Effects of Gypenosides on Acute Stress in Mice

Abstract

The effects of gypenosides (GPS) on electric footshock (EF)-induced acute stress in mice were investigated. Mice were treated orally with GPS (30-400 mg/kg) once a day for 5 days. After 2 days of GPS treatment, mice were exposed to EF stimuli (intensity, 2 mA; interval, 10 s; duration, 3 min) for acute stress for 3 days. Spontaneous locomotor activity was increased by acute EF stress, which was decreased by treatment with GPS (100 and 400 mg/kg). In addition, the increased levels of dopamine and serotonin by acute EF stress in the brain were reduced by treatment with GPS (100 and 400 mg/kg). The serum levels of corticosterone increased by acute EF stress were also reduced by GPS (100 and 400 mg/kg). These results suggest that GPS shows the ameliorating effects on acute EF stress by modulating the activity of dopaminergic and serotonergic neurons, and the serum levels of corticosterone. Clinical trials of GPS need to be conducted further so as to develop promising anti-stress agents.

Introduction

Various stresses can cause physical changes as well as mental performances. Acute stress can make human and animals exciting to protect against the nociceptive stimuli, which increase the heart rate and blood pressure (Kovacs et al., 2005). In contrast, chronic stress has been associated with the many illnesses, including anxiety disorders and depression (Kendler et al., 1999). Both acute and chronic stresses activate the hypothalamic-pituitary-adrenal (HPA) axis, which are characterized by a sudden rise in adrenocorticotrophic hormone followed by the release of glucocorticoids, such as corticosterone and cortisol (Keeney et al., 2006; Rivier and Plotsky, 1986). In addition, the brain levels of dopamine and serotonin are increased under the conditions of acute stress, whereas the repeated and chronic stress leads to decrease in dopamine and serotonin levels in the brain (Sheikh et al., 2007).

Many stress models, including electric footshock (EF) stimulus, forced swimming, noise stimulus, restraint and immobilization have been employed to examine the stressful responses in mice and rats (Xie et al., 2008). Spontaneous locomotor activity increases after being exposed to acute stress (Katz et al., 1981). In contrast, locomotor activity, grip strength, body weight and endurance decrease after exposure to chronic stress (Retana-Marquez et al., 2003).

Gynostemma pentaphyllum Makino (Cucurbitaceas, GP) is a traditional medicinal herb that has shown various effects on diabetes, fatigue, hyperlipidemia, immunity, oxidative stress and tumor (Razmovski-Namovski et al., 2005). Recently, it has been reported that ethanol extract from GP (GP-EX) had an anti-stress function by improving the loss of body weight and the reduction of grip strength which were induced by chronic EF stress, as well as an immunomodulatory effect in mice (Choi et al., 2008; Im et al., 2012). GP-EX also had an ameliorating effect on chronic stress-induced anxiety disorders, which were evaluated by the elevated plus-maze and marble burying tests (Choi et al., 2013). In addition, GP-EX had a protective effect against neurotoxicity in the 6-hydroxydopamine (6-OHDA)-lesioned rat model of Parkinson’s disease (PD) (Choi et al., 2010). Gypenosides (GPS) are the dammarane-type gynosaponin-enriched components isolated from GP (Razmovski-Namovski et al., 2005). GPS shows a neuroprotective effect in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model of PD (Wang et al., 2010a).

In this study, the main GPS was obtained from GP (Shang et al., 2006; Wang et al., 2010b), and the pharmacological effects of GPS on acute EF stressinduced behaviors in mice were investigated in order to further define the anti-stress function of GP-EX. After being exposed to acute stress by electric EF stimuli, we examined the behavioral changes using the spontaneous locomotor test and the biochemical changes on the levels of dopamine, serotonin and corticosterone.

 

Experimental

Materials – GPS was purchased from Ankang Dongke Maidisen Nature Pharmaceutical Co. (Xi’an, China) (Shang et al., 2006; Wang et al., 2010b). Dopamine, serotonin, isoproterenol and 5-hydroxyindoleacetic acid (HIAA) were purchased from Sigma Co. (St. Louis, MO, USA). A corticosterone kit was purchased from USCN Life Sci. (E0504m, Wuhan, China). All other chemicals were of HPLC grade.

Animals – Mice (ICR, male, 20 - 25 g) were purchased from Samtako Co. (Animal Breeding Center, Osan, Korea). Animals were housed in a temperature (23 ± 2 ℃) and humidity (50 ± 2%) controlled environment with a 12 h light/dark cycle (lights on at 07:00), and with ad libitum access to standard mouse food and water. The present study was performed in accordance with the guidelines for the care and use of laboratory animals of Chungbuk National University Laboratory Animal Research Center (approval number: CBNU-481-12-01).

Experimental design and the exposure to acute EF stress – Mice were randomly divided into the groups containing 8 - 12 animals. The control groups received saline (0.9%). GPS was the groups which were treated orally with GPS (30 - 400 mg/kg) for 5 days once a day including a 2 day-adaptation period. Stress was the groups which, after 2 days of GPS treatment, were exposed to the acute EF stimuli (intensity, 2 mA; interval, 10 s; duration, 3 min) in an electrified shock chamber at 14:00 every day for 3 days using an electric shock generator (Seil Electric Co., Daejeon, Korea). During the periods of acute stress, mice were treated with GPS approximately 2 h before the exposure of EF stress. After the final treatment with GPS and behavioral tests, mice were anaesthetized and sacrificed to obtain brain tissues and serum for biochemical analyses.

The spontaneous locomotor activity test – Spontaneous locomotor activity was measured every day using a tilting-type ambulometer (Model AMB-10, O'Hara, Tokyo, Japan). Each mouse was placed in a round cage (diameter, 20 cm; depth, 18 cm) and the numbers of horizontal movements were detected automatically for 30 min.

Measurement of dopamine and serotonin levels – After the final behavioral tests, the whole brain tissues were homogenized in perchloric acid (1 M, 300 μl) and isoproterenol (100 pmol, internal standard) or HIAA (300 pmol, internal standard) and the homogenates were centrifuged at 12,000 × g at 4 ℃ for 20 min. The supernatants were filtered using pore filters (Millex-GV, 0.45 μm, Waters, Milford, MA, USA) and the filtrate (100 μl) was injected into an HPLC system (Satoh et al., 2008; Yanagisa et al., 1982).

Measurement of corticosterone – After the final behavioral tests, blood was collected from the heart of sacrificed mice and centrifuged at 12,000 × g at 4 ℃ for 15 min to obtain serum. The serum levels of corticosterone were assessed using an enzyme-linked immunosorbent assay kit.

Statistical analysis – Data were analyzed using a oneway analysis of variance (ANOVA) followed by a Tukey’s test for evaluating the dose-dependent effects of GPS. Two-way ANOVA followed by Tukey’s test was also employed to evaluate the effects of GPS on acute EF stress. All data were expressed as means ± S.E.M. with p values < 0.05 being considered statistically significant.

 

Results

Effects of GPS on spontaneous locomotor activity – Treatment with GPS (30 - 400 mg/kg) did not alter the counts of spontaneous locomotor activity at day-3, compared with the control groups (Fig. 1). In contrast, the counts of spontaneous locomotor activity after being exposed to acute EF stress were increased by 16.8% (p < 0.05) at day-3, compared with the control groups (n = 12). However, the counts of spontaneous locomotor activity were reduced by 3.2%, 5.0%, 10.9% (p < 0.05), 12.3% (p < 0.05) and 13.1% (p < 0.05) by treatment with GPS (30, 50, 100, 200 and 400 mg/kg) respectively for 5 days, compared with the acute EF-stressed groups (n = 12) (Fig. 1). In addition, at day-2, the counts of spontaneous locomotor activity were increased by 7.9% by acute EF stress, and they were reduced by 0.7 - 6.1% by treatment with GPS (30 - 400 mg/kg), but it was not significant (data not shown).

Effects of GPS on the levels of dopamine and serotonin in the brain – Treatment with GPS (30 - 400 mg/kg) for 5 days did not alter the levels of dopamine and serotonin in the brain, compared with the control groups (Fig. 2). The levels of dopamine significantly were increased by 38.1% (p < 0.01) after exposure to acute EF stress for 3 days, compared with the control groups (n = 8), and they were recovered by 4.3%, 10.6%, 17.6% (p < 0.05), 18.5% (p < 0.05) and 23.7% (p < 0.05), respectively by treatment with GPS (30, 50, 100, 200 and 400 mg/kg), compared with the acute EF-stressed groups (n = 8) (Fig. 2).

Fig. 1.Effects of GPS on spontaneous locomotor activity after exposure to acute EF stress in mice. Mice (ICR, male, 25 - 30 g) were treated orally with GPS (30 - 400 mg/kg) or saline (0.9%) once a day for 5 days. After 2 days of GPS treatment, mice were also exposed to EF stimuli (2 mA, with an interval and duration of 10 s for 3 min) for 3 days (Stress groups). Spontaneous locomotor activity was performed as described in the Experimental section. The results are expressed as means ± S.E.M. (n = 12). *p < 0.05 compared with the control groups; #p < 0.05 compared with the acute EF-stressed groups (one-way ANOVA followed by Tukey’s test). §p < 0.05 compared with the un-stressed groups (two-way ANOVA followed by Tukey’s test).

Fig. 2.Effects of GPS on the levels of dopamine in the brain. Mice (ICR, male, 25 - 30 g) were treated orally with GPS (30 - 400 mg/kg) or saline (0.9%) once a day for 5 days. Mice were also exposed to EF stimuli (2 mA, with an interval and duration of 10 s for 3 min) for 3 days (Stress groups). The brains were removed and the levels of dopamine were determined by an HPLC method as described in the Experimental section. The levels of dopamine in the control groups were 7.2 ± 1.1 ng/mg tissue in the brain. The results are expressed as means ± S.E.M. (n = 8). *p < 0.01 compared with the control groups; #p < 0.05 compared with the chronic EF-stressed groups (one-way ANOVA followed by Tukey’s test). §p < 0.05 compared with the unstressed groups (two-way ANOVA followed by Tukey’s test).

Fig. 3.Effects of GPS on the levels of serotonin in the brain. The brains were removed and the levels of serotonin were determined by an HPLC method as described in the Experimental section. The levels of serotonin in the control groups were 4.1 ± 0.5 ng/mg tissue in the brain. For further comments, see Fig. 2.

Fig. 4.Effects of GPS on the levels of corticosterone in the serum. The blood samples were collected and the serum levels of corticosterone were determined by an enzyme-linked immunosorbent assay kit as described in the Experimental section. The levels of corticosterone in the control groups were 156.3 ± 10.2 ng/ml in the serum. The results are expressed as means ± S.E.M. (n = 8). For further comments, see Fig. 2.

In addition, the levels of serotonin were increased by 38.9% (p < 0.01) after exposure to acute EF stress, compared with the control groups (n = 8) (Fig. 3). However, the increased levels of serotonin by acute EF stress were reduced by 4.2%, 15.5% (p < 0.05), 18.9% (p < 0.05), 20.2% (p < 0.05) and 21.9% (p < 0.05), respectively by treatment with GPS (30, 50, 100, 200 and 400 mg/kg), compared with the acute EF-stressed groups (n = 8) (Fig. 3).

Effects of GPS on the levels of corticosterone in the serum – The serum levels of corticosterone were not altered by treatment with GPS (30 - 400mg/kg) for 5 days, compared with the control groups (Fig. 4). The levels of corticosterone in the serum were increased by 37.2% (p < 0.01) by exposure to acute EF stress, compared with the control groups (n = 8) (Fig. 4). However, the increased levels of corticosterone were decreased by 3.2%, 6.7%, 15.4% (p < 0.05), 17.0% (p < 0.05) and 19.1% (p < 0.05), respectively by treatment with GPS (30, 50, 100, 200 and 400 mg/kg), compared with the acute EF-stressed groups (n = 8) (Fig. 4).

 

Discussion

Recently, GP-EX has been found to have an ameliorating effect on chronic EF stress-induced anxiety disorders in mice (Choi et al., 2013). GP-EX has been reported to have approximately 90 kinds of GPS (Razmovski-Naumovski et al., 2005). In present study, the ameliorating effects of GPS on acute EF stressinduced behaviors were investigated in order to define the main functional components of GP-EX.

In the initial excited state of acute stress, the locomotor behaviors could be increased due to an increase in muscle tone and blood pressure (Kovacs et al., 2005). The acute stress also increases grip strength due to the excited states (Benaroya-Milshtein et al., 2004). In this study, the spontaneous locomotor activity were increased after being exposed to acute EF stress for 3 days, and they were ameliorated by treatment with GPS (100 - 400 mg/kg) in a dose-dependent manner (Fig. 1). The changes of grip strength increased by acute EF stress were also reduced by treatment with GPS (200 - 400 mg/kg), but it was not significant (data not shown).

It has been suggested that spontaneous locomotor activity is associated with dopaminergic neurons of the brain as the dopamine receptor blocking agents can reduce this activity (Anden et al., 1970). The acute stressinduced changes of grip strength are also associated with dopamine levels and dopaminergic neurons in the brain (Dunnett et al., 1998; Kirby et al., 1995). Acute stress increases the brain levels of dopamine and serotonin, while chronic stress reduces dopamine and serotonin levels in the brain (Fujino et al., 2002; Sheikh et al., 2007; Sorg et al., 1991). In addition, both acute and chronic stresses lead to the increase in tyrosine hydroxylase activity and the release of dopamine (Kvetnansky et al., 1970). The serum levels of corticosterone are increased by both acute and chronic stresses, which keep the sympathetic nervous systems exciting (Murakami et al., 2005).

In this study, treatment with GPS reduced the levels of dopamine and serotonin that were increased by acute EF stress in a dose-dependent manner (Figs. 2 and 3). The serum levels of corticosterone, which were increased by acute EF stress, were also decreased by treatment with GPS (100 - 400 mg/kg) in a dose-dependent manner (Fig. 4). Together, these results suggest that GPS has the capability to reduce the excitability via inhibiting the activation of dopaminergic and serotonergic neurons, and the serum levels of corticosterone by HPA axis in mice.

It is suggested that the stressful stimuli induce the production of reactive oxygen species and increase the release of catecholamines and glucocorticoids (Kandel, 2000). GPS has been found to protect aortic endothelial cells against oxidative damage (Tanner et al., 1999). GPS also exhibits a protective effect on glutamate-induced oxidative neurotoxicity in PC12 cells (Shang et al., 2006) and on the MPTP-induced mouse model of PD (Wang et al., 2010a). In addition, GP-EX has a protective effect against neurotoxicity in the 6-OHDA-lesioned rat model of PD (Choi et al., 2010). It is therefore possible to explain that the responses induced by acute EF stress might be relieved by treatment with GPS through the antioxidative activity in rodents.

Furthermore, GPS and GP-EX recover the brain levels of dopamine and serotonin which are reduced by chronic EF stress in mice (Choi et al, 2008; 2013). In contrast, the increased dopamine and serotonin levels by acute EF stress were reduced by treatment with GPS (Figs. 2-4). These results suggest that GPS and GP-EX have an adaptogenic effect on the modulation of monoaminerelated neuronal functions, including anxiety disorders and stress.

In this study, treatment with GPS (50 - 400 mg/kg) did not exhibit the adverse effects, such as weight loss, diarrhea, vomiting and death. The values of LD50 of total GPS are 755-838 mg/kg (injected into the abdominal cavity) and 402 (± 18.2 mg/kg, i.p.) in mice (Guo and Wang, 1993). The water extract (750mg/kg) of GP has also been shown not to produce any significant toxicity in rats during a 6-month period of treatment (Attawish et al., 2004).

In conclusion, GPS (100 - 400 mg/kg) had the ameliorating effects on acute EF stress in mice, which was evaluated by measuring the spontaneous locomotor activity and the levels of dopamine, serotonin and corticosterone. Clinical trials of GPS need to be conducted further so as to develop promising anti-stress agents.