Antinociceptive Effects of Ginsenosides Injected Intracerebroventricularly or Intrathecally in Substance P-Induced Pain Model

• Abstract
• Abbreviations
• Introduction
• Methods and Materials
• Experimental animals
• lntracerebroventricular (i. c. v.) and intrathecal (i. t.) injections
• Drug treatment and substance P-induced nociceptive behavioral test
• Rotarod test
• Statistical analysis
• Drugs
• Results
• Effects of ginsenosides treated i. c. v. on the nociceptive behavior induced by i. t. substance P
• Effects of ginsenosides treated i. t. on the nociceptive behavior induced by i. t. substance P
• Effect of ginsenosides treated i. c. v. or i. t. on the rotarod test
• Discussion
• References

Abstract

We have examined the effects of several ginsenosides (R_b1, R_b2, R_c, R_d, R_e, R_f, R_g1 and R_g3) adminstered intracerebroventricularly (i. c. v.) or intrathecally (i. t.) on the nociceptive behavior induced by substance P (0.7 μg) injected i. t. Among the several ginsenosides studied, R_b2, R_c, R_d, and R_e, but not R_b1, R_f, R_g1 and R_g3, treated i. c. v. (50 μg) attenuated the nociceptive behavior induced by substance P injected i. t. On the other hand, we found that i. t. treatment with 50 μg of R_b1, R_b2, R_d, or R_f effectively attenuated the nociceptive behavior induced by i. t. injected substance P. However, the i. t. treatment with the same doses of R_c, R_e, R_g1 or R_g3 was not effective for antagonizing i. t. injected substance P-induced nociceptive behavior. Our results show that ginsenosides R_b2, R_c, R_d, or R_e injected supraspinally exert a antinociceptive effect in the substance P-induced pain model. Furthermore, R_b1, R_b2, R_d, or R_f treated spinally produce antinociception in the substance P-induced pain model.

Abbreviations

i.c.v.:intracerebroventricular

i.t.:intrathecal

S.E.M.:Standard error of the means

Introduction

Recent studies have shown that ginsenosides administered systemically (i. p.), intracerebroventricularly (i. c. v.) or intrathecally (i. t.) are involved in the regulation of nociception in various pain models. For example, the action of ginseng total saponin, the active component of the root of Panax ginseng used in far-east Asian traditional medicine, in the regulation of nociception as well as responsible ginsenosides of the ginseng total saponin has not been fully characterized. Our group as well as others have previously observed that various doses of several ginsenosides injected i. t. dose-dependently attenuate antinociception induced by U50,488H (a κ opioid receptor agonist) administered i. c. v. in the tail-flick test, suggesting that ginsenosides administered spinally appear to be responsible for blocking the antinociception induced by U50,488H administered supraspinally [1], [2]. Also, we have reported that total ginseng saponin or ginsenosides dose-dependently attenuate antinociception induced by morphine administered i. c. v. or systemically in the tail-flick test [1], [3]. In contrast, Shin et al. [4] have reported that ginsenosides, such as R_c, R_d and R_e, produce antinociception induced by formalin injected subcutaneously into the plantar of the hindpaw of mice. However, the exact reasons for this dual function of ginsenosides in the regulation of nociception are not well known.

Substance P located in the primary nerve ending is known as an important neurotransmitter for the pain transmission. The nociceptive behavior induced by substance P injected i. t. has been used as a pain model [5], [6]. However, the roles or functions of ginsenosides, when administered supraspinally or spinally in the regulation of the nociceptive behavior induced by substance P administered spinally have not been well characterized yet. Thus, the present study was designed to determine the possible modulatory role of various kinds of ginsenosides (R_b1, R_b2, R_c, R_d, R_e, R_f, R_g1 and R_g3) (Fig. [1]) injected supraspinally or spinally in the regulation of the nociceptive behavior induced by substance P injected spinally.

Methods and Materials

Experimental animals

Male ICR mice (20 - 25 g) from MJ Ltd. (Seoul, Korea) were used for all experiments. Animals were housed 5 per cage in a room maintained at 22 ± 0.5 °C with an alternating 12 h light-dark cycle. Food and water were available ad libitum. Each mouse was used only once. These experiments were approved by the University of Hallym Animal Care and Use Committee. All procedures were conducted in accordance with the ”Guide for Care and Use of Laboratory Animals” published by the National Institutes of Health and the ethical guidelines of the International Association for the Study of Pain.

lntracerebroventricular (i. c. v.) and intrathecal (i. t.) injections

I.c. v. injections were made according to the procedure of Haley and McCormick [7]. Briefly, each mouse was injected at the bregma with a 50 μL Hamilton microsyringe fitted with a 26-gauge needle that was inserted to a depth of 2.4 mm. The bregma could be found about 1 - 3 mm rostral to a line drawn between the anterior base of the ears after having felt the suture line by lightly rubbing with the point of needle. The i. t. administration was performed following the method described by Hylden and Wilcox [5], [8] using a 30-gauge needle connected to a 25 μL Hamilton syringe. I.c. v. and i. t. injection volumes were 5μL. In two separate groups of animals, the injection sites were verified by injecting a similar volume of 1 % methylene blue solution and determining the distribution of the injected dye in the ventricular space or in the spinal cord. The dye injected i. c. v. was found to be distributed through the ventricular spaces and reached the ventral surface of the brain and upper cervical portion of the spinal cord. The dye injected i. t. was distributed both rostrally and caudally but for a short distance (about 0.5 cm from the injection site) and no dye was found visually in the brain. The success rate for the injections was consistently found to be over 95 %, before the experiments were done.

Drug treatment and substance P-induced nociceptive behavioral test

The i. t. substance P-induced nociceptive behavioral test was performed by the following procedures. Vehicle (control) or 50 μg of ginsenoside R_b1, R_b2, R_c, R_d, R_e, R_f, R_g1 and R_g3 were treated i. c. v. or i. t. 10 min prior to i. t. injection of substance P (0.7 μg). Immediately after i. t. injection with substance P, the mice were placed in an observation chamber and their nociceptive behavioral responses were recorded during 30 min. The cumulative response time(s) of scratching and biting episodes directed toward the lumbar and caudal region of spinal cord were considered as indicative of nociception and were measured. Morphine (0.5 μg/5 μL, dissolved in saline or 20 % DMSO) administered i. c. v. or i. t. was used as a positive control.

Rotarod test

To examine that ginsenosides caused motor abnormality, the rotarod test was performed [9]. The apparatus consisted of a bar, with a diameter of 2.5 cm, subdivided into six compartments by disks 25 cm in diameter (Ugo Basile, Italy). The bar ro1ated at a constant speed of 14 revolutions/min. Mice were treated i. c. v. or i. t. with vehicle or ginsenosides. After 10 and 30 min, the rotarod test was conducted. The time that they remained on the rotating bar (maximum of 60 sec) was recorded.

Statistical analysis

The data are presented as the mean ± S.E.M. Statistical analysis was carried out by one-way analysis of variance (ANOVA) with Dunnett’s post-hoc test using GraphPad Prism version 3.02 for Windows (GraphPad Software, San Diego, CA, USA). P values less than 0.05 were considered to indicate statistical significance.

Drugs

Ginsenosides (R_b1, R_b2, R_c, R_d, R_e, R_f, R_g1, and R_g3) were obtained from Korea Ginseng and Tobacco Research Institute (Taejon, Korea). The purities of R_b1, R_b2, R_c, R_d, R_e, R_f, R_g1, and Rg3 used in the present study were 98.63, 98.13, 95.97, 97.87, 99.23, 99.33, 98.10, and 99.00 %, respectively. Substance P was purchased from Sigma-Aldrich Korea (purity: > 99 %). Morphine hydrochloride was purchased from Sam-Sung Pharm. Co. (Seoul, Korea). All drugs except R_d used for injection were dissolved in sterile saline (0.9 % NaCl solution). Ginsenoside R_d was prepared in saline containing 20 % dimethyl sulfoxide (DMSO), which was used as a vehicle control.

Results

Effects of ginsenosides treated i. c. v. on the nociceptive behavior induced by i. t. substance P

Groups of mice were treated i. c. v. with 20 % DMSO (vehicle control) or 50 μg/5 μL of various kinds of ginsenosides (R_b1, R_b2, R_c, R_d, R_e, R_f, R_g1 and R_g3). After 10 min, the mice were injected i. t. with substance P (0.7 μg). Then, the nociceptive behavior induced by substance P was measured for 30 min. In the positive control test, substance P-induced nociceptive behavior was almost completely reduced by morphine administration (0.5 μg, i. c. v.). There was no significant difference between morphine dissolved in saline and in 20 % DMSO. As shown in Fig. [2], substance P injected i. t. showed nociceptive behavior in a vehicle-treated control group of mice. Among the several ginsenosides, only R_b2, R_c, R_d, or R_e treated i. c. v. attenuated the nociceptive behavior induced by i. t. substance P. However, R_b1, R_f, R_g1 and R_g3 did not alter the substance P-induced nociceptive behavior.

Effects of ginsenosides treated i. t. on the nociceptive behavior induced by i. t. substance P

Groups of mice were treated i. t. with 20 % DMSO (vehicle control) or 50 μg/5 μL of various kinds of ginsenosides (R_b1, R_b2, R_c, R_d, R_e, R_f, R_g1 and R_g3). After 10 min, the mice were injected i. t. with substance P (0.7 μg). Then, the nociceptive behavior induced by substance P was measured for 30 min. In the positive control test, substance P-induced nociceptive behavior was almost completely reduced by morphine administration (0.5 μg, i. t.). There was no significant difference between morphine dissolved in saline and in 20 % DMSO. As shown in Fig. [3], only R_b1, R_b2, R_d, or R_f treated i. t. attenuated the nociceptive behavior induced by i. t. substance P. However, R_c, R_e, R_g1 and R_g3 did not alter the substance P-induced nociceptive behavior.

Effect of ginsenosides treated i. c. v. or i. t. on the rotarod test

Groups of mice were treated i. c. v. or i. t. with 20 % DMSO (vehicle control) or 50 μg/5 μL of various kinds of ginsenosides (R_b1, R_b2, R_c, R_d, R_e, R_f, R_g1 and R_g3). After 10 and 30 min, the rotarod test was performed. The administration of any ginsenoside produced no significant changes in rotarod performance compared with control. Representative data are demonstrated in Tables [1] and [2].

Discussion

In the present study, we examined whether ginsenosides (R_b1, R_b2, R_c, R_d, R_e, R_f, R_g1 and R_g3) have the antinociceptive effect on the nociceptive behavior elicited by substance P injected spinally. We found that the i. c. v. treatment with R_b2, R_c, R_d, or R_e attenuates the nociceptive behavior induced by i. t. substance P. In addition, i. t. treatment with R_b1, R_b2, R_d, or R_f exerted an inhibitory action against substance P-induced nociceptive behavior. Our results suggest that ginsenosides R_b2, R_c, R_d, or R_e injected supraspinally exert an antinociceptive effect in the substance P-induced pain model. Furthermore, R_b1, R_b2, R_d, or R_f treated i. t. produce antinociception in the substance P-induced pain model.

In recent studies, Nah and colleagues have demonstrated that total ginseng saponin produces antinociception in capsaicin- and substance P-induced pain models [10], [11]. Although ginseng total saponin administered systemically produces antinociception in these pain models, it is not known which components of ginseng total saponin are responsible for the production of antinociception. We found in the present study that several ginsenosides treated i. c. v. (R_b2, R_c, R_d, and R_e) or i. t. (R_b1, R_b2, R_d, or R_f) are the major components of ginseng total saponin responsible for the production of antinociception in the substance P-induced pain model. Mogil et al. [12] have reported that systemic administration of R_f attenuated acetic acid-induced writhing response or tonic phase in the formalin-induced pain, without affecting nociception in tail-flick and hot-plate tests, and the acute phase nociceptive behavior in the formalin test. Moreover, Shin et al. [4] have reported that R_c, R_d, and R_e administered systemically produce antinociception in the formalin-induced pain model. These findings are, in part, in line with that observed in the present study in that, in the substance P-induced nociceptive behavioral test, antinociception is produced by i. t. injection with R_d or R_f as well as i. c. v. injection with R_c, R_d or R_e.

Although several ginsenosides produce antinociception in certain pain models [10], [11], ginseng total saponin or ginsenosides does not affect the basal latency of tail-flick or hot-plate responses [4], [12]. Furthermore, ginseng total saponin or ginsenosides exert an antagonistic action against opioid-induced antinociception in the tail-flick test. For example, ginseng total saponin administered systemically shows inhibitory action against opioid-induced antinociception [1], [13]. Additionally, our laboratory has recently reported that various doses (from 0.1 to 1 μg) of ginsenosides R_b1, R_b2, R_c, R_d, and R_g1, but not R_e, injected i. t. dose-dependently attenuate antinociception induced by U50,488H (a κ opioid receptor agonist) administered supraspinally [2]. We also reported that ginsenosides R_b2, R_c, R_d and R_g1, but not R_b1 and R_e, injected i. t. dose-dependently attenuate antinociception induced by morphine administered i. c. v. [3]. Furthermore, it has been reported that the antagonism of U50,488H-induced antinociception in mice treated with ginseng total saponin was abolished by treatment with a serotonin precursor, 5-hydroxytryptophan, in the tail-flick test [1]. However, the exact reasons of differential functions of each ginsenoside in the modulation of nociception in various pain models are not exactly understood at the present time.

References

• 1 Kim H S, Oh K W, Rheu H M, Kim S R. Antagonism of U-50,488H-induced antinociception by ginseng total saponins is dependent on serotonergic mechanisms.  Pharmacol Biochem Behav. 1992;  42 587-93
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  CrossrefPubMedSearch in Google Scholar
• 11 Yoon S R, Nah J J, Shin Y R, Kim S K, Nam K Y, Choi H S, Nah S Y. Ginsenosides induce differential antinociception and inhibit substance P induced-nociceptive response in mice.  Life Sci. 1998;  62 PL319-25
  CrossrefPubMedSearch in Google Scholar
• 12 Mogil J S, Shin Y R, McCleskey E W, Kim S C, Nah S Y. Ginsenoside Rf, a trace component of ginseng root, produces antinociception in mice.  Brain Res. 1998;  792 218-28
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• 13 Nemmani K V, Ramarao P. Role of benzodiazepine-GABAA receptor complex in attenuation of U-50,488H-induced analgesia and inhibition of tolerance to its analgesia by ginseng total saponin in mice.  Life Sci. 2002;  70 1727-40
  CrossrefPubMedSearch in Google Scholar

Professor Hong-Won Suh, Ph. D.

Department of Pharmacology

College of Medicine

Hallym University

1 Okchun-dong

Chunchon

Kangwon Do 200-702

South Korea

Phone: +82-33-248-2614

Fax: +82-33-248-2612

Email: hwsuh@hallym.ac.kr

References

• 1 Kim H S, Oh K W, Rheu H M, Kim S R. Antagonism of U-50,488H-induced antinociception by ginseng total saponins is dependent on serotonergic mechanisms.  Pharmacol Biochem Behav. 1992;  42 587-93
  CrossrefPubMedSearch in Google Scholar
• 2 Suh H W, Song D K, Huh S O, Kim Y R. Modulatory role of ginsenosides injected intrathecally or intracerebroventricularly in the production of antinociception induced by kappa-opioid receptor agonist administered intracerebroventricularly in the mouse.  Planta Med. 2000;  66 412-7
  Thieme ConnectPubMedSearch in Google Scholar
• 3 Suh H W, Song D K, Kim Y R. Effects of ginsenosides injected intrathecally or intracerebroventricularly on antinociception induced by morphine administrated intracerebroventricularly in the mouse.  Gen Pharmacol. 1997;  29 873-7
  CrossrefPubMedSearch in Google Scholar
• 4 Shin Y R, Jung O M, Nah J J, Nam K Y, Kim C Y, Nah S Y. Ginsenosides that produce differential antinociception in mice.  Gen Pharmacol. 1999;  32 53-9
  PubMedSearch in Google Scholar
• 5 Hylden J LK, Wilcox G L. Intrathecal substance P elicits a caudally-directed biting and scratching behavior in mice.  Brain Res. 1981;  217 212-5
  CrossrefPubMedSearch in Google Scholar
• 6 Hunskaar S, Rasmer O B, Hole K. Acetylsalicylic acid, paracetamol and morphine inhibit behavioral responses to intrathecally administered substance P or capsaicin.  Life Sci. 1998;  37 835-41
  PubMedSearch in Google Scholar
• 7 Haley T J, McCormick W G. Pharmacological effects produced by intracerebral injections of drugs in the conscious mouse.  Br J Pharmacol. 1957;  12 12-5
  PubMedSearch in Google Scholar
• 8 Hylden J LK, Wilcox G L. Intrathecal morphine in mice: a new technique.  Eur J Pharmacol. 1980;  67 313-6
  CrossrefPubMedSearch in Google Scholar
• 9 Dunham N W, Miya T S. A note on a simple apparatus for detecting neurological deficit in rats and mice.  J Am Pharm Assoc. 1957;  46 208-9
  PubMedSearch in Google Scholar
• 10 Nah J J, Hahn J H, Chung S, Choi S, Kim Y I, Nah S Y. Effect of ginsenosides, active components of ginseng, on capsaicin-induced pain-related behavior.  Neuropharmacology. 2000;  39 2180-4
  CrossrefPubMedSearch in Google Scholar
• 11 Yoon S R, Nah J J, Shin Y R, Kim S K, Nam K Y, Choi H S, Nah S Y. Ginsenosides induce differential antinociception and inhibit substance P induced-nociceptive response in mice.  Life Sci. 1998;  62 PL319-25
  CrossrefPubMedSearch in Google Scholar
• 12 Mogil J S, Shin Y R, McCleskey E W, Kim S C, Nah S Y. Ginsenoside Rf, a trace component of ginseng root, produces antinociception in mice.  Brain Res. 1998;  792 218-28
  CrossrefPubMedSearch in Google Scholar
• 13 Nemmani K V, Ramarao P. Role of benzodiazepine-GABAA receptor complex in attenuation of U-50,488H-induced analgesia and inhibition of tolerance to its analgesia by ginseng total saponin in mice.  Life Sci. 2002;  70 1727-40
  CrossrefPubMedSearch in Google Scholar

Professor Hong-Won Suh, Ph. D.

Department of Pharmacology

College of Medicine

Hallym University

1 Okchun-dong

Chunchon

Kangwon Do 200-702

South Korea

Phone: +82-33-248-2614

Fax: +82-33-248-2612

Email: hwsuh@hallym.ac.kr