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Quercetin Enhances Exercise-Mediated Neuroprotective Effects in Brain Ischemic Rats

CHANG, HENG-CHIH1; YANG, YEA-RU1; WANG, PAULUS S.2,3,4; WANG, RAY-YAU1

Medicine & Science in Sports & Exercise: October 2014 - Volume 46 - Issue 10 - p 1908–1916
doi: 10.1249/MSS.0000000000000310
BASIC SCIENCES
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Purpose Reactive oxygen species are markedly increased after ischemia and play important roles in the mechanism of ischemia-reperfusion injury. Regulating the oxidative stress response after brain ischemia provides a potential therapeutic strategy. Quercetin is a natural flavonoid that exhibits antioxidant properties. However, the mechanisms by which it protects cells are not fully understood. Exercise training also reduces oxidative stress and enhances brain recovery. The purpose of this study was to determine whether combined exercise training with quercetin treatment could result in better neuroprotection and functional recovery in rats subjected to brain ischemia.

Methods Rats were randomly assigned to the following groups: middle cerebral artery occlusion (MCAO) with rest control, MCAO with quercetin, MCAO with exercise, or MCAO with exercise and quercetin. To determine the effect of PI3K/Akt pathway in quercetin and exercise-mediated neuroprotection, two additional groups, a group of MCAO with quercetin and PI3K/Akt inhibitor (LY294002) and a group of MCAO with exercise, quercetin, and PI3K/Akt inhibitor, were added in this study. Motor function was examined at the 24th hour and 14th day post-MCAO. Brain samples were used to measure the expression of antioxidative and antiapoptotic proteins as well as to measure the infarct volume.

Results Treatment with either exercise or quercetin significantly decreased oxidative stress and infarct volume, increased antioxidative and antiapoptotic signaling, and improved motor function. Exercise training combined with quercetin treatment resulted in better outcomes than either treatment alone. PI3K/Akt inhibition eliminated the protective effects of exercise training and quercetin treatment.

Conclusion Quercetin enhances exercise-mediated functional recovery after brain ischemia via up-regulation of PI3K/Akt activity to promote antioxidative and antiapoptotic signaling.

1Department of Physical Therapy and Assistive Technology, National Yang-Ming University, Taipei, TAIWAN; 2Department and Institute of Physiology, National Yang-Ming University, Taipei, TAIWAN; 3Graduate Institute of Basic Medical Science, PhD Program for Aging, College of Medicine, China Medical University, Taichung, TAIWAN; and 4Department of Biotechnology, College of Health Science, Asia University, Taichung, TAIWAN

Address for correspondence: Ray-Yau Wang, Ph.D., P.T., Department of Physical Therapy and Assistive Technology, National Yang-Ming University, 155, Section 2, Li-Nong Street, Taipei, Taiwan; E-mail: rywang@ym.edu.tw.

Submitted for publication July 2013.

Accepted for publication February 2014.

The brain is quite susceptible to oxidative damage (9,18), and the ability of neurons to combat oxidative stress is limited (2). Several studies have indicated that reactive oxygen species are markedly increased after ischemia, suggesting that oxidative stress plays an important role in the mechanism of ischemia–reperfusion injury (18,32). The production of free radicals can lead to the consumption of endogenous antioxidants, such as glutathione (GSH), which has been suggested to be critical for the survival of cells in the brain (26). Maintaining the balance between oxidative stress defense and reactive oxygen species production is important for neurons to function normally and to recover from injury. In addition, the integrity and coordination of the central nervous system is important for motor function. One of the neural bases for recovery after stroke is known to be neuronal plasticity (3). However, reactive oxygen species limits neuronal plasticity (22). As a result, regulating the oxidative stress response to protect neurons from oxidative stress is a plausible potential therapeutic strategy after brain ischemia.

Exercise training is a regular therapeutic intervention that improves recovery in stroke survivors. In animal studies, it is proved that the exercise training reduces reactive oxygen species production and increases GSH expression in both young and old rats (13), protects the blood–brain barrier after brain ischemia (43), and increases the brain oxidative stress response (29). It has been noted in several studies that neuroprotective effects that benefited from the exercise after brain ischemia are mainly associated with the activation of the PI3K/Akt pathway (4,24). The activation of PI3K/Akt pathway reduces mitochondrial dysfunction and cell apoptosis by increasing Bad phosphorylation and Bcl-2 expression to reduce the release of free Bax and cytochrome c as well as the caspase 3 proteolysis (41). In addition, Bcl-2 has been suggested to be a key regulator of mitochondrial GSH levels (36). As such, antioxidative and antiapoptotic mechanisms are suggested to be the two main factors contributing to the protective effects of exercise after brain ischemia. Furthermore, recent studies report that combined exercise training with pharmaceutical agents results in improved functional recovery after brain ischemia (21,31). These studies imply that combined exercise training with antioxidant treatment may result in better outcomes than single treatment alone after brain ischemia.

Quercetin is a natural flavonoid commonly found in vegetables, fruits, and tea. It is found that quercetin is capable of protecting cells against injury in organs, including the brain, as it can permeate the blood–brain barrier (10,40). A previous study has indicated that quercetin treatment during the acute phase of brain ischemia significantly increases the expression of antioxidants and reduces cell death (1). Other studies also have reported that quercetin exerts protective effects after brain ischemia (20,38) and reduces programmed cell death in a rat model of Parkinson’s disease (17). However, the mechanisms by which quercetin regulates antioxidants after brain ischemia are not fully understood.

Furthermore, little is known regarding the combined effects of exercise and quercetin in the brain recovery mechanisms after an ischemia. This study aimed to determine whether combined exercise training with daily quercetin supplementation could result in better functional recovery in ischemic rat brains. The expression levels of antioxidative and antiapoptotic factors were measured in the present study.

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MATERIALS AND METHODS

Experimental animals.

Eighty-four adult Sprague–Dawley rats (8 wk of age, body weight = 300–350 g) were used. All experimental procedures were approved by the Institutional Animal Care and Use Committee of National Yang-Ming University, Taipei, Taiwan (IACUC-991247). The procedures were in accordance with the policy statement of the American College of Sports Medicine on research with experimental animals. Rats were housed in a temperature-controlled room (22°C ± 1°C) with daily artificial illumination for 12 h between 7:00 a.m. and 7:00 p.m. and received food and water ad libitum. Rats were randomly assigned to one of the four groups: middle cerebral artery occlusion (MCAO) with rest control (C, n = 14), MCAO with quercetin treatment (Q, n = 14), MCAO with exercise training (E, n = 14), and MCAO with exercise training and quercetin treatment (EQ, n = 14). To determine the involvement of the PI3K/Akt pathway in quercetin and exercise-mediated protection, two additional groups, MCAO with quercetin and PI3K/Akt inhibitor (LY294002) treatment (Q + I, n = 14) and MCAO with exercise training, quercetin, and PI3K/Akt inhibitor treatment (EQ + I, n = 14), were added. Our previous study has indicated that the predominant damaged area in our rat brain ischemia model is the right motor cortex and striatum (35). In this study, the right motor cortex and striatum were examined and analyzed. Half of the rats in each group were used to examine the motor function, protein expression, levels of lipid peroxidation (LP), GSH concentration, and activities of GSH peroxidase (GPx) and GSH reductase (GRx) in the right motor cortex and striatum. The remaining half of the rats in each group were used to measure the infarct volume for comparison.

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Intracerebroventricular cannulation.

A stainless steel cannula was inserted intracerebroventricularly into rats in the Q + I and EQ + I groups for intracerebroventricular injection of the PI3K/Akt inhibitor. Rats were anesthetized with pentobarbital (50 mg·kg−1) and placed on a stereotaxic frame. The skull was exposed, and a cannula was implanted after boring a hole above the cerebral ventricle (AP: –0.36 mm, RL: +1.5 mm, V: –4.4 mm from Bregma) as described by Paxinos and Watson (28). After fixing the cannula on the skull, the rats were allowed to recover for 7 d and then underwent MCAO.

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MCAO procedure.

MCAO was induced on the right side of the brain using standard microsurgical techniques as described previously (4,37). In brief, the right middle cerebral artery trunk was occluded above the rhinal fissure using 10-0 suture needle, and both common carotid arteries were occluded using nontraumatic aneurysm clips for 60 min under pentobarbital (50 mg·kg−1) anesthesia. A previous study has showed that the occlusion of the bilateral common carotid arteries in addition to the MCA is essential to induce unilateral brain ischemia in rats because of the abundant communicative circulation between the cerebral arteries (6). The complete interruption and reperfusion of blood flow was confirmed using an operating microscope. The needle and aneurysm clips were removed at the 60th minute after the induction of ischemia. The wound was sutured, and the rats were returned to their cages after recovery from anesthesia. Neurological examination was performed at the 24th hour post-MCAO based on a neurological grading scale (0–4), as described by Menzies et al. (25). Median neurological scores were 2 (range: 1–3), 3 (range: 1–3), 2 (range: 1–3), 3 (range: 1–3), 3 (range: 1–3), and 2 (range: 1–3) in the C, Q, Q + I, E, EQ, and EQ + I groups, respectively. The neurological scores did not differ significantly between the groups, and no rats died during the experimental period.

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Exercise training.

A motor-driven treadmill (T410E; Singa Co., Taiwan) was used for exercise training. Treadmill training was initiated at the 24th hour after the induction of brain ischemia. The training protocols were 20 m·min−1 and 30 min·d−1 for 14 consecutive days (4). Rats in the C, Q, and Q + I groups did not receive exercise training, whereas those in the E, EQ, and EQ + I groups exercised on the treadmill.

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Injection of quercetin and the PI3K inhibitor.

Each day for 14 d, starting at the 24th hour after the induction of brain ischemia, rats in the C and E groups received intraperitoneal injection of 100 μL of 0.1 M PBS as control injection, and rats in the Q and Q + I groups received quercetin injection. Rats in the EQ and EQ + I groups received quercetin injection immediately after 30 min of exercise training each day for 14 d. Quercetin was purchased from Sigma-Aldrich (United States, product no. Q4951, purity > 98%) and was suspended in 0.1% DMSO in PBS (30 mg·kg−1). The PI3K/Akt inhibitor, LY294002 (dissolved in 5% DMSO in PBS), was administered via intracerebroventricular injection (10 μg per 5 μL) 30 min before quercetin injection in the Q + I and EQ + I groups.

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Motor function measurement.

The foot fault-placing, parallel bar-crossing, and ladder-climbing tests were performed to assess motor performance (8). To determine the effects of exercise training and quercetin treatment, rats in the E, EQ, and EQ + I groups were tested at the 24th hour post-MCAO (before the first training) and at the 14th day post-MCAO (1 h after the final training). Rats in the C, Q, and Q + I groups were tested at the same time points, except that they did not receive exercise training during the 14 experimental days. Each motor function test was performed three times with a 5-min rest in between, and the average score was used for comparison. Before the induction of brain ischemia, all of the rats were acclimated to these motor tests by performing three repetitions per day for 3 d to ensure they could perform the test.

The foot fault-placing test was used to examine forelimb function (8). Rats were encouraged to traverse a grid surface for 1 min. Mistakes, denoted by their affected (left) forelimb fell through one of the openings in the grid, were recorded. The error is defined and calculated by mistakes per meter. Fewer errors indicate better forelimb function.

The parallel bar-crossing test was performed to examine hindlimb function (8). Rats were encouraged to walk on a parallel bar for 1 min. Instances in which the affected (left) hind paw slipped over the rod, both hind paws were placed on the same bar, or the rats fell or swung from the rods were recorded as mistakes. The error is defined and calculated by mistakes per meter. Fewer errors indicate better hindlimb function.

The ladder-climbing test was conducted to examine the coordination of all four limbs (8). Rats were encouraged to climb a vertical ladder for 1 min. The number of rungs climbed in the 1-min period was recorded. More rungs climbed indicate better coordination of all four limbs.

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Sample preparation.

Half of the rats in each group were sacrificed at the 15th day post-MCAO under deep pentobarbital anesthesia (100 mg·kg−1), and the right motor cortex and striatum were isolated, rinsed twice with cold PBS, weighed, and homogenized using lysis buffer containing 50 mM Tris–HCl (pH 8.0), 150 mM NaCl, 0.02% sodium azide, 0.1% SDS, 1% Nonidet P-40, 1 mM sodium orthovanadate, 1 mM sodium fluoride, 10 μg·mL−1 aprotinin, 10 μg·mL−1 leupeptin, and 1 mM phenylmethylsulfonyl fluoride. Forty microliters of lysate from each sample was used to examine the LP level. The remaining lysate was centrifuged at 12,500g for 30 min at 4°C. The supernatant was collected to examine protein expression, the GSH level, and the activities of GPx and GRx. The total protein concentration of supernatant was determined via the Bradford-red protein assay.

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Protein measurement.

Equivalent amount of protein (30 mg) from each sample was resolved using 8% or 12% SDS–polyacrylamide gels and transferred onto polyvinylidene fluoride membranes (Millipore, USA). After being blocked with 0.1% Tween 20/Tris-buffered saline (TBST) containing 5% nonfat milk at room temperature for 1 h, the membranes were probed using primary antibodies, including rabbit antiphosphorylated Akt (p-Akt, 1:1000), antitotal Akt (t-Akt, 1:2000), anti-p-Bad (1:1000), anti-t-Bad (1:1000), anti-Bcl-2 (1:12000), anti–cytochrome c (1:1000), and mouse anti–beta actin (1:20000), diluted in TBST at 4°C overnight. After being washed with TBST, the membranes were incubated in horseradish peroxidase-conjugated goat antirabbit or antimouse IgG secondary antibody (1:6000) diluted in TBST at room temperature for 1 h. All antibodies were purchased from Cell Signaling (Millipore, USA). The signals were visualized using a Western blot chemiluminescence reagent (Amersham Biosciences, Hong Kung). The quantitative protein band density of each sample was detected and assayed by the Image Gauge software (Fujifilm, Japan) and presented as percentage to the C group.

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Measurement of GSH level and the activities of GPx and GRx.

The GSH level was determined according to the method of a previous study (30). First, 10 μL of the sample supernatant or a standard concentration of GSH was mixed with 10 μL of 0.2 N perchloric acid, followed by the addition of 740 μL of 0.1 M PBS (pH 7.5), 20 μL of 0.6 mM 5,5′-dithio-bis-2-nitrobenzoic acid, 70 μL of 0.2 mg·mL−1 nicotinamide adenine dinucleotide phosphate (NADPH), and 130 μL of double distillated water to a total volume of 980 μL. After mixing, 20 μL of 20 U·mL−1 GRx was added to initiate the assay. The formation of 5′-thio-2-nitrobenzoic acid was measured at 412 nm. The expression of GSH in each sample was presented as nanomoles per milligram of protein.

The activity of GPx was determined according to the method of a previous study (27). The reaction mixture consisted of 0.05 M PBS (pH 7.0), 1 mM EDTA, 1 mM sodium azide, 1 EU·mL−1 GRx, 1 mM GSH, 0.2 mM NADPH, and 0.25 mM hydrogen peroxide. To measure the activity of GPx in each sample, 0.02 mL of the tissue supernatant was added to 0.98 mL of the reaction mixture, and the disappearance of NADPH at 340 nm was recorded at room temperature. The activity was calculated as units per milligram of protein using a molar extinction coefficient of 6.229 × 103 M−1·cm−1.

The activity of GRx was determined according to the method of a previous study (27). The assay mixture consisted of 0.1 M PBS (pH 7.6), 0.1 mM NADPH, 0.5 mM EDTA, and 1 mM GSSG. To measure the activity of GRx in each sample, 0.02 mL of the tissue supernatant was added to 0.98 mL of the assay mixture, and the disappearance of NADPH at 340 nm was recorded at room temperature. The activity was calculated as n-units per milligram of protein using a molar extinction coefficient of 6.229 × 103 M−1·cm−1.

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LP measurement.

The LP level was determined according to the method of a previous study (16). From each sample, 40 μL of the tissue lysate was incubated at 37°C for 1 h and then mixed with 40 μL of 5% chilled trichloroacetic acid and 40 μL of 0.67% thiobarbituric acid. The mixture was centrifuged at 4000g for 15 min, and 50 μL of the supernatant was transferred to another microcentrifuge tube and boiled for 10 min. The LP was determined by measuring the formation of thiobarbituric acid-reactive substances (TBARS) at 523 nm. The expression level of LP was expressed as nanomoles of TBARS formed per milligram of protein using a molar extinction coefficient of 1.56 × 105 M−1·cm−1.

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Infarct volume measurement.

The remaining half of the rats in each group were sacrificed under anesthesia (pentobarbital, 50 mg·kg−1) via transcardial perfusion with 100 mL of 0.1 M PBS (pH 7.2). The brain was removed and dissected into 2-mm coronal sections using a brain slicer. The fresh brain slices were immersed in a 2% 2,3,5-triphenyltetrazolium chloride saline solution at 37°C for 10 min, followed by fixation in 10% phosphate-buffered formalin. Unstained areas of each brain slice, defined as infarct areas, were detected using an image analyzer (Image-Pro Plus, Total Smart Technology, Taiwan). The total measured infarct volume (MV) of each brain was calculated via the summation of the infarct volume of each brain slice. The right hemisphere volume (RV) and the left hemisphere volume (LV) were measured and calculated, and the corrected infarct volume was calculated and used as previously described: LV − (RV − MV) (37). The corrected infarct volume was then presented as the percentage of the volume of the left hemisphere.

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Statistical analysis.

All data were expressed as means ± SEM. Two-way (quercetin × exercise) ANOVA was used to determine the main effects of exercise and quercetin and their interactive effects on the infarct volume, protein expression, GSH concentration, activities of GPx and GRx, and LP level among the C, Q, E, and EQ groups. Post hoc one-way ANOVA was used to determine the difference between the groups of C, Q, E, and EQ. The independent t-test was performed to determine the differences between the Q and the Q + I groups and between the EQ and the EQ + I groups to elucidate the involvement of the PI3K/Akt pathway. The differences in motor performance were examined via two-way repeated-measurement (group–time) ANOVA. The post hoc Tukey test was used to determine the differences between the groups. Significance was set at p < 0.05.

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RESULTS

The results of motor function test are shown in Figure 1. Quercetin and exercise both significantly improved motor function in all of the tests 14 d post-MCAO compared with the motor function of the C group. Cotreatment with exercise and quercetin resulted in improved motor performances compared with quercetin or exercise alone, except that there was no significant difference between the EQ and the E groups on the parallel bar-crossing test. The PI3K/Akt inhibitor eliminated the effects of quercetin and co-treatment with exercise and quercetin on motor performance in all of the tests.

FIGURE 1

FIGURE 1

Two-way ANOVA revealed the main effects of quercetin and exercise on the expression levels of GSH, GPx, GRx, and LP in the affected (right) cortex and striatum and on the infarct volume (quercetin: P < 0.01 for the expression levels of GSH, GPx, GRx, and LP in the cortex and striatum and the infarct volume; exercise: P < 0.01 for the expression levels of GSH, GPx, GRx, and LP in the cortex and striatum and the infarct volume). There were no interactions detected between exercise and quercetin. Post hoc analysis revealed that treatment with either quercetin or exercise increased the expression levels of GSH, GPx, and GRx and decreased the LP level and the infarct volume compared with the results of the C group. The combination of exercise and quercetin treatment resulted in greater expression levels of GSH, GPx, and GRx and a lower LP level and infarct volume compared with that in the Q and E groups (Table 1). These results indicated the effect of combined exercise and quercetin treatment on the expression levels of antioxidative compounds was greater than either treatment alone after brain ischemia.

TABLE 1

TABLE 1

Two-way ANOVA revealed the main effects of quercetin and exercise training on the expression levels of p-Akt in the affected cortex and striatum (quercetin: P < 0.01 in the cortex and striatum; exercise: P < 0.01 in the cortex and striatum). There was no interaction between quercetin and exercise. Post hoc analysis revealed that treatment with either quercetin or exercise significantly increased the expression levels of p-Akt in the affected cortex and striatum relative to those in the C group. The combination of exercise and quercetin treatment resulted in higher p-Akt expression than either quercetin or exercise alone (Fig. 2).

FIGURE 2

FIGURE 2

Two-way ANOVA revealed the main effects of quercetin and exercise training on the expression levels of p-Bad and Bcl-2 in the affected cortex and striatum (quercetin: P < 0.01 for p-Bad and Bcl-2 expression levels in the cortex and striatum; exercise: P < 0.01 for p-Bad and Bcl-2 expression levels in the cortex and striatum). There were no interactions between exercise and quercetin. Post hoc analysis revealed that treatment with either quercetin or exercise significantly increased the expression levels of p-Bad and Bcl-2 in the affected cortex and striatum compared with the levels in the C group. The combination of exercise with quercetin treatment resulted in higher p-Bad and Bcl-2 expression levels than either quercetin or exercise alone (Figs. 3 and 4).

FIGURE 3

FIGURE 3

FIGURE 4

FIGURE 4

Furthermore, the main effects of exercise and quercetin on the expression levels of cytochrome c in the affected cortex and striatum were also detected (quercetin: P < 0.01 in the cortex and striatum; exercise: P < 0.01 in the cortex and striatum). There was no interaction between quercetin and exercise. Post hoc analysis revealed that treatment with either quercetin or exercise significantly decreased the expression levels of cytochrome c in the cortex and striatum relative to the levels in the C group. The combination of exercise with quercetin treatment resulted in significantly lower cytochrome c expression than either quercetin or exercise alone (Fig. 5). These results indicated the effect of combined exercise and quercetin treatment on the expression levels of antiapoptotic factors was greater than either treatment alone after brain ischemia.

FIGURE 5

FIGURE 5

The PI3K/Akt inhibitor eliminated the effects of quercetin and cotreatment of exercise and quercetin on all tested parameters compared with those of comparable groups (Q and EQ groups). These results indicated that both exercise and quercetin used the PI3K/Akt pathway as a common signal transduction pathway to regulate antioxidative and antiapoptotic signaling (increases in p-Bad, Bcl-2, GSH, GPx, and GRx and decreases in cytochrome c and LP expression levels), which were contributed to the neuroprotection and the improvement of motor function.

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DISCUSSION

The degree of neuronal damage after an ischemic insult determines the outcomes of stroke survivors. Preventing more neurons from becoming injured may provide improved outcomes after brain ischemia. Exercise training is one therapeutic intervention for stroke survivors, and its neuroprotective effects have been reported by many studies (5,8,24). Recent studies have demonstrated that exercise combined with pharmaceutical agents produces improved functional recovery after brain ischemia (21,31). These studies indicate that combined treatment can result in better outcomes than exercise training alone. Oxidative stress is known to be markedly increased after a brain ischemic insult, and the brain is susceptible to oxidative damage (9,18). Thus, combined exercise training with an antioxidant or a free radical scavenger may provide an additional neuroprotective effect after brain ischemia.

Quercetin is a potent free radical scavenger, and its therapeutic potential after brain ischemia is supported by previous studies (1,38). In the present study, quercetin or exercise treatment alone prevented neuronal death and improved motor function after brain ischemia in rats. Combining exercise training with quercetin treatment further improved functional outcomes than either treatment alone. This study indicated that the oxidative stress responses, including GSH and its enzyme activities, were significantly increased after treatment with either quercetin or exercise, and combined treatment resulted in even greater expressions of these antioxidants. The increased activities of GPx and GRx may promote the recycling of GSH and facilitate the degradation of reactive oxygen species, leading to a significantly decreased LP level and infarct volume, as demonstrated in this study. These results suggested that the protective effects of exercise and quercetin treatment were attributable to the up-regulation of antioxidative signaling pathways, such as the GSH system.

Furthermore, the expression of GSH and the activities of GPx and GRx induced by quercetin and exercise were associated with the activation of the PI3K/Akt pathway. The PI3K/Akt pathway is thought to restore the imbalance between GSH and GSH/GSSG and regulate the function of GPx after the induction of oxidative stress (19,34). This study demonstrated that treatment with a PI3K/Akt inhibitor eliminated the effects of quercetin and co-treatment of exercise and quercetin on the expression of GSH and the activities of GPx and GRx, and no differences were detected between the two groups treated with the PI3K/Akt inhibitor. In addition, the neuroprotective effects of exercise after brain ischemia have also been suggested to be related to the activation of the PI3K/Akt pathway (4,24). The PI3K/Akt pathway may constitute a common signaling pathway by which quercetin and exercise protect neurons from damage after brain ischemia. Thus, the combination of exercise and quercetin treatment can induce even greater activation of the PI3K/Akt pathway to up-regulate the antioxidant GSH system and contribute to neuroprotective effects. Quercetin is also reported to attenuate apoptosis via activation of the BDNF-TrkB-mediated Akt pathway (38). Our previous study has also revealed that exercise training increases BDNF and p-Akt expression in the ischemic rat brain (5). Thus, the combined treatment of exercise training and quercetin administration may also increase the levels of additional neurotrophic factors to protect neurons from injury, resulting in increased functional recovery.

The present study showed that the activation of p-Akt signaling by treatment with quercetin and exercise alone and in combination up-regulated the expression levels of p-Bad and Bcl-2, and decreased the expression of cytochrome c on the affected cortex and striatum. The importance of p-Akt-mediated induction of these antiapoptotic signals after quercetin and exercise treatments was also confirmed by the treatment of PI3K/Akt inhibitor. However, previous studies have reported that quercetin and exercise increase mitochondrial biogenesis and increase cytochrome c release in normal brain and muscle (7,33). The responses of cells under normal and pathological conditions may be different. Under a pathological condition, such as brain ischemia, the permeability of the mitochondrial membrane is abnormally increased, resulting in extensive release of cytochrome c to induce cell apoptosis (14). A previous study has demonstrated that exercise increases mitochondrial biogenesis and results in functional improvement after brain ischemia (42). Another study has indicated that the PI3K/Akt-mediated interaction between Bad and Bcl (XL) plays an important role in maintaining mitochondrial integrity (41). Therefore, the increased mitochondrial biogenesis mediated by quercetin and exercise may contribute to the regulation of the abnormal mitochondrial membrane permeability that is induced by brain ischemia. Thus, the release of cytochrome c after exercise and quercetin treatment in ischemic brains was lower than that in controls, as demonstrated in the present study.

The present study showed that exercise training alone was more effective than the quercetin treatment alone. One possible explanation is that exercise itself is a whole body activity and exercise can induce multiple physiological changes in the body, including blood circulation and the endocrine system, among others (23), which may not be easily achieved by treatment with quercetin alone.

Quercetin has also been found to reduce chronic oxidative stress-mediated impairments in synaptic plasticity in the hippocampus of rats (15) and is able to alleviate the reduction in long-term potentiation and improve the learning and memory ability of brain ischemia rats (39). These results suggest that treatment with quercetin after brain ischemia may enhance not only the neuroprotective effects of exercise but also neuronal plasticity.

A previous study has found that quercetin increases brain and muscle mitochondrial biogenesis and enhances exercise tolerance in healthy animals (7). However, Gomez-Cabrera et al. (12) have reported that the combination of antioxidant administration with exercise decreases exercise performance and mitochondrial biogenesis in the muscle of animals. These studies indicate that oxidative stress response plays a role in the physiological responses of normal brain and muscle to exercise. These prior studies may not apply to the present pathological scenario, as the brain ischemia markedly induces reactive oxygen species and neurons are susceptible to oxidative stress (9,18). Therefore, the combined exercise training with an antioxidant to reduce oxidative stress and to promote functional improvement can become a potential therapeutic strategy for brain ischemia.

Although this and other studies have demonstrated that intraperitoneal injection of quercetin exerts neuroprotective effects (1,20), quercetin by intraperitoneal injection may not be the most effective. Galindo et al. have demonstrated that administration of quercetin via oral gavage results in better protective effects on cardiovascular function than administration via intraperitoneal injection (11). A different route of administration of quercetin may result in a different result and its effectiveness in brain ischemia requires further study.

In conclusion, this study provides a basic evidence for clinical applications that combined exercise and quercetin treatment can result in better functional outcomes than either treatment alone. The combined therapeutic strategy is feasible to become a very effective treatment for stroke survivors during rehabilitation.

This study was supported by the National Science Council (grant no. NSC101-2314-B-010-002-MY2) and the Ministry of Education, Aim for the Top University Plan (grant no. 101AC-P508) of the Republic of China.

The authors have no conflicts of interest to report, and the results of the present study do not constitute an endorsement by the American College of Sports Medicine.

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Keywords:

QUERCETIN; EXERCISE; OXIDATIVE STRESS DEFENSE; ANTIAPOPTOTIC SIGNALING; BRAIN ISCHEMIA; MOTOR FUNCTION

© 2014 American College of Sports Medicine