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

Authors: Chao Wang; Chunfang Liu; Hongye Wan; Danhua Wang; Danni Sun; Tengfei Xu; Yue Yang; Yakun Qu; Ying Xu; Xianghong Jing; Junling Liu; Shuping Chen; Zhiqiang Liu; Na Lin
Journal: BioMed Research International
 (2015)

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## Abstract

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 of the 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.


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## Body

## 1. Introduction

Chronic inflammatory pain ensues when the normal process of inflammation does not resolve, resulting in excess proinflammatory cytokines and chemoattractants that can eventually lead to peripheral and central sensitization [1]. Indeed, hypersensitivities to heat, cold, and mechanical stimuli are well-documented characteristic symptoms of inflammatory and neuropathic pain both in the clinical setting and in experimental animal models [2, 3]. It can be either recurrent or persistent and is a serious developmental health concern that can interfere significantly with daily functioning [4]. Over the past decade, flourishing research has revealed that members of the transient receptor potential (TRP) ion channel family in nociceptor peripheral terminals and dorsal root ganglions (DRGs) are fundamental molecules that detect noxious stimuli and transduce a diverse range of physical and chemical energy into action potentials in somatosensory nociceptors and contribute to the initiation and maintenance of hypersensitivity under inflammatory and neuropathic pain conditions [5]. Among them, TRPV1, TRPA1, and TRPM8 ion channels have attracted the most attentions. It has been shown that deleting or inhibiting the activities of these ion channels is associated with reduced inflammatory nociceptive responses [5–7], and inflammatory hypersensitivity also appears to be dependent on the expression of TRPs [8–10]. Thus, pain-evoking TRP channels emerge as promising therapeutic targets for a wide variety of pain and inflammatory conditions [5–7].Treatment of chronic inflammatory pain still remains a major challenge in clinical practice because of its heterogeneous etiology and the complex underlying pathophysiology mechanisms. The most commonly used analgesics, such as opioids, nonsteroidal anti-inflammatory drugs (NSAIDs), and anticonvulsants (for neuropathic pain), produce serious side effects [11, 12]. Therefore, there is an urgent need to develop novel and safer pain medicines to meet the needs of patients. In this context, natural products that present fewer side effects emerge as interesting therapeutic resources for the development of new drugs for the management of certain chronic pain states.Wu-tou decoction (WTD), a classic Traditional Chinese Medicine (TCM) formula from Chinese medical sage Zhongjing Zhang, has been recorded inJin Gui Yao Lue for more than 1,000 years. It was formulated according to TCM theory especially for the treatment of cold pattern of arthromyodynia. Clinically, WTD has been used effectively to treat joint diseases. Previous studies have shown that WTD possess the anti-inflammatory effect by inhibiting proinflammatory cytokines such as IL-1β and TNF-α and regulating TLR2/TRAF6/Faslg signal pathway in complete Freund’s adjuvant- (CFA-) induced arthritis rats [13, 14]. However, it remains unclear whether WTD could inhibit inflammatory pain. In this study, we investigated the antinociceptive activity of WTD on cutaneous inflammation induced by CFA in mice, and its possible mechanism of the action associated with TRP ion channels was also explored.


## 2. Materials and Methods

This study was supported by the Research Ethics Committee of China Academy of Chinese Medical Sciences, Beijing, China (Permit number: 2013-61). All animals were treated in accordance with the guidelines and regulations for the use and care of animals at the Center for Laboratory Animal Care, China Academy of Chinese Medical Sciences. All efforts were made to demonstrate consistent effects of the drug treatments and minimize the suffering of animals.


### 2.1. Preparation of WTD

WTD is a mixture of five traditional drugs (Table1). The drugs were purchased from Beijing Huamiao Chinese Medicine Engineering Development Center (Beijing, China) and authenticated by Professor Shilin Hu, China Academy of Chinese Medical Sciences. WTD was prepared according to our previous studies [14, 15]. All the components of WTD were dried and homogenized to fine powders; then the 84 g powdered materials were immersed in 840 mL deionized water for 1 h and then heated to refluxing for 1.5 h. Water as 8 times of the above total weight was added for another 1.5 h refluxing after being filtered. The filtered extraction solutions were combined and concentrated to 50 mL and then kept at −20°C for preparation. The final concentration is 1.68 g/mL (crude herbs) stock solution. This stock solution was diluted with distilled water to the proper concentration before usein vivo and the following chemical experiments. Take 1 mL and dilute it 1000 times as the analyte sample with internal standards. The sample was filtered through a 0.22 μm filter membrane and was quantified by UPLC-MS/MS analysis. Standards of ephedrine, berberine, paeoniflorin, liquiritin, glycyrrhizic acid, calycosin-7-glucoside, formononetin, dioscin, and liquiritigenin were obtained from the Chinese Authenticating Institute of Material and Biological Products (Beijing, China). Standards of benzoylmesaconine, benzoylhypacoitine, aconitine, and benzoylaconitine were purchased from Lan Yuan Biological Technology Co., Ltd. (Shanghai, China). The purity of all compounds was more than 98% (determined by HPLC). They were prepared at a proper concentration in methanol. HPLC-grade acetonitrile and formic acid were obtained from Fisher Scientific (Loughborough, UK). Ultrapure water was prepared by the Milli-Q plus (Milford, MA, USA) water purification system.Table 1
Composition of WTD.
Pharmaceutical name
Botanical source/family
Part used
Traditional actions/uses
Amount (g)
Glycyrrhiza Radix Preparata
Glycyrrhiza uralensis(Leguminosae)
Root and rhizome (processed)
Supplements spleen, replenishes qi, harmonizes all drugs
9
Ephedrae Herba
Ephedra sinensis(Ephedrae)
Grass stem
Resolves surface, regulates water metabolism
9
Paeoniae Radix Alba
Paeonia lactiflora Pall.
Root
Treats gastrointestinal disorders, relieves pains
9
Aconiti Radix Cocta
Ranunculaceae
Mother root (processed)
Expel wind and cold and eliminate dampness to relieve pain
6
Astragali Radix
Astragalus membranaceus(Leguminosae)
Dried root
Supplements qi, increases yang, delivers water
9The UPLC-MS/MS system used was a Waters Acquity-Ultra Performance LC system (Waters, USA) coupled with Xevo TQ MS spectrometer with electrospray ion source (ESI) (Waters, Manchester, UK). Chromatographic separation was performed on a Waters Acquity UPLC BEH C18 column (1.7μm, 2.1 * 50 mm) kept at 35°C. Acetonitrile (A) and 0.1% aqueous formic acid (v/v) (B) were used as the mobile phase. The gradient elution with the flow rate of 0.3 mL/min was performed as follows: 10% A at 0–2 min, 10–15% A at 2–7 min, 15–30% A at 7–15 min, 30–39% A at 15–21 min, and 39% up to 100% A at 21–25 min. The sample injected volume was 5 μL. The MS analysis was carried out by the ESI source in both positive and negative ion mode. The source temperature was 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 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, respectively, and in negative ion mode, they were set at 2.0 KV and 5.0 V, respectively. The multiple reaction monitoring (MRM) transitions for fourteen analytes as well as their respective cone voltages and collision energies are listed in Table 2.Table 2
Ion transitions and instrumental parameters for their LC-MS/MS quantification in MRM mode.
Analytes
Ion mode
Quantitation transition (m
/
z)
Cone voltage (V)
Collision energy (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.


### 2.2. Analysis of Water Extract of WTD

The content of eleven main components in WTD used in this trial was quantified by liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), and the calibration curves, calibration ranges, and content in 1 g WTD of these compounds are performed in Table3.Table 3
Calibration curves, concentration ranges, and content of 11 main components in WTD.
Sample
Calibration curves
R
2
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


### 2.3. Animals

Male ICR mice (30–35 g) were obtained from Laboratory Animal Center of Academy of Military Medical Sciences, Beijing, China (License number: SCXK 2012-0004). They were kept in a temperature controlled environment (22
±
1°C), 55
±
5% relative humidity with a 12 h : 12 h light-dark cycle, and fed with standard chow, for at least one week before any manipulations. Animals were habituated to the laboratory conditions for at least 1 h before testing and all experiments were performed during the light phase of the cycle.


### 2.4. CFA-Induced Chronic Inflammatory Pain

ICR mice were divided separately into 6 groups randomly with the equal number (n
=
11): the normal control group (control), the CFA-induced inflammatory pain model group (CFA), the WTD (1.58, 3.15, and 6.30 g/kg, p.o., resp.) treated groups, and the ibuprofen (0.14 g/kg, p.o.) treated group. The dosage of ibuprofen used was equivalent conversion from rats as previously reported [16–18]. Six groups of mice were orally administrated by syringe feeding with distilled water (20 mL/kg) or WTD or ibuprofen daily for 15 days 24 h after CFA injection, respectively. Mice received an intraplantar injection of 20 μL of CFA (Sigma-Aldrich, St. Louis, MO) to the right hind paw. Control group received 20 μL of vehicle (1% tween 80 plus saline) intraplantar injection. CFA caused hind paw mechanical and thermal hypersensitivities, evaluated 24 hours after CFA injection.


#### 2.4.1. Measurement of Mechanical Hypersensitivity

Mice were acclimatized in individual clear boxes on wire-mesh platform, to allow access to the ventral surface of the hind paws, and mechanical hypersensitivity was assessed by the sensitivity to the application of von Frey hairs (Stoelting Co., Chicago, USA). The von Frey filaments (0.008–4.0 g) were presented perpendicularly to the plantar surface of the injected paw and held in this position for 5–8 s with enough force to cause a slight bend in the filament. Positive responses included an abrupt withdrawal of the hind paw or flinching behaviour immediately following removal of the stimulus. A median paw withdrawal threshold was determined using an adaptation of Dixon’s up-down method [19]. The 50% paw withdrawal threshold was analyzed at 0, 1, 2, 4, and 24 h and 3, 5, 7, 9, 11, 13, and 15 days after WTD administration, respectively.


#### 2.4.2. Measurement of Cold Hypersensitivity

Thermal hypersensitivity to cold was evaluated as described previously [20]. Briefly, with the aid of syringe (1 mL), 20 μL acetone was sprayed gently from a short distance to the middle of the plantar surface of hind paw. The amount of time that mice were licking and/or shaking the hind paw was recorded during 5 min after acetone application and was used as an index of nocifensive responsiveness for cold hypersensitivity. The licking and/or shaking time was measured at 0, 1, 2, 4, 6, 8, and 10 h and 3, 5, 7, 9, 11, 13, and 15 days after WTD administration, respectively.


#### 2.4.3. Measurement of the Heat Hypersensitivity

Thermal hyperalgesia of the hind paw was assessed using the plantar test (Ugo Basile Srl, Comerio VA, Italy), according to what is previously described [21]. Briefly, mice were acclimatized to an apparatus consisting of individual perspex boxes; an infrared radiant heat (40 W) source was directed to middle part of the plantar surface of the hind paw. The cut-off time was set at 20 s to prevent tissue damage. Paw withdrawal latency (PWL) was measured at 0, 0.5, 1, 1.5, 2, 3, 4, and 6 h and 3, 5, 7, 9, 11, 13, and 15 days after WTD administration, respectively, and three trials for each hind paw. The interval of PWL measurement was five minutes.


### 2.5. Capsaicin-Induced Nociception

To evaluate the possible involvement of TRPV1 channel on WTD antinociceptive effect, mice were submitted to a test using capsaicin, a specific activator of TRPV1, as previously described [22, 23]. Mice were pretreated with vehicle (1% Tween-80 plus saline, 20 mL/kg, p.o.), AMG9810 (a selective TRPV1 antagonist, 30 mg/kg, i.p.), or WTD (6.30 g/kg, p.o.) 1 h (for p.o. administration) or 0.5 h (for i.p. administration) prior to the injection of 20 μL capsaicin (2 μg/paw) in the plantar surface of the right hind paw, respectively. Immediately after the capsaicin injection, mice were placed into clear observation boxes and the nociceptive response was evaluated as the time of lifting and spent licking the injected paw during 5 min.


### 2.6. Cinnamaldehyde-Induced Nociception

To evaluate the possible involvement of TRPA1 channel in WTD antinociceptive effect, mice were submitted to a test using cinnamaldehyde, a specific activator of TRPA1, as previously described [23, 24]. Mice were pretreated with vehicle (20 mL/kg, p.o.), 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. administration) prior to the injection of 20 μL of cinnamaldehyde (TRPA1 activator, 1.3 μg/paw) in the plantar surface of the right hind paw. Immediately after the cinnamaldehyde injection, mice were placed into clear observation boxes and the nociceptive response was evaluated as the time of lifting and spent licking the injected paw during 5 min.


### 2.7. Icilin-Induced Jumping and Wet-Dog-Shake Behaviors

To evaluate the possible involvement of TRPM8 channel in WTD antinociceptive effect, mice were submitted to a test using icilin, a specific activator of TRPM8, as previously described [25]. Briefly, 12.5 mg/mL icilin was suspended in 1% Tween-80/distilled water and sonicated. Icilin was intraperitoneally administered at a concentration of 50 mg/kg. Mice were orally pretreated with vehicle (20 mL/kg) or WTD (6.30 g/kg) 1 h prior to the injection of vehicle (10 mL/kg, i.p.) or icilin (50 mg/kg, i.p.). Immediately after the icilin treatment, jumping and wet-dog-shakes (WDS) behaviors were counted for 20 min.


### 2.8. Western Blot Analysis

Mice were sacrificed by cervical dislocation under ether anesthesia 15 days after the WTD and ibuprofen administration. Plantar surfaces of injured hind paw skin were dissected and stored at −80°C for western blot analysis. The expression of TRPV1, TRPA1, and TRPM8 in hind paw skins tissues was determined. The western blot protocol and semiquantitative analysis were carried out following the protocol of our previous study [26]. The following primary antibodies were used: TRPV1 (rabbit polyclonal antibody, dilution 1 : 100, Alomone Labs, Israel), TRPA1 (rabbit polyclonal antibody, dilution 1 : 100, Alomone Labs, Israel), TRPM8 (rabbit polyclonal antibody, dilution 1 : 100, Alomone Labs, Israel), and GAPDH (internal control, rabbit polyclonal antibody, dilution 1 : 1000, Beyotime, China). A goat-anti-rabbit antibody conjugated to horseradish peroxidase (1 : 5000, Thermo Fisher Scientific, USA) was used as the second antibody. TRPV1, TRPA1, and TRPM8 protein levels were normalized against GAPDH. All experiments were done for four times.


### 2.9. Immunohistochemical Staining

The immunohistochemical staining protocol was carried out following the protocol of our previous study [26]. Primary antibodies: TRPV1 (rabbit polyclonal antibody, 1 : 100, Alomone Labs, Israel), TRPA1 (rabbit polyclonal antibody, 1 : 100, Alomone Labs, Israel), and TRPM8 (rabbit polyclonal antibody, 1 : 100, Alomone Labs, Israel); and secondary antibody (goat-anti-rabbit 1 : 100, Boster, China) was used and stained with DAB (Dako, Denmark). The percentage of immunopositive cells was analyzed by counting the number of immunopositive neurons and multiplying by 100/number of total number of neurons. A total of 2 DRGs were measured in each animal, and the number of immunoreactive neuronal profiles was counted in a blinded fashion. The percentage of immunopositive cells was used for statistic index.


### 2.10. Statistical Analysis

Data obtained from the mechanical allodynia and thermal hyperalgesia experiments were analyzed by repeated measures one-way analysis of variance (ANOVA) followed by post hoc Dunnett’s Multiple Comparison Test. The data in immunohistochemical staining experiments were analyzed using the Mann-Whitney test. A value ofP
<
0.05 was taken to be significant.


## 2.1. Preparation of WTD

WTD is a mixture of five traditional drugs (Table1). The drugs were purchased from Beijing Huamiao Chinese Medicine Engineering Development Center (Beijing, China) and authenticated by Professor Shilin Hu, China Academy of Chinese Medical Sciences. WTD was prepared according to our previous studies [14, 15]. All the components of WTD were dried and homogenized to fine powders; then the 84 g powdered materials were immersed in 840 mL deionized water for 1 h and then heated to refluxing for 1.5 h. Water as 8 times of the above total weight was added for another 1.5 h refluxing after being filtered. The filtered extraction solutions were combined and concentrated to 50 mL and then kept at −20°C for preparation. The final concentration is 1.68 g/mL (crude herbs) stock solution. This stock solution was diluted with distilled water to the proper concentration before usein vivo and the following chemical experiments. Take 1 mL and dilute it 1000 times as the analyte sample with internal standards. The sample was filtered through a 0.22 μm filter membrane and was quantified by UPLC-MS/MS analysis. Standards of ephedrine, berberine, paeoniflorin, liquiritin, glycyrrhizic acid, calycosin-7-glucoside, formononetin, dioscin, and liquiritigenin were obtained from the Chinese Authenticating Institute of Material and Biological Products (Beijing, China). Standards of benzoylmesaconine, benzoylhypacoitine, aconitine, and benzoylaconitine were purchased from Lan Yuan Biological Technology Co., Ltd. (Shanghai, China). The purity of all compounds was more than 98% (determined by HPLC). They were prepared at a proper concentration in methanol. HPLC-grade acetonitrile and formic acid were obtained from Fisher Scientific (Loughborough, UK). Ultrapure water was prepared by the Milli-Q plus (Milford, MA, USA) water purification system.Table 1
Composition of WTD.
Pharmaceutical name
Botanical source/family
Part used
Traditional actions/uses
Amount (g)
Glycyrrhiza Radix Preparata
Glycyrrhiza uralensis(Leguminosae)
Root and rhizome (processed)
Supplements spleen, replenishes qi, harmonizes all drugs
9
Ephedrae Herba
Ephedra sinensis(Ephedrae)
Grass stem
Resolves surface, regulates water metabolism
9
Paeoniae Radix Alba
Paeonia lactiflora Pall.
Root
Treats gastrointestinal disorders, relieves pains
9
Aconiti Radix Cocta
Ranunculaceae
Mother root (processed)
Expel wind and cold and eliminate dampness to relieve pain
6
Astragali Radix
Astragalus membranaceus(Leguminosae)
Dried root
Supplements qi, increases yang, delivers water
9The UPLC-MS/MS system used was a Waters Acquity-Ultra Performance LC system (Waters, USA) coupled with Xevo TQ MS spectrometer with electrospray ion source (ESI) (Waters, Manchester, UK). Chromatographic separation was performed on a Waters Acquity UPLC BEH C18 column (1.7μm, 2.1 * 50 mm) kept at 35°C. Acetonitrile (A) and 0.1% aqueous formic acid (v/v) (B) were used as the mobile phase. The gradient elution with the flow rate of 0.3 mL/min was performed as follows: 10% A at 0–2 min, 10–15% A at 2–7 min, 15–30% A at 7–15 min, 30–39% A at 15–21 min, and 39% up to 100% A at 21–25 min. The sample injected volume was 5 μL. The MS analysis was carried out by the ESI source in both positive and negative ion mode. The source temperature was 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 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, respectively, and in negative ion mode, they were set at 2.0 KV and 5.0 V, respectively. The multiple reaction monitoring (MRM) transitions for fourteen analytes as well as their respective cone voltages and collision energies are listed in Table 2.Table 2
Ion transitions and instrumental parameters for their LC-MS/MS quantification in MRM mode.
Analytes
Ion mode
Quantitation transition (m
/
z)
Cone voltage (V)
Collision energy (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.


## 2.2. Analysis of Water Extract of WTD

The content of eleven main components in WTD used in this trial was quantified by liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), and the calibration curves, calibration ranges, and content in 1 g WTD of these compounds are performed in Table3.Table 3
Calibration curves, concentration ranges, and content of 11 main components in WTD.
Sample
Calibration curves
R
2
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


## 2.3. Animals

Male ICR mice (30–35 g) were obtained from Laboratory Animal Center of Academy of Military Medical Sciences, Beijing, China (License number: SCXK 2012-0004). They were kept in a temperature controlled environment (22
±
1°C), 55
±
5% relative humidity with a 12 h : 12 h light-dark cycle, and fed with standard chow, for at least one week before any manipulations. Animals were habituated to the laboratory conditions for at least 1 h before testing and all experiments were performed during the light phase of the cycle.


## 2.4. CFA-Induced Chronic Inflammatory Pain

ICR mice were divided separately into 6 groups randomly with the equal number (n
=
11): the normal control group (control), the CFA-induced inflammatory pain model group (CFA), the WTD (1.58, 3.15, and 6.30 g/kg, p.o., resp.) treated groups, and the ibuprofen (0.14 g/kg, p.o.) treated group. The dosage of ibuprofen used was equivalent conversion from rats as previously reported [16–18]. Six groups of mice were orally administrated by syringe feeding with distilled water (20 mL/kg) or WTD or ibuprofen daily for 15 days 24 h after CFA injection, respectively. Mice received an intraplantar injection of 20 μL of CFA (Sigma-Aldrich, St. Louis, MO) to the right hind paw. Control group received 20 μL of vehicle (1% tween 80 plus saline) intraplantar injection. CFA caused hind paw mechanical and thermal hypersensitivities, evaluated 24 hours after CFA injection.


### 2.4.1. Measurement of Mechanical Hypersensitivity

Mice were acclimatized in individual clear boxes on wire-mesh platform, to allow access to the ventral surface of the hind paws, and mechanical hypersensitivity was assessed by the sensitivity to the application of von Frey hairs (Stoelting Co., Chicago, USA). The von Frey filaments (0.008–4.0 g) were presented perpendicularly to the plantar surface of the injected paw and held in this position for 5–8 s with enough force to cause a slight bend in the filament. Positive responses included an abrupt withdrawal of the hind paw or flinching behaviour immediately following removal of the stimulus. A median paw withdrawal threshold was determined using an adaptation of Dixon’s up-down method [19]. The 50% paw withdrawal threshold was analyzed at 0, 1, 2, 4, and 24 h and 3, 5, 7, 9, 11, 13, and 15 days after WTD administration, respectively.


### 2.4.2. Measurement of Cold Hypersensitivity

Thermal hypersensitivity to cold was evaluated as described previously [20]. Briefly, with the aid of syringe (1 mL), 20 μL acetone was sprayed gently from a short distance to the middle of the plantar surface of hind paw. The amount of time that mice were licking and/or shaking the hind paw was recorded during 5 min after acetone application and was used as an index of nocifensive responsiveness for cold hypersensitivity. The licking and/or shaking time was measured at 0, 1, 2, 4, 6, 8, and 10 h and 3, 5, 7, 9, 11, 13, and 15 days after WTD administration, respectively.


### 2.4.3. Measurement of the Heat Hypersensitivity

Thermal hyperalgesia of the hind paw was assessed using the plantar test (Ugo Basile Srl, Comerio VA, Italy), according to what is previously described [21]. Briefly, mice were acclimatized to an apparatus consisting of individual perspex boxes; an infrared radiant heat (40 W) source was directed to middle part of the plantar surface of the hind paw. The cut-off time was set at 20 s to prevent tissue damage. Paw withdrawal latency (PWL) was measured at 0, 0.5, 1, 1.5, 2, 3, 4, and 6 h and 3, 5, 7, 9, 11, 13, and 15 days after WTD administration, respectively, and three trials for each hind paw. The interval of PWL measurement was five minutes.


## 2.4.1. Measurement of Mechanical Hypersensitivity

Mice were acclimatized in individual clear boxes on wire-mesh platform, to allow access to the ventral surface of the hind paws, and mechanical hypersensitivity was assessed by the sensitivity to the application of von Frey hairs (Stoelting Co., Chicago, USA). The von Frey filaments (0.008–4.0 g) were presented perpendicularly to the plantar surface of the injected paw and held in this position for 5–8 s with enough force to cause a slight bend in the filament. Positive responses included an abrupt withdrawal of the hind paw or flinching behaviour immediately following removal of the stimulus. A median paw withdrawal threshold was determined using an adaptation of Dixon’s up-down method [19]. The 50% paw withdrawal threshold was analyzed at 0, 1, 2, 4, and 24 h and 3, 5, 7, 9, 11, 13, and 15 days after WTD administration, respectively.


## 2.4.2. Measurement of Cold Hypersensitivity

Thermal hypersensitivity to cold was evaluated as described previously [20]. Briefly, with the aid of syringe (1 mL), 20 μL acetone was sprayed gently from a short distance to the middle of the plantar surface of hind paw. The amount of time that mice were licking and/or shaking the hind paw was recorded during 5 min after acetone application and was used as an index of nocifensive responsiveness for cold hypersensitivity. The licking and/or shaking time was measured at 0, 1, 2, 4, 6, 8, and 10 h and 3, 5, 7, 9, 11, 13, and 15 days after WTD administration, respectively.


## 2.4.3. Measurement of the Heat Hypersensitivity

Thermal hyperalgesia of the hind paw was assessed using the plantar test (Ugo Basile Srl, Comerio VA, Italy), according to what is previously described [21]. Briefly, mice were acclimatized to an apparatus consisting of individual perspex boxes; an infrared radiant heat (40 W) source was directed to middle part of the plantar surface of the hind paw. The cut-off time was set at 20 s to prevent tissue damage. Paw withdrawal latency (PWL) was measured at 0, 0.5, 1, 1.5, 2, 3, 4, and 6 h and 3, 5, 7, 9, 11, 13, and 15 days after WTD administration, respectively, and three trials for each hind paw. The interval of PWL measurement was five minutes.


## 2.5. Capsaicin-Induced Nociception

To evaluate the possible involvement of TRPV1 channel on WTD antinociceptive effect, mice were submitted to a test using capsaicin, a specific activator of TRPV1, as previously described [22, 23]. Mice were pretreated with vehicle (1% Tween-80 plus saline, 20 mL/kg, p.o.), AMG9810 (a selective TRPV1 antagonist, 30 mg/kg, i.p.), or WTD (6.30 g/kg, p.o.) 1 h (for p.o. administration) or 0.5 h (for i.p. administration) prior to the injection of 20 μL capsaicin (2 μg/paw) in the plantar surface of the right hind paw, respectively. Immediately after the capsaicin injection, mice were placed into clear observation boxes and the nociceptive response was evaluated as the time of lifting and spent licking the injected paw during 5 min.


## 2.6. Cinnamaldehyde-Induced Nociception

To evaluate the possible involvement of TRPA1 channel in WTD antinociceptive effect, mice were submitted to a test using cinnamaldehyde, a specific activator of TRPA1, as previously described [23, 24]. Mice were pretreated with vehicle (20 mL/kg, p.o.), 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. administration) prior to the injection of 20 μL of cinnamaldehyde (TRPA1 activator, 1.3 μg/paw) in the plantar surface of the right hind paw. Immediately after the cinnamaldehyde injection, mice were placed into clear observation boxes and the nociceptive response was evaluated as the time of lifting and spent licking the injected paw during 5 min.


## 2.7. Icilin-Induced Jumping and Wet-Dog-Shake Behaviors

To evaluate the possible involvement of TRPM8 channel in WTD antinociceptive effect, mice were submitted to a test using icilin, a specific activator of TRPM8, as previously described [25]. Briefly, 12.5 mg/mL icilin was suspended in 1% Tween-80/distilled water and sonicated. Icilin was intraperitoneally administered at a concentration of 50 mg/kg. Mice were orally pretreated with vehicle (20 mL/kg) or WTD (6.30 g/kg) 1 h prior to the injection of vehicle (10 mL/kg, i.p.) or icilin (50 mg/kg, i.p.). Immediately after the icilin treatment, jumping and wet-dog-shakes (WDS) behaviors were counted for 20 min.


## 2.8. Western Blot Analysis

Mice were sacrificed by cervical dislocation under ether anesthesia 15 days after the WTD and ibuprofen administration. Plantar surfaces of injured hind paw skin were dissected and stored at −80°C for western blot analysis. The expression of TRPV1, TRPA1, and TRPM8 in hind paw skins tissues was determined. The western blot protocol and semiquantitative analysis were carried out following the protocol of our previous study [26]. The following primary antibodies were used: TRPV1 (rabbit polyclonal antibody, dilution 1 : 100, Alomone Labs, Israel), TRPA1 (rabbit polyclonal antibody, dilution 1 : 100, Alomone Labs, Israel), TRPM8 (rabbit polyclonal antibody, dilution 1 : 100, Alomone Labs, Israel), and GAPDH (internal control, rabbit polyclonal antibody, dilution 1 : 1000, Beyotime, China). A goat-anti-rabbit antibody conjugated to horseradish peroxidase (1 : 5000, Thermo Fisher Scientific, USA) was used as the second antibody. TRPV1, TRPA1, and TRPM8 protein levels were normalized against GAPDH. All experiments were done for four times.


## 2.9. Immunohistochemical Staining

The immunohistochemical staining protocol was carried out following the protocol of our previous study [26]. Primary antibodies: TRPV1 (rabbit polyclonal antibody, 1 : 100, Alomone Labs, Israel), TRPA1 (rabbit polyclonal antibody, 1 : 100, Alomone Labs, Israel), and TRPM8 (rabbit polyclonal antibody, 1 : 100, Alomone Labs, Israel); and secondary antibody (goat-anti-rabbit 1 : 100, Boster, China) was used and stained with DAB (Dako, Denmark). The percentage of immunopositive cells was analyzed by counting the number of immunopositive neurons and multiplying by 100/number of total number of neurons. A total of 2 DRGs were measured in each animal, and the number of immunoreactive neuronal profiles was counted in a blinded fashion. The percentage of immunopositive cells was used for statistic index.


## 2.10. Statistical Analysis

Data obtained from the mechanical allodynia and thermal hyperalgesia experiments were analyzed by repeated measures one-way analysis of variance (ANOVA) followed by post hoc Dunnett’s Multiple Comparison Test. The data in immunohistochemical staining experiments were analyzed using the Mann-Whitney test. A value ofP
<
0.05 was taken to be significant.


## 3. Results

### 3.1. WTD Attenuates CFA-Induced Mechanical Hypersensitivity in Mice

To investigate the knowledge on the antinociceptive activity of WTD, a chronic inflammatory model of nociception was used. For this, mechanical hypersensitivity was evaluated from day 1 to day 15 after an intraplantar injection of CFA in mice. As shown in Figure1(a), CFA caused significant mechanical hypersensitivity characterized by the reduced paw withdrawal threshold compared to control group. Oral administration of WTD (1.58, 3.15, and 6.30 g/kg) was able to significantly reverse mechanical hypersensitivity that lasted up to 3 hours in high dose group. The maximum effect was observed 1 h posttreatment. And this antinociceptive effect was maintained while WTD (1.58, 3.15, and 6.30 g/kg) was orally administered daily, until the 15th day posttreatment. Oral administration of ibuprofen (0.14 g/kg) was able to reduce mechanical hypersensitivity with a time-course effect profile similar to 1.58 g/kg WTD administration.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 of WTD 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 (0.14 g/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.
(a)
(b)
(c)To investigate whether WTD has side effects on animals, tolerance experiment was first examined. Our data showed that the prolonged treatment with orally administrated WTD (6.30 g/kg) once a day for 11 days significantly reduced the mechanical hypersensitivity induced by CFA (Figure1(b)). When treatment was interrupted for 2 days, mechanical hypersensitivity in WTD treated group was reestablished. On the 14th day the treatment was restarted, and WTD was able to reduce mechanical hypersensitivity with a time-course effect profile similar to the 1st day. These results excluded the possibility of the development of tolerance effect of WTD. Additionally, body weight of mice was also explored. The results showed that WTD treatment for 15 days did not induce significant body weight change compared with CFA group and also with control group. Conversely, ibuprofen decreased body weight with significance on the 15th day compared to CFA group (Figure 1(c)).


### 3.2. WTD Inhibits CFA-Induced Cold Hypersensitivity in Mice

Hypersensitivity to cold stimulus was evaluated at 1st (0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, and 10 h), 3rd, 5th, 7th, 9th, 11th, 13th, and 15th day after CFA injection. As shown in Figure2, mice that received oral administration of WTD (1.58, 3.15, or 6.30 g/kg) exhibited a reduction in cold hypersensitivity induced by CFA that began at 30 minutes post-WTD treatment and was maintained for more than 6 hours in high dose group. The most pronounced effect was observed 1 h after its administration, when WTD (6.30 g/kg) inhibition reached 70
±
9%. Also, the prolonged treatment with orally administrated WTD (1.58, 3.15, and 6.30 g/kg) once a day for 15 days significantly 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 obvious effect on cold hypersensitivity.Figure 2
Effect of WTD on cold hypersensitivity induced by CFA in mice. 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. On 1st day, evaluations for cold hypersensitivity were done 0.5, 1, 2, 4, 6, 8, and 10 hours posttreatment; all other evaluations were done 1 hour posttreatment. Data are represented as the mean ± SEM (n
=
11). P
*
<
0.05 and P
*
*
*
<
0.001 versus the CFA group, respectively.


### 3.3. WTD Reduces CFA-Induced Heat Hypersensitivity in Mice

Hypersensitivity to heat stimulus was explored in CFA-induced mice. The time-course effect of WTD treatment on heat hypersensitivity was similar to that on mechanical and cold hypersensitivities. This antinociceptive effect began 30 minutes and lasted up to 2 hours after WTD treatment (Figure3). The maximum effect on 1st day was observed at 1 h posttreatment, when WTD (6.30 g/kg) inhibition reached 73
±
8%, and the inhibition on days 13 and 15 was about 100%. Ibuprofen (0.14 g/kg) could significantly inhibit heat hypersensitivity and to the similar extent as 1.58 g/kg WTD 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 6.30 g/kg, resp.), ibuprofen (0.14 g/kg), or water daily for 15 days. On 1st day, evaluations for heat hypersensitivity were done 0.5, 1, 1.5, 2, 3, 4, and 6 hours posttreatment; all other evaluations were done 1 hour posttreatment. Data are represented as the mean ± SEM (n
=
8). P
*
<
0.05, P
*
*
<
0.01, and P
*
*
*
<
0.001 versus the CFA group, respectively.


### 3.4. WTD Suppresses the Activities of TRPs in Mice

To obtain insights into the mechanisms of WTD mediated beneficial effects, the activities of TRPV1, TRPA1, and TRPM8 in mice were investigated. As shown in Figure4(a), the TRPV1 activator capsaicin induced obvious licking and lifting behaviors characteristics of nociception in mice, which were significantly 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 (59
±
6%) is more remarkable than that (47
±
4%) of AMG9810 (Figure 4(a)).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 (2μg/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.
(a)
(b)
(c)As shown in Figure4(b), the TRPA1 activator cinnamaldehyde also induced obvious nociceptive behaviors in mice, and oral administration of WTD (6.30 g/kg) significantly reduced the licking time, lifting time, and licking plus lifting time induced by cinnamaldehyde with similar effect to those of camphor, the TRPA1 antagonist.Intraplantar injection of icilin (30μg/paw, 20 μL) did not induce obvious nociceptive behaviors compared to control group (data not shown), which is consistent with previous report [27]. Then, icilin intraperitoneally 50 mg/kg was used to explore the inhibiting characteristics of WTD on TRPM8 ion channel. As shown in Figure 4(c), icilin induced high numbers of jumping and WDS behaviors, and oral administration of WTD (6.30 g/kg) strikingly decreased these numbers compared to icilin control group, with inhibitions of 78
±
5% for jumping behaviors, 72
±
7% for WDS behaviors, and 78
±
8% for jumping plus WDS behaviors, respectively.


### 3.5. WTD Downregulates the Protein Expression of TRPs in DRG and Skins of Injured Paws from CFA-Induced Mice

Since overexpression of TRP channels is associated with inflammatory hypersensitivity [8–10], the protein expression of TRPV1, TRPA1, and TRPM8 was further examined in CFA-induced mice. Immunohistochemical staining analysis demonstrated that the numbers of TRPV1- and TRPA1-positive neurons in DRGs increased remarkably in CFA group compared to control group, while oral administration of WTD 1.58, 3.15, and 6.30 g/kg decreased the percentage of these positive neurons, with significance in WTD (3.15 and 6.30 g/kg) treated groups (Figure 5). Western blot analysis verified that WTD (1.58, 3.15, and 6.30 g/kg) treatment significantly decreased the production of TRPV1 and TRPA1 in the skins of injured paws following CFA-induced inflammation in a dose-related manner (Figure 6). However, TRPM8 protein expression both in DRGs and skins was not changed significantly after WTD treatment, and this was also true for CFA group.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 the mean ± 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.
(a)
(b)
(c)
(d)Figure 6
Effect of WTD on the expression of TRPV1 (a), TRPA1 (b), and TRPM8 (c) in skins of injured paw of inflammatory pain mice by 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 as the mean ± 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.
(a)
(b)
(c)


## 3.1. WTD Attenuates CFA-Induced Mechanical Hypersensitivity in Mice

To investigate the knowledge on the antinociceptive activity of WTD, a chronic inflammatory model of nociception was used. For this, mechanical hypersensitivity was evaluated from day 1 to day 15 after an intraplantar injection of CFA in mice. As shown in Figure1(a), CFA caused significant mechanical hypersensitivity characterized by the reduced paw withdrawal threshold compared to control group. Oral administration of WTD (1.58, 3.15, and 6.30 g/kg) was able to significantly reverse mechanical hypersensitivity that lasted up to 3 hours in high dose group. The maximum effect was observed 1 h posttreatment. And this antinociceptive effect was maintained while WTD (1.58, 3.15, and 6.30 g/kg) was orally administered daily, until the 15th day posttreatment. Oral administration of ibuprofen (0.14 g/kg) was able to reduce mechanical hypersensitivity with a time-course effect profile similar to 1.58 g/kg WTD administration.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 of WTD 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 (0.14 g/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.
(a)
(b)
(c)To investigate whether WTD has side effects on animals, tolerance experiment was first examined. Our data showed that the prolonged treatment with orally administrated WTD (6.30 g/kg) once a day for 11 days significantly reduced the mechanical hypersensitivity induced by CFA (Figure1(b)). When treatment was interrupted for 2 days, mechanical hypersensitivity in WTD treated group was reestablished. On the 14th day the treatment was restarted, and WTD was able to reduce mechanical hypersensitivity with a time-course effect profile similar to the 1st day. These results excluded the possibility of the development of tolerance effect of WTD. Additionally, body weight of mice was also explored. The results showed that WTD treatment for 15 days did not induce significant body weight change compared with CFA group and also with control group. Conversely, ibuprofen decreased body weight with significance on the 15th day compared to CFA group (Figure 1(c)).


## 3.2. WTD Inhibits CFA-Induced Cold Hypersensitivity in Mice

Hypersensitivity to cold stimulus was evaluated at 1st (0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, and 10 h), 3rd, 5th, 7th, 9th, 11th, 13th, and 15th day after CFA injection. As shown in Figure2, mice that received oral administration of WTD (1.58, 3.15, or 6.30 g/kg) exhibited a reduction in cold hypersensitivity induced by CFA that began at 30 minutes post-WTD treatment and was maintained for more than 6 hours in high dose group. The most pronounced effect was observed 1 h after its administration, when WTD (6.30 g/kg) inhibition reached 70
±
9%. Also, the prolonged treatment with orally administrated WTD (1.58, 3.15, and 6.30 g/kg) once a day for 15 days significantly 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 obvious effect on cold hypersensitivity.Figure 2
Effect of WTD on cold hypersensitivity induced by CFA in mice. 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. On 1st day, evaluations for cold hypersensitivity were done 0.5, 1, 2, 4, 6, 8, and 10 hours posttreatment; all other evaluations were done 1 hour posttreatment. Data are represented as the mean ± SEM (n
=
11). P
*
<
0.05 and P
*
*
*
<
0.001 versus the CFA group, respectively.


## 3.3. WTD Reduces CFA-Induced Heat Hypersensitivity in Mice

Hypersensitivity to heat stimulus was explored in CFA-induced mice. The time-course effect of WTD treatment on heat hypersensitivity was similar to that on mechanical and cold hypersensitivities. This antinociceptive effect began 30 minutes and lasted up to 2 hours after WTD treatment (Figure3). The maximum effect on 1st day was observed at 1 h posttreatment, when WTD (6.30 g/kg) inhibition reached 73
±
8%, and the inhibition on days 13 and 15 was about 100%. Ibuprofen (0.14 g/kg) could significantly inhibit heat hypersensitivity and to the similar extent as 1.58 g/kg WTD 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 6.30 g/kg, resp.), ibuprofen (0.14 g/kg), or water daily for 15 days. On 1st day, evaluations for heat hypersensitivity were done 0.5, 1, 1.5, 2, 3, 4, and 6 hours posttreatment; all other evaluations were done 1 hour posttreatment. Data are represented as the mean ± SEM (n
=
8). P
*
<
0.05, P
*
*
<
0.01, and P
*
*
*
<
0.001 versus the CFA group, respectively.


## 3.4. WTD Suppresses the Activities of TRPs in Mice

To obtain insights into the mechanisms of WTD mediated beneficial effects, the activities of TRPV1, TRPA1, and TRPM8 in mice were investigated. As shown in Figure4(a), the TRPV1 activator capsaicin induced obvious licking and lifting behaviors characteristics of nociception in mice, which were significantly 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 (59
±
6%) is more remarkable than that (47
±
4%) of AMG9810 (Figure 4(a)).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 (2μg/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.
(a)
(b)
(c)As shown in Figure4(b), the TRPA1 activator cinnamaldehyde also induced obvious nociceptive behaviors in mice, and oral administration of WTD (6.30 g/kg) significantly reduced the licking time, lifting time, and licking plus lifting time induced by cinnamaldehyde with similar effect to those of camphor, the TRPA1 antagonist.Intraplantar injection of icilin (30μg/paw, 20 μL) did not induce obvious nociceptive behaviors compared to control group (data not shown), which is consistent with previous report [27]. Then, icilin intraperitoneally 50 mg/kg was used to explore the inhibiting characteristics of WTD on TRPM8 ion channel. As shown in Figure 4(c), icilin induced high numbers of jumping and WDS behaviors, and oral administration of WTD (6.30 g/kg) strikingly decreased these numbers compared to icilin control group, with inhibitions of 78
±
5% for jumping behaviors, 72
±
7% for WDS behaviors, and 78
±
8% for jumping plus WDS behaviors, respectively.


## 3.5. WTD Downregulates the Protein Expression of TRPs in DRG and Skins of Injured Paws from CFA-Induced Mice

Since overexpression of TRP channels is associated with inflammatory hypersensitivity [8–10], the protein expression of TRPV1, TRPA1, and TRPM8 was further examined in CFA-induced mice. Immunohistochemical staining analysis demonstrated that the numbers of TRPV1- and TRPA1-positive neurons in DRGs increased remarkably in CFA group compared to control group, while oral administration of WTD 1.58, 3.15, and 6.30 g/kg decreased the percentage of these positive neurons, with significance in WTD (3.15 and 6.30 g/kg) treated groups (Figure 5). Western blot analysis verified that WTD (1.58, 3.15, and 6.30 g/kg) treatment significantly decreased the production of TRPV1 and TRPA1 in the skins of injured paws following CFA-induced inflammation in a dose-related manner (Figure 6). However, TRPM8 protein expression both in DRGs and skins was not changed significantly after WTD treatment, and this was also true for CFA group.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 the mean ± 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.
(a)
(b)
(c)
(d)Figure 6
Effect of WTD on the expression of TRPV1 (a), TRPA1 (b), and TRPM8 (c) in skins of injured paw of inflammatory pain mice by 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 as the mean ± 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.
(a)
(b)
(c)


## 4. Discussion

WTD is known to be used effectively to treat joint diseases in clinic. Previous studies indicated that WTD possess anti-inflammatory effect [13, 14]; however, its actions on pain remain unclear. Data presented in this study indicate that WTD can potently attenuate hypersensitivities to mechanical, cold, and heat stimuli in CFA-induced mice. Moreover, WTD inhibits the activities of TRPV1, TRPA1, and TRPM8 ion channels and reduces the protein expression of TRPV1 and TRPA1 in DRGs and skins of inflamed paw. Thus, our data show for the first time that WTD possess antinociceptive activity and the possible mechanism of its analgesic action is associated with the inhibition of activities and protein expression of TRP ion channels in inflammatory conditions.Intraplantar injection of CFA to rodents is a widely used model to study persistent chronic inflammatory pain, which mimics a chronic inflammatory condition. A characteristic symptom of this model of nociception is that it displays hyperalgesia to mechanical, cold, and heat stimulation [2, 3]. In this study, we analyzed the effect of WTD on inflammatory pain induced by intraplantar injection of CFA in mice. Our results showed that oral administration of WTD at doses of 1.58~6.30 g/kg (0.25~1-fold equivalent to patient dosage daily) could remarkably reduce mechanical and thermal (cold and heat) hypersensitivities caused by intraplantar CFA injection. Specially, the antinociceptive effect of WTD treatment on cold hypersensitivity is evident, lasted up to 6 h, and with maximum inhibitions of over 95% from day 9 to day 15 after administration in WTD (6.30 g/kg) treated group, partly accounting for its effective treatment for patients with cold pattern of arthromyodynia. It is known that tolerance and other side effects such as body weight loss and ulceration of the gastric mucosa limit analgesic drugs to be used clinically [11, 12]. In our study, the antinociceptive effect of WTD was not susceptible to tolerance and the prolonged treatment of WTD did not cause significant change in body weight of mice. This effect was different from that of ibuprofen, which could induce remarkable body weight loss and gastric mucosa as previously reported [28, 29].The TRP ion channels are highly involved in the transduction and sensitization in primary afferent somatosensory neurons and play an integral role in pain and neurogenic inflammation via sensory nerve activation, at either central or peripheral level [5–7]. Among them, TRPV1 is a heat transducer in normal and pathological conditions, activated by temperatures above 42°C, deleting or inhibiting the activity of this heat-sensitive channel results in reducing inflammatory heat and mechanical hyperalgesia [30–32]. TRPA1 is a cold sensor, activated by temperatures below 17°C and involved in pathophysiological cold hypersensitivity since the use of TRPA1 antisense oligodeoxynucleotide reverses the cold hypersensitivity after CFA-induced inflammation [10, 33]. Meanwhile, TRPA1 channel seems to be important in the maintenance of mechanical hypersensitivity, because TRPA1 knockout mice show deficits in the development of inflammatory mechanical hyperalgesia [9, 34]. TRPM8 is also a cold sensor, deleting or blocking the channel resulting in the reduction of inflammation-induced cold hypersensitivity [35, 36]. Considering the close connections of these ion channels with mechanical and thermal hypersensitivities in inflammatory pain conditions, it is reasonable to think that the antihypersensitivity effect of WTD may be mediated by TRPs. To test this hypothesis, we evaluated the antinociceptive effect of WTD against specific activators of these three ion channels. The results from our study showed that capsaicin, a selective activator of TRPV1, and cinnamaldehyde, a selective activator of TRPA1, both induced spontaneous nociceptive responses in mice, which were reversed by WTD with similar effect to that of antagonist AMG9810 or camphor, respectively. Meanwhile, WTD strikingly reversed the jumping and WDS behaviors to intraperitoneal injection of icilin, a TRPM8 activator. These findings were consistent with our results on CFA model, when WTD could reverse the mechanical, cold, and heat hypersensitivities.It has been demonstrated that the increased levels of TRPV1 and TRPA1 protein in skins, DRG, and trigeminal neurons play a relevant role in the maintenance of inflammatory hyperalgesia [8–10, 37]. In our present study, WTD significantly reduced the protein expression of TRPV1 and TRPA1 both in DRGs and skins of injured paws, which may partly account for WTD antinociceptive action. Similar to previous report [14], our present experiment found that the levels of nerve growth factor (NGF), IL-1β, and TNF-α were inhibited by WTD in skins of the inflamed paws in CFA-induced mice (data not shown). Since NGF is known to increase the expression of TRPV1 and TRPA1 in inflammatory conditions [8–10, 37], and IL-1β and TNF-α inducing the upregulation of NGF in CFA-induced inflamed sites have been identified [38], thus, it seems to be the suppression of these factors by WTD contributing to the downregulation of TRPV1 and TRPA1 expression in DRG or peripheral tissue. Interestingly, an unexpected finding was that WTD treatment did not influence the production of TRPM8 both in DRGs and skins, and this was true for CFA group, supported partly by previous observation [10]. Further studies are needed to clarify the exact role of TRPM8 in the antinociceptive mechanism of WTD.In conclusion, our data offered the convincing evidence for the first time that WTD has an antinociceptive activity in chronic inflammation conditions, attenuating mechanical, cold, and heat hypersensitivity. This antinociceptive effect may result in part from the inhibiting activities of TRPV1, TRPA1, and TRPM8. And the suppression of TRPV1 and TRPA1 protein expression 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.



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*Source: 328707-2015-03-09.xml*