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Paeoniflorin inhibits excitatory amino acid agonist-and high-dose morphine-induced nociceptive behavior in mice via modulation of N-methyl-D-aspartate receptors
- Yuh-Fung Chen†1, 2Email author,
- Ming-Ming Lee†3,
- Hsun-Lang Fang1,
- Jhao-Guei Yang4,
- Yu-Chien Chen5 and
- Huei-Yann Tsai2
© The Author(s). 2016
Received: 19 December 2015
Accepted: 19 July 2016
Published: 26 July 2016
Pain, the most common reasons for physician consultation, is a major symptom in many medical conditions that can significantly interfere with a person’s life quality and general functioning. Almost all painkillers have its untoward effects. Therefore, seeking for a safe medication for pain relieve is notable nowadays. Paeonia lactiflora is a well-known traditional Chinese medicine. Paeoniflorin is an active component found in Paeonia lactiflora, which has been reported to inhibit formalin-induced nociceptive behavior in mice. Aims of this present study were to investigate effects of paeoniflorin on excitatory amino acid agonist- or high-dose morphine-induced nociceptive behaviors in mice.
Paeoniflorin (100, 200, 500 nmol, i.c.v.) alone and combined with glutamatergic antagonists (MK-801 14.8 pmol, or NBQX 5 nmol, i.t.) inhibited nociception. Those agents also inhibited the clonic seizure-like excitation induced by high-dose morphine (250 nmol, i.t) in mice. Antisense oligodeoxynucleotides of NMDA receptor subunits NR1, NR2A, NR2B significantly enhanced the inhibition of paeoniflorin on excitatory amino acid-and high-dose morphine-induced nociception. Docking energy data revealed that paeoniflorin had stronger binding activity in NR2A and NR2B than NR2C of NMDA receptors.
Results of this study indicate that paeoniflorin-induced inhibition of excitatory amino acid agonist- and high-dose morphine-induced nociceptive behaviors might be due to modulation of NMDA receptors, specifically the NR2B subunit.
Glutamate is an excitatory amino acid (EAA) and a crucial neurotransmitter involved in nociceptive signaling and pain modulation in the central nervous system [1–5]. Glutamate and glutamatergic receptors locate in the central and peripheral nervous systems [6, 7]. The excitation of glutamate is partially mediated through the ionotropic glutamate receptors (iGluRs) including N-methyl-D-aspartate (NMDA), α-amino-3-hydroxy-5-methylisoxazole-4- propionic acid (AMPA), and kainate receptors [8, 9]. NMDA receptors, among the iGluRs, have received the most attention for they mediate central and peripheral sensitization during pain states [10, 11]. Results obtained from animal behavioral studies suggest that NMDA receptor antagonists have antinociception effects [12–14]. Pretreatment with NMDA receptor antagonist effectively attenuates formalin-induced nociception in rats . Besides, experiments show that high doses of morphine produce hyperalgesia, allodynia and myoclonic seizures in mice and rats [16–18]. Morphine-induced hyperalgesia is a state of nociceptive sensitization caused by exposure to morphine . It has been reported that such effects are mediated by NMDA receptor (NMDAR) and can be reversed by an NMDA receptor antagonist but not by naltrexone .
NMDAR is composed of the NR1, NR2 (A, B, C, and D) and NR3 (A and B) subunits. Several reports are indicating that the NR2B subunit is involved in mediating pain [21–23] and learning. It is well-established that the NMDA receptor NR2B subunit is an important contributor to pain mechanisms. Identifying selective NR2B antagonistic drugs is a priority in pain management .
Paeoniflorin (PF) was purchased from Nacalai tesque (Kyoto, Japan). L-Glutamic acid hydrochloride (glutamate), N-methyl-D-aspartic acid (NMDA), (±)-α-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid hydrobromide (AMPA), (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d] cyclohepten-5,10-imine hydrogen maleate (MK-801), 2,3-Dioxo-6-nitro-1,2,3,4-tetrahydrobenzo [f] quinoxaline-7-sulfonamide disodium (NBQX) were purchased from Research Biochemical Incorporated (Natick, MA, USA). Anti-rabbit HRP-IgG was purchased from Abcam (Cambridge, UK). Morphine hydrochloride was purchased from Food and Drug Administration, Ministry of Health and Welfare (Taipei, Taiwan). Formalin was purchased from Merck (Darmstadt, Germany). Antisense ODN subunits: NR1, NR2A, NR2B, NR2C, and β-actin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Zoletil® was purchased from Virbac Laboratories (Carros, France). The anti-NR2B antibody was purchased from R & D Systems (Minneapolis, MN, USA).
Animals and treatment
Male ICR mice (18–25 g) were purchased from the National Laboratory Animal Center, Taipei, Taiwan. Mice were housed 5 per cage at a constant temperature (22 ± 1 °C) and relative humidity (60 %) under a regular light–dark schedule (light 7:00 AM to 7:00 PM) and with free access to food and water. Each animal was used only once. The experimental protocol was approved by Animal Care and Use Committee, China Medical University (permit number 102–224) and conducted in agreement with the Ethical guidelines for investigations of experimental pain in conscious animals . All drugs for intrathecal (i.t.) and intracerebroventricular (i.c.v.) administration were dissolved and diluted to appropriate concentrations in artificial CSF (ACSF), except that formalin solution was diluted with normal saline. Drug solutions were freshly prepared before the experiment and given in a volume of 5 μl in ACSF.
Drug administration schedule
Effects of PF on formalin-induced licking and biting behavior
Mice were allowed to acclimatize for 30 min before drug injection. Twenty μl of formalin (1 %) was intraplantar injected into the dorsal surface of the hind paw of the mouse using a 31 gauge needle. Immediately after a formalin injection, animals were individually placed in the observation chamber, and a mirror was arranged at a 45° angle under the cage to allow clear observation of the paws of the animals. PF (100, 200, 500 nmol, i.c.v.) was administered 15 min before intraplantar injection of 20 μl 1 % formalin. MK-801 (14.8 pmol, i.t.) was injected 5 min before intraplantar formalin injection (Fig. 2a). The total time (seconds) spent on licking and biting of the injected paw during periods of 0–5 min (early phase), and 10–30 min (late phase) were measured as indicators of nociceptive behavior [40, 41].
Effects of PF on EAA agonists-induced biting and scratching behavior
Intrathecal injections of EAA agonists, such as glutamate (500 nmol), NMDA (122 pmol) and AMPA (12.5 pmol), result in behavior characterized by caudally directed biting and scratching in mice [2, 37, 38, 42, 43]. PF (100, 200, 500 nmol, i.c.v.) was administered 15 min before intrathecal injection of EAA agonists (in 5 μl ACSF). MK-801 (14.8 pmol, i.t.) and NBQX (5 nmol) were injected 5 min before intrathecal injection of EAA agonists. After intrathecal injection of EAA agonists, each mouse was then placed in the observation chamber. The time spent on biting or scratching induced by EAA agonists was observed and recorded during the first 2 min (Fig. 2b).
Effects of PF on the high dose morphine-induced clonic seizure-like excitatory behavior in mice
PF (100, 200, 500 nmol, i.c.v.) and MK-801 (14.8 pmol, i.t.) were administered 15 min and 5 min before intrathecal injection of morphine, respectively. Mice were intrathecally administered morphine (250 nmol) and were immediately placed in an observation chamber. The onset and the total number of clonic seizure-like excitatory behavior were observed and recorded for 60 min . The of drug treatment schedule is shown in Fig. 2c.
Effects of PF and antisense ODN subunits on the high dose morphine-induced clonic seizure-like excitation behavior
Mice were administered various antisense ODNs: NR1, NR2A, NR2B, and NR2C (15nM dissolved in ACSF, i.c.v.) 24 h before injection of morphine (250 nmol, i.t.). Different concentrations of PF (100, 200, 500 nmol, i.c.v.) were administered 15 min before morphine administration. The onset and the total number of clonic seizure-like excitatory responses in mice were observed and recorded for 60 min.
Effects of PF and antisense ODN subunits on NMDA-induced biting and scratching behavior
Mice were administered various antisense ODNs: NR1, NR2A, NR2B, and NR2C  (15nM dissolved in 5 μl ACSF, i.c.v.) once a day for 1, 3 and 7 days. NMDA (122 pmol, i.t.) was administered. Different doses of PF (100, 200, 500 nmol) were used to investigate the effect of antisense ODN subunits on NMDA-induced biting and scratching behavior. The onset and the total number of biting and scratching behavior in mice were observed and recorded for 2 min.
Effects of PF and antisense ODN of NR2B subunit on NMDA receptor in brain cortex by immunohistochemical detection
Mice were deeply anesthetized by an intraperitoneal injection 50 mg/kg of zoletil® and sacrificed. Brain cortex was quickly removed and soaked in 4 % paraformaldehyde to dehydrate and fix for overnight to form a paraffin-embed tissue. Tissue was sliced into 5 μm thickness with a microtome. Brain slices were incubated with the anti-NMDA receptor subunit 2B (NR2B) antibody (R&D Systems, Minneapolis, MN, USA) overnight and immunohistochemical labeled using a NovoLink Polymer Detection System Kit (Leica Microsystems Inc., Newcastle Upon Tyne, UK). The positive NR2B staining cells within the cortex were detected and photographed using an inverted microscope as previously described .
Effects of PF and antisense of ODN NR2B on protein levels of NMDA Receptor in mouse brain by western blotting
Mice were anesthetized and sacrificed. Brain tissue of mouse was quickly removed on the ice. A 10 % homogenate was prepared in lysis buffer, centrifuged at 13,000 (rpm) for 15 min at 4 °C. Total protein was prepared with RIPA protein lysis buffer, and the concentration of protein was determined by the Bradford method using Bio-Rad protein assay dye reagent (Amresco, OH, USA). SDS-PAGE separated the cell lysates containing 30 μg of protein and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, MA, USA). Five percent non-fat milk in PBST buffer was used to block non-specific binding sites. The PVDF membranes were incubated overnight at 4 °C with specific primary antibodies for NR2B (R&D Systems, Minneapolis, MN, USA) and β-actin (Sigma-Aldrich, MO, USA). The membranes were then washed with PBST buffer and incubated with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, CA, USA). Immunoreactive proteins were detected using a Western Blotting Chemiluminescence Reagent Plus kit (Millipore, MA, USA) and developed on Kodak Bio-MAX light film (Eastman Kodak, Rochester, NY, USA) as previously described .
Effects of PF docked in the active sites of NMDA receptor subtypes
Each simulation and calculation was launched under Discovery Studio (Discovery Studio Modeling 2.0, Accelrys, San Diego, CA, USA). The receptor structure of NMDA including NR1, NR2A, and NR2B were obtained from protein data bank (PDB ID: 1Y1M, 2A5S, and 1FTK, respectively). Waters and ligands were removed from the crystal structures before the minimization of whole crystal structure and the docking of the ligand to the receptors. Paeoniflorin was prepared (ChemOffice 2006, Cambridge Scientific Computing, Cambridge, Massachusetts, USA), including sketch and minimization (MM2 force field) before docking procedure. After the comparable performance, the protocol of Dock Ligands (Ligandfit) was used to predict the binding mode of this compound, and the scoring functions of the docking results were calculated automatically.
Where E is energy. The lower binding free energy demonstrated, the better stability.
Results are expressed as mean ± standard error. Data were analyzed by one-way ANOVA followed by Dunnett’s test. P < 0.05 was considered significant.
Effects of PF and MK-801 on formalin-induced licking and biting behavior
Effects of PF Combined with MK-801 and NBQX on EAA agonists-induced biting and scratching behavior
Effects of PF with MK-801 on high dose morphine-induced clonic seizure-like excitatory behavior
Effects of PF and antisense ODN of NMDA receptor subunits on high dose morphine-induced clonic seizure-like excitation behavior
Effects of PF and antisense ODN subunits of NMDA receptor on NMDA-induced biting and scratching behavior
Effects of PF and antisense ODN NR2B subunit on NR2B-receptor abundance in mouse cortex
Effects of PF and antisense ODN NR2B subunit on protein expression of NMDA receptor
Effects of PF docked in the active sites of NMDA receptor subtypes
Paeoniflorin, an active component from Paeonia lactifloria, is converted to paeonimetabolines by human intestinal bacteria . Only very trace amount of paeoniflorin in rat hippocampus was detected by high-performance liquid chromatography after intravenous administration of Paeoniae Radix extract  Since the bioavailability for oral administration of paeoniflorin is low, the intracerebroventricular injection is applied in the present study to acquire high potency of paeoniflorin. Previously, it was reported that PF inhibited formalin-induced nociception in mice . It has been suggested that formalin-induced nociception could be blocked by NMDA antagonists [13, 49]. In the present study, we determined if PF could inhibit formalin-induced licking and biting behavior by acting on the NMDA receptor.
PF produced significant inhibition of both neurogenic (early phase) and inflammatory (late phase) pain responses caused by formalin injection in mice. Co-administration of PF increased the antinociceptive effect of MK-801 on formalin-induced nociception. The enhancement of PF on the inhibitory effects of MK-801 on formalin-induced licking behavior is significant both in early phase and in late phase.
However, PF did not affect 100 nmol trans-(±) - ACPD-induced nociceptive behavior (data not shown). Since trans-(±)-ACPD is an mGluRs agonist, effects of PF effects may be mediated primarily by acting on iGluRs but not mGluRs.
To provide more direct evidence concerning the interaction of PF with iGluRs, we determined if PF could diminish EAA agonist-induced nociceptive behavior in mice. PF attenuated biting and scratching behavior induced by glutamate, NMDA, and AMPA in a dose-dependent manner. Co-administration of PF augmented effects of MK-801 on reducing nociceptive behavior induced by the 3 EAA agonists. MK-801 is a NMDA receptor antagonist in the glutamate category involved with the central nervous system . Co-administration of PF and MK-801 revealed a synergistic inhibitory effect on formalin-induced licking behavior. The effect observed from the combination of PF and MK-801 might be due to their effect on NMDA receptor with different binding sites.
Our results indicate that the antinociception effects of PF are due in part to targeting the glutamatergic system, especially via interaction with iGluRs. Such conclusion is supported by evidence showing that PF produced significant attenuation of the biting and scratching behavior induced by NMDA, AMPA, and glutamate. Besides, the spinal NMDA receptors may be primarily involved in eliciting the licking and biting behavior that followed intrathecal injection of high-dose morphine.
PF not only delayed the onset but also decreased the total number of clonic seizure-like excitation induced by morphine, the antinociception caused by PF was due to an interaction with iGluRs, more specifically via an interaction with the NMDA receptor.
Impaired glutaminergic neurotransmission has been implicated in several neurological diseases such as acute stroke, trauma, epilepsy, schizophrenia, depression, chronic pain and opioid dependence . The excitatory effect of glutamate is thought to be mediated in part through NMDA receptors [3, 4, 10]. Substances which block NMDA receptors could have potential clinical use in pain management. However, antagonists that completely block NMDA receptors cause numerous side effects, such as ataxia, motor incoordination, memory impairment and psychotomimetic effects. Therefore, developing new substances that can inhibit excitation, and maintain the normal physiological function of NMDA receptors would be ideal candidates for the treatment of NMDA-induced diseases. Such agents might be predicted to be devoid of CNS side effects at doses producing potent antinociception at peripheral NMDA receptors . The antinociception effects of PF on amino acid agonists and high-dose morphine-induced nociceptive behavior are mediated by targeting iGluRs, especially modulation of the NMDA 2B receptor. The NMDA receptor family consists of NR1, NR2 (A, B, C, and D), and NR 3 (A and B) subunits, which determine the characteristics of native NMDA receptors. Among NMDA receptor subunits, the NR2B subunit for pain is particularly noteworthy. A drug that has NR2B selective antagonistic properties, may be effectively used for the treatment of chronic pain . In this study, it revealed that NR2B was significantly inhibited by PF and antisense ODN for NR2B. PF significantly increased the suppressive effects of the antisense oligodeoxynucleotide (ODN) subunits, NR1, NR2A, NR2B on EAA and high dose morphine-induced nociceptive behavior. PF targeting NR2A and NR2B subunits was confirmed in in silico experiments. Docking energy data revealed that paeoniflorin had stronger binding activity in NR2A and NR2B than NR2C of NMDA receptors. Notably, the result of docking procedure was corresponding to the experiment of an animal model.
Remarkably, no side effects such as ataxia, motor incoordination, memory impairment and psychomimetic effects were found in mice during the period of antisense ODNs of NMDAR subunits co-administered with PF. This observation reveals the superiority of PF.
ACSF, artificial CSF; AMPA, α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid; EAA, excitatory amino acid; GluRs, glutamate receptor; Glutamate, L-glutamic acid hydrochloride; i.c.v., intracerebroventrical injection; i.t., intrathecal injection; IACUC, institutional animal care and use committee; iGluRs, iontropic glutamate receptors; mGluRs, metabotropic glutamate receptors; MK-801, (5R,10S)-(+)-5-methyl- 10,11-dihydro-5H-dibenzo [a,d] cyclohepten-5,10-imine hydrogen maleate; NBQX, 2,3-Dioxo-6-nitro-1,2,3,4-tetrahydrobenzo [f] quinoxaline-7-sulfonamide disodium; NMDA, N-methyl-D-asparate; NMDAR, NMDA receptor; ODN, oligodeoxynucleotide; PF, paeoniflorin; PVDF, polyvinylidene fluoride
Authors would like to thank Professor Wei Wang, China Medical University for kindly photographed Paeonia lactiflora Pallas plant.
This study was supported by the Ministry of Science and Technology (NSC89-2320-B-039-045), the Department of Chinese Medicine and Pharmacy, Ministry of Health and Welfare (CCMP89-RD-035), and China Medical University (CMU101-ASIA-06), Taiwan, R.O.C.
Availability of data and materials
Data are contained within this article and the supporting materials can be obtained upon request via email to the corresponding author.
YFC and MML conceived for the study, participated in its design, and drafted the manuscript. HLF performed the animal studies and western blot analysis. JGY and YCC performed the molecular docking analysis. HYT helped to draw figures and revised the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
The experimental protocol was approved by the Institutional Animal Care and Use Committee of China Medical University (permit number 102–224). All experiments were conducted with the Ethical guidelines for investigations of experimental pain in conscious animals (Pain. 1983; 16 (2):109–10).
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