Effect of ginsenoside Rg3 on tyrosine hydroxylase and related mechanisms in the forced swimming-induced fatigue rats

Journal of Ethnopharmacology 150 (2013) 138–147

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Effect of ginsenoside Rg3 on tyrosine hydroxylase and related mechanisms in the forced swimming-induced fatigue rats Yuxia Xu a,b, Peng Zhang c, Chu Wang a,b, Ye Shan a,b, Dandan Wang a,b, Fenglei Qian c, Mengwei Sun c, Cuiqing Zhu a,b,n a

State Key Laboratory of Medical Neurobiology, School of Basic Medical Sciences, Fudan University, 138 Yixueyuan Road, Shanghai 200032, PR China Institutes of Brain Science, School of Basic Medical Sciences, Fudan University, 138 Yixueyuan Road, Shanghai 200032, PR China c Shanghai Research Institute of Sports Science, 87 Wuxing Road, Shanghai 200030, PR China b

art ic l e i nf o

a b s t r a c t

Article history: Received 9 April 2013 Received in revised form 9 July 2013 Accepted 6 August 2013 Available online 28 August 2013

Ethnopharmacological relevance: Ginsenoside Rg3 has shown multiple pharmacological activities and been considered as one of the most promising approaches for fatigue treatment. However, little is known about the cellular and molecular mechanisms of Rg3 on anti-fatigue and the effect of Rg3 on dopaminergic system has not been reported yet. The major aim of this study is to investigate the effect of Rg3 on TH expression and the related biochemical parameters, such as PKAα, ERK1/2, Akt and αsynuclein in brain of fatigue rats. Materials and methods: Weight-loaded forced swimming was performed to establish an animal model of fatigue. Rg3 (10 mg/kg, 50 mg/kg and 100 mg/kg) was intragastrically administrated before swimming. The effect of Rg3 on the expression and phosphorylation of TH and TH-related proteins in fatigue rats or in SH-SY5Y cells was assessed with western blotting. HPLC was used to examine the level of DA and DOPAC in the fatigue rats tissues. Results: TH and phosphorylated TH were decreased in different brain regions of which ventral midbrain were less affected in weight-loaded forced swimming rats. Pretreatment with Rg3 significantly suppressed fatigue-induced decrease expression of TH and TH phosphorylation. Also treatment with Rg3 reversed the decrease expression of PKAα as well as the phosphorylation of ERK1/2 and Akt which were induced by weight-loaded forced swimming. Moreover, weight-loaded swimming could induce the increase expression of α-synuclein in hippocampus and midbrain, while suppressed α-synuclein expression in striatum and prefrontal cortex. Furthermore, Rg3 could induce the increase of TH expression and phosphorylation which was accompanied with elevated expression and phosphorylation of related kinase proteins in vitro, while the inhibitors of kinase proteins could suppress these effects of Rg3. In addition, HPLC results showed that Rg3 could reverse the weight-loaded swimming-induced increase of DOPAC/DA ratio. Conclusion: Our data suggest that fatigue can induce the decrease of DA which might partially result from the change of TH expression and phosphorylation, and Rg3 can reverse these fatigue-induced changes. The underling mechanisms may include the activity changes of PKAα, ERK1/2, Akt and α-synuclein. & 2013 Elsevier Ireland Ltd. All rights reserved.

Keywords: Rg3 Fatigue Tyrosine hydroxylase PKAα α-Synuclein

1. Introduction The causes of fatigue are believed to be of peripheral and central origin. Fatigue symptoms are related to central nervous system (CNS) dysfunction including reduced activities and muscle endurance and impaired coordination, learning and memory (Afari and Buchwald, 2003). Central fatigue is used as n Corresponding author at: State Key Laboratory of Medical Neurobiology, School of Basic Medical Sciences, Fudan University, 138 Yixueyuan Road, Shanghai 200032, PR China. Tel.: þ 86 21 542 37858; fax: þ86 21 641 74579. E-mail addresses: [email protected] (Y. Xu), [email protected] (C. Zhu).

0378-8741/$ - see front matter & 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jep.2013.08.016

the term that defines reduction in voluntary activation of the skeletal muscles, whereas peripheral fatigue refers to changes developed distal to the neuromuscular junction. During a sustained effort central and peripheral fatigue develops gradually (Nybo and Nielsen, 2001), but the exercise task is usually terminated when the muscle still possess the capability to produce the force (Enoka and Stuart, 1992; Loscher et al., 1996; McKenzie et al., 1997) which indicate that loss of CNS impetus is the point of fatigue. Given that the muscle contraction is initiated by the central nervous system, it is reasonable to assume that alterations in CNS may contribute to the progress of fatigue.

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There is an evidence to show that changes in cerebral metabolism, hyperthermia and monoamine neurotransmission (Meeusen et al., 2006; Park et al., 2002) may be mechanistically linked to prolonged exercise-induced fatigue. In the mechanisms of central fatigue, the significance of the monoamines serotonin (5-hydroxytryptamine, 5-HT) and dopamine (DA) has been concerned. It has been supposed that central fatigue may occur due to a failure in the integration of the limbic input including hippocampus and the motor functions within the basal ganglia, in which the malfunction of dopaminergic system and the serotonin pathway were most importantly involved (Chaudhuri and Behan, 2000). Pharmacological manipulations that increased 5-HT concentration during treadmill running to fatigue could hasten the onset of fatigue (Ahlenius et al., 1997; Bailey et al., 1993; Yamamoto et al., 2012), while blocking 5-HT activity delayed the time to fatigue. In contrast, manipulations that increased DA concentration in brain delay the onset of fatigue (Hasegawa et al., 2008; Novaes et al., 2012), while reducing DA activity hasten time to fatigue (Davis et al., 2003; Kalinski et al., 2001). Therefore, a low ratio of 5-HT/DA favors improved performance while a high ratio decreases motivation and augments lethargy, consequently decreasing performance (Davis and Bailey, 1997). Although the decrease of DA in fatigue has been confirmed, the mechanisms of DA change and the expression of tyrosine hydroxylase (TH) and TH activity, the rate limiting enzyme in DA synthesis, has not been clarified yet. Until now, both pharmacological and nutritional manipulations have been studied which aims to influence the neurotransmitters 5-HT and DA in brain to delay the onset of fatigue. Many herb compounds have been found to be effective in anti-fatigue. Ginseng, the root of the Panax species, has been used in traditional Chinese medicine for thousands of years (Gillis, 1997). The major biologically active components of ginseng are ginsenosides which have been traditionally used as tonic medicine to treat many disorders such as debility, aging, stress and fatigue (Barton et al., 2010; Lewis et al., 1999; Tang et al., 2008). Ginsenoside Rg3, a minor ginsenoside from the Panax ginseng, has been shown multiple pharmacological activities including anti-tumor, immunity enhancement (Wang et al., 1999) and memory improvement (Bao et al., 2005). As reported, Rg3 at the higher doses showed significant neuroprotective effect in rats against focal cerebral ischemic injury (He et al., 2012). Also, Rg3 could relief fatigue in the weight-loaded swimming test (Tang et al., 2008). However, the mechanism of Rg3 on the exercise-related central fatigue and the effect of Rg3 on dopaminergic system in brain have not been clarified. This study aimed to investigate the expression and phosphorylation of TH in different brain regions in weightloaded swimming-induced fatigue and also to elucidate the Rg3 treatment effect on TH. The activity of TH could be regulated by numerous factors (Adell and Artigas, 2004). The phosphorylation of key seryl residues in the TH regulatory domain was important for TH activity (Hakansson et al., 2004). Although other sites of phosphorylation have been recognized in TH, only Ser19, Ser31 and Ser40 could be regulated in vivo (Haycock, 1990). Phosphorylation Ser40 of TH was considered to be the most important in regulation of TH activity (Dunkley et al., 2004; Fujisawa and Okuno, 2005). A variety of protein kinases and phosphatases have been implicated in TH phosphorylation at Ser40 (Bobrovskaya et al., 2007; Dunkley et al., 2004; Gelain et al., 2007), among which cAMP-dependent protein kinase A (PKA) (Dunkley et al., 2004) and other kinases such as extracellular signal-regulated kinases (ERK1/2) are important in regulation of TH phosphorylation (Salvatore et al., 2001). The PKA-dependent phosphorylation of TH at Ser40 increases the catalytic activity of TH, further increasing dopamine synthesis in a positive-feedback loop (Harada et al., 1996). Moreover, TH Ser40

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could be phosphorylated by several kinase of which only PKA has been shown to directly phosphorylate Ser40 in situ (Haycock, 1996). Extracellular signal-regulated kinase (ERK), one member of mitogen-activated extracellular kinase (MAPK) family, transduces a broad range of extracellular stimuli into diverse intracellular responses. It has been reported that ERK can regulate TH activity through phosphorylating Ser31 and Ser40 sites (Lindgren et al., 2002; Salvatore et al., 2001). Furthermore, ERK1/2 could phosphorylate Ser31 and then increase TH activity in chromaffin cells (Bobrovskaya et al., 2001; Haycock et al., 1992). In addition, the phosphatidylinositol 3-kinase (PI3-K)/Akt signaling pathway plays an important role in neuronal survival (Brunet et al., 2001). Previous studies have shown that phosphorylation of Akt is essential for dopaminergic neurons to survive and maintain DA synthesis (Chong et al., 2005; Ries et al., 2006). Beside the modulation of protein kinases, the process of TH phosphorylation is also regulated by several other factors, of which α-synuclein is a key negative modulator. α-synuclein could reduce TH activity by directly binding TH or via activation of protein phosphatases and subsequently inducing TH dephosphorylation (Peng et al., 2005; Perez et al., 2002). However, it is still not known whether these protein kinases are involved in fatigue and whether they play some roles in the antifatigue effect of Rg3. In this study, we demonstrated that the expression and phosphorylation of TH were decreased in weight-loaded forced swimming rats, accompanied by a low level of DA in brain. The mechanisms might be related to the expression and phosphorylation of related regulating factors indicated above. Results also revealed that Rg3 could reverse weight-loaded swimminginduced decrease of TH and phosphorylation of TH and could reverse the changes of related regulating factors.

2. Methods and materials 2.1. Reagents Antibodies to the following targets were used: mouse monoclonal anti-β-actin, mouse monoclonal anti-TH antibodies and H89 were from Sigma. Rabbit polyclonal anti-phospho Ser40-TH and rabbit polyclonal anti-phospho Ser129-synuclein were from Chemicon Company. Mouse anti-α-synuclein was from BD Transduction Laboratories. Rabbit anti-phospho Thr202/Tyr204-ERK1/2, total ERK1/2, rabbit anti-phospho Ser473-Akt and rabbit anti-Akt antibodies were obtained from Cell Signaling Technology Inc. Rabbit polyclonal anti-PKAα, GAPDH, PD98059 and LY290042 were from Santa Cruz. All the other chemical reagents used were of the high grade available commercially. 2.2. Animal treatment Adult male Sprague–Dawley rats weighing 300 710 g were purchased from Shanghai Experimental Animal center, Chinese Academy of Sciences. The animals were cared complied with the Provisions and General Recommendation of the Chinese Experimental Animals Administration Legislation and were approved by the Science and Technology Department of Shanghai. All of the experiments were conducted to minimize the sufferings caused by the experimental procedures. Rats were housed at a room temperature of 22 72 1C under humidity (50 75%) and light (lights on at 08:00 h and off at 20:00 h) conditions with food and water made available ad libitum. The animals were divided into five groups (8 rats/group) (1) normal control group (CON); (2) fatigue þnormal saline group (NS); (3) fatigue þlow-dose Rg3 group (Rg3, 10 mg/kg); (4) fatigue þmiddle-dose Rg3 group (Rg3, 50 mg/kg)

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and (5) fatigue þhigh-dose Rg3 treatment group (Rg3, 100 mg/kg). Rg3 (purity: 94.28%) was purchased from Dalian Fusheng Pharmaceutical Company, Dalian, China and was dissolved in distilled saline water with 0.5% carboxymethylcellulose (Sigma) suspension at different concentrations. Rg3 was intragastrically administrated. NS control group received an administration of same amount of normal saline in above solution. 2.3. Weight-loaded forced swimming Weight-loaded forced swimming was performed to induce fatigue according to the methodology as described previously (Dhir and Kulkarni, 2008; Tanaka et al., 2003) with some modifications. The rats swam with a load of steel rings that weighed approximately 8% of their body weight and were attached to rat tails. Rats were individually placed in cylindrical recipients (diameter 1.6 m) filled with water (22 71 1C) to a depth of 30 cm. Thirty minutes after administration of Rg3 or NS, rats were forced to swim in the recipient once a day for 7 consecutive days, whereas the rats in normal control group were left in cage without swimming. Fatigue was defined as the point at which the animals were no longer swimming but keep silence in water (Tanaka et al., 2003; Tang et al., 2008). On the 7th day of the experiment, the body weight and swimming time after swimming were analyzed and then the animals were sacrificed. 2.4. Cell culture and treatment Human neuroblastoma SH-SY5Y cells were cultured in DMEM supplemented with 10% (v/v) fetal bovine serum, 100 U/ml penicillin, and 100 μM streptomycin at 37 1C and 5% CO2. Ginsenoside Rg3 was dissolved in dimethylsulfoxide and added to DMEM medium. After exposure to Rg3 for 12 h, cells were washed twice with cold PBS and lysed in ice-cold lysis buffer (50 mM Tris–HCl, 1% NP-40, 0.25% Na-deoxycholate, 150 mM NaCl and 1 mM EGTA) with a mixture of protease inhibitors (Complete Protease Inhibitor, Roche, Molecular Biochemicals) for 30 min with frequent vortexing on ice. For another experiment, cells were pretreated with 20 μM PD98059 (ERK inhibitor), 20 μM LY294002 (Akt inhibitor) or 20 μM H-89 (PKA inhibitor) for 30 min respectively, and then cotreated with Rg3 (1 μM) for 12 h. The protein concentrations of the supernatants were determined using the Bradford method and then analyzed by western blot. 2.5. Western blot analysis The brain tissue samples were homogenized in radioimmunoprecipitation assay buffer with a mixture of protease inhibitors (Complete Protease Inhibitor, Roche, Molecular Biochemicals) at

4 1C and lysed for 30 min on ice. Samples were then centrifuged at 4 1C, and the protein concentration of supernatant was quantified by Bradford assay. Protein samples were resolved by SDS–polyacrylamide gel electrophoresis. Proteins were transferred to polyvinyldifluoridine (PVDF; Millipore), which was submerged in blocking buffer TBS containing 5% BSA for 2 h at room temperature and then incubated overnight at 4 1C with primary antibody. The membranes were then repeatedly washed in TBS containing 0.1% Tween-20 and exposed to HRP-conjugated anti-rabbit or mouse IgG for 1 h at room temperature. After three additional washes, western blotting luminal reagent was used to visualize peroxidase activity. Films were scanned and subsequently analyzed by measuring optical densities of immunostained bands using an imageprocessing and analysis system (Image J software, NIH, USA). 2.6. HPLC assay As soon as the fatigue point was reached, rats were anesthetized with chloral hydrate. After decapitation, the brains were quickly removed and washed with ice-cold saline. Four regions were carefully dissected: prefrontal cortex, hippocampus, striatum and midbrain, as described by Glowinski and Iversen (1966). The brain tissues were weighed and homogenized in 0.1 M perchloric acid. After centrifugation (13,000 rpm, 20 min, 4 1C), the supernatant was filtered through a Millipore membrane (0.22 μm pore-size) and stored thereafter at  80 1C until measured by high-pressure liquid chromatography (HPLC). Twenty microlitres were injected into the HPLC-EC system for analysis (Agilent 1200 Infinity; USA). Quantification of dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) was made by comparing the peak area to a standard curve. 2.7. Statistical analysis All of the experiments described in this study were repeated at least three times with independent treatments, and all data were presented as the mean 7SEM. The significance of the differences was analyzed via one-way ANOVA. Mean values were considered to be statistically significant at P o0.05.

3. Results 3.1. Effects of Rg3 on swimming time and body weight It is essential to confirm the success of animal model of fatigue, so in this study we first investigated the establishment of fatigue. At the last day of weight-loaded swimming, body weight of animals was analyzed. Results showed that there were no significant differences in the body weight of the swimming rats in NS

Fig. 1. Effect of Rg3 on forced swimming time and body weight of animals. Rats were forced swimming for 7 consecutive days after intragastrically administrated with normal saline (NS) or different dosage of Rg3 (10 mg/kg, 50 mg/kg and 100 mg/kg). The body weight at the terminal time point was tested (A). Moreover, the swimming time in the last day was also collected and analyzed (B). Body weight and swimming time are shown as a percentage of the value of the control group. Values were represented as the mean 7 SEM. #Po 0.05; ##Po 0.01 compared with the normal control group (CON).nPo 0.05; nnPo 0.01 compared with NS group (n¼ 8/group).

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or Rg3 pretreatment groups as illustrated in Fig. 1A. However, rats being forced swimming for 7 consecutive days showed a significant decrease in body weight in NS or Rg3 treatment groups compared with which in the normal control groups. As the length of the swimming time to exhaustion indicated the degree of fatigue, we then analyzed the swimming time of different group rats at the last swimming day. As shown in Fig. 1B, there was a marked enhancement in running performance in Rg3 treatment group, measured as an obvious increase in time to fatigue compared with the NS control group. These results indicated that weight-loaded forced swimming can induce the occurrence of fatigue, while Rg3 treatment can improve the swimming performance and relieve fatigue.

3.2. Effects of Rg3 on the expression and phosphorylation of TH We next investigated whether Rg3 could modulate the expression and phosphorylation of TH in different brain regions of forced swimming rats. Western blot analyses showed that no significant expression change of TH after weight-loaded forced swimming was detected in ventral midbrain where most dopaminergic neurons were distributed (Fig. 2D). Meanwhile, the detection of TH in striatum, a brain region receiving a large amount of dopaminergic projection from the substantia nigra, showed a mild but significant decrease of TH expression (Fig. 2C). Moreover, the levels of TH in prefrontal cortex (Fig. 2A) and hippocampus (Fig. 2B) were also significantly decreased after 7 consecutive days weight-loaded swimming. The effect of Rg3 on TH expression appeared different among these four brain regions. Pretreatment with 10 mg/kg Rg3 already caused a significant increase of TH in prefrontal cortex, which was

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even more than that of normal control rats. The dosage of 50 mg/ kg exerted the most prominent effect on upregulation of TH in all these brain regions, but the effect with higher dosage of 100 mg/ kg was nearly same to/or less effective than that with 50 mg/kg in different brain regions. These results indicate that Rg3 may ameliorate forced swimming-induced decrease expression of TH in different brain regions at different extent. Next, we investigated the phosphorylation of TH with a highly specific antibody against phosphorylation Ser40 (p-Ser40) of TH. Immunoblotting assay results showed that p-Ser40 TH was readily detectable under basal condition. As could be seen in Fig. 2E–H, the suppression of p-Ser40 TH induced by weight-loaded swimming was significant in dopaminergic terminal distributed brain regions including striatum, prefrontal cortex and hippocampus. In contrast, there was no significant change in ventral midbrain where most dopaminergic neurons distributed. In groups pretreated with different concentrations of Rg3, the p-Ser40 TH was significantly increased at various extents in all four brain regions. Therefore, fatigue can induce the decrease phosphorylation of TH in dopaminergic innervated brain regions, while Rg3 pretreatment can reverse fatigue-induced decrease of TH phosphorylation in different brain regions. 3.3. Effects of Rg3 on the expression of PKAα To further elucidate the mechanism of TH phosphorylation in this study, we then examined whether weight-loaded swimminginduced downregulation of TH phosphorylation is related to PKA. Our results showed that the expression of PKAα, the functional subunit of PKA, was downregulated in various degrees in four detected brain regions. In these brain regions, the decrease of

Fig. 2. Effect of Rg3 on TH expression and TH phosphorylation. Rats were intragastrically administrated with normal saline (NS) or Rg3 (10 mg/kg, 50 mg/kg and 100 mg/kg) and then forced swimming to fatigue. Lysis of different brain regions (prefrontal cortex, hippocampus, striatum and ventral midbrain) was for western blotting analysis using the antibodies against TH (A, B, C and D) and phosphor Ser40-TH (E, F, G and H). Relative density ratio in control level was expressed as 100 arbitrary units. Actin was a loading control. The results represented means 7 SEM of five experiments. nPo 0.05; nnP o0.01 compared with the normal control group (CON). #Po 0.05; ##Po 0.01 compared with the NS group.

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Fig. 3. Effect of Rg3 on PKAα expression. Rats were intragastrically administrated with normal saline (NS) or Rg3 (10 mg/kg, 50 mg/kg and 100 mg/kg) and then forced swimming to fatigue. Lysis of different brain regions in prefrontal cortex (A), hippocampus (B), striatum (C) and ventral midbrain (D) was for western blotting analysis using the antibody against PKAα. Relative density ratio in control level was expressed as 100 arbitrary units. Actin was a loading control. The results represented means 7 SEM of four experiments. nPo 0.05; nnP o0.01 compared with the normal control group (CON). #Po 0.05; ##P o 0.01 compared with the NS group.

Fig. 4. Effect of Rg3 on phosphorylation of ERK1/2 and Akt. Representative parallel immunoblots from normal control (CON), forced swimming rats pretreated saline (NS) and Rg3 (10 mg/kg, 50 mg/kg and 100 mg/kg) groups were evaluated by western blotting with four rats for each condition in four brain regions. The expression of p-ERK1/2 and total ERK1/2 (A, B, C and D), and p-Akt and total Akt (E, F, G and H) was examined. Relative density ratio in control level was expressed as 100 arbitrary units. The histogram represented the means 7SEM of data from four independent experiments. nPo 0.05; nnPo 0.01 compared with the normal control group (CON). #Po 0.05; ## Po 0.01 compared with the NS group.

PKAα in hippocampus was very limited and did not reach statistic significance (Fig. 3B). Meanwhile the decrease in ventral midbrain was also relatively mild (Fig. 3D). In contrast, it was dramatically

decreased in striatum (Fig. 3C) and prefrontal cortex (Fig. 3A), especially in striatum. Rg3 treatment could enhance the level of PKAα. It should be taken notice that different dosages of Rg3

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induced upregulation expression of PKAα to various extents in four brain regions. In hippocampus, 100 mg/kg Rg3 upregulated PKAα most effectively and 50 mg/kg Rg3 was most effective in ventral midbrain and striatum. Whereas 10 mg/kg Rg3 pretreatment already caused a highest level of PKAα in prefrontal cortex which was even higher than that of the normal level, 50 mg/kg and 100 mg/kg Rg3 were relatively less effective. These results suggest that change expression of PKAα may affect TH activity in mechanisms of fatigue and may play roles in mediating Rg3 pharmacological effect.

phosphorylated Akt were also appeared higher molecular weight in immunoblotting. Maximum effect on upregulation of Akt phosphorylation was generally achieved with 50 mg/kg Rg3, while 100 mg/kg Rg3 was more effective only in prefrontal cortex but less effective for other brain regions. Taken together, these results indicated that fatigue could induce the inhibition of ERK1/2 and Akt, whereas Rg3 could upregulate the activation of these two kinases.

3.4. Effects of Rg3 on phosphorylation of ERK1/2 and Akt

To explore whether α-synuclein is involved in fatigue mechanism and Rg3 effect, we then analyzed the expression and phosphorylation of α-synuclein. As shown in Fig. 5, the level of αsynuclein was significantly decreased in prefrontal cortex (Fig. 5A) and striatum (Fig. 5C) in weight-loaded forced swimming rats, while it was increased in hippocampus (Fig. 5B) and ventral midbrain (Fig. 5D). At the dosage of 10 mg/kg, Rg3 treatment could apparently decreased α-synuclein expression. However, 50 mg/kg Rg3 treatment resulted in significantly increased expression of α-synuclein even more than that of normal control and 100 mg/kg Rg3 treatment alleviated the upregulative effect on αsynuclein expression in general. In that the activity of α-synuclein in modulation of TH depends on its phosphorylation, we next took advantage of a specific Ser129 phosphorylation dependent α-synuclein antibody to tested the change of α-synuclein phosphorylation. Our results showed that Ser129 phosphorylated α-synuclein was detected in the normal brain tissues. After 7 days weight-loaded swimming, Ser129 phosphorylated α-synuclein was decreased significantly in prefrontal cortex (Fig. 5E) and striatum (Fig. 5G), whereas it was slightly increased in hippocampus (Fig. 5F) and ventral midbrain (Fig. 5H). These changes displayed a basic consistency with the change trend of α-synuclein expression. In contrast, the changes of

In the present study, we also tested the ERK1/2 which also can regulate TH activity by phosphorylation. The results showed that the pattern of basal phosphorylated (activated) ERK1/2 in the four brain regions appeared different (Fig. 4A–D). In weight-loaded forced swimming rats, the ERK1/2 phosphorylation was significantly decreased in all four brain regions. Pretreatment with Rg3 significantly reversed the decrease of phosphorylated ERK1/2 with various extents in four brain regions. The upregulative effect of Rg3 was more evident in prefrontal cortex (Fig. 4A). It should be noted that the most effective dosage of Rg3 for different brain regions was diverse, which appeared 10 mg/kg Rg3 for hippocampus and 50 mg/kg Rg3 for other three brain regions. In the following experiments we further explored whether Rg3 affected the expression and phosphorylation (the activated Akt) of Akt in weight-loaded forced swimming condition. Western blot results showed a significant decrease of Akt phosphorylation in all four detected brain regions, but no obvious change of Akt expression. Pretreatment with Rg3 significantly increased the phosphorylation of Akt, while Rg3 had no evident effect on total Akt expression (Fig. 4E–H). The upregulative effect was more obvious in prefrontal cortex than other brain regions and large amount of

3.5. Effects of Rg3 on expression and phosphorylation of α-synuclein

Fig. 5. Effect of Rg3 on α-synuclein expression and α-synuclein phosphorylation. Representative parallel immunoblots from control (CON), normal saline (NS) group and Rg3 (10 mg/kg, 50 mg/kg and 100 mg/kg) groups were evaluated for α-synuclein (A, B, C and D) and phosphor-Ser129 α-synuclein (E, F, G and H) with four rats for each condition in selected four brain regions. Relative density ratio in control level was expressed as 100 arbitrary units. Actin was a loading control. The histogram represented the means 7 SEM of data from four independent experiments. nPo 0.05; nnPo 0.01 compared to the normal control group (CON). #P o0.05; ##P o0.01 compared to the NS group.

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Ser129 phosphorylated α-synuclein in rats pretreated with Rg3 were complicated, which were not always consistent with the changes of α-synuclein expression. In brief, as shown in Fig. 5, it could be preliminarily concluded that pretreatment of Rg3 could increase phosphorylation of α-synuclein and even more higher than that of control rats. This effect of Rg3 was depended on the different dosages and the different brain regions.

3.6. Effects of Rg3 on the activation of TH-related biochemical parameters in vitro The above data showed that Rg3 could regulate TH and its related proteins in brain, while the specific effect of Rg3 on dopaminergic neurons was still elusive. To further determine the role of Rg3 on TH, we used human dopaminergic neuroblastoma SH-SY5Y cells to study the effect of Rg3 on expression and activation of TH-related biochemical parameters. The results showed that compared with normal control cells, Rg3 treatment could significantly induce the increase expression and phosphorylation of TH (Fig. 6A), especially with low concentration of Rg3 (0.1 μM). In addition, Rg3 also induced an increase of α-synuclein expression and phosphorylation, AKT phosphorylation and PKAα expression (Fig. 6C–E). Although p-ERK1/2 was not increased under treatment with 0.1 μM Rg3, it was increased with 1 μM Rg3 (Fig. 6B). With the increase of Rg3 concentration, the effect on

TH was declined (Fig. 6A). Meanwhile the effects of Rg3 on ERK1/2, Akt, α-synuclein and PKAα were also decreased. To further identify the involvement of signaling pathways proteins in Rg3 effect, we analyzed the effects of the specific ERK inhibitor PD98059, the Akt inhibitor LY294002 and PKA inhibitor H89 on Rg3-induced effects. We found that all these inhibitors could block the upregulation of TH expression and phosphorylation induced by Rg3 (Fig. 6F and G). Meanwhile, PD98059 and LY294002 significantly inhibited the phosphorylation of ERK1/2 and Akt respectively (Fig. 6H and I). In addition, only ERK inhibitor PD98059 could decrease Rg3 induced upregulation of α-synuclein phosphorylation (Fig. 6F and J). These results further indicated that Rg3 may exert direct effect on dopaminergic neurons and then activate TH possibly through ERK1/2, Akt,αsynuclein and PKAα mechanisms.

3.7. Effects of Rg3 on DA content in brain To assess the effect on the levels of DA, DA contents in brain tissue extracts were measured by HPLC with electrochemical detection. In weight-loaded swimming rats, DA content was significantly decreased in the prefrontal cortex, hippocampus, striatum and ventral midbrain compared with the normal control animals (Fig. 7A), which was consistent with the inhibition of TH expression and phosphorylation (Fig. 2). Rg3 pretreatment could increase DA content with varying degrees in four brain regions,

Fig. 6. Effect of Rg3 on the activation of TH-related proteins in SH-SY5Y cells. Human SH-SY5Y cells were treated with different concentration of Rg3 (0.1 μM, 1 μM, 10 μM and 100 μM). The whole cell extracts were prepared after treatment for 12 h in the presence or absence of indicated concentrations of Rg3 and the changes in the expression levels of indicated proteins were assessed by western blotting. Representative parallel immunoblots from control and Rg3 group were shown as: total TH and phosphorylated TH (A), total ERK1/2 and phosphorylated ERK1/2 (B), total Akt and phosphorylated Akt (C), total α-synuclein and p-α-synuclein (D) and PKAα (E). Cells were preincubated with PD98059 (PD), LY294002 (LY) or/and H-89 with the indicated concentration for 30 min and further incubated for 12 h after the addition of Rg3 (1 μM), and then the cell lysis was analyzed by western blot (F–J). GAPDH was a loading control. Relative density ratio in control level was expressed as 100 arbitrary units. The histogram represented the means 7 SEM of data from four independent experiments. nP o 0.05; nnP o 0.01 compared with the normal control group (CON). #Po 0.05; ##P o 0.01, compared with the Rg3 treatment group.

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Fig. 7. Effect of Rg3 on the content of DA and DOPAC in weight-loaded forced swimming rats. Contents of DA (A), DOPAC (B) and DOPAC/DA ratio (C) were shown as a percentage of the corresponding value of the control group. The values were means 7 SEM. nP o0.05; nnPo 0.01, compared with the normal control group (CON). #Po 0.05; ## Po 0.01, compared with the NS group (n¼ 8/group).

which tended to or was even higher than that of the control levels. We also tested DA metabolic product DOPAC by HPLC. We observed the increase of DOPAC and DOPAC/DA ratio (Fig. 7B and C) in weight-loaded forced swimming rats, as well as a decrease in Rg3 treated rats, which suggested that forced swimming and Rg3 treatment might also affect DA release and its turnover.

4. Discussion Fatigue has traditionally been attributed to the occurrence of a “metabolic end point”. Peripheral candidates for the occurrence of fatigue include muscle, cardiovascular, metabolic dysfunction and cancer (Hofman et al., 2007; Meeusen and Roelands, 2010). Moreover, the CNS also takes important roles in decrease motivation, augment lethargy and consequently decrease performance in exercise (Davis and Bailey, 1997). It is well accepted that the CNS fatigue is related to disrupting the balance of monoamine neurotransmitters. In our study, fatigue rat also appeared a low DA level in detected brain regions. Most dopaminergic neurons in brain are located in ventral midbrain. The dopaminergic system in basal ganglia performs crucial regulation of cortical motor output (Chaudhuri and Behan, 2000), in which striatum receives large amount of dopaminergic inputs from substantia nigra of midbrain. Moreover, prefrontal cortex and hippocampus which their functions are regulated by dopaminergic innervations are devoted to regulation of focused attention (Chase et al., 2012; Clark et al., 2012; Rao et al., 2008). Based on the function of dopaminergic system in the above brain regions, we then focus on the four brain areas including the prefrontal cortex, hippocampus, striatum and ventral midbrain to investigate the molecular mechanisms involved in fatigue and Rg3 effects. In this study, fatigue induced by weight-loaded swimming was accompanied by a decrease of TH protein level in prefrontal cortex, hippocampus and striatum where the dopaminergic nerve terminals were distributed, but no significant change of TH level was detected in midbrain where dopaminergic neurons were located. This inconsistence of results at different brain areas might result from the increase degradation of TH in nerve terminals or the decrease transportation of TH to nerve terminals affected by microenvironment of these brain regions in fatigue. The activity of TH can be regulated by phosphorylation, of which Ser40 is the most important site (Dunkley et al., 2004). In the present study, the exploration of p-Ser40 TH showed a significant decrease in the dopaminergic innervating brain regions including prefrontal cortex, hippocampus and striatum, which suggests that weight-loaded swimming can induce the decrease activation of TH in these brain regions. The detection of PKAα in our study showed a dramatic downregulation of PKAα in

prefrontal cortex and striatum, but no significant change in hippocampus and ventral midbrain. In addition, the phosphorylated active ERK1/2 was suppressed in all detected brain regions accompanied by the increase expression of total ERK1/2. Based on these findings, it is reasonable to presume that forced swimminginduced fatigue can inhibit different kinases at different brain regions which might subsequently result in the decrease of TH activity through downphosphorylation of TH. Although it is still not clear whether Akt directly participate in regulation of TH phosphorylation, several Akt isoforms have been identified in the substantia nigra (Ries et al., 2006). As reported, Akt is essential for the survival of dopaminergic neurons (Brunet et al., 2001; Chong et al., 2005). In the present study, we have found that phosphorylated Akt was significantly decreased following forced swimming, while total Akt level did not show significant change. It should be noted that the decrease of phosphorylated p-Akt was more significant in prefrontal cortex, hippocampus and striatum, but less in ventral midbrain which imply that fatigue can more inclined to affect the dopaminergic terminals or the cells in dopaminergic projected brain regions. TH activity is regulated not only by kinases but also by some other binding proteins, such as α-synuclein. Previous results have shown that overexpression of α-synuclein could reduce TH activity and then influence dopamine synthesis (Perez et al., 2002). Phosphorylation of α-synuclein at Ser129 suppresses the function of α-synuclein in regulating TH activity (Lou et al., 2010). In this study, α-synuclein expression and phosphorylation were significantly decreased in prefrontal cortex and striatum, but not in hippocampus and ventral midbrain in weight-loaded swimming rats. This observation is consistent with previous study which showed acute cold water swimming could induce a significant decrease of Ser129 phosphorylated α-synuclein in striatum (Hirai et al., 2004). Since dopaminergic neurons are mainly located in midbrain, the increase expression of α-synuclein, especially the unphosphorylation form in midbrain induced by weight-loaded swimming, may potentially inhibit TH activity in dopaminergic neurons and then cause a decrease of dopamine level in this brain region. Because α-synuclein is not only expressed in dopaminergic neurons but also in other cells, the change expression of αsynuclein in prefrontal cortex, hippocampus and striatum is not unique to dopaminergic nerve terminals. So, the change of αsynuclein may also affect other neuronal functions such as exocytosis of synaptic vesicles (Larsen et al., 2006). Panax ginseng has been used to treat many disorders (Gillis, 1997). Rg3, a ginsenoside from the Panax ginseng, has shown multiple pharmacological activities including anti-fatigue function. In the present results, Rg3 treatment could attenuate the decrease expression and phosphorylation of TH induced by forced swimming, and could increase the DA level. Moreover, Rg3 also

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regulated the TH in cultured SH-SY5Y cells. Previous results showed that Rd, which has a similar chemical structure to Rg3, can pass the blood-brain barrier and protects from neuronal insults (Ye et al., 2011). Moreover, treatment with another ginsenoside Rg1 could reduce 6-OHDA or MPTP-induced neurotoxicity as well as reverse the decrease of dopamine content in striatum (Wang et al., 2009). Therefore, Rg3 may play roles in regulation of TH at central level. In this study, Rg3 treatment could induce a significant increase of PKAα and phosphorylated active ERK1/2, and these effects were also dependent on different dosages and different brain regions. Moreover, Rg3 could upregulate phosphorylation of Akt which previously implies a neuroprotective effect of Rg3 on dopaminergic neurons (Mannaa et al., 2006). In addition treatment with 10 mg/kg Rg3 could decrease α-synuclein expression in all four detected brain regions, while higher dosage of Rg3 could increase the expression of α-synuclein as well as its phosphorylation. Therefore, the mechanism of TH regulation via α-synuclein by Rg3 might be complicated. Of note, in this study we found that Rg3 could regulate TH and all these TH-related proteins in cultured cells in vitro. Moreover, pretreatment with inhibitors of these kinase proteins could partially inhibit this effect of Rg3. From these results we can infer that PKAα, ERK1/2 and Akt may devote to the increase of TH activity and DA biosynthesis in Rg3 treated rats under fatigue conditions. An important aspect of the present study is that Rg3 did not show the anti-fatigue effect in a dose-dependent manner. The experiment results are consistent with those of other reports which showed that medium-dose and high-dose of Rg3 could significantly prolong the weight loaded swimming time of mice, but the difference between the two dose groups was not significant (Tang et al., 2008). This phenomenon might result from that the effects of ginsenosides on central nervous system have a two-way effect (Kim et al., 1998). Moreover, the effects of ginsenosides on cell viability demonstrate a dose-response plateau and appear to be cytotoxic to neuronal cells at higher concentrations. Furthermore, as reported, the changes of DA content in the studied different brain areas including hypothalamus and hippocampus were also different with each other in running rats (Balthazar et al., 2009). These results also suggest that Rg3 might exert diverse effects on modulating DA level dependent on different brain regions and different doses. In addition, DA metabolic product DOPAC was increased in different brain regions with different extents which could be reversed by Rg3, it might suggest that that under the condition of fatigue, the DA turnover is improperly activated, which also devote to the decrease of DA content in brain. On other hand, the effect of Rg3 on increase DA might also related its regulation of DA degradation or turnover. In conclusion, weight-loaded forced swimming can induce the decrease of DA level in brain, which is related to the decrease of TH expression and phosphorylation. Treatment with Rg3 could increase the expression and phosphorylation of TH and increase DA level. The underlining mechanisms of fatigue and Rg3 antifatigue effects are related to the signaling pathways including PKAα, ERK1/2, Akt and α-synuclein.

Acknowledgment Project was also supported by the Shanghai Foundation for Development of Science and Technology, China (Grant no. 10DZ1976000). This work was also supported in part by the National Natural Science Foundation of China (Grant nos. 81200974 and 81071017). The authors report no biomedical financial interests or potential conflicts of interest.

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