Wu-Tou Decoction Inhibits Chronic Inflammatory Pain in Mice: Participation of TRPV1 and TRPA1 Ion Channels

Hindawi Publishing Corporation BioMed Research International Volume 2015, Article ID 328707, 12 pages http://dx.doi.org/10.1155/2015/328707

Research Article Wu-Tou Decoction Inhibits Chronic Inflammatory Pain in Mice: Participation of TRPV1 and TRPA1 Ion Channels

Chao Wang,1 Chunfang Liu,1 Hongye Wan,1 Danhua Wang,1 Danni Sun,1 Tengfei Xu,2,3 Yue Yang,1 Yakun Qu,1 Ying Xu,1 Xianghong Jing,4 Junling Liu,4 Shuping Chen,4 Zhiqiang Liu,2 and Na Lin1

1 Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China 2National Center for Mass Spectrometry in Changchun & Jilin Province Key Laboratory for Traditional Chinese Medicine Chemistry and Mass Spectrometry & Chemical Biology Laboratory, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, China 3University of Chinese Academy of Sciences, Beijing 100049, China 4Institute Acupuncture & Moxibustion, China Academy of Chinese Medical Sciences, Beijing 100700, China

Correspondence should be addressed to Na Lin; [email protected]

Received 5 January 2015; Accepted 20 February 2015

Academic Editor: Akio Hiura

Copyright © 2015 Chao Wang et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Wu-tou decoction (WTD) is a classic traditional Chinese medicine formula and has been used effectively to treat joint diseases clinically. Previous reports indicated that WTD possesses anti-inflammatory activity; however, its actions on pain have not been clarified. Here, we investigated the antinociceptive activity of WTD in CFA-induced mice, and its possible mechanism ofthe action associated with transient receptor potential (TRP) ion channels was also explored. Our results showed that 1.58, 3.15, and 6.30 g/kg WTD significantly attenuated mechanical, cold, and heat hypersensitivities. Moreover, WTD effectively inhibited spontaneous nociceptive responses to intraplantar injections of capsaicin and cinnamaldehyde, respectively. WTD also effectively suppressed jumping and wet-dog-shake behaviors to intraperitoneal injection of icilin. Additionally, WTD significantly reduced protein expression of TRPV1 and TRPA1 in dorsal root ganglia and skins of injured paw. Collectively, our data demonstrate firstly that WTD exerts antinociceptive activity in inflammatory conditions by attenuating mechanical, cold, and heat hypersensitivities. This antinociceptive effect may result in part from inhibiting the activities of TRPV1, TRPA1, and TRPM8, and the suppression of TRPV1 and TRPA1 protein by WTD was also highly effective. These findings suggest that WTD might be an attractive and suitable therapeutic agent for the management of chronic inflammatory pain.

1. Introduction that members of the transient receptor potential (TRP) ion channel family in nociceptor peripheral terminals and Chronic inflammatory pain ensues when the normal process dorsal root ganglions (DRGs) are fundamental molecules of inflammation does not resolve, resulting in excess proin- that detect noxious stimuli and transduce a diverse range flammatory cytokines and chemoattractants that can eventu- of physical and chemical energy into action potentials in ally lead to peripheral and central sensitization [1]. Indeed, somatosensory nociceptors and contribute to the initiation hypersensitivities to heat, cold, and mechanical stimuli are and maintenance of hypersensitivity under inflammatory well-documented characteristic symptoms of inflammatory and neuropathic pain conditions [5]. Among them, TRPV1, and neuropathic pain both in the clinical setting and in TRPA1,andTRPM8ionchannelshaveattractedthemost experimental animal models [2, 3]. It can be either recurrent attentions. It has been shown that deleting or inhibiting the or persistent and is a serious developmental health concern activities of these ion channels is associated with reduced that can interfere significantly with daily functioning4 [ ]. inflammatory nociceptive responses5 [ –7], and inflamma- Over the past decade, flourishing research has revealed tory hypersensitivity also appears to be dependent on the 2 BioMed Research International expression of TRPs [8–10]. Thus, pain-evoking TRP channels experiments. Take 1 mL and dilute it 1000 times as the analyte emerge as promising therapeutic targets for a wide variety of sample with internal standards. The sample was filtered pain and inflammatory conditions [5–7]. through a 0.22 𝜇m filter membrane and was quantified Treatment of chronic inflammatory pain still remains a by UPLC-MS/MS analysis. Standards of ephedrine, berber- major challenge in clinical practice because of its heteroge- ine, paeoniflorin, liquiritin, glycyrrhizic acid, calycosin-7- neous etiology and the complex underlying pathophysiology glucoside, formononetin, dioscin, and liquiritigenin were mechanisms.Themostcommonlyusedanalgesics,suchas obtained from the Chinese Authenticating Institute of Mate- opioids, nonsteroidal anti-inflammatory drugs (NSAIDs), rial and Biological Products (Beijing, China). Standards and anticonvulsants (for neuropathic pain), produce serious of benzoylmesaconine, benzoylhypacoitine, aconitine, and side effects [11, 12]. Therefore, there is an urgent need to benzoylaconitine were purchased from Lan Yuan Biological develop novel and safer pain medicines to meet the needs of Technology Co., Ltd. (Shanghai, China). The purity of all patients. In this context, natural products that present fewer compounds was more than 98% (determined by HPLC). side effects emerge as interesting therapeutic resources for They were prepared at a proper concentration in methanol. the development of new drugs for the management of certain HPLC-grade acetonitrile and formic acid were obtained from chronic pain states. Fisher Scientific (Loughborough, UK). Ultrapure water was Wu-tou decoction (WTD), a classic Traditional Chi- prepared by the Milli-Q plus (Milford, MA, USA) water nese Medicine (TCM) formula from Chinese medical sage purification system. Zhongjing Zhang, has been recorded in Jin Gui Yao Lue The UPLC-MS/MS system used was a Waters Acquity- for more than 1,000 years. It was formulated according to Ultra Performance LC system (Waters, USA) coupled with TCM theory especially for the treatment of cold pattern of Xevo TQ MS spectrometer with electrospray ion source (ESI) arthromyodynia. Clinically, WTD has been used effectively (Waters, Manchester, UK). Chromatographic separation was to treat joint diseases. Previous studies have shown that WTD performed on a Waters Acquity UPLC BEH C18 column ∘ possess the anti-inflammatory effect by inhibiting proinflam- (1.7 𝜇m, 2.1 ∗ 50 mm) kept at 35 C. Acetonitrile (A) and 0.1% matory cytokines such as IL-1𝛽 and TNF-𝛼 and regulating aqueous formic acid (v/v) (B) were used as the mobile phase. TLR2/TRAF6/Faslg signal pathway in complete Freund’s The gradient elution with the flow rate of 0.3 mL/min was adjuvant- (CFA-) induced arthritis rats [13, 14]. However, it performed as follows: 10% A at 0–2 min, 10–15% A at 2–7 min, remains unclear whether WTD could inhibit inflammatory 15–30% A at 7–15 min, 30–39% A at 15–21 min, and 39% up to pain.Inthisstudy,weinvestigatedtheantinociceptiveactivity 100% A at 21–25 min. The sample injected volume was 5 𝜇L. ofWTDoncutaneousinflammationinducedbyCFAinmice, TheMSanalysiswascarriedoutbytheESIsourceinboth and its possible mechanism of the action associated with TRP positiveandnegativeionmode.Thesourcetemperaturewas ∘ ∘ ion channels was also explored. 110 C, and the desolvation gas temperature was 300 C. The flow rates of cone and desolvation gas were set at 30 L/h and 2. Materials and Methods 600 L/h, respectively. The voltages of capillary and extraction cone in positive ion mode were set at 2.5 kV and 5.0 V,respec- This study was supported by the Research Ethics Committee tively, and in negative ion mode, they were set at 2.0 KV and of China Academy of Chinese Medical Sciences, Beijing, 5.0 V, respectively. The multiple reaction monitoring (MRM) China (Permit number: 2013-61). All animals were treated in transitions for fourteen analytes as well as their respective accordance with the guidelines and regulations for the use cone voltages and collision energies are listed in Table 2. andcareofanimalsattheCenterforLaboratoryAnimalCare, China Academy of Chinese Medical Sciences. All efforts were 2.2. Analysis of Water Extract of WTD. The content of eleven made to demonstrate consistent effects of the drug treatments main components in WTD used in this trial was quantified by and minimize the suffering of animals. liquid chromatography-tandem mass spectrometry (UPLC- MS/MS), and the calibration curves, calibration ranges, and 2.1. Preparation of WTD. WTD is a mixture of five traditional content in 1 g WTD of these compounds are performed in drugs (Table 1). The drugs were purchased from Beijing Table 3. Huamiao Chinese Medicine Engineering Development Cen- ter (Beijing, China) and authenticated by Professor Shilin 2.3. Animals. MaleICRmice(30–35g)wereobtainedfrom Hu, China Academy of Chinese Medical Sciences. WTD was Laboratory Animal Center of Academy of Military Medical prepared according to our previous studies [14, 15]. All the Sciences, Beijing, China (License number: SCXK 2012-0004). They were kept in a temperature controlled environment components of WTD were dried and homogenized to fine ∘ powders; then the 84 g powdered materials were immersed in (22 ± 1 C), 55 ± 5% relative humidity with a 12 h : 12 h light- 840 mL deionized water for 1 h and then heated to refluxing dark cycle, and fed with standard chow, for at least one week for1.5h.Wateras8timesoftheabovetotalweightwas before any manipulations. Animals were habituated to the added for another 1.5 h refluxing after being filtered. The laboratory conditions for at least 1 h before testing and all filtered extraction solutions were combined and concentrated experiments were performed during the light phase of the ∘ to 50 mL and then kept at −20 Cforpreparation.Thefinal cycle. concentration is 1.68 g/mL (crude herbs) stock solution. This stock solution was diluted with distilled water to the proper 2.4. CFA-Induced Chronic Inflammatory Pain. ICRmicewere concentration before use in vivo and the following chemical divided separately into 6 groups randomly with the equal BioMed Research International 3

Table 1: Composition of WTD.

Botanical Pharmaceutical name Part used Traditional actions/uses Amount (g) source/family Glycyrrhiza Radix Glycyrrhiza uralensis Root and rhizome Supplements spleen, replenishes qi, harmonizes 9 Preparata (Leguminosae) (processed) all drugs Ephedra sinensis Ephedrae Herba Grass stem Resolves surface, regulates water metabolism 9 (Ephedrae) Paeoniae Radix Alba Paeonia lactiflora Pall. Root Treats gastrointestinal disorders, relieves pains 9 Mother root Expel wind and cold and eliminate dampness to Aconiti Radix Cocta Ranunculaceae 6 (processed) relieve pain Astragalus Astragali Radix membranaceus Dried root Supplements qi, increases yang, delivers water 9 (Leguminosae)

Table 2: Ion transitions and instrumental parameters for their LC-MS/MS quantification in MRM mode.

Analytes Ion mode Quantitation transition (𝑚/𝑧)Conevoltage(V)Collisionenergy(eV) Benzoylmesaconine + 590.33 > 77.02 52 78 Aconitine + 646.39 > 586.34 50 34 Benzoylhypacoitine + 574.32 > 542.35 46 34 Benzoylaconitine + 604.41 > 105.04 58 54 Hypaconitine + 616.36 > 556.31 42 32 Ephedrine + 166.07 > 133.05 14 20 Calycosin-7-glucoside + 447.11 > 285.06 18 18 ∗ Reserpine (IS) +609.33> 174.07 40 42 ∗ Berberine (IS) + 336.10 > 320.12 32 32 Glycyrrhizic acid − 821.65 > 351.08 48 28 Liquiritin − 417.22 > 255.03 24 20 Formononetin − 267.10 > 252.03 28 22 Liquiritigenin − 255.10 > 119.09 30 24 ∗ Dioscin (IS) − 867.67 > 721.66 58 32 ∗ IS: internal standard. number (𝑛=11): the normal control group (control), force to cause a slight bend in the filament. Positive responses the CFA-induced inflammatory pain model group (CFA), included an abrupt withdrawal of the hind paw or flinching the WTD (1.58, 3.15, and 6.30 g/kg, p.o., resp.) treated behaviour immediately following removal of the stimulus. A groups, and the ibuprofen (0.14 g/kg, p.o.) treated group. The median paw withdrawal threshold was determined using an dosage of ibuprofen used was equivalent conversion from adaptation of Dixon’s up-down method [19]. The 50% paw rats as previously reported [16–18]. Six groups of mice were withdrawal threshold was analyzed at 0, 1, 2, 4, and 24 h orally administrated by syringe feeding with distilled water and 3, 5, 7, 9, 11, 13, and 15 days after WTD administration, (20 mL/kg) or WTD or ibuprofen daily for 15 days 24 h after respectively. CFA injection, respectively. Mice received an intraplantar injection of 20 𝜇L of CFA (Sigma-Aldrich, St. Louis, MO) 2.4.2. Measurement of Cold Hypersensitivity. Thermal hyper- to the right hind paw. Control group received 20 𝜇Lof sensitivity to cold was evaluated as described previously [20]. vehicle (1% tween 80 plus saline) intraplantar injection. CFA Briefly, with the aid of syringe (1 mL), 20 𝜇L acetone was caused hind paw mechanical and thermal hypersensitivities, sprayed gently from a short distance to the middle of the evaluated 24 hours after CFA injection. plantar surface of hind paw. The amount of time that mice were licking and/or shaking the hind paw was recorded 2.4.1. Measurement of Mechanical Hypersensitivity. Mice during 5 min after acetone application and was used as an were acclimatized in individual clear boxes on wire-mesh index of nocifensive responsiveness for cold hypersensitivity. platform,toallowaccesstotheventralsurfaceofthehind The licking and/or shaking time was measured at 0, 1, 2,4, paws, and mechanical hypersensitivity was assessed by the 6, 8, and 10 h and 3, 5, 7, 9, 11, 13, and 15 days after WTD sensitivity to the application of von Frey hairs (Stoelting administration, respectively. Co., Chicago, USA). The von Frey filaments (0.008–4.0 g) were presented perpendicularly to the plantar surface of the 2.4.3. Measurement of the Heat Hypersensitivity. Thermal injected paw and held in this position for 5–8 s with enough hyperalgesia of the hind paw was assessed using the plantar 4 BioMed Research International

Table 3: Calibration curves, concentration ranges, and content of 11 main components in WTD.

2 Sample Calibration curves 𝑅 Dynamic ranges (ng/mL) Content in WTD (ug/g) Benzoylmesaconine 𝑦 = 0.100691 ∗𝑥+ 11.8616 0.9904 250–6250 22.6723 Aconitine 𝑦 =1.28789∗𝑥−0.0142279 0.9942 1–75 0.0114 Benzoylhypacoitine 𝑦 = 0.530931 ∗𝑥+ 1.13486 0.9974 8–240 0.3827 Benzoylaconitine 𝑦 = 0.00548907 ∗𝑥−0.101374 0.9974 125–5250 60.4727 Hypaconitine 𝑦 = 0.504712 ∗𝑥+ 0.769754 0.9929 5–220 0.1208 Ephedrine 𝑦 = 0.124394 ∗𝑥+ 2.0736 0.9971 125–4500 41.698 Calycosin-7-glucoside 𝑦 = 0.402752 ∗𝑥+ 2.43032 0.9938 27–675 0.9628 Glycyrrhizic acid 𝑦 =7.73147∗𝑥−231.514 0.9954 200–1800 97.7193 Liquiritin 𝑦 = 54.3054 ∗𝑥−34.0128 0.9957 20–1400 22.6723 Formononetin 𝑦 = 68.0618 ∗𝑥+ 107.86 0.9903 10–390 0.2514 Liquiritigenin 𝑦 =75.7498∗𝑥+ 107.016 0.9934 12–290 2.4887

test (Ugo Basile Srl, Comerio VA, Italy), according to what WTD antinociceptive effect, mice were submitted to a test is previously described [21]. Briefly, mice were acclimatized using icilin, a specific activator of TRPM8, as previously to an apparatus consisting of individual perspex boxes; an described [25]. Briefly, 12.5 mg/mL icilin was suspended in 1% infrared radiant heat (40 W) source was directed to middle Tween-80/distilled water and sonicated. Icilin was intraperi- part of the plantar surface of the hind paw. The cut-off time toneally administered at a concentration of 50 mg/kg. Mice wassetat20stopreventtissuedamage.Pawwithdrawal were orally pretreated with vehicle (20 mL/kg) or WTD latency(PWL)wasmeasuredat0,0.5,1,1.5,2,3,4,and6h (6.30 g/kg) 1 h prior to the injection of vehicle (10 mL/kg, and 3, 5, 7, 9, 11, 13, and 15 days after WTD administration, i.p.) or icilin (50 mg/kg, i.p.). Immediately after the icilin respectively, and three trials for each hind paw. The interval treatment, jumping and wet-dog-shakes (WDS) behaviors of PWL measurement was five minutes. were counted for 20 min.

2.5. Capsaicin-Induced Nociception. To evaluate the possible 2.8. Western Blot Analysis. Mice were sacrificed by cervical involvement of TRPV1 channel on WTD antinociceptive dislocation under ether anesthesia 15 days after the WTD effect, mice were submitted to a test using capsaicin, a and ibuprofen administration. Plantar surfaces of injured ∘ specific activator of TRPV1, as previously described [22, 23]. hindpawskinweredissectedandstoredat−80 Cfor Mice were pretreated with vehicle (1% Tween-80 plus saline, western blot analysis. The expression of TRPV1, TRPA1, 20 mL/kg, p.o.), AMG9810 (a selective TRPV1 antagonist, and TRPM8 in hind paw skins tissues was determined. The 30 mg/kg, i.p.), or WTD (6.30 g/kg, p.o.) 1 h (for p.o. adminis- western blot protocol and semiquantitative analysis were tration) or 0.5 h (for i.p. administration) prior to the injection carried out following the protocol of our previous study of 20 𝜇L capsaicin (2 𝜇g/paw) in the plantar surface of the [26]. The following primary antibodies were used: TRPV1 right hind paw, respectively. Immediately after the capsaicin (rabbit polyclonal antibody, dilution 1 : 100, Alomone Labs, injection, mice were placed into clear observation boxes and Israel), TRPA1 (rabbit polyclonal antibody, dilution 1 : 100, the nociceptive response was evaluated as the time of lifting Alomone Labs, Israel), TRPM8 (rabbit polyclonal antibody, and spent licking the injected paw during 5 min. dilution 1 : 100, Alomone Labs, Israel), and GAPDH (internal control, rabbit polyclonal antibody, dilution 1 : 1000, Bey- 2.6. Cinnamaldehyde-Induced Nociception. To evaluate the otime, China). A goat-anti-rabbit antibody conjugated to possible involvement of TRPA1 channel in WTD antinocicep- horseradish peroxidase (1 : 5000, Thermo Fisher Scientific, tive effect, mice were submitted to a test using cinnamalde- USA) was used as the second antibody. TRPV1, TRPA1, and hyde, a specific activator of TRPA1, as previously described TRPM8 protein levels were normalized against GAPDH. All [23, 24]. Mice were pretreated with vehicle (20 mL/kg, p.o.), experiments were done for four times. WTD (6.30 g/kg, p.o.), or camphor (a TRPA1 antagonist, 7.6 mg/kg, s.c.) 1 h (for p.o. administration) or 0.5 h (for s.c. 2.9. Immunohistochemical Staining. The immunohistochem- administration) prior to the injection of 20 𝜇L of cinnamalde- ical staining protocol was carried out following the protocol hyde (TRPA1 activator, 1.3 𝜇g/paw) in the plantar surface of of our previous study [26]. Primary antibodies: TRPV1 (rab- the right hind paw. Immediately after the cinnamaldehyde bit polyclonal antibody, 1 : 100, Alomone Labs, Israel), TRPA1 injection, mice were placed into clear observation boxes and (rabbit polyclonal antibody, 1 : 100, Alomone Labs, Israel), the nociceptive response was evaluated as the time of lifting and TRPM8 (rabbit polyclonal antibody, 1 : 100, Alomone and spent licking the injected paw during 5 min. Labs, Israel); and secondary antibody (goat-anti-rabbit 1 : 100, Boster, China) was used and stained with DAB (Dako, Den- 2.7. Icilin-Induced Jumping and Wet-Dog-Shake Behaviors. mark). The percentage of immunopositive cells was analyzed To evaluate the possible involvement of TRPM8 channel in by counting the number of immunopositive neurons and BioMed Research International 5

1.6 1.6 1.4 1.4 1.2 1.2 1.0 ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ 1.0 0.8 0.8 ∗∗ 0.6 ∗∗∗ ∗∗∗ 0.6 ∗∗∗ ∗ threshold (g) threshold 0.4 ∗∗∗ ∗∗∗ threshold (g) threshold 0.4 ∗∗ 0.2 0.2 withdrawal 50% paw 50% paw withdrawal 50% paw 0.0 0.0 0 12342435791112130123424 0 1 2 3 4 24 3 5 7 9 11 13 15 Hours posttreatment Repeated daily treatment Hours posttreatment Repeated daily No Hours posttreatment 1st day 1st day treatment WTD 14th day Control CFA administration WTD 1.58 g/kg WTD 3.15 g/kg Control WTD 6.30 g/kg WTD 6.30 g/kg Ibuprofen 0.14 g/kg CFA (a) (b) 42 40 # 38 ∗ 36 34 Body (g) weight 32 0481215 Time posttreatment (days)

Control CFA WTD 1.58 g/kg WTD 3.15 g/kg WTD 6.30 g/kg Ibuprofen 0.14 g/kg (c)

Figure 1: Effect of WTD on mechanical hypersensitivity induced by CFA in mice. (a) Effect of WTD on mechanical hypersensitivity. Mice were orally administrated with WTD (1.58, 3.15, and 6.30 g/kg, resp.), ibuprofen (0.14 g/kg), or water daily for 15 days. (b) WTD has no tolerance effect. CFA-induced inflammatory pain mice were administrated with WTD (6.30 g/kg, p.o.) daily for 11 days. Treatment ofWTD was interrupted for 2 days and then reestablished on 14th day. On 1st and 14th days, evaluations for mechanical hypersensitivity were done 0, 1, 2, 3, 4, and 24 hours posttreatment; all other evaluations were done 1 hour posttreatment. (c) Effect of WTD (1.58, 3.15, and 6.30 g/kg, resp., p.o.) and ibuprofen (0.14g/kg, p.o.) on body weights change 15 days posttreatment. Data are represented as the mean ± SEM (𝑛=11). # ∗ ∗∗ ∗∗∗ 𝑃 < 0.05 versus the control group; 𝑃 < 0.05, 𝑃 < 0.01,and 𝑃 < 0.001 versus the CFA group, respectively.

multiplying by 100/number of total number of neurons. A CFAinmice.AsshowninFigure 1(a), CFA caused significant total of 2 DRGs were measured in each animal, and the mechanical hypersensitivity characterized by the reduced number of immunoreactive neuronal profiles was counted in paw withdrawal threshold compared to control group. Oral a blinded fashion. The percentage of immunopositive cells administration of WTD (1.58, 3.15, and 6.30 g/kg) was able to was used for statistic index. significantly reverse mechanical hypersensitivity that lasted up to 3 hours in high dose group. The maximum effect was 2.10. Statistical Analysis. Data obtained from the mechan- observed 1 h posttreatment. And this antinociceptive effect ical allodynia and thermal hyperalgesia experiments were was maintained while WTD (1.58, 3.15, and 6.30 g/kg) was analyzed by repeated measures one-way analysis of variance orally administered daily, until the 15th day posttreatment. (ANOVA) followed by post hoc Dunnett’s Multiple Com- Oral administration of ibuprofen (0.14 g/kg) was able to parison Test. The data in immunohistochemical staining reduce mechanical hypersensitivity with a time-course effect experiments were analyzed using the Mann-Whitney test. A profile similar to 1.58 g/kg WTD administration. value of 𝑃 < 0.05 was taken to be significant. To investigate whether WTD has side effects on animals, tolerance experiment was first examined. Our data showed 3. Results that the prolonged treatment with orally administrated WTD (6.30 g/kg) once a day for 11 days significantly reduced the 3.1. WTD Attenuates CFA-Induced Mechanical Hypersensitiv- mechanical hypersensitivity induced by CFA (Figure 1(b)). ity in Mice. To investigate the knowledge on the antinocicep- When treatment was interrupted for 2 days, mechanical tive activity of WTD, a chronic inflammatory model of noci- hypersensitivity in WTD treated group was reestablished. ception was used. For this, mechanical hypersensitivity was On the 14th day the treatment was restarted, and WTD was evaluated from day 1 to day 15 after an intraplantar injection of able to reduce mechanical hypersensitivity with a time-course 6 BioMed Research International effectprofilesimilartothe1stday.Theseresultsexcludedthe 40 possibility of the development of tolerance effect of WTD. 35 Additionally, body weight of mice was also explored. The 30 results showed that WTD treatment for 15 days did not induce 25 ∗ ∗ ∗∗ significant body weight change compared with CFA group 20 ∗ and also with control group. Conversely, ibuprofen decreased 15 ∗∗∗ ∗∗∗ body weight with significance on the 15th day compared to 10 CFA group (Figure 1(c)). 5 Shaking and licking time (s) licking and Shaking 0 3.2. WTD Inhibits CFA-Induced Cold Hypersensitivity in Mice. 0 0.5 1 2 4 6 8 10 3 5 7 9 11 13 15 Hypersensitivity to cold stimulus was evaluated at 1st (0.5 h, Hours posttreatment 1st Repeated daily 1 h, 2 h, 4 h, 6 h, 8 h, and 10 h), 3rd, 5th, 7th, 9th, 11th, 13th, and day treatment Control CFA 15th day after CFA injection. As shown in Figure 2,micethat WTD 1.58 g/kg WTD 3.15 g/kg received oral administration of WTD (1.58, 3.15, or 6.30 g/kg) WTD 6.30 g/kg Ibuprofen 0.14 g/kg exhibited a reduction in cold hypersensitivity induced by CFA that began at 30 minutes post-WTD treatment and was main- Figure 2: Effect of WTD on cold hypersensitivity induced by CFA tained for more than 6 hours in high dose group. The most in mice. Mice were orally administrated with WTD (1.58, 3.15, and pronounced effect was observed 1 h after its administration, 6.30 g/kg, resp.), ibuprofen (0.14 g/kg), or water daily for 15 days. On 1st day, evaluations for cold hypersensitivity were done 0.5, 1, 2, 4, 6, when WTD (6.30 g/kg) inhibition reached 70 ± 9%. Also, 8, and 10 hours posttreatment; all other evaluations were done 1 hour the prolonged treatment with orally administrated WTD posttreatment. Data are represented as the mean ± SEM (𝑛=11). ∗ ∗∗∗ (1.58, 3.15, and 6.30 g/kg) once a day for 15 days significantly 𝑃 < 0.05 and 𝑃 < 0.001 versus the CFA group, respectively. reduced the cold hypersensitivity induced by CFA (peak on day 15 with inhibition of 97 ± 1% for high dose group). However, ibuprofen treatment did not decrease the licking and shaking time for nociceptive response, indicating no 15 obvious effect on cold hypersensitivity. 13 11 ∗∗∗ 3.3. WTD Reduces CFA-Induced Heat Hypersensitivity in 9 Mice. Hypersensitivity to heat stimulus was explored in CFA- 7 ∗∗∗ ∗∗ induced mice. The time-course effect of WTD treatment Latency time (s) 5 ∗ ∗ on heat hypersensitivity was similar to that on mechanical 3 and cold hypersensitivities. This antinociceptive effect began 1 30 minutes and lasted up to 2 hours after WTD treatment 0 0.5 1 1.5 2 3 4 6 3 5 7 9 11 13 15 (Figure 3). The maximum effect on 1st day was observed at Hours posttreatment 1st Repeated daily 1 h posttreatment, when WTD (6.30 g/kg) inhibition reached day treatment 73 ± 8%, and the inhibition on days 13 and 15 was about Control CFA 100%. Ibuprofen (0.14 g/kg) could significantly inhibit heat WTD 1.58 g/kg WTD 3.15 g/kg hypersensitivity and to the similar extent as 1.58 g/kg WTD WTD 6.30 g/kg Ibuprofen 0.14 g/kg treatment (Figure 3). Figure 3: Effect of WTD on heat hypersensitivity induced by CFA in mice. Mice were orally administrated with WTD (1.58, 3.15, and 3.4. WTD Suppresses the Activities of TRPs in Mice. To obtain 6.30 g/kg, resp.), ibuprofen (0.14 g/kg), or water daily for 15 days. On insights into the mechanisms of WTD mediated beneficial 1stday,evaluationsforheathypersensitivityweredone0.5,1,1.5,2,3, effects, the activities of TRPV1, TRPA1, and TRPM8 in mice 4, and 6 hours posttreatment; all other evaluations were done 1 hour were investigated. As shown in Figure 4(a), the TRPV1 acti- posttreatment. Data are represented as the mean ± SEM (𝑛=8). ∗ ∗∗ ∗∗∗ vator capsaicin induced obvious licking and lifting behaviors 𝑃 < 0.05, 𝑃 < 0.01,and 𝑃 < 0.001 versus the CFA group, characteristics of nociception in mice, which were signifi- respectively. cantly reduced by oral administration of WTD (6.30 g/kg). The inhibitions of WTD on licking time and lifting time are similar to those of the TRPV1 antagonist and AMG9810 (30 mg/kg), and the inhibition on the licking plus lifting time Intraplantar injection of icilin (30 𝜇g/paw, 20 𝜇L) did not (59±6%) is more remarkable than that (47±4%) of AMG9810 induce obvious nociceptive behaviors compared to control (Figure 4(a)). group (data not shown), which is consistent with previous As shown in Figure 4(b), the TRPA1 activator cinnamal- report [27]. Then, icilin intraperitoneally 50 mg/kg was used dehyde also induced obvious nociceptive behaviors in mice, to explore the inhibiting characteristics of WTD on TRPM8 and oral administration of WTD (6.30 g/kg) significantly ion channel. As shown in Figure 4(c),icilininducedhigh reduced the licking time, lifting time, and licking plus lifting numbers of jumping and WDS behaviors, and oral adminis- time induced by cinnamaldehyde with similar effect to those tration of WTD (6.30 g/kg) strikingly decreased these num- of camphor, the TRPA1 antagonist. bers compared to icilin control group, with inhibitions of BioMed Research International 7

120 120

90 90

∗∗∗ 60 && ∗∗∗ 60 ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ Reaction time (s) ∗∗∗ Reaction time (s) ∗∗∗ 30 ∗∗∗ ∗∗∗ 30 ∗∗∗ ∗∗∗

0 0 Licking Lifting Licking + Licking Lifting Licking + lifting lifting Control WTD 6.30 g/kg Control WTD 6.30 g/kg AMG9810 30 mg/kg Camphor 7.6 mg/kg (a) (b) 100

∗∗∗ 20 ∗∗∗ ∗∗∗ Number of evoked behaviors

0 Jumping WDS Jumping + WDS Control WTD 6.30 g/kg (c)

Figure 4: Effect of WTD on the activities of TRPV1 (a), TRPA1 (b), and TRPM8 (c) ion channels in mice. (a) Effect of WTD (6.30 g/kg, p.o.) and the TRPV1 antagonist AMG9810 (30 mg/kg, i.p.) on capsaicin-induced (2 𝜇g/paw) nociception. (b) Effect of WTD (6.30 g/kg, p.o.) and the TRPA1 antagonist camphor (7.6 mg/kg, s.c.) on cinnamaldehyde-induced (1.3 𝜇g/paw) nociception. (c) Effect of WTD (6.30 g/kg, p.o.) ∗∗∗ on icilin-induced (50 mg/kg, i.p.) jumping and WDS behaviors. Data are represented as the mean ± SEM (𝑛=6). 𝑃 < 0.001 versus the && control group; 𝑃 < 0.01 versus the AMG9810 group.

78 ± 5% for jumping behaviors, 72 ± 7%forWDSbehaviors, theskinsofinjuredpawsfollowingCFA-inducedinflamma- and 78 ± 8% for jumping plus WDS behaviors, respectively. tion in a dose-related manner (Figure 6). However, TRPM8 protein expression both in DRGs and skins was not changed 3.5. WTD Downregulates the Protein Expression of TRPs in significantly after WTD treatment, and this was also true for DRG and Skins of Injured Paws from CFA-Induced Mice. CFA group. Since overexpression of TRP channels is associated with inflammatory hypersensitivity [8–10], the protein expression 4. Discussion of TRPV1, TRPA1, and TRPM8 was further examined in CFA-induced mice. Immunohistochemical staining analysis WTD is known to be used effectively to treat joint diseases demonstrated that the numbers of TRPV1- and TRPA1- in clinic. Previous studies indicated that WTD possess anti- positive neurons in DRGs increased remarkably in CFA inflammatory effect13 [ , 14]; however, its actions on pain group compared to control group, while oral administration remain unclear. Data presented in this study indicate that of WTD 1.58, 3.15, and 6.30 g/kg decreased the percentage of WTD can potently attenuate hypersensitivities to mechani- these positive neurons, with significance in WTD (3.15 and cal, cold, and heat stimuli in CFA-induced mice. Moreover, 6.30 g/kg) treated groups (Figure 5). Western blot analysis WTD inhibits the activities of TRPV1, TRPA1, and TRPM8 verified that WTD (1.58, 3.15, and 6.30 g/kg) treatment sig- ion channels and reduces the protein expression of TRPV1 nificantly decreased the production of TRPV1 and TRPA1 in and TRPA1 in DRGs and skins of inflamed paw. Thus, our 8 BioMed Research International

Control CFA WTD 6.30 g/kg 1 TRPV 1 TRPA 8 TRPM

10 𝜇m

(a) 80 ## 60 ### ∗ ∗ 60 ∗∗ ∗∗ 40

40 neurons (%) neurons neurons (%) neurons 20 TRPA1-positive TRPA1-positive TRPV1-positive 20

0 0 Control CFA WTD WTD WTD Control CFA WTD WTD WTD 1.58 g/kg 3.15 g/kg 6.30 g/kg 1.58 g/kg 3.15 g/kg 6.30 g/kg (b) (c)

Figure 5: Continued. BioMed Research International 9

20 neurons (%) neurons

TRPM8-positive 10

0 Control CFA WTD WTD WTD 1.58 g/kg 3.15 g/kg 6.30 g/kg (d)

Figure 5: Effect of WTD on the expression of TRPV1, TRPA1, and TRPM8 in DRGs of inflammatory pain mice by immunohistochemical staining. Mice were orally administrated with WTD (1.58, 3.15, and 6.30 g/kg, resp.) or water daily for 15 days. (a) Localization of positive TRPV1, TRPA1, and TRPM8 neurons in DRGs of mice from control, CFA, and WTD groups, respectively. ((b) and (c)) The numbers of TRPV1- and TRPA1-positive neurons significantly increased in DRGs in CFA group, while WTD significantly reduced their expression. (c) No significant difference of the number of TRPM8-positive neurons was observed among the five groups. Data are represented as themean ± ## ### ∗ ∗∗ SEM (𝑛=8). 𝑃 < 0.01 and 𝑃 < 0.001 versus the control group, respectively. 𝑃 < 0.05 and 𝑃 < 0.01 versus the CFA group, respectively.

∘ data show for the first time that WTD possess antinociceptive by temperatures above 42 C, deleting or inhibiting the activ- activity and the possible mechanism of its analgesic action ity of this heat-sensitive channel results in reducing inflam- is associated with the inhibition of activities and protein matory heat and mechanical hyperalgesia [30–32]. TRPA1 ∘ expression of TRP ion channels in inflammatory conditions. is a cold sensor, activated by temperatures below 17 Cand Intraplantar injection of CFA to rodents is a widely used involved in pathophysiological cold hypersensitivity since model to study persistent chronic inflammatory pain, which the use of TRPA1 antisense oligodeoxynucleotide reverses mimics a chronic inflammatory condition. A characteristic the cold hypersensitivity after CFA-induced inflammation symptom of this model of nociception is that it displays [10, 33]. Meanwhile, TRPA1 channel seems to be important hyperalgesia to mechanical, cold, and heat stimulation [2, 3]. in the maintenance of mechanical hypersensitivity, because In this study, we analyzed the effect of WTD on inflammatory TRPA1 knockout mice show deficits in the development of pain induced by intraplantar injection of CFA in mice. Our inflammatorymechanicalhyperalgesia[9, 34]. TRPM8 is also results showed that oral administration of WTD at doses a cold sensor, deleting or blocking the channel resulting in the of 1.58∼6.30 g/kg (0.25∼1-fold equivalent to patient dosage reduction of inflammation-induced cold hypersensitivity35 [ , daily)couldremarkablyreducemechanicalandthermal(cold 36]. Considering the close connections of these ion channels and heat) hypersensitivities caused by intraplantar CFA injec- with mechanical and thermal hypersensitivities in inflam- tion. Specially, the antinociceptive effect of WTD treatment matory pain conditions, it is reasonable to think that the on cold hypersensitivity is evident, lasted up to 6 h, and with antihypersensitivity effect of WTD may be mediated by TRPs. maximum inhibitions of over 95% from day 9 to day 15 Totest this hypothesis, we evaluated the antinociceptive effect after administration in WTD (6.30 g/kg) treated group, partly of WTD against specific activators of these three ion channels. accounting for its effective treatment for patients with cold The results from our study showed that capsaicin, a selective patternofarthromyodynia.Itisknownthattoleranceand activator of TRPV1, and cinnamaldehyde, a selective activator othersideeffectssuchasbodyweightlossandulcerationof of TRPA1, both induced spontaneous nociceptive responses the gastric mucosa limit analgesic drugs to be used clinically in mice, which were reversed by WTD with similar effect [11, 12]. In our study, the antinociceptive effect of WTD was to that of antagonist AMG9810 or camphor, respectively. not susceptible to tolerance and the prolonged treatment of Meanwhile, WTD strikingly reversed the jumping and WDS WTD did not cause significant change in body weight of mice. behaviors to intraperitoneal injection of icilin, a TRPM8 This effect was different from that of ibuprofen, which could activator. These findings were consistent with our results on induce remarkable body weight loss and gastric mucosa as CFA model, when WTD could reverse the mechanical, cold, previously reported [28, 29]. and heat hypersensitivities. The TRP ion channels are highly involved in the trans- It has been demonstrated that the increased levels of duction and sensitization in primary afferent somatosensory TRPV1 and TRPA1 protein in skins, DRG, and trigeminal neurons and play an integral role in pain and neurogenic neurons play a relevant role in the maintenance of inflam- inflammation via sensory nerve activation, at either central matory hyperalgesia [8–10, 37]. In our present study, WTD or peripheral level [5–7]. Among them, TRPV1 is a heat significantly reduced the protein expression of TRPV1 and transducer in normal and pathological conditions, activated TRPA1 both in DRGs and skins of injured paws, which may 10 BioMed Research International

0.8 0.30

0.24 ### 0.6 ###

∗ 0.18 ∗ ∗ ∗∗ 0.4 ∗∗ 0.12 TRPA1/GAPDH TRPV1/GAPDH ∗∗∗ 0.2 0.06

0.0 0.00

TRPV1 TRPA1

GAPDH GAPDH

Control CFA WTD WTD WTD Control CFA WTD WTD WTD 1.58 g/kg 3.15 g/kg 6.30 g/kg 1.58 g/kg 3.15 g/kg 6.30 g/kg (a) (b) 0.4

0.3

0.2 TRPM8/GAPDH 0.1

TRPM8

GAPDH

Control CFA WTD WTD WTD 1.58 g/kg 3.15 g/kg 6.30 g/kg (c)

Figure 6: Effect of WTD on the expression of TRPV1 (a), TRPA1 (b), and TRPM8 (c) in skins of injured paw of inflammatory pain miceby western blot. Mice were orally administrated with WTD (1.58, 3.15, and 6.30 g/kg, resp.), ibuprofen (0.14 g/kg), or water daily for 15 days. The protein expression of TRPV1 and TRPA1 significantly increased in CFA group, while WTD dose-dependently decreased their expression. No significant difference of TRPM8 protein expression was observed among the five groups. Data are represented asthemean ± SEM (𝑛=4). ## ### ∗ ∗∗ ∗∗∗ 𝑃 < 0.01 and 𝑃 < 0.001 versus the control group, respectively; 𝑃 < 0.05, 𝑃 < 0.01,and 𝑃 < 0.001 versus the CFA group, respectively. BioMed Research International 11 partly account for WTD antinociceptive action. Similar to neurons as targets for novel analgesics,” British Journal of previous report [14], our present experiment found that the Pharmacology,vol.171,no.10,pp.2508–2527,2014. levels of nerve growth factor (NGF), IL-1𝛽,andTNF-𝛼 were [6] M. A. Schumacher, “Transient receptor potential channels in inhibited by WTD in skins of the inflamed paws in CFA- pain and inflammation: therapeutic opportunities,” Pain Prac- induced mice (data not shown). Since NGF is known to tice,vol.10,no.3,pp.185–200,2010. increase the expression of TRPV1 and TRPA1 in inflamma- [7] J.-D. Brederson, P. R. Kym, and A. Szallasi, “Targeting TRP tory conditions [8–10, 37], and IL-1𝛽 and TNF-𝛼 inducing channels for pain relief,” European Journal of Pharmacology,vol. the upregulation of NGF in CFA-induced inflamed sites have 716, no. 1–3, pp. 61–76, 2013. been identified [38], thus, it seems to be the suppression of [8]R.-R.Ji,T.A.Samad,S.-X.Jin,R.Schmoll,andC.J.Woolf, these factors by WTD contributing to the downregulation of “p38 MAPK activation by NGF in primary sensory neurons TRPV1 and TRPA1 expression in DRG or peripheral tissue. after inflammation increases TRPV1 levels and maintains heat Interestingly, an unexpected finding was that WTD treatment hyperalgesia,” Neuron,vol.36,no.1,pp.57–68,2002. did not influence the production of TRPM8 both in DRGs [9]D.S.M.daCosta,F.C.Meotti,E.L.Andrade,P.C.Leal,E. and skins, and this was true for CFA group, supported partly M. Motta, and J. B. Calixto, “The involvement of the transient by previous observation [10]. 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