apoptosis

Cellular apoptosis in the brain tissue or in the cell cultures from different groups was analyzed by a TUNEL assay or Hoechst staining. The procedure was performed according to the manufacturerʼs instructions (detailed information can be found as Supporting Information).

Western blot analysis for NOX2 and NOX4 protein expression

The procedures for sample preparation and Western blot were described previously [32]. Briefly, samples containing 40–60 µg of protein were subjected to 8 % SDS-PAGE gel and the proteins were transferred onto polyvinylidene fluoride membranes. Blots were incubated with primary antibodies against NOX2, NOX4, or β-actin followed by horseradish peroxidase (HRP)-coupled secondary antibodies. The signals of bands were measured by Luminata™ Crescendo Western HRP substrate through the Molecular Imager ChemiDoc XRS System. The densitometric quantification was conducted with Image J.

Measurement of activities of NOX and caspase-3

NOX activity was measured by a commercially available kit following the manufacturerʼs instructions. Briefly, the supernatant of brain tissue homogenates was incubated with oxidized cytochrome c in a quartz cuvette at 30 °C for 3 min, and then the NOX substrate (NADPH) was added to the reaction mixture and incubated for 15 min. The change of absorbance at 550 nm was monitored by a spectrophotometer. NOX activity was estimated by calculating cytochrome c reduction per min.

Measurement of caspase-3 activity was performed according to the manufacturerʼs instructions. In brief, 10 µl of brain tissue (dissections of ischemic hemisphere) homogenates were mixed with 90 µl of reaction solution containing caspase-3 substrate (Ac-DEVD-pNA) and incubated for 60 min at 37 °C. The absorbance was read at 405 nm. The enzyme activity was expressed as U/g protein, and 1 U of enzyme was defined as the amount of enzyme required to cleave 1.0 nmol Ac-DEVD-pNA per hour at 37 °C.

Measurement of H₂O₂ content

The detection of H₂O₂ is based on the oxidation of ferrous (Fe²⁺) to ferric ion (Fe³⁺) in the presence of xylenol orange. In a sulfuric acid solution, the Fe³⁺ complexes were combined xylenol orange dye to yield a purple product with a maximum absorbance at 560 nm. For measurement of the H₂O₂ level, 50 µL of supernatant of brain tissue or NG108–15 cell homogenates and 100 µL of work solution (0.25 mM ammonium ferrous II sulfate, 25 mM H₂SO₄, 100 mM sorbitol, 125 µM xylenol orange) were mixed and incubated at room temperature for 20 min. The change of absorbance at 560 nm was monitored and the level of H₂O₂ was calculated according to a standard curve made from the standard solutions provided by the supplier.

Lactate dehydrogenase release assay

Culture medium in the cell experiments was collected for the LDH release (an indicator of cellular damage) assay using a colorimetric assay kit according to the manufacturerʼs instructions. The released LDH was measured with a coupled enzymatic reaction that resulted in the conversion of a tetrazolium salt into a red color formazan by diaphorase. In brief, 20 µL of culture medium were mixed with 60 µL of LDH work solution and incubated at 30 °C for 25 min. The absorbance was recorded at 490 nm. The percentage of cell damage was calculated according to a formula provided by the kit supplier.

Statistical analysis

SPSS software (Version 19.0) was used for statistical analysis. Data were expressed as mean ±SEM. Differences in measured values among the multiple groups were analyzed by the analysis of variance with Bonferroniʼs multiple comparison tests. Differences were considered significant when p < 0.05.

Supporting information

Detailed information on the experimentʼs setup and on measuring methods are available online as Supporting Information

Acknowledgements

This work was supported by the Major Research Plan of the National Natural Science Foundation of China (No. 91 439 104 to Jun Peng), the National Nature Science Foundation of China (No. 81 373 409 to Jun Peng, No. 81 370 250 to Qi-Lin Ma), the Hunan Provincial Natural Science Foundation of China (No. 13JJ2008 to Jun Peng, No. 2015JJ2156 to Xiu-Ju Luo), and the Doctoral Fund of Ministry of Education of China (No. 20 120 162 110 056 to Jun Peng).

Conflict of Interest

None declared.

^* These authors contributed equally to this work.

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• 19 Zhao H, Wang R, Tao Z, Yan F, Gao L, Liu X, Wang N, Min L, Jia Y, Zhao Y, Ji X, Luo Y. Activation of T-LAK-cell-originated protein kinase-mediated antioxidation protects against focal cerebral ischemia-reperfusion injury. FEBS J 2014; 281: 4411-4420
  CrossrefPubMedSearch in Google Scholar
• 20 Slemmer JE, Shacka JJ, Sweeney MI, Weber JT. Antioxidants and free radical scavengers for the treatment of stroke, traumatic brain injury and aging. Curr Med Chem 2008; 15: 404-414
  CrossrefPubMedSearch in Google Scholar
• 21 Chen RJ, Jinn TR, Chen YC, Chung TY, Yang WH, Tzen JT. Active ingredients in Chinese medicines promoting blood circulation as Na⁺/K⁺ -ATPase inhibitors. Acta Pharmacol Sin 2011; 32: 141-151
  CrossrefPubMedSearch in Google Scholar
• 22 Fang C, Ren X, Zhou H, Gong ZC, Shen L, Bai J, Yin JY, Qu J, Li XP, Zhou HH, Liu ZQ. Effects of eNOS rs1799983 and ACE rs4646994 polymorphisms on the therapeutic efficacy of salvianolate injection in Chinese patients with coronary heart disease. Clin Exp Pharmacol Physiol 2014; 41: 558-564
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• 23 Wang Z, Wei X, Liu K, Zhang X, Yang F, Zhang H, He Y, Zhu T, Li F, Shi W, Zhang Y, Xu H, Liu J, Yi F. NOX2 deficiency ameliorates cerebral injury through reduction of complexin II-mediated glutamate excitotoxicity in experimental stroke. Free Radic Biol Med 2013; 65: 942-951
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• 24 Yang ZB, Tan B, Li TB, Lou Z, Jiang JL, Zhou YJ, Yang J, Luo XJ, Peng J. Protective effect of vitexin compound B-1 against hypoxia/reoxygenation-induced injury in differentiated PC12 cells via NADPH oxidase inhibition. Naunyn Schmiedebergs Arch Pharmacol 2014; 387: 861-871
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• 25 ten Freyhaus H, Huntgeburth M, Wingler K, Schnitker J, Baumer AT, Vantler M, Bekhite MM, Wartenberg M, Sauer H, Rosenkranz S. Novel Nox inhibitor VAS2870 attenuates PDGF-dependent smooth muscle cell chemotaxis, but not proliferation. Cardiovasc Res 2006; 71: 331-341
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• 26 Sun QA, Hess DT, Wang B, Miyagi M, Stamler JS. Off-target thiol alkylation by the NADPH oxidase inhibitor 3-benzyl-7-(2-benzoxazolyl) thio-1,2,3-triazolo[4,5-d] pyrimidine (VAS2870). Free Radic Biol Med 2012; 52: 1897-1902
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• 27 Ahmad A, Khan MM, Javed H, Raza SS, Ishrat T, Khan MB, Safhi MM, Islam F. Edaravone ameliorates oxidative stress associated cholinergic dysfunction and limits apoptotic response following focal cerebral ischemia in rat. Mol Cell Biochem 2012; 367: 215-225
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• 28 Nakase T, Yoshioka S, Suzuki A. Free radical scavenger, edaravone, reduces the lesion size of lacunar infarction in human brain ischemic stroke. BMC Neurol 2011; 11: 39
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• 29 Wu S, Sena E, Egan K, Macleod M, Mead G. Edaravone improves functional and structural outcomes in animal models of focal cerebral ischemia: a systematic review. Int J Stroke 2014; 9: 101-106
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• 30 Lapchak PA. A critical assessment of edaravone acute ischemic stroke efficacy trials: is edaravone an effective neuroprotective therapy?. Expert Opin Pharmacother 2011; 11: 1753-1763
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• 31 Fu SH, Zhang HF, Yang ZB, Li TB, Liu B, Lou Z, Ma QL, Luo XJ, Peng J. Alda-1 reduces cerebral ischemia/reperfusion injury in rat through clearance of reactive aldehydes. Naunyn Schmiedebergs Arch Pharmacol 2014; 387: 87-94
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• 32 Zhang HF, Li TB, Liu B, Lou Z, Zhang JJ, Peng JJ, Zhang XJ, Ma QL, Peng J, Luo XJ. Inhibition of myosin light chain kinase reduces NADPH oxidase-mediated oxidative injury in rat brain following cerebral ischemia/reperfusion. Naunyn Schmiedebergs Arch Pharmacol, advance online publication 29 April 2015
  PubMed

Correspondence

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  CrossrefPubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar
• 25 ten Freyhaus H, Huntgeburth M, Wingler K, Schnitker J, Baumer AT, Vantler M, Bekhite MM, Wartenberg M, Sauer H, Rosenkranz S. Novel Nox inhibitor VAS2870 attenuates PDGF-dependent smooth muscle cell chemotaxis, but not proliferation. Cardiovasc Res 2006; 71: 331-341
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• 26 Sun QA, Hess DT, Wang B, Miyagi M, Stamler JS. Off-target thiol alkylation by the NADPH oxidase inhibitor 3-benzyl-7-(2-benzoxazolyl) thio-1,2,3-triazolo[4,5-d] pyrimidine (VAS2870). Free Radic Biol Med 2012; 52: 1897-1902
  CrossrefPubMedSearch in Google Scholar
• 27 Ahmad A, Khan MM, Javed H, Raza SS, Ishrat T, Khan MB, Safhi MM, Islam F. Edaravone ameliorates oxidative stress associated cholinergic dysfunction and limits apoptotic response following focal cerebral ischemia in rat. Mol Cell Biochem 2012; 367: 215-225
  CrossrefPubMedSearch in Google Scholar
• 28 Nakase T, Yoshioka S, Suzuki A. Free radical scavenger, edaravone, reduces the lesion size of lacunar infarction in human brain ischemic stroke. BMC Neurol 2011; 11: 39
  CrossrefPubMedSearch in Google Scholar
• 29 Wu S, Sena E, Egan K, Macleod M, Mead G. Edaravone improves functional and structural outcomes in animal models of focal cerebral ischemia: a systematic review. Int J Stroke 2014; 9: 101-106
  CrossrefPubMedSearch in Google Scholar
• 30 Lapchak PA. A critical assessment of edaravone acute ischemic stroke efficacy trials: is edaravone an effective neuroprotective therapy?. Expert Opin Pharmacother 2011; 11: 1753-1763
  PubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar
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  PubMed

• Supplementary Material