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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

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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 et al. BMC Complement Altern Med.

Abstract

Background: 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.

Results: 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.

Conclusions: 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.

Keywords: Antisense oligodeoxynucleotides; Excitatory amino acid agonists; High-dose morphine; NMDA receptor; NR2B; Nociceptive behavior; Paeoniflorin.

Figures

Fig. 1
Fig. 1
Plant (a) medicinal part (b) and the active component (c) of Paeonia lactiflora Pallas
Fig. 2
Fig. 2
Schedule of drug treatment and experimental processes. (a) 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. (b) PF (100, 200, 500 nmol, i.c.v.) was administered 150min before intrarthecal 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. The time spnet on biting or scretching induced by EAA agonists was observed and recorded during the first 2 min. (c) PF (100, 200, 500 nmol, i.c.v.) were administered 15 min and 5 min intrathecal injection of morphine, respectively. Mice were intrathecally administered morphine (250 nmol). The onset and the total number of clonic seizure-like excitatory behavior were observed and recorded for 60 min
Fig. 3
Fig. 3
Effects of PF and MK-801 on formalin-induced licking and biting behavior in mice. a: represents early phase (0–5 min) and b represents late phase (10–30 min) of formalin-induced nociception. PF (100, 200, 500 nmol, i.c.v.) was administered 15 min before intraplantar injection of 20 μl 1 % formalin. MK-801 (pmol, i.t.) was injected 5 min before intraplantar formalin injection. The control group received ACSF. Total time of each mouse spent on licking and biting of the injected paw was recorded. Data represents mean ± S.E. (n = 12). *** p < 0.001 compared with the control group; ### p < 0.001 compared with the MK-801-treated group, respectively
Fig. 4
Fig. 4
Effects of PF, MK-801, and NBQX on EAA agonist-induced biting and scratching behavior in mice. a: treatment with PF + MK-801 on glutamate-induced behavior; b: treatment with PF + MK-801 on NMDA-induced behavior; c: treatment with PF + MK-801 or PF + NBQX on AMPA-induced behavior. PF (100, 200, 500 nmol, i.c.v.) was administered 15 min before excitatory amino acid injection. MK-801 (MK 14.8 pmol, i.t.) and NBQX (5 nmol, i.t.) were administered 5 min before excitatory amino acid injection. The control group received ACSF. The time spent on biting or scratching induced by excitatory amino acids during the first 2 min was recorded. Data represent mean ± S.E. (n = 12). * p < 0.05, *** p < 0.001 compared with the control group; # p < 0.05, ## p < 0.01, ### p < 0.001 compared with the MK-treated group, respectively
Fig. 5
Fig. 5
Effects of PF and MK-801 on high dose morphine-induced clonic seizure- like excitation. PF (100, 200, or 500 nmol, i.c.v.) was administered 15 min before injection of morphine (250 nmol, i.t.). MK-801 (14.8 pmol, i.t.) was injected 5 min before excitatory amino acid injection. Control mice received ACSF. The onset time a and total numbers of clonic seizure-like excitation episodes b induced by morphine injection was recorded for 60 min. Data represent mean ± S.E. (n = 12). *** p < 0.001 compared with the control group; # p < 0.05, ### p < 0.001 compared with the MK-801 treated group, respectively
Fig. 6
Fig. 6
Effect of PF and antisense ODN of NMDA receptor subunits on high dose morphine-induced clonic seizure-like excitation in mice. Antisense ODN subunits: NR1, NR2A, NR2B, NR2C (15nM, i.c.v.) were administered 24 h before morphine (250 nmol, i.t.) administration. PF (100, 200, 500 nmol, i.c.v.) was administered 15 min before morphine administration. a: onset, b: total numbers of morphine-induced clonic seizure-like excitation during the first 60 min after morphine administration was recorded. Data were shown as mean ± S.E. (n = 12). * p < 0.05, *** p < 0.001 compared with the morphine group. # p < 0.05, ## P < 0.01, ### P < 0.001 compared with itself antisense group
Fig. 7
Fig. 7
Effects of PF and antisense ODN subunits of NMDA receptor on the NMDA-induced biting and scratching behavior in mice. a: The time course effect of antisense ODNs on 1, 3, 7 days. Antisense ODNs: NR1, NR2A, NR2B, NR2C (15nM, i.c.v.) were administered 1, 3, 7 days before NMDA (122 pmol, i.t.). b: The effects of 2B and 2B combined with PF on biting and scratching behavior in NMDA-treated mice. PF 100, 200, 500 nmol, i.c.v.) were administered 15 min before of NMDA. Antisense ODNs (15 nM, i.c.v.) were administered 1, 3, 7 days before NMDA administration. The time spent on biting or scratching behavior during the first 2 min after NMDA administration was recorded. Data are shown as mean ± S.E. (n = 12). * p < 0.05, ** p < 0.01, *** p < 0.001 compared with the NMDA group; # P < 0.05, ## P < 0.01, ### P < 0.001 compared with the antisense 2B group, respectively
Fig. 8
Fig. 8
Effects of PF and antisense ODN of NMDA receptor subunit, NR2B on the NMDA receptor in mouse cortex by immunohistochemical assay. Antisense ODN subunit of NMDA receptor, NR2B (15 nM, i.c.v.) was administered once daily for 1–7 days. PF (100, 200, 500 nmol, i.c.v.) was administered 15 min before mice were sacrificed. The arrow was showing the NR2B positive cells. a: Normal control; b-d): treated with NR2B Day 1–7; e-f: PF 100–500 nmol combined treatment with NR2B in 7 days; h-j: treated with PF 500 nmol Day 1 ~ 7. Scare bar: 30 μm. (400×)
Fig. 9
Fig. 9
Effects of PF and antisense ODN NR2B on NMDA receptor protein expression in mouse brain. Antisense ODN, NR2B (15 nmol, i.c.v.) was administered 1, 3, 7 days. PF (500 nmol, i.c.v.) was administered 15 min prior to antisense ODN, NR2B. Protein level of NR2B was analyzed by Western blotting. β-actin was used as a control of protein loading. * p < 0.05 compared with the control group, # p < 0.05 compared with the antisense NR2B group itself on different days
Fig. 10
Fig. 10
Effects of PF docked in the active sites of NMDA receptor subtypes. a: NR1 gaining −59.53 kcal/mol binding energy, which had one hydrogen bond with Glu406 (2.56 Å) and one with Thr518 (2.51 Å). b: NR2A gaining −128.49 kcal/mol binding energy, which had two hydrogen bonds with Lys484 (1.90 Å and 2.29 Å), one with Ser685 (2.32 Å) and one with Thr686 (2.35 Å). c: NR2B gaining −106.58 kcal/mol binding energy, which had one hydrogen bond with Lys485 (1.96 Å)
Fig. 11
Fig. 11
Paeoniflorin inhibits excitatory amino acid agonist- and high-dose morphine- induced nociceptive behavior in mice via modulation of NMDA receptor

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