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Synergistic Interaction Between Intrathecal Ginsenosides and Morphine on Formalin-Induced Nociception in Rats
The Journal of Pain, Vol 12, No 7 (July), 2011: pp 774-781 Available online at www.sciencedirect.com
Synergistic Interaction Between Intrathecal Ginsenosides and Morphine on Formalin-Induced Nociception in Rats Myung Ha Yoon,*,y,z Kwang Soo Kim,* Hyung Gon Lee,* Chang Mo Kim,* Woong Mo Kim,* Jeong Il Choi,* and Yeo Ok Kim* * Department of Anesthesiology and Pain Medicine, Chonnam National University, Medical School, Gwangju, Korea. y The Brain Korea 21 Project, Center for Biomedical Human Resources at Chonnam National University, Gwangju, Korea. z Chonnam National University Research Institute of Medical Sciences, Gwangju, Korea.
Abstract: We defined the nature of the pharmacological interaction between ginsenosides and morphine in a nociceptive state and clarified the role of the different types of opioid receptor in the effects of ginsenosides. An intrathecal catheter was placed in male Sprague-Dawley rats. Pain was induced by formalin injection into the hindpaw. Isobolographic analysis was used to evaluate drug interactions. Furthermore, a nonselective opioid receptor antagonist (naloxone), a m opioid receptor antagonist (CTOP), a d opioid receptor antagonist (naltrindole), and a k opioid receptor antagonist (GNTI) were given intrathecally to verify the involvement of the opioid receptors in the antinociceptive effects of ginsenosides. Both ginsenosides and morphine produced antinociceptive effects in the formalin test. Isobolographic analysis revealed a synergistic interaction after intrathecal delivery of the ginsenosides-morphine mix. Intrathecal CTOP, naltrindole, and GNTI reversed the antinociceptive effects of ginsenosides. RT-PCR indicated that opioid receptors’ mRNA was detected in spinal cord of na€ıve rats and the injection of formalin had no effect on the expression of opioid receptors’ mRNA. Taken together, our results indicate synergistic antinociception following intrathecal coadministration of a ginsenosides/morphine mix in the formalin test, and that m, d, and k opioid receptors are involved in the antinociceptive mechanism of ginsenosides. Perspective: This article concerns the antinociceptive activity of ginsenosides, which increases antinociception by morphine. Thus, a spinal combination of ginsenosides and morphine may be useful in the management of acute pain as well as facilitated state pain. ª 2011 by the American Pain Society Key words: Antinociception, drug interaction, ginsenosides, morphine, opioid receptors, spinal cord.
G
inseng, the root of Pannax ginseng C.A. Meyer, has long been used in Oriental traditional medicine to improve the weakened physical status brought on by stress or disease.13 Ginsenosides, ginseng saponins, are the major component responsible for the effects of ginseng and more than 20 different ginsenosides have been found todate.18 Therefore, many different kinds of ginsenosides may be involved in the effects of ginseng. Several lines of evidence suggest that ginsenosides are effective against various nociceptive states. In particular, Received November 15, 2010; Revised December 16, 2010; Accepted December 31, 2010. Address reprint requests to Myung Ha Yoon, Professor in Department of Anesthesiology and Pain Medicine, Chonnam National University, Medical School, 8 Hakdong, Donggu, Gwangju 501-757, Korea. E-mail: [email protected] 1526-5900/$36.00 ª 2011 by the American Pain Society doi:10.1016/j.jpain.2010.12.012
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intrathecal ginsenosides attenuate formalin-induced, substance P-induced, and capsaicin-induced pain behaviors in mice.3,17,38 These findings suggest that ginseng may play an important role in the modulation of nociception at the level of the spinal cord. According to several previous reports, Ca21 channels are the pharmacological sites of action of ginsenosides,19,20,25 and alpha2, muscarinic, opioid, and GABA receptors are not involved in ginsenoside activity.19,29 However, a recent study has shown that intrathecal ginsenosides are effective in treating postoperative pain simulated by paw incision in rats and that naloxone attenuates the antinociceptive effects of ginsenosides,28 indicating that opioid receptors may play an important role in the mechanism of action of ginsenosides at the spinal level. Thus, the involvement of opioid receptors in the actions of ginsenosides remains to be determined.
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Methods
a 30‑gauge needle. The formalin injection produces specific pain behavior, which is readily discriminated and characterized by rapid and brief withdrawal or flexing of the injected paw. This behavior has been called a flinching response. Such pain behavior was thus quantified by periodically counting the number of flinches of the injected paw after injection. The number of flinches was counted for 1-minute periods at 1 and 5 minutes and in 5-minute intervals from 10 to 60 minutes. Formalininduced flinching behavior is biphasic; the initial acute phase (0–9 minutes) is followed by a relatively short quiescent period, which is then followed by a prolonged tonic response (10–60 minutes). At the end of the experiment, the rats were sacrificed by sevoflurane overdose.
Animal Preparation
Experimental Protocol
The experimental protocol was approved by The Institutional Animal Care and Use Committee, Medical Science of Chonnam National University. Adult male Sprague-Dawley rats, weighing 250 to 300 g each, were used. The animals were housed 4 to a cage and kept in a vivarium, maintained at 22 C, with a 12/ 12‑hour alternating light/dark cycle, and were given food and water ad libitum. All test drugs were administered intrathecally. Thus, rats were implanted with an intrathecal catheter under sevoflurane anesthesia, as described previously.34 An 8.5-cm polyethylene-10 catheter was advanced caudally through an incision in the cisternal membrane to the thoracolumbar level of the spinal cord. The exterior portion of the catheter was secured at the skull by subcutaneous tunneling and closed with a 28-gauge wire. The skin was sutured with 3-0 silk. After catheter implantation, rats were housed individually. Only animals with no evidence of neurological deficit after catheter implantation were included in the study and they were housed individually. The behavioral study occurred 5 days after intrathecal catheterization.
Five days after intrathecal cannulation, the rats were placed in a restraint cylinder for the experiment. After acclimatization for 15 to 20 minutes, the rats were allocated to receive one of the experimental drugs. The same volume of the vehicle (saline or DMSO) was used as the control. Each animal was tested only once. All experimental tests were made with the investigator blinded as to treatment condition of each animal.
As is widely known, the antinociceptive effects induced by opiate drugs occur through their action at opioid receptors, located in the central nervous system, including the spinal cord.6 Thus, spinal opioid receptor agonists are effective and are widely used in the management of various types of pain.15,21,23,40 The aims of the present study were to investigate the effect of ginsenosides in rat formalin-induced nociception in the spinal cord, and to determine the characteristics of the drug interaction between ginsenosides and morphine. We also examined the involvement of opioid receptor types on the effect of ginsenosides at the spinal level.
Drugs The following drugs were used: ginsenosides, morphine sulfate (Sigma, St Louis, MO), CTOP (D-Phe-CysTyr-D-Trp-Orn-Yhr-NH2; Tocris Cookson, Bristol, UK), naloxone [(5a)-4,5-Epoxy-3,14-dihydro-17-(2-prophenyl) morphinan-6-one hydrochloride, Tocris] naltrindole [17-(Cyclopropylmethyl)-6,7-dehydro-4,5a-epoxy-3, 14dihydroxy-6,7-20 ,30 -indolomorphian hydrochloride; Tocris], and GNTI [50 -Guanidinyl-17-(cyclopropylmethyl)6,7-dehydro-4,5a-epoxy-3,14-dihydroxy- 6,7-20 ,30 -indolomorphian dihydrochloride; Tocris]. Ginsenosides were provided by the Korea Ginseng and Tobacco Research Institute (Daejon, Korea). The ginsenosides were dissolved in dimethylsulfoxide (DMSO), whereas all other drugs were dissolved in distilled water. Intrathecal administration of these agents was performed using a hand-driven, gear-operated syringe pump. All drugs were delivered in a volume of a 10-mL solution.
Nociceptive Test Antinociception was assessed using the formalin test.36 Briefly, 50 mL 5% formalin solution was injected subcutaneously into the plantar surface of a hind paw using
Effects of Ginsenosides and Morphine Rats received intrathecal saline and increasing doses of either ginsenosides (30, 100, 300 mg, n = 28) or morphine (1, 3, 10, 30 mg, n = 30) 10 min before formalin injection, and the effects of ginsenosides and morphine were examined. Each ED50 value (effective dose producing a 50% reduction in control formalin response) for the agents was calculated separately in 2 phases.
Drug Interaction To define the properties of the pharmacological interaction between ginsenosides and morphine in the formalin test, isobolographic analysis was used (n = 48).36 This method is based on comparing doses determined to be equally effective. Initially, each ED50 value was determined from the dose-response curves of the 2 agents alone. Then, a dose-response curve was obtained with concurrent delivery of the 2 drugs in a constant dose ratio, based on the ED50 values of the single agent. Thus, separate groups received: ginsenosides ED50 1 morphine ED50; (ginsenosides ED50 1 morphine ED50)/2; (ginsenosides ED50 1 morphine ED50)/4; and (ginsenosides ED50 1 morphine ED50)/8. From the dose-response curves of the combined drugs, the ED50 values of the mixture were calculated, and the dose combinations were used to plot the isobologram. The isobologram was constructed by plotting the ED50 values of the single agents on the x- and y-axes, respectively. The theoretical additive dose combination was calculated. From the variance of the total dose, the individual variances for the combined agents were obtained. In combination, a total fraction value was calculated to describe the magnitude of the interaction. Total fraction value = (ED50 of drug 1 combined with drug 2)/(ED50 for drug 1 given alone) 1 (ED50 of drug 2 combined with drug 1)/(ED50 for drug 2 given alone).
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Values close to 1 indicate an additive interaction, values >1 indicate an antagonistic interaction, and values <1 indicate a synergistic interaction. The formalin test was done 10 minutes after administration of mixtures of the 2 drugs.
Opioid Receptor Types and Ginsenosides To determine the contribution of opioid receptor types on the effect of ginsenosides, rats were pretreated with several opioid receptor antagonists. These antagonists were given intrathecally 10 minutes before the administration of intrathecal ginsenosides (300 mg) and the formalin test was done 10 minutes after ginsenosides delivery. Maximal doses of opioid receptor antagonists that did not affect the control formalin response or cause behavioral abnormalities were determined from pilot experiments and previous studies.5,7,39 These experiments were conducted in 2 phases. The opioid receptor antagonists used in this study were as follows: a nonselective opioid receptor antagonist (naloxone, .3 mg, n = 10), a m opioid receptor antagonist (CTOP, 15 mg, n = 11), a d opioid receptor antagonist (naltrindole, 10 mg, n = 11), and a k opioid receptor antagonist (GNTI, 50 mg, n = 10).
General Behavior To examine the behavioral changes due to ginsenosides and morphine, the highest dose of each drug was given intrathecally to additional rats (n = 10). Motor function was assessed by the placing/stepping reflex and the righting reflex.37 The first reflex was evoked by drawing the dorsum of each hindpaw across the edge of the table. Healthy rats generally try to put the paw ahead, into a position for walking. The other reflex was evaluated by placing the rat horizontally with its back on the table. Healthy rats perform an immediate and coordinated twisting of the body into an upright position. The pinna reflex and corneal reflex were also evaluated with a paper string.37 Each reflex was evoked by stimulation of the ear canal or the cornea with a paper string. Healthy rats spontaneously shake their heads or blink, respectively. Normal behavior was judged as present or absent.
Detection of Opioid Receptors Opioid receptor types (m, d, k) mRNA expression was measured in the dorsal spinal cord of na€ıve, formalininjected, and ginsenosides-delivered rats (n = 12) using reverse transcriptase polymerase chain reaction (RT-PCR). Ginsenosides (300 mg) were administered intrathecally 10 minutes before the formalin test. At 5 and 35 minutes after formalin injection, rats were killed by decapitation and the spinal cord was quickly removed and stored at –80 C. The area of the spinal cord from L4 to L6 was dissected and total RNA in the spinal cord was isolated according to the manufacturer’s protocol for the RNeasy kit (Cat. No.74104, Qiagen, Hilden, Germany). Purified total RNA was quantified spectrophotometrically at A260. The isolated RNA (1 mg per sample) was reverse-transcribed using the Omniscript RT kit (Cat. No.205111, Qiagen), following the manufacturer’s instructions. Previously published
primer sets were used for the rat opioid receptor.4,24,33 The PCR conditions were standardized for each type of receptor by using PCR PreMix (Cat. No.K-2012, Bioneer, Daejon, Korea) containing 1PCR buffer, 1.5 mM MgCl2, 250 mM dNTPs, 20 pmol each of forward and reverse oligonucleotide primers coding for rat m (m-1: 50 -GAC CGT TTC CTG GCA CTT CT-30 , m-2: 50 -TAG GGC AAT GGA GCA GTT TCT-30 ), or d (d-1: 50 -ATC TTC ACG CTC ACC ATG AT-30 , d-2: 50 -CGG TCC TTC TCC TTG GAG CC-30 ), or k (k-1: 50 -GAA GTT GGT TTT GCC TGT GT-30 , k-2: 50 -TGA GTA GGG GCA GCA AGT GT-30 ), followed by 40 cycles of thermal cycling (45 sec at 94 C, 45 sec at 63 C, and 45 sec at 72 C) for m and 35 cycles (1 minute at 95 C, 1 minute at 55 C, and 1 minute at 72 C) for d and 40 cycles (30 seconds at 95 C, 30 seconds at 54 C, and 30 seconds at 72 C) for k. The final cycle was followed by a 5-minute extension step at 72 C before reducing the temperature to 4 C for storage. Amplification of rat b-actin (GenBank accession no. NM_031144) was used as an internal and loading control. The PCR products (10 ml per lane) were separated by gel electrophoresis in a 1.5% agarose gel containing .5 mg/mL ethidium bromide, observed under ultraviolet light and photographed. Densitometry was performed using Multi gauge V3.0 (Life-science, Fujifilm Global, Tokyo, Japan) and analysis software to determine the ratio between opioid receptor types (m, d, k) and b-actin.
Statistical Analyses Data are expressed as means 6 SEM. The time-response data are presented as the number of flinches. The doseresponse data are presented as a percentage of the control in each phase. The numbers of flinches were converted to a percentage of the control to calculate the ED50 values for each drug. Percentage of the control = [(Sum of phase 1 (2) count with drug)/(Sum of control phase 1 (2) count)] 100. The dose-response data were analyzed using 1-way analysis of variance (ANOVA) with the Scheffe post hoc test. The dose-response lines were fitted using least-squares linear regression, and the ED50 and its 95% confidence intervals were calculated using the method reported by Tallarida.32 The difference between theoretical ED50 and experimental ED50, and the antagonism of ginsenosides were analyzed using the t-test. P values < .05 were deemed to indicate statistical significance.
Results Behavioral Responses to Ginsenosides and Morphine The placing/stepping reflex, the righting reflex, and the pinna and corneal reflexes were present after intrathecal administration of ginsenosides and morphine at the highest doses used here.
Effects of Intrathecal Ginsenosides and Morphine on Formalin Stimulation The sum of the number of flinches in the saline and DMSO control groups did not differ from each other in
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each phase (saline: DMSO; 18 6 2:18 6 1 in phase 1, 152 6 11: 151 6 13 in phase 2). Fig 1 shows the time course of intrathecal ginsenosides and morphine, administered 10 minutes before the formalin injection. Intrathecal ginsenosides and morphine dose dependently inhibited the flinching response during phase 1 and phase 2 in the formalin test (Fig 2). The phase 1 ED50 values (95% confidence intervals) of ginsenosides and morphine were 117.5 (59.6–231.6) and 9.2 mg (5.1–16.5 mg), respectively. The ED50 values (95% confidence intervals) of ginsenosides and morphine for phase 2 were 108.6 (67.9–173.5) and 3 mg (1.6–5.8 mg), respectively.
Interactions Between Ginsenosides and Morphine Isobolographic analysis revealed a synergistic interaction after combined administration of ginsenosides and morphine during phase 1 and 2 in the formalin test (Fig 3). The experimental ED50 value was significantly lower than the theoretical ED50 value. Accordingly, the ED50 values (95% confidence intervals) of ginsenosides in the mixture of ginsenosides and morphine for phases 1 and 2 were 34.9 (20.1–60.7) and 19.6 mg (10.2–37.6 mg), respectively. The total fraction values for the mixture of
Figure 2. Dose-response curve of intrathecal ginsenosides and morphine for flinching in the formalin test. Data are presented as percentages of the control. Ginsenosides and morphine produced a dose-dependent inhibition of flinches in phase 1 (A) and phase 2 (B). Each line represents means 6 SEM of 6 to 7 rats. Compared to control, *P < .05, yP < .01, zP < .001. ginsenosides and morphine in phases 1 and 2 were .54 and .35, indicating a synergistic interaction.
Opioid Receptor Types and Ginsenosides The antinociceptive effect of intrathecal ginsenosides was antagonized by intrathecal naloxone (Fig 4). In addition, the antinociception of ginsenosides was antagonized by CTOP, naltrindole, and GNTI (Fig 4).
Detection of Opioid Receptors RT-PCR showed the presence of m, d, and k opioid receptor types mRNA in the na€ıve rat lumbar spinal cord. All 3 types of spinal opioid receptor mRNA levels measured at 5 and 35 minutes after formalin injection were not different from those of na€ıve rats. Additionally, intrathecal ginsenosides had no effect on the mRNA levels of all of spinal opioid receptors expressed in formalin-injected rats (Fig 5). Figure 1. Time effect curve of intrathecal ginsenosides (A) and morphine (B) for flinching in the formalin test. Each drug was administered 10 minutes before formalin injection. Formalin was injected at time 0. Data are presented as the number of flinches. Each line represents means 6 SEM of 6 to 7 rats.
Discussion Subcutaneous injection of formalin induces 2 different nociceptive states: acute nociception (phase 1), followed
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Figure 3. Isobologram for the interaction between intrathecal ginsenosides and morphine during phase 1 (A) and phase 2 (B) in the formalin test. The ED50 values for each agent are plotted on the x- and y-axes, respectively. Horizontal and vertical bars indicate confidence intervals. The straight line connecting each ED50 value is the theoretical additive line and the point on the line is the theoretical additive ED50. The experimental ED50 point was significantly different than the theoretical ED50 point, indicating a synergistic interaction. by a facilitated state (phase 2) over time. The phase 1 response results from the direct stimulation of nociceptors of the primary afferent, and corresponds to a high level of activity in the primary afferent. The phase 2 response seems to originate from activation of dorsal horn neurons by a continuous low level of activity in the primary afferents. Behaviorally, the phase 2 response appears to be prominent, with intensified pain. The merit of this formalin pain model is that investigators may sequentially assess the effects of various analgesic agents on these 2 pain types at once. The present study demonstrated that intrathecal ginsenosides and morphine reduce the flinching response during phases 1 and 2 in the formalin test. These observations are consistent with previous findings.21,35,38 Interestingly, the ED50 of ginsenosides in phase 1 was close to that of phase 2 in the formalin test. In contrast, phase 1 exhibited 3-fold higher ED50 than phase 2 with morphine. These findings suggest that the effectiveness of spinal ginsenosides was similar in acute pain and the facilitated state, while spinal morphine was more effective in the facilitated state than acute pain.
Interaction Between Ginsenosides and Morphine
Figure 4. Antagonistic effects of intrathecal naloxone (.3 mg), CTOP (15 mg), naltrindole (10 mg), and GNTI (50 mg) on the antinociceptive action of intrathecal ginsenosides (300 mg) during phase 1 (A) and phase 2 (B) in the formalin test. Naloxone, CTOP, naltrindole, and GNTI were given 10 minutes before ginsenoside administration, and the formalin test was done 10 minutes after ginsenoside delivery. Data are presented as percentages of the control. All compounds reversed the effect of ginsenosides in both phases. Each bar represents means 6 SEM of 5 to 6 rats. Compared to ginsenosides, *P < .01, yP < .001.
Furthermore, the ratio of phase 1 ED50 to phase 2 ED50 of ginsenosides was about 2 when ginsenosides were combined with morphine. These data suggest that the antinociception of ginsenosides seems to be more potentiated by morphine in the facilitated state than in acute pain. Ginsenosides are the main pharmacologically active molecules of ginseng. Various types of ginsenosides are found in ginseng depending on their chemical constitution.18 Chemically, ginsenosides have a 4-ring, steroidlike structure with sugar moieties attached and show properties similar to acetylcholine, adrenaline, histamine, and opioids,10 which suggests that the antinociceptive action of ginsenosides may involve similar mechanisms as these chemicals. Previous experiments have also suggested that the nicotinic acetylcholine receptor may be involved in the mechanism of action of ginsenosides.2,11,12,27,31 In addition, several lines of evidence show that ginsenosides inhibit voltage-dependent Ca21 channels in sensory neurons,16,19,20,25 suggesting that the modulation of Ca21 channels by ginsenosides could be
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Figure 5. The expression of opioid receptors in the spinal cord of rats. Data are shown from each experiment. RT-PCR analysis revealed m (234 bp, (A)), d (356 bp, (B)), k (276 bp, (C)) opioid receptors mRNA, and b-actin (500 bp, (D)) in na€ıve state. M, PCR marker; lane 1, na€ıve; lane 2, 5 minutes after formalin injection; lane 3, 35 minutes after formalin injection; lane 4, 5 minutes after formalin injection 1 ginsenosides; lane 5, 35 minutes after formalin injection 1 ginsenosides. Quantitative analysis indicated that neither formalin injection nor ginsenosides administration affected the levels of spinal opioid receptors mRNA.
the pharmacological basis of ginsenosides antinociception. On the other hand, neither the inhibitory effect on Ca21 currents nor the antinociception of ginsenosides were blocked by alpaha2, muscarinic, opioid, or GABA receptor antagonists.16,19,29 No specific binding of ginsenosides to alpha1, alpha2, or betaadrenoceptors, or serotonin, GABA, or muscarinic receptors has been detected.42 Thus, it seems unlikely that the effect of ginsenosides is mediated by those receptors. However, in the current study, the antinociception of ginsenosides was attenuated by naloxone. Furthermore, m, d, and k opioid receptor antagonists decreased the effect of ginsenosides. This interaction between ginsenosides and opioid receptors is supported by recent work that showed that the antinociceptive action of ginsenosides in a postoperative pain model was reversed by naloxone at the spinal level.28 Thus, these observations suggest that opioid receptors may be involved in the action of ginsenosides at the spinal level, and that m‑, d‑, and k-type opioid receptors may be involved in mediating the effects of ginsenosides. The ginsenosides used in this study were total ginseng saponins, which are a mixture of various types of individual ginsenoside. Thus, many different molecules may have contributed to the effects noted. Although m, d, and k opioid receptor antagonists all blocked the effects of ginsenosides in this study, it has not been determined whether it is
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a single molecular entity that is acting on all types of opioid receptor or different chemicals for each opioid receptor type. On the other hand, the results of this study indicated that formalin injection did not alter the levels of spinal m, d, and k opioid receptor types’ mRNA. Furthermore, intrathecal ginsenosides failed to change the mRNA levels of 3 opioid receptor types. These findings suggest that neither formalin-induced nociception nor ginsenosides affected directly the synthesis of spinal opioid receptors’ mRNA. Previous studies have shown that no alteration in m, d, and k opioid binding, and all 3 opioid receptor types’ mRNA was observed in the spinal cord after injection of carrageenan or complete Freund’s adjuvant, respectively.8,22 However, other studies demonstrated an increase, a decrease, or no change in spinal opioid receptors expression after carrageenan or complete Freund’s adjuvant injection.1,9,41 Thus, it could be assumed that all 3 spinal opioid receptor types are involved in the modulation of nociception, but their roles may be different according to a pathological state. Therefore, further research needs to investigate such discrepancy. In the present study, intrathecal morphine reduced the flinching response in both phases of the formalin test. Thus, morphine acts in the spinal cord, where presynaptic and, to a lesser extent, postsynaptic opioid receptors modulate nociceptive transmission.14 An isobolographic analysis revealed a synergistic interaction between intrathecal ginsenosides and morphine during phases 1 and 2 in the formalin test. These results indicate that combining ginsenosides with morphine reinforces the antinociceptive effect of each drug, in acute pain and the facilitated state evoked by formalin injection. The present study demonstrates for the first time an interaction between ginsenosides and morphine at the spinal level. Although the pharmacological interaction between the 2 kinds of drugs is too complex to characterize at this point, several explanations may be possible for the synergism. First, the drugs may interact by altering the kinetics of the other. One agent may alter the actions of the other agent at a receptor or channel. Thus, ginsenosides may affect opioid receptors or a related channel, such as a potassium channel, thereby increasing the antinociceptive effects of the mixture. Second, functional interactions may result from distinct drug effects at separate anatomical sites that may act independently as well as together to inhibit spinal nociceptive processing.26 It has been reported that ginsenosides and morphine possess both pre- and post-synaptic actions in the spinal cord.14,38 Thus, simultaneous engagement of pre- and post-synaptic mechanisms may augment the antinociceptive action produced by either drug acting at one site independently,30 leading to synergism. Spinal ginsenosides are not yet available clinically. However, in the future they may be used alone or in combination with other drugs to treat acute pain and the facilitated state, because the combination may result in a decreased dose of either drug or an increased maximum achievable effect.
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In conclusion, the present study shows that intrathecal ginsenosides and morphine attenuate acute pain and the facilitated state evoked by formalin injection. Ginsenosides interact with morphine in a synergistic manner in both pain states. Thus, a mixture
of ginsenosides and morphine may be another option in the management of pain. Finally, spinal m‑, d‑, and k‑type opioid receptors appear to be involved in the antinociceptive effects of intrathecal ginsenosides.
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25. Rhim H, Kim H, Lee DY, Oh TH, Nah SY: Ginseng and ginsenoside Rg3, a newly identified active ingredient of ginseng, modulate Ca21 channel currents in rat sensory neurons. Eur J Pharmacol 436:151-158, 2002
12. Lee JH, Jeong SM, Lee BH, Kim DH, Kim JI, Lee SM, Nah SY: Differential effect of bovine serum albumin on ginsenoside metabolite-induced inhibition of alpha3beta4 nicotinic acetylcholine receptor expressed in Xenopus oocytes. Arch Pharm Res 26:868-873, 2003
26. Roerig SC, Fujimoto JM: Multiplicative interaction between intrathecally and intracerebroventricularly administered mu opioid agonists but limited interactions between delta and kappa agonists for antinociception in mice. J Pharmacol Exp Ther 249:762-768, 1989
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27. Sala F, Mulet J, Choi S, Jung SY, Nah SY, Rhim H, Valor LM, Criado M, Sala S: Effects of ginsenoside Rg2 on human neuronal nicotinic acetylcholine receptors. J Pharmacol Exp Ther 301:1052-1059, 2002
35. Yoon MH, Bae HB, Choi JI, Kim SJ, Chung ST, Kim CM: Lack of reciprocity between opioid and 5-HT(3) receptors for antinociception in rat spinal cord. Pharmacology 77: 195-202, 2006
28. Shin DJ, Yoon MH, Lee HG, Kim WM, Park BY, Kim YO, Huang LJ, Cui JH: The effect of treatment with intrathecal ginsenosides in a rat model of postoperative pain. Korean J Pain 20:100-105, 2007
36. Yoon MH, Choi JI: Pharmacologic interaction between cannabinoid and either clonidine or neostigmine in the rat formalin test. Anesthesiology 99:701-707, 2003
29. Shin YH, Jung OM, Nah JJ, Nam KY, Kim CY, Nah SY: Ginsenosides that produce differential antinociception in mice. Gen Pharmacol 32:653-659, 1999 30. Solomon RE, Gebhart GF: Synergistic antinociceptive interactions among drugs administered to the spinal cord. Anesth Analg 78:1164-1172, 1994 31. Tachikawa E, Kudo K: Proof of the mysterious efficacy of ginseng: Basic and clinical trials: Suppression of adrenal medullary function in vitro by ginseng. J Pharmacol Sci 95: 140-144, 2004 32. Tallarida RJ: Drug Synergism and Dose-Effect Data Analysis. New York, NY, Chapman & Hall/CRC, 2000, pp 21-71 33. Tongjaroenbuangam W, Jongkamonwiwat N, Phansuwan-Pujito P, Casalotti SO, Forge A, Dodson H, Govitrapong P: Relationship of opioid receptors with GABAergic neurons in the rat inferior colliculus. Eur J Neurosci 24:1987-1994, 2006 34. Yaksh TL, Rudy TA: Chronic catheterization of the spinal subarachnoid space. Physiol Behav 17:1031-1036, 1976
37. Yoon MH, Choi JI, Jeong SW: Antinociception of intrathecal cholinesterase inhibitors and cholinergic receptors in rats. Acta Anaesthesiol Scand 47:1079-1084, 2003 38. Yoon SR, Nah JJ, Shin YH, Kim SK, Nam KY, Choi HS, Nah SY: Ginsenosides induce differential antinociception and inhibit substance P induced-nociceptive response in mice. Life Sci 62:PL 319-325, 1998 39. Yu SQ, Lundeberg T, Yu LC: Involvement of oxytocin in spinal antinociception in rats with inflammation. Brain Res 983:13-22, 2003 40. Zahn PK, Gysbers D, Brennan TJ: Effect of systemic and intrathecal morphine in a rat model of postoperative pain. Anesthesiology 86:1066-1077, 1997 41. Zaringhalam J, Manaheji H, Mghsoodi N, Farokhi B, Mirzaiee V: Spinal mu-opioid receptor expression and hyperalgesia with dexamethasone in chronic adjuvant-induced arthritis in rats. Clin Exp Pharmacol Physiol 35:1309-1315, 2008 42. Zhang JT, Qu ZW, Liu Y, Deng HL: Preliminary study on antiamnestic mechanism of ginsenoside Rg1 and Rb1. Chin Med J 103:932-938, 1990
Synergistic Interaction Between Intrathecal Ginsenosides and Morphine on Formalin-Induced Nociception in Rats Myung Ha Yoon,*,y,z Kwang Soo Kim,* Hyung Gon Lee,* Chang Mo Kim,* Woong Mo Kim,* Jeong Il Choi,* and Yeo Ok Kim* * Department of Anesthesiology and Pain Medicine, Chonnam National University, Medical School, Gwangju, Korea. y The Brain Korea 21 Project, Center for Biomedical Human Resources at Chonnam National University, Gwangju, Korea. z Chonnam National University Research Institute of Medical Sciences, Gwangju, Korea.
Abstract: We defined the nature of the pharmacological interaction between ginsenosides and morphine in a nociceptive state and clarified the role of the different types of opioid receptor in the effects of ginsenosides. An intrathecal catheter was placed in male Sprague-Dawley rats. Pain was induced by formalin injection into the hindpaw. Isobolographic analysis was used to evaluate drug interactions. Furthermore, a nonselective opioid receptor antagonist (naloxone), a m opioid receptor antagonist (CTOP), a d opioid receptor antagonist (naltrindole), and a k opioid receptor antagonist (GNTI) were given intrathecally to verify the involvement of the opioid receptors in the antinociceptive effects of ginsenosides. Both ginsenosides and morphine produced antinociceptive effects in the formalin test. Isobolographic analysis revealed a synergistic interaction after intrathecal delivery of the ginsenosides-morphine mix. Intrathecal CTOP, naltrindole, and GNTI reversed the antinociceptive effects of ginsenosides. RT-PCR indicated that opioid receptors’ mRNA was detected in spinal cord of na€ıve rats and the injection of formalin had no effect on the expression of opioid receptors’ mRNA. Taken together, our results indicate synergistic antinociception following intrathecal coadministration of a ginsenosides/morphine mix in the formalin test, and that m, d, and k opioid receptors are involved in the antinociceptive mechanism of ginsenosides. Perspective: This article concerns the antinociceptive activity of ginsenosides, which increases antinociception by morphine. Thus, a spinal combination of ginsenosides and morphine may be useful in the management of acute pain as well as facilitated state pain. ª 2011 by the American Pain Society Key words: Antinociception, drug interaction, ginsenosides, morphine, opioid receptors, spinal cord.
G
inseng, the root of Pannax ginseng C.A. Meyer, has long been used in Oriental traditional medicine to improve the weakened physical status brought on by stress or disease.13 Ginsenosides, ginseng saponins, are the major component responsible for the effects of ginseng and more than 20 different ginsenosides have been found todate.18 Therefore, many different kinds of ginsenosides may be involved in the effects of ginseng. Several lines of evidence suggest that ginsenosides are effective against various nociceptive states. In particular, Received November 15, 2010; Revised December 16, 2010; Accepted December 31, 2010. Address reprint requests to Myung Ha Yoon, Professor in Department of Anesthesiology and Pain Medicine, Chonnam National University, Medical School, 8 Hakdong, Donggu, Gwangju 501-757, Korea. E-mail: [email protected] 1526-5900/$36.00 ª 2011 by the American Pain Society doi:10.1016/j.jpain.2010.12.012
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intrathecal ginsenosides attenuate formalin-induced, substance P-induced, and capsaicin-induced pain behaviors in mice.3,17,38 These findings suggest that ginseng may play an important role in the modulation of nociception at the level of the spinal cord. According to several previous reports, Ca21 channels are the pharmacological sites of action of ginsenosides,19,20,25 and alpha2, muscarinic, opioid, and GABA receptors are not involved in ginsenoside activity.19,29 However, a recent study has shown that intrathecal ginsenosides are effective in treating postoperative pain simulated by paw incision in rats and that naloxone attenuates the antinociceptive effects of ginsenosides,28 indicating that opioid receptors may play an important role in the mechanism of action of ginsenosides at the spinal level. Thus, the involvement of opioid receptors in the actions of ginsenosides remains to be determined.
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Methods
a 30‑gauge needle. The formalin injection produces specific pain behavior, which is readily discriminated and characterized by rapid and brief withdrawal or flexing of the injected paw. This behavior has been called a flinching response. Such pain behavior was thus quantified by periodically counting the number of flinches of the injected paw after injection. The number of flinches was counted for 1-minute periods at 1 and 5 minutes and in 5-minute intervals from 10 to 60 minutes. Formalininduced flinching behavior is biphasic; the initial acute phase (0–9 minutes) is followed by a relatively short quiescent period, which is then followed by a prolonged tonic response (10–60 minutes). At the end of the experiment, the rats were sacrificed by sevoflurane overdose.
Animal Preparation
Experimental Protocol
The experimental protocol was approved by The Institutional Animal Care and Use Committee, Medical Science of Chonnam National University. Adult male Sprague-Dawley rats, weighing 250 to 300 g each, were used. The animals were housed 4 to a cage and kept in a vivarium, maintained at 22 C, with a 12/ 12‑hour alternating light/dark cycle, and were given food and water ad libitum. All test drugs were administered intrathecally. Thus, rats were implanted with an intrathecal catheter under sevoflurane anesthesia, as described previously.34 An 8.5-cm polyethylene-10 catheter was advanced caudally through an incision in the cisternal membrane to the thoracolumbar level of the spinal cord. The exterior portion of the catheter was secured at the skull by subcutaneous tunneling and closed with a 28-gauge wire. The skin was sutured with 3-0 silk. After catheter implantation, rats were housed individually. Only animals with no evidence of neurological deficit after catheter implantation were included in the study and they were housed individually. The behavioral study occurred 5 days after intrathecal catheterization.
Five days after intrathecal cannulation, the rats were placed in a restraint cylinder for the experiment. After acclimatization for 15 to 20 minutes, the rats were allocated to receive one of the experimental drugs. The same volume of the vehicle (saline or DMSO) was used as the control. Each animal was tested only once. All experimental tests were made with the investigator blinded as to treatment condition of each animal.
As is widely known, the antinociceptive effects induced by opiate drugs occur through their action at opioid receptors, located in the central nervous system, including the spinal cord.6 Thus, spinal opioid receptor agonists are effective and are widely used in the management of various types of pain.15,21,23,40 The aims of the present study were to investigate the effect of ginsenosides in rat formalin-induced nociception in the spinal cord, and to determine the characteristics of the drug interaction between ginsenosides and morphine. We also examined the involvement of opioid receptor types on the effect of ginsenosides at the spinal level.
Drugs The following drugs were used: ginsenosides, morphine sulfate (Sigma, St Louis, MO), CTOP (D-Phe-CysTyr-D-Trp-Orn-Yhr-NH2; Tocris Cookson, Bristol, UK), naloxone [(5a)-4,5-Epoxy-3,14-dihydro-17-(2-prophenyl) morphinan-6-one hydrochloride, Tocris] naltrindole [17-(Cyclopropylmethyl)-6,7-dehydro-4,5a-epoxy-3, 14dihydroxy-6,7-20 ,30 -indolomorphian hydrochloride; Tocris], and GNTI [50 -Guanidinyl-17-(cyclopropylmethyl)6,7-dehydro-4,5a-epoxy-3,14-dihydroxy- 6,7-20 ,30 -indolomorphian dihydrochloride; Tocris]. Ginsenosides were provided by the Korea Ginseng and Tobacco Research Institute (Daejon, Korea). The ginsenosides were dissolved in dimethylsulfoxide (DMSO), whereas all other drugs were dissolved in distilled water. Intrathecal administration of these agents was performed using a hand-driven, gear-operated syringe pump. All drugs were delivered in a volume of a 10-mL solution.
Nociceptive Test Antinociception was assessed using the formalin test.36 Briefly, 50 mL 5% formalin solution was injected subcutaneously into the plantar surface of a hind paw using
Effects of Ginsenosides and Morphine Rats received intrathecal saline and increasing doses of either ginsenosides (30, 100, 300 mg, n = 28) or morphine (1, 3, 10, 30 mg, n = 30) 10 min before formalin injection, and the effects of ginsenosides and morphine were examined. Each ED50 value (effective dose producing a 50% reduction in control formalin response) for the agents was calculated separately in 2 phases.
Drug Interaction To define the properties of the pharmacological interaction between ginsenosides and morphine in the formalin test, isobolographic analysis was used (n = 48).36 This method is based on comparing doses determined to be equally effective. Initially, each ED50 value was determined from the dose-response curves of the 2 agents alone. Then, a dose-response curve was obtained with concurrent delivery of the 2 drugs in a constant dose ratio, based on the ED50 values of the single agent. Thus, separate groups received: ginsenosides ED50 1 morphine ED50; (ginsenosides ED50 1 morphine ED50)/2; (ginsenosides ED50 1 morphine ED50)/4; and (ginsenosides ED50 1 morphine ED50)/8. From the dose-response curves of the combined drugs, the ED50 values of the mixture were calculated, and the dose combinations were used to plot the isobologram. The isobologram was constructed by plotting the ED50 values of the single agents on the x- and y-axes, respectively. The theoretical additive dose combination was calculated. From the variance of the total dose, the individual variances for the combined agents were obtained. In combination, a total fraction value was calculated to describe the magnitude of the interaction. Total fraction value = (ED50 of drug 1 combined with drug 2)/(ED50 for drug 1 given alone) 1 (ED50 of drug 2 combined with drug 1)/(ED50 for drug 2 given alone).
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Values close to 1 indicate an additive interaction, values >1 indicate an antagonistic interaction, and values <1 indicate a synergistic interaction. The formalin test was done 10 minutes after administration of mixtures of the 2 drugs.
Opioid Receptor Types and Ginsenosides To determine the contribution of opioid receptor types on the effect of ginsenosides, rats were pretreated with several opioid receptor antagonists. These antagonists were given intrathecally 10 minutes before the administration of intrathecal ginsenosides (300 mg) and the formalin test was done 10 minutes after ginsenosides delivery. Maximal doses of opioid receptor antagonists that did not affect the control formalin response or cause behavioral abnormalities were determined from pilot experiments and previous studies.5,7,39 These experiments were conducted in 2 phases. The opioid receptor antagonists used in this study were as follows: a nonselective opioid receptor antagonist (naloxone, .3 mg, n = 10), a m opioid receptor antagonist (CTOP, 15 mg, n = 11), a d opioid receptor antagonist (naltrindole, 10 mg, n = 11), and a k opioid receptor antagonist (GNTI, 50 mg, n = 10).
General Behavior To examine the behavioral changes due to ginsenosides and morphine, the highest dose of each drug was given intrathecally to additional rats (n = 10). Motor function was assessed by the placing/stepping reflex and the righting reflex.37 The first reflex was evoked by drawing the dorsum of each hindpaw across the edge of the table. Healthy rats generally try to put the paw ahead, into a position for walking. The other reflex was evaluated by placing the rat horizontally with its back on the table. Healthy rats perform an immediate and coordinated twisting of the body into an upright position. The pinna reflex and corneal reflex were also evaluated with a paper string.37 Each reflex was evoked by stimulation of the ear canal or the cornea with a paper string. Healthy rats spontaneously shake their heads or blink, respectively. Normal behavior was judged as present or absent.
Detection of Opioid Receptors Opioid receptor types (m, d, k) mRNA expression was measured in the dorsal spinal cord of na€ıve, formalininjected, and ginsenosides-delivered rats (n = 12) using reverse transcriptase polymerase chain reaction (RT-PCR). Ginsenosides (300 mg) were administered intrathecally 10 minutes before the formalin test. At 5 and 35 minutes after formalin injection, rats were killed by decapitation and the spinal cord was quickly removed and stored at –80 C. The area of the spinal cord from L4 to L6 was dissected and total RNA in the spinal cord was isolated according to the manufacturer’s protocol for the RNeasy kit (Cat. No.74104, Qiagen, Hilden, Germany). Purified total RNA was quantified spectrophotometrically at A260. The isolated RNA (1 mg per sample) was reverse-transcribed using the Omniscript RT kit (Cat. No.205111, Qiagen), following the manufacturer’s instructions. Previously published
primer sets were used for the rat opioid receptor.4,24,33 The PCR conditions were standardized for each type of receptor by using PCR PreMix (Cat. No.K-2012, Bioneer, Daejon, Korea) containing 1PCR buffer, 1.5 mM MgCl2, 250 mM dNTPs, 20 pmol each of forward and reverse oligonucleotide primers coding for rat m (m-1: 50 -GAC CGT TTC CTG GCA CTT CT-30 , m-2: 50 -TAG GGC AAT GGA GCA GTT TCT-30 ), or d (d-1: 50 -ATC TTC ACG CTC ACC ATG AT-30 , d-2: 50 -CGG TCC TTC TCC TTG GAG CC-30 ), or k (k-1: 50 -GAA GTT GGT TTT GCC TGT GT-30 , k-2: 50 -TGA GTA GGG GCA GCA AGT GT-30 ), followed by 40 cycles of thermal cycling (45 sec at 94 C, 45 sec at 63 C, and 45 sec at 72 C) for m and 35 cycles (1 minute at 95 C, 1 minute at 55 C, and 1 minute at 72 C) for d and 40 cycles (30 seconds at 95 C, 30 seconds at 54 C, and 30 seconds at 72 C) for k. The final cycle was followed by a 5-minute extension step at 72 C before reducing the temperature to 4 C for storage. Amplification of rat b-actin (GenBank accession no. NM_031144) was used as an internal and loading control. The PCR products (10 ml per lane) were separated by gel electrophoresis in a 1.5% agarose gel containing .5 mg/mL ethidium bromide, observed under ultraviolet light and photographed. Densitometry was performed using Multi gauge V3.0 (Life-science, Fujifilm Global, Tokyo, Japan) and analysis software to determine the ratio between opioid receptor types (m, d, k) and b-actin.
Statistical Analyses Data are expressed as means 6 SEM. The time-response data are presented as the number of flinches. The doseresponse data are presented as a percentage of the control in each phase. The numbers of flinches were converted to a percentage of the control to calculate the ED50 values for each drug. Percentage of the control = [(Sum of phase 1 (2) count with drug)/(Sum of control phase 1 (2) count)] 100. The dose-response data were analyzed using 1-way analysis of variance (ANOVA) with the Scheffe post hoc test. The dose-response lines were fitted using least-squares linear regression, and the ED50 and its 95% confidence intervals were calculated using the method reported by Tallarida.32 The difference between theoretical ED50 and experimental ED50, and the antagonism of ginsenosides were analyzed using the t-test. P values < .05 were deemed to indicate statistical significance.
Results Behavioral Responses to Ginsenosides and Morphine The placing/stepping reflex, the righting reflex, and the pinna and corneal reflexes were present after intrathecal administration of ginsenosides and morphine at the highest doses used here.
Effects of Intrathecal Ginsenosides and Morphine on Formalin Stimulation The sum of the number of flinches in the saline and DMSO control groups did not differ from each other in
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each phase (saline: DMSO; 18 6 2:18 6 1 in phase 1, 152 6 11: 151 6 13 in phase 2). Fig 1 shows the time course of intrathecal ginsenosides and morphine, administered 10 minutes before the formalin injection. Intrathecal ginsenosides and morphine dose dependently inhibited the flinching response during phase 1 and phase 2 in the formalin test (Fig 2). The phase 1 ED50 values (95% confidence intervals) of ginsenosides and morphine were 117.5 (59.6–231.6) and 9.2 mg (5.1–16.5 mg), respectively. The ED50 values (95% confidence intervals) of ginsenosides and morphine for phase 2 were 108.6 (67.9–173.5) and 3 mg (1.6–5.8 mg), respectively.
Interactions Between Ginsenosides and Morphine Isobolographic analysis revealed a synergistic interaction after combined administration of ginsenosides and morphine during phase 1 and 2 in the formalin test (Fig 3). The experimental ED50 value was significantly lower than the theoretical ED50 value. Accordingly, the ED50 values (95% confidence intervals) of ginsenosides in the mixture of ginsenosides and morphine for phases 1 and 2 were 34.9 (20.1–60.7) and 19.6 mg (10.2–37.6 mg), respectively. The total fraction values for the mixture of
Figure 2. Dose-response curve of intrathecal ginsenosides and morphine for flinching in the formalin test. Data are presented as percentages of the control. Ginsenosides and morphine produced a dose-dependent inhibition of flinches in phase 1 (A) and phase 2 (B). Each line represents means 6 SEM of 6 to 7 rats. Compared to control, *P < .05, yP < .01, zP < .001. ginsenosides and morphine in phases 1 and 2 were .54 and .35, indicating a synergistic interaction.
Opioid Receptor Types and Ginsenosides The antinociceptive effect of intrathecal ginsenosides was antagonized by intrathecal naloxone (Fig 4). In addition, the antinociception of ginsenosides was antagonized by CTOP, naltrindole, and GNTI (Fig 4).
Detection of Opioid Receptors RT-PCR showed the presence of m, d, and k opioid receptor types mRNA in the na€ıve rat lumbar spinal cord. All 3 types of spinal opioid receptor mRNA levels measured at 5 and 35 minutes after formalin injection were not different from those of na€ıve rats. Additionally, intrathecal ginsenosides had no effect on the mRNA levels of all of spinal opioid receptors expressed in formalin-injected rats (Fig 5). Figure 1. Time effect curve of intrathecal ginsenosides (A) and morphine (B) for flinching in the formalin test. Each drug was administered 10 minutes before formalin injection. Formalin was injected at time 0. Data are presented as the number of flinches. Each line represents means 6 SEM of 6 to 7 rats.
Discussion Subcutaneous injection of formalin induces 2 different nociceptive states: acute nociception (phase 1), followed
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Figure 3. Isobologram for the interaction between intrathecal ginsenosides and morphine during phase 1 (A) and phase 2 (B) in the formalin test. The ED50 values for each agent are plotted on the x- and y-axes, respectively. Horizontal and vertical bars indicate confidence intervals. The straight line connecting each ED50 value is the theoretical additive line and the point on the line is the theoretical additive ED50. The experimental ED50 point was significantly different than the theoretical ED50 point, indicating a synergistic interaction. by a facilitated state (phase 2) over time. The phase 1 response results from the direct stimulation of nociceptors of the primary afferent, and corresponds to a high level of activity in the primary afferent. The phase 2 response seems to originate from activation of dorsal horn neurons by a continuous low level of activity in the primary afferents. Behaviorally, the phase 2 response appears to be prominent, with intensified pain. The merit of this formalin pain model is that investigators may sequentially assess the effects of various analgesic agents on these 2 pain types at once. The present study demonstrated that intrathecal ginsenosides and morphine reduce the flinching response during phases 1 and 2 in the formalin test. These observations are consistent with previous findings.21,35,38 Interestingly, the ED50 of ginsenosides in phase 1 was close to that of phase 2 in the formalin test. In contrast, phase 1 exhibited 3-fold higher ED50 than phase 2 with morphine. These findings suggest that the effectiveness of spinal ginsenosides was similar in acute pain and the facilitated state, while spinal morphine was more effective in the facilitated state than acute pain.
Interaction Between Ginsenosides and Morphine
Figure 4. Antagonistic effects of intrathecal naloxone (.3 mg), CTOP (15 mg), naltrindole (10 mg), and GNTI (50 mg) on the antinociceptive action of intrathecal ginsenosides (300 mg) during phase 1 (A) and phase 2 (B) in the formalin test. Naloxone, CTOP, naltrindole, and GNTI were given 10 minutes before ginsenoside administration, and the formalin test was done 10 minutes after ginsenoside delivery. Data are presented as percentages of the control. All compounds reversed the effect of ginsenosides in both phases. Each bar represents means 6 SEM of 5 to 6 rats. Compared to ginsenosides, *P < .01, yP < .001.
Furthermore, the ratio of phase 1 ED50 to phase 2 ED50 of ginsenosides was about 2 when ginsenosides were combined with morphine. These data suggest that the antinociception of ginsenosides seems to be more potentiated by morphine in the facilitated state than in acute pain. Ginsenosides are the main pharmacologically active molecules of ginseng. Various types of ginsenosides are found in ginseng depending on their chemical constitution.18 Chemically, ginsenosides have a 4-ring, steroidlike structure with sugar moieties attached and show properties similar to acetylcholine, adrenaline, histamine, and opioids,10 which suggests that the antinociceptive action of ginsenosides may involve similar mechanisms as these chemicals. Previous experiments have also suggested that the nicotinic acetylcholine receptor may be involved in the mechanism of action of ginsenosides.2,11,12,27,31 In addition, several lines of evidence show that ginsenosides inhibit voltage-dependent Ca21 channels in sensory neurons,16,19,20,25 suggesting that the modulation of Ca21 channels by ginsenosides could be
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Figure 5. The expression of opioid receptors in the spinal cord of rats. Data are shown from each experiment. RT-PCR analysis revealed m (234 bp, (A)), d (356 bp, (B)), k (276 bp, (C)) opioid receptors mRNA, and b-actin (500 bp, (D)) in na€ıve state. M, PCR marker; lane 1, na€ıve; lane 2, 5 minutes after formalin injection; lane 3, 35 minutes after formalin injection; lane 4, 5 minutes after formalin injection 1 ginsenosides; lane 5, 35 minutes after formalin injection 1 ginsenosides. Quantitative analysis indicated that neither formalin injection nor ginsenosides administration affected the levels of spinal opioid receptors mRNA.
the pharmacological basis of ginsenosides antinociception. On the other hand, neither the inhibitory effect on Ca21 currents nor the antinociception of ginsenosides were blocked by alpaha2, muscarinic, opioid, or GABA receptor antagonists.16,19,29 No specific binding of ginsenosides to alpha1, alpha2, or betaadrenoceptors, or serotonin, GABA, or muscarinic receptors has been detected.42 Thus, it seems unlikely that the effect of ginsenosides is mediated by those receptors. However, in the current study, the antinociception of ginsenosides was attenuated by naloxone. Furthermore, m, d, and k opioid receptor antagonists decreased the effect of ginsenosides. This interaction between ginsenosides and opioid receptors is supported by recent work that showed that the antinociceptive action of ginsenosides in a postoperative pain model was reversed by naloxone at the spinal level.28 Thus, these observations suggest that opioid receptors may be involved in the action of ginsenosides at the spinal level, and that m‑, d‑, and k-type opioid receptors may be involved in mediating the effects of ginsenosides. The ginsenosides used in this study were total ginseng saponins, which are a mixture of various types of individual ginsenoside. Thus, many different molecules may have contributed to the effects noted. Although m, d, and k opioid receptor antagonists all blocked the effects of ginsenosides in this study, it has not been determined whether it is
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a single molecular entity that is acting on all types of opioid receptor or different chemicals for each opioid receptor type. On the other hand, the results of this study indicated that formalin injection did not alter the levels of spinal m, d, and k opioid receptor types’ mRNA. Furthermore, intrathecal ginsenosides failed to change the mRNA levels of 3 opioid receptor types. These findings suggest that neither formalin-induced nociception nor ginsenosides affected directly the synthesis of spinal opioid receptors’ mRNA. Previous studies have shown that no alteration in m, d, and k opioid binding, and all 3 opioid receptor types’ mRNA was observed in the spinal cord after injection of carrageenan or complete Freund’s adjuvant, respectively.8,22 However, other studies demonstrated an increase, a decrease, or no change in spinal opioid receptors expression after carrageenan or complete Freund’s adjuvant injection.1,9,41 Thus, it could be assumed that all 3 spinal opioid receptor types are involved in the modulation of nociception, but their roles may be different according to a pathological state. Therefore, further research needs to investigate such discrepancy. In the present study, intrathecal morphine reduced the flinching response in both phases of the formalin test. Thus, morphine acts in the spinal cord, where presynaptic and, to a lesser extent, postsynaptic opioid receptors modulate nociceptive transmission.14 An isobolographic analysis revealed a synergistic interaction between intrathecal ginsenosides and morphine during phases 1 and 2 in the formalin test. These results indicate that combining ginsenosides with morphine reinforces the antinociceptive effect of each drug, in acute pain and the facilitated state evoked by formalin injection. The present study demonstrates for the first time an interaction between ginsenosides and morphine at the spinal level. Although the pharmacological interaction between the 2 kinds of drugs is too complex to characterize at this point, several explanations may be possible for the synergism. First, the drugs may interact by altering the kinetics of the other. One agent may alter the actions of the other agent at a receptor or channel. Thus, ginsenosides may affect opioid receptors or a related channel, such as a potassium channel, thereby increasing the antinociceptive effects of the mixture. Second, functional interactions may result from distinct drug effects at separate anatomical sites that may act independently as well as together to inhibit spinal nociceptive processing.26 It has been reported that ginsenosides and morphine possess both pre- and post-synaptic actions in the spinal cord.14,38 Thus, simultaneous engagement of pre- and post-synaptic mechanisms may augment the antinociceptive action produced by either drug acting at one site independently,30 leading to synergism. Spinal ginsenosides are not yet available clinically. However, in the future they may be used alone or in combination with other drugs to treat acute pain and the facilitated state, because the combination may result in a decreased dose of either drug or an increased maximum achievable effect.
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In conclusion, the present study shows that intrathecal ginsenosides and morphine attenuate acute pain and the facilitated state evoked by formalin injection. Ginsenosides interact with morphine in a synergistic manner in both pain states. Thus, a mixture
of ginsenosides and morphine may be another option in the management of pain. Finally, spinal m‑, d‑, and k‑type opioid receptors appear to be involved in the antinociceptive effects of intrathecal ginsenosides.
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