antinociceptive actions of honokiol and magnolol on glutamatergic and inflammatory pain

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Open AccessResearchAntinociceptive actions of honokiol and magnolol on glutamatergic and inflammatory painYi-Ruu Lin1, Hwei-Hsien Chen1, Yu-Chin Lin2, Chien-Hsin Ko3 and Ming-Huan Chan*1

Address: 1Institute of Pharmacology and Toxicology, Tzu Chi University, Hualien, Taiwan, 2Department of Biotechnology, Transworld Institute of Technology, Yunlin, Taiwan and 3Department of Chinese Medicine, Buddhist Tzu Chi General Hospital, Hualien, Taiwan

Email: Yi-Ruu Lin - [email protected]; Hwei-Hsien Chen - [email protected]; Yu-Chin Lin - [email protected]; Chien-Hsin Ko - [email protected]; Ming-Huan Chan* - [email protected]

* Corresponding author

AbstractThe antinociceptive effects of honokiol and magnolol, two major bioactive constituents of the barkof Magnolia officinalis, were investigated on animal paw licking responses and thermal hyperalgesiainduced by glutamate receptor agonists including glutamate, N-methyl-D-aspartate (NMDA), andmetabotropic glutamate 5 receptor (mGluR5) activator (RS)-2-chloro-5-hydroxyphenylglycine(CHPG), as well as inflammatory mediators such as substance P and prostaglandin E2 (PGE2) inmice. The actions of honokiol and magnolol on glutamate-induced c-Fos expression in the spinalcord dorsal horn were also examined. Our data showed that honokiol and magnolol blockedglutamate-, substance P- and PGE2-induced inflammatory pain with similar potency and efficacy.Consistently, honokiol and magnolol significantly decreased glutamate-induced c-Fos proteinexpression in superficial (I-II) laminae of the L4-L5 lumbar dorsal horn. However, honokiol wasmore selective than magnolol for inhibition of NMDA-induced licking behavioral and thermalhyperalgesia. In contrast, magnolol was more potent to block CHPG-mediated thermalhyperalgesia. These results demonstrate that honokiol and magnolol effectively decreased theinflammatory pain. Furthermore, their different potency on inhibition of nociception provoked byNMDA receptor and mGluR5 activation should be considered.

BackgroundIn inflammatory pain, the neurotransmitters (glutamateand serotonin), neuromodulators (substance P) andinflammatory mediators (bradykinin, histamine andprostaglandin) released from the primary afferents anddorsal horn neurons under tissue damage play an impor-tant role in eliciting the hyperactivity on nociceptivebehaviors [1-5]. A number of evidence has further sup-ported the development of central hyperexcitability andpersistent pain relating to the activation of N-methyl-D-

aspartate (NMDA) receptors [6]. The glutamatergic systemis well known to involve in the inflammatory hyperalge-sia, since NMDA and non-NMDA receptor antagonistsattenuate the nociceptive behaviors which are associatedwith inflammatory responses in various animal models[7]. Recently, we have observed that honokiol and mag-nolol, isolated from the bark of Magnolia officinals [8,9],possess the ability to block the glutamate receptor-medi-ated intracellular cation signals [10] and inhibit gluta-mate-induced cell damage [11]. According to the above

Published: 16 October 2009

Journal of Biomedical Science 2009, 16:94 doi:10.1186/1423-0127-16-94

Received: 1 April 2009Accepted: 16 October 2009

This article is available from: http://www.jbiomedsci.com/content/16/1/94

© 2009 Lin et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Journal of Biomedical Science 2009, 16:94 http://www.jbiomedsci.com/content/16/1/94


evidence, it is proposed that honokiol and magnolol mayhave the potent analgesic activity via blockade of gluta-mate receptors.

Magnolol has been reported to inhibit acetic acid-inducednociceptive response, and to reduce the hind-paw edemainduced by carrageenan, compound 48/80, and poly-myxin B in mice [12]. Our previous study also demon-strates that honokiol and magnolol block theinflammatory phase of the overt nociception induced byformalin. However, they do not produce analgesia in tail-flick, hot-plate paw-licking and the neurogenic phase offormalin-induced licking response [13]. Thus, we suggestthat honokiol and magnolol may not affect the thermalpain, but reduce the inflammatory pain. Therefore, theantinociceptive mechanisms of honokiol and magnololparticularly in inflammatory pain require further investi-gation.

In the present study, we evaluated the antinociceptiveactions of honokiol and magnolol on pain behaviorsinduced by glutamate receptor agonists including gluta-mate, NMDA, and mGluR5 agonist (RS)-2-chloro-5-hydroxyphenylglycine (CHPG), and inflammatory medi-ators such as substance P and prostaglandin E2 (PGE2),and also characterized their effects on glutamate-inducedc-Fos expression in the spinal cord. Our results showedthat honokiol and magnolol produced antinociceptiveeffects against glutamate-, NMDA-, CHPG-, substance P-and PGE2-induced inflammatory pain. Honokiol wasmore selective for inhibition of NMDA-induced lickingbehavioral and thermal hyperalgesia. Alternatively, mag-nolol was more potentially to block CHPG-mediated ther-mal hyperalgesia. Furthermore, honokiol and magololalso significantly decreased c-Fos expression induced byglutamate in the spinal cord.

Materials and methodsAnimalsMale NMRI mice (8-9 weeks, 33-39 g) were supplied fromthe Laboratory Animal Center of Tzu Chi University(Hualien, Taiwan) and housed 4-5 animals per cage in a12 hr light/dark cycle with free access to food and water.The experimental protocol was approved by Review Com-mittee of the Tzu Chi University for the use of animal.

Glutamate-induced nociceptive testInvestigation of excitatory amino acid (EAA), glutamate,involving in nociception has been described previously[14]. Briefly, a volume of 20 l of glutamate (300 g) wasintraplantarly (i.pl.) injected into the ventral surface ofthe hind paw. Animals were observed individually for 30min following glutamate injection. The amount of timespent licking the injected paw was recorded with a chro-nometer and was considered as indicative of nociception.

For examining the antinociceptive effects, honokiol andmagnolol (5 and 10 mg/kg, i.p.) were applied to animals20 min before glutamate injection (i.pl.). Control animalsreceived a similar volume of vehicle (corn oil, 10 ml/kg).

NMDA- and PGE2-induced nociceptive testNMDA (150 g) or PGE2 (0.5 g) were intraplantarly(i.pl.) injected into the ventral surface of the hind paw.Animals were observed individually for 5 min followingNMDA or PGE2 injection. The amount of time spent lick-ing the injected paw was recorded with a chronometerand was considered as indicative of nociception. Animalswere pretreated with vehicle, honokiol and magnolol (0.1to 1.0 g/paw, i.pl.) 15 min before NMDA or PGE2 injec-tion.

Measurement of thermal hyperalgesiaThermal hypersensitivity was determined by animal hindpaw withdrawal response in hot water (47.0 ± 0.1°C). Inorder to calm down the animal's anxiety, each mouse waswrapped in a sock before any testing. Then the hind pawwas immersed into a water bath (47.0 ± 0.1°C) to meas-ure the hind paw withdrawal latency. The initial paw-withdrawal latency (T0) was measured before any nocice-ptive chemical treatment and the cut-off time was limitedto 10 sec in order to minimize tissue damage. The nocice-ptive chemicals such as glutamate, NMDA, CHPG, PGE2or substance P were given as an intraplantar injection intoone hind paw, which was then immersed into hot waterfor the observation of paw withdrawal latency (T1) at 5,15, 30, 60 and 120 min after chemical injection. For ameasurement of antinociceptive properties, honokiol andmagnolol were intraperitoneally (5 or 10 mg/kg) injected20 min before glutamate injection, or intraplantarly (0.05to 1.0 g/paw) treated 15 min before NMDA, CHPG,PGE2 or substance P injection. Control animals receivedan injection of an appropriate volume of vehicle. Datawere expressed as a mean value of paw-withdrawallatency which was calculated as T0 - T1.

c-Fos immunohistochemistryMice were pretreated with vehicle (corn oil), honokiol ormagnolol (10 mg/kg, i.p.) 20 minutes before injection ofsaline or glutamate into the plantar surface. Ninety min-utes after injection of saline or glutamate, mice weredeeply anaesthetized with thiopental (50 mg/kg, i.p.),then perfused transcardially with heparin solution (1000USP/ml in 0.1 M PBS), and subsequently perfused with100 ml of cold fixative (4% paraformaldehyde in 0.1 MPBS). The L4-L5 segments of spinal cord were removed,postfixed, and cryoprotected with 30% sucrose in 0.1 MPBS. Spinal cord tissues were then frozen and cut with acryostat in frontal sections (50 m thickness) which werecollected in 0.1 M PBS for immunohistochemistry asdescribed previously [15]. Rabbit primary antibody (Ab)



for c-Fos (Santa Cruz, 1:5000) was used to identify thenumber of c-Fos labeled neurons of laminas I-II in ran-domly selected sections (4-5 sections per mouse).

c-Fos immunoreactivity was observed in the cell nucleiand shown as a brownish round dot in the spinal cord sec-tions under a light microscope. Quantification of Fos pos-itive nuclei were counted with the aid of a Nikon Eclipse800 microscope, equipped with a Polaroid DMC digitalcamera (1600 × 1200 dpi in 8 bits) with 100× magnifica-tion using the Image Pro Plus 4.5 morphometry program(Media Cybernetics, Silver, MD). Following backgroundsubtraction, the threshold was adjusted so that pale- anddeep-stained nuclei could be equally recognized by thecounting program. Cell counts were made with the help ofthe Image Pro Plus 4.5 software and manually counted byan observer who was blind to the group assignment. Thenumber of c-Fos-stained nuclei was counted in four sec-tions per animal in superficial dorsal horn (laminae I andII) of the L4-L5 lumbar dorsal horn ipsilateral to the for-malin-injected site.

MaterialsHonokiol and magnolol were purchased from NacalaiTesque (Kyoto, Japan) and dissolved in corn oil as a stocksolution. c-Fos antibody was purchased from Santa CruzBiotechnology (Delaware Avenue, CA, USA). CHPG,metabotropic glutamate 5 receptor (mGluR5) agonist,was purchased from Tocris (Northpoint Fourth WayAvonmouth, UK). Other chemicals were obtained fromSigma (St. Louis, MO, USA).

Data analysisMost data are expressed as means ± S.E.M. Statistical sig-nificance of difference between groups was determined bytwo way repeated measures ANOVA, two-way ANOVA orone-way ANOVA followed by a Student-Newman-Keulspost-hoc test. A P value of less than 0.05 was consideredstatistically significant.

ResultsEffects of honokiol and magnolol on nociceptive responses and thermal hyperalgesia induced by glutamate receptor agonistsTo test whether the antinociceptive actions of honokioland magnolol are involved in glutamatergic regulation,we assessed the effects of honokiol and magnolol onglutamate and NMDA induced licking response and ther-mal hyperalgesia in mice. As shown in Figure 1, honokioland magnolol applied by intraperitoneal injection (i.p.)at 10 mg/kg significantly inhibited the glutamate-inducedcumulative licking time and thermal hyperalgesia. Moreo-ver, intraplantar application (i.pl.) of honokiol and mag-nolol (0.1-1.0 g/paw) also concentration-dependentlyreduced the total licking time and thermal hyperalgesia

induced by NMDA [(F(3,144) = 27.17, P < 0.001) andF(3,136) = 3.875, P = 0.017, respectively] (Fig. 2). How-ever, magnolol at lower dose (0.1 g/paw) did not signif-icantly alter NMDA-induced nociceptive responses andthermal hyperalgesia (Fig. 2B and 2D).

The following study was undertaken to investigatewhether the analgesia elicited by honokiol and magnololis associated with the inhibition of metabotropic gluta-mate receptor (mGluR) activity. CHPG (1 g/paw), aselective mGlu5 receptor agonist, was observed to inducea significant increase in the latency of thermal hyperalge-sia. However, the thermal hyperalgesic effects of CHPGwere inhibited by honokiol (0.1-1 g/paw) and magnolol(0.05-0.5 g/paw). Importantly, magnolol at lower does(0.05 g/paw) could reduce CHPG-induced thermalhyperalgisia (Fig. 3).

Effects of honokiol and magnolol on inflammatory mediator-induced nociceptive responses and thermal hyperalgesiaThe intraplantar injection of substance P (0.5 g/paw)and PGE2 (0.5 g/paw) significantly decreased the pawwithdrawal latency, which was gradually disappeared 2 hafter injection (Fig. 4 and Fig. 5C, 5D). The thermal hyper-algesia induced by substance P was dose-dependentlyinhibited by honokiol (0.5-1.0 g/paw, i.pl.) and mag-nolol (0.5-1.0 g/paw, i.pl.) [F(2, 109) = 9.04, P = 0.002and F(2, 92) = 42.5, P < 0.001, respectively] (Fig. 4).

Furthermore, honokiol (0.5-1.0 g/paw, i.pl.) and mag-nolol (0.5-1.0 g/paw, i.pl.) inhibited the nociceptivebehaviors and thermal hyperalgesia [F(2, 84) = 1.182, P <0.001 and F(2, 84) = 19.6, P < 0.001, respectively]induced by intraplantar injection of PGE2 (Fig. 5).

Effect of honokiol and magnolol on glutamate-induced c-Fos expressionThe highest counts of c-Fos-positive cells were observed atsuperficial (I-II) laminae of the L4-L5 lumbar dorsal hornipsilateral to the glutamate-injected site (Fig. 6B). In con-trast, there were little or no c-Fos-positive cells in the con-tralateral spinal cord of glutamate injection or ipsilateralof saline-injected site (Fig. 6A). Honokiol (10 mg/kg) andmagnolol (10 mg/kg) significantly decreased the gluta-mate-induced c-Fos protein expression in superficial spi-nal cord dorsal horn (Fig. 6).

DiscussionOur previous report has indicated that honokiol and mag-nolol possess the ability to inhibit formalin-inducedinflammatory nociception [13]. The present results fur-ther demonstrated that honokiol and magnolol attenu-ated glutamate-, NMDA-, mGluR5 agonist CHPG-,substance P- and PGE2-induced nociceptive responses.



The inhibitory actions of honokiol on NMDA-inducedlicking behavioral and thermal hyperalgesia were morepotent than magnolol. In contrast, magnolol had higherpotency than honokiol to block CHPG-mediated thermalhyperalgesia. Importantly, both honokiol and magnololsignificantly decreased glutamate-induced c-Fos proteinexpression in superficial (I-II) laminae of the L4-L5 lum-bar dorsal horn. Taken together, these results indicatedthat the analgesic actions of honokiol and magnolol maybe related to the blockade of glutamate receptor, NMDAreceptor and mGlu5 receptor activation, and inflamma-tion-mediated nociception.

Accumulating evidence has demonstrated that the excita-tory amino acid glutamate and the glutamatergic recep-

tors, both ionotropic and metabotropic glutamatereceptors (iGluR and mGluRs), are critically contributedto nociceptive neurotransmission under the developmentand maintenance of pain responsiveness [16-19]. Inresponse to noxious stimuli or tissue injury, excitatoryamino acids are released in peripheral neurons or spinalcord [20,21]. Then activation of glutamate receptors, espe-cially NMDA receptors, is involved in the development ofspinal hyperexicitability and persistent pain transmission[6]. Consistently, NMDA receptor antagonists arereported to inhibit the spread of pain sensation and thehyperexcitability of spinal cord nociceptive neuronsinduced by C-fiber stimulation [22,23]. Furthermore,modulation of mGluRs can regulate peripheral nociceptorfunction. Importantly, activation of mGluR5 is contrib-

Effects of honokiol and magnolol on glutamate-induced nociceptive responses and thermal hyperalgesia in miceFigure 1Effects of honokiol and magnolol on glutamate-induced nociceptive responses and thermal hyperalgesia in mice. Honokiol (5 and 10 mg/kg), magnolol (5 and 10 mg/kg) and vehicle (corn oil) were intraperitoneally administered 20 min prior to glutamate (300 g) injection into hindpaw, and the total time spent licking the hindpaw was measured for 30 min after intraplantar injection of glutamate (A, B). The paw withdrawal latency in hot water at 47°C was measured during 5-120 min after intraplantar injection of glutamate (C, D). Each point represents the mean ± S.E.M. (n = 6-8). * indicates significantly dif-ferent from vehicle group, *p < 0.05.

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uted to the maintenance of peripheral nociceptive proc-esses which are associated with inflammatory pain butnot in physiological pain [24]. Peripheral injection ofmGluR5 antagonist, 2-methyl-6-(phenylethynyl)-pyrid-ine (MPEP), inhibits the inflammatory, but not the neu-rogenic phase of nociception induced by formalin [25].However, activation of group III mGluRs, mGluR4 andmGluR7, just evokes the antinociceptive action on neuro-pathic pain [26]. In this regard, the inhibitory effects ofhonokiol and magonol on inflammatory pain mediatedby mGluR5 activation were examined in this study. Hereour results demonstrated that honokiol and magnololreduced glutamate-, NMDA- and CHPG (mGluR5 ago-nist)-induced licking behavioral and thermal hyperalge-sia, suggesting that the antinociceptive actions of

honokiol and magnolol may be related to the inhibitionof glutamate receptors including both NMDA and mGlu5receptors.

It has been demonstrated that noxious stimuli-inducedrelease of glutamate from nociceptive afferents in spinalcord, leading to the activation of either iGluR or mGluRon the postsynapse [21]. Fos, a nuclear phosphoproteininduced by a mammalian c-Fos protooncogene, isregarded as a maker of spinal activated neurons after nox-ious stimulation, such as thermal, mechanical and chem-ical stimuli [27]. Because c-Fos protein also contributes tobehavioral hyperalgesia, the c-Fos expression in spinalcord after glutamate stimulation and the influences ofhonokiol and magnolol on glutamate-induced c-Fos

Effects of honokiol and magnolol on NMDA-induced nociceptive responses and thermal hyperalgesia in miceFigure 2Effects of honokiol and magnolol on NMDA-induced nociceptive responses and thermal hyperalgesia in mice. Honokiol (0.1-1.0 g/paw), magnolol (0.1-1.0 g/paw) and vehicle were intraplantarly administered 20 min prior to NMDA (150 g) injection into hindpaw, and the total time spent licking the hindpaw was measured for 5 min after intraplantar injec-tion of NMDA (A, B). The paw withdrawal latency in hot water at 47°C was measured during 5-120 min after intraplantar injection of NMDA (C, D). Each point represents the mean ± S.E.M. (n = 8-16). * indicates significantly different from vehicle group, *p < 0.05.

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Effects of honokiol and magnolol on CHPG-induced thermal hyperalgesia in miceFigure 3Effects of honokiol and magnolol on CHPG-induced thermal hyperalgesia in mice. Honokiol (0.1-1.0 g/paw), mag-nolol (0.05-0.5 g/paw) and vehicle were administered 20 min prior to CHPG (1 g) injection into hindpaw. The paw with-drawal latency in the 47°C water was measured during 5-120 min after intraplantar injection of CHPG. Each point represents the mean ± S.E.M. (n = 7-19). * indicates significantly different from vehicle group, *p < 0.05.

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Effects of honokiol and magnolol on substance P-induced thermal hyperalgesia in miceFigure 4Effects of honokiol and magnolol on substance P-induced thermal hyperalgesia in mice. Honokiol (0.5 or 1.0 g/paw), magnolol (0.5 or 1.0 g/paw) and vehicle were administered 20 min prior to substance-P (0.5 g) injection into hindpaw. The paw withdrawal latency in the 47°C water was measured during 5-120 min after intraplantar injection of substance P. Each point represents the mean ± S.E.M. (n = 7-9). * indicates significantly different from vehicle group, *p < 0.05.

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expression were determined in this study. Here our datashowed that honokiol and magnolol attenuated gluta-mate-induced c-Fos protein expression in superficial lam-inae of dorsal horn of the spinal cord. This inhibition onc-Fos expression was correlated to reduction in glutamate-induced nociceptive response and thermal hyperalgesia.The present finding is also in agreement with the inhibi-tory actions of NMDA and mGlu5 receptor antagonists onc-Fos expression in spinal neurons after noxious stimula-tion [28,29]. It is important to note that the antinocicep-tive potency of honokiol is higher than magnolol onNMDA-induced nociception, although their antinocicep-tive potency is similar to glutamate-induced nociceptionand c-Fos expression. These results are consistent to ourprevious observation for both compounds on blockade ofglutamate- and NMDA-induced increases in [Ca2+]i or

neurotoxicity in cerebellar granule cells [10,11]. In con-trast, the antinociceptive potency of magnolol is morespecific than honokiol on CHPG-induced thermal hyper-algesia. Therefore, our data further indicate that honokioland magnolol may have the differential analgesic actionson NMDA and mGlu5 receptors. However, their influenceon the activity of other subtypes of GluR is still unknownand required further study.

During an inflammatory event, nociception generation isa consequence of a complex interaction between anumber of inflammatory mediators, including prostag-landins (PG), in inflamed tissues and the spinal cord.Notably, PGE2 is the prostanoids most relevant for theinduction and maintenance of the inflammatory pain byeither increasing the primary nociceptor responses or

Effects of honokiol and magnolol on PGE2-induced nociceptive responses and thermal hyperalgesia in miceFigure 5Effects of honokiol and magnolol on PGE2-induced nociceptive responses and thermal hyperalgesia in mice. Honokiol (0.5 or 1.0 g/paw), magnolol (0.5 or 1.0 g/paw) and vehicle were administered 20 min prior to PGE2 (0.5 g) injec-tion into hindpaw, and the total time spent licking the hindpaw was measured for 5 min after intraplantar injection of PGE2 (A, B). The paw withdrawal latency in the 47°C water was measured during 5-120 min after intraplantar injection of PGE2 (C, D). Each point represents the mean ± S.E.M. (n = 7-8). * indicates significantly different from vehicle group, *p < 0.05.

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Effects of honokiol and magnolol on glutamate-induced c-Fos protein expression in the spinal dorsal horn of the side ipsilateral to the injectionFigure 6Effects of honokiol and magnolol on glutamate-induced c-Fos protein expression in the spinal dorsal horn of the side ipsilateral to the injection. Photomicrographs illustrating the four experimental groups are represented: corn oil i.p.+saline i.pl. (A), corn oil i.p.+glutamate i.pl. (B), honokiol (10 mg/kg) i.p.+glutamate i.pl. (C), and magnolol (10 mg/kg) i.p.+glutamate i.pl. (D). Summary data of glutamate-induced c-Fos protein expression after honokiol and magnolol treatment in superficial (I-II) spinal cord (E). Values are means ± S.E.M. (n = 4). # indicates significantly different from corn oil-saline group, #P < 0.05; * indicates significantly different from corn oil-glutamate group, *P < 0.05. Statistical analysis was determined by one-way ANOVA followed by a Student-Newman-Keuls post-hoc test. Scale bar = 100 m.

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changing the spinal nociceptive signaling [30]. The nocic-eptive behavior and mechanical allodynia caused by PGE2are attributed to the activation of G-protein-coupled EPreceptors located in intrinsic spinal neurons (EP2) or pri-mary afferent neurons (EP1, EP3, and EP3) and proteinkinase dependent mechanisms [31,32]. Prostaglandinsare also reported to enhance the release of glutamate andsubstance P from peripheral afferent fiber terminals,involving an increase in levels of cAMP and an increase inCa2+ and Na+ conductance [33]. Furthermore, PGE2-induced hyperalgesia is abolished in NMDA receptorknock-out mice [34]. The present data showed thathonokiol and magnolol not only inhibited the nocicep-tive response and thermal hyperlgesia induced by PGE2but also blocked these responses induced by NMDA andglutamate. Thus, the blockade of glutamate receptors byhonokiol and magnolol could contribute to their antino-ciception on PGE2-induced pain responses. However, theantinociceptive principles for interfering at the EP recep-tors or the following signaling transduction in PGE2-induced thermal hyperalgesia can not be excluded. Fur-ther study is required to figure out the analgesic mecha-nisms of honokiol and magnolol on PGE2-mediatednociceptive and inflammatory responses.

Upon noxious stimulation, tachykinins such as substanceP and neurokinin A that are released from primary affer-ent fibers and excite spinal cord dorsal horn neurons bindto neurokinin receptors. It is believed that substance Pinduced signaling pathway is involved in the inflamma-tory process and in the transmission of sensory nocicep-tive information [30,35,36]. Although the release ofsubstance P is facilitated by prostaglandins, substance Pand glutamate conversely increase prostaglandin release[31]. It was observed that honokiol and magnolol signifi-cantly reduced substance P-mediated nociceptive signals,suggesting that the multiple antinociceptive actions ofhonokiol and magnolol may be via interaction with sub-stance P-induced nociception and pain transmission inaddition to prostaglandins and glutamate-mediated noci-ceptive responses. However, their pharmacological mech-anisms still require corroboration in depth.

In conclusion, the present study demonstrates thathonokiol and magnolol produce the antinociceptionagainst glutamate-, NMDA-, CHPG-, PGE2- and substanceP-induced pain, and consistently decrease glutamate-induced c-Fos protein expression in superficial (I-II) lam-inae of the L4-L5 lumbar dorsal horn. It is noted thathonokiol preferentially modulates the NMDA receptor-mediated nociception, whereas magnolol is more potentthan honokiol to inhibit mGluR5-mediated response.Therefore, these results indicate that honokiol and mag-nolol may possess the therapeutic potential to treat theinflammatory pain and have the differential analgesic

mechanisms on NMDA receptor and mGluR5 activationunder nociceptive neurotransmission.

Competing interestsThe authors declare that they have no competing interests.

Authors' contributionsYRL carried out the nociceptive tests and immunohisto-chemical assay, measured the thermal hyperalgesia, anddrafted the manuscript. HHC participated in the design ofthe study and sequence alignment. YCL and CHK partici-pated in the coordination and sequence alignment. MHCconceived of the study and helped to draft the manuscript.All authors read and approved the final manuscript.

AcknowledgementsThis research was supported by a grant NSC 97-2320-B320-002-MY3 from the National Science Council, Taiwan.

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antinociceptive actions of honokiol and magnolol on glutamatergic and inflammatory pain

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