Analgesic Effect of Chinese Herbal Formula Hua-Jian-Ba-Du Ointment on Visceral Pain in Mice Induced by Acetic Acid

Abstract

Introduction. Visceral pain is one of the most important pains caused by cancer or other diseases, and most of the medications may lead to tolerance, addiction, and toxic side effects. Hua-Jian-Ba-Du Ointment (HJBDO), which is a commonly used conjugate based on traditional Chinese medicine theory, has been effective against visceral pain. Here, we verify the efficacy and underlying mechanism of HJBDO in an acetic-acid induced visceral pain model. Methods. Mice were subjected to acetic acid with or without HJBDO. Hua-Jian-Ba-Du Ointment at low (7.5 mL/kg•d), moderate (15 mL/kg•d), and high (30 mL/kg•d) dosages was applied on the abdomen, 3 times per day for 3 days. The acetic acid writhing test was used to evaluate antinociception. Interleukin-2 (IL-2) in serum, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and prostaglandin E₂ (PGE₂) in peritoneal fluid were detected by ELISA. 5-hydroxytryptamine (5-HT), norepinephrine (NE), dopamine (DA), and β-endorphin (β-EP) were examined by high performance liquid chromatography and radioimmunoassay, respectively. N-methyl-D-aspartic acid receptor (NMDAR1) and c-fos expressions in both rostral ventromedial medulla (RVM) and spinal dorsal horn were determined by western blot. Results. Hua-Jian-Ba-Du Ointment at 3 dosage levels produced dose-dependent antinociception and shortened the latent time. Hua-Jian-Ba-Du Ointment at high or moderate dosage inhibited the release of TNF-α, IL-6, and PGE₂, as well as increased the release of IL-2. Hua-Jian-Ba-Du Ointment could also increase NE and 5-HT contents and decrease the NE content. No effect of HJBDO at 3 dosages on the DA system was detected. Furthermore, HJBDO could suppress the expressions of NMDAR and c-fos in both RVM and spinal dorsal horn. Conclusion. Our results exhibited the analgesic effect of HJBDO on visceral pain in mice, and this effect might be mediated by the regulation of inflammation and neurotransmitters.

Introduction

Visceral pain constitutes the most common form of clinically treated pain, especially in cancerous pain. When there is local and metastatic spread of cancer, complications arise and pain is an inevitable outcome. ¹ In step 3 of the analgesic ladder recommended by the World Health Organization, morphine is the drug of choice for the management of moderate to severe cancer pain. ² However, emerging data have evidenced that adequate analgesia with morphine was not achieved in 10% to 30% of patients with cancer pain. ³ Although fentanyl provides pain relief and has improved the quality of life of many cancer patients living with chronic pain, ⁴ the medications may lead to tolerance, addiction, and toxic side effects. Currently, no drug that effectively targets a particular mechanism has been developed; however, recent clinical and experimental studies have found that complementary and alternative medicine (CAM) demonstrates a unique functional efficacy in the treatment of visceral pain.^5,6 A recent cross-sectional descriptive survey in Europe indicated that the CAMs more frequently provided to cancer patients were acupuncture (55.3%), homeopathy (40.4%), herbal medicine (38.3%), and traditional Chinese medicine (36.2%). ⁷

As a CAM, traditional Chinese medicine (TCM) has attracted the attention of the medical community and is widely used in the treatment of pain.^8-11 According to the TCM theory, cancer pain refers to body pain caused by poison invasion of the meridian-collateral system or toxin blockage of Qi and blood. The pathogenesis of TCM holds that after several kinds of external evil invasion, toxins gather in the visceral or meridian-collateral system, which cause Qi hoisting disorder, poor blood circulation, and stagnant meridians and eventually lead to condensation and pain. According to the pathogenesis of TCM, Hua-Jian-Ba-Du Ointment (HJBDO), a formula that has been used for visceral pain in cancer patients in the First Teaching Hospital of Tianjin University of Traditional Chinese Medicine, is composed of Jianghuang (Turmeric Rhizoma Curcumae Longae), Wugong (Scolopendra subspinipes mutilans L Koch), Dahuang (Radix et Rhizoma Rhei), Xixin (Herba Asari), Bingpian (Borneol Borneolum), Chuanwu (Common Monkshood Mother Root; Radix Aconiti), and Huangbai (Amur Corktree Bark; Cortex Phellodendri). Hua-Jian-Ba-Du Ointment is formulated based on the theory of clearing away heat and toxic materials, eliminating mass, and relieving sclerosis. Hua-Jian-Ba-Du Ointment was demonstrated to ameliorate cancer pain and improve the quality of life of patients.^12,13 Further multiple-centered clinical trials are being undertaken to confirm its efficacy. However, few experiments have been conducted in animal models with respect to the role of HJBDO in visceral pain. The present study aimed to address the effects and underlying mechanisms of HJBDO with the acetic acid mouse model.

Methods

Animals

Male Kunming mice weighing 25 to 35 g were housed, 4 or 5 per cage, at a controlled temperature (22°C ± 2°C) under a 12-hour light/dark cycle. Animals had free access to food and water and were used for 1 procedure only. Afterward, they were killed under diethylether anesthesia. The experiments were performed in the light cycle between 8 and 12 am. The study protocol was approved by the Institutional Animal Care and Use Committee of Tianjin University of Traditional Chinese Medicine and in accordance with the principles outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Hua-Jian-Ba-Du Ointment Preparation

The composition of HJBDO was listed in Table 1. All of these herbal materials (Heyanling Chinese Herbal Medicine Company, Ltd, Beijing, China) followed the standards described in the Pharmacopoeia of Chinese Medicine. To prepare the herbal Ointment, the mixture of the 7 herbs was boiled in water and filtered to acquire the herbal extract and then mixed with a dibutyl phthalate, polyvinyl formal acetal, ethanol-acetone (1:3) mixture. Using these procedures, the total concentration of the HJBDO should be 0.5249 g/mL based on data from processing and stability studies.

Table 1.

The Components of Hua-Jian-Ba-Du Ointment.

	Chinese Herb Name in Pinyin [Botanic Name]	Part	Human Dose (Raw Herb)
1	Wugong [Scolopendra subspinipes mutilans L Koch]	Dried body of centipede	10 g
2	Dahuang [Radix et Rhizoma Rhei]	Rhizome and root	10 g
3	Jianghuang [Turmeric Rhizoma Curcumae Longae]	Rhizome and root	10 g
4	Xixin [Herba Asari]	Rhizome and root	10 g
5	Bingpian [Borneol Borneolum]	Processed products of dipterocarp resins	10 g
6	Chuanwu [Common Monkshood Mother Root Radix Aconiti]	Parent root	10 g
7	Huangbai [Amur Corktree Bark Cortex Phellodendri]	Bark	10 g
Total	7 herbs	NA	70 g

Drugs and Animal Treatment

Acetic acid (Sigma, St. Louis, Missouri, USA) was freshly dissolved in distilled water and used to induce visceral pain. Abdominal hair about 3 cm in diameter was removed from mice, which were randomly divided into 5 groups: control group, model group, low-dosage HJBDO treatment group, moderate-dosage HJBDO treatment group, and high-dosage HJBDO treatment group. Mice in the moderate-dosage HJBDO treatment group were applied with 15 mL/kg•d Ointment, 3 times per day for 3 days, which was calculated according to the body surface area from the daily human clinical dosage. The animals in the high-dosage and low-dosage groups received HJBDO in the same manner, but at a dose 2-fold that of moderate and half that of moderate, respectively (30 mL/kg•d, 7.5 mL/kg•d).

Acetic Acid-Induced Writhing

While placed in individual transparent boxes, the animals were allowed to habituate to the laboratory surroundings. Acetic acid (0.6%) was injected intraperitoneally (10 mL/kg of body weight) 30 minutes after the last administration of HJBDO, and the number of writhings (as a measure of visceral pain) was counted for 30 minutes by 2 separate lab assistants. Their latency was defined as writhing for the first time (latent time). Writhing inhibition rate (%) was calculated by the formula: (mean writhing number in the model group – mean writhing number in the HJBDO group) / mean writhing number in the model group × 100%. Nine animals were used in each experimental group.

Enzyme-Linked Immunosorbent Assay

Mouse serum and peritoneal fluid were prepared from peripheral blood and peritoneal lavage fluid. Interleukin-2 (IL-2) level in serum and tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and prostaglandin E₂ (PGE₂) levels were measured using a mouse enzyme-linked immunosorbent assay (ELISA) kit (Abcam, Cambridge, UK) according to the instructions. The concentrations of IL-2, TNF-α, IL-6, and PGE₂ were calculated according to the standard curve. Five animals were used in each experimental group.

Radioimmunoassay Assay

The dorsal raphe nucleus tissue of mice was removed and homogenized with 1 mol/L glacial acetic acid, followed by a centrifugation of 3000×g at 4°C for 20 minutes. A β-endorphin (β-EP) ¹²⁵I radioimmunoassay kit (Sino-UK Institute of Biological Technology, Beijing, China) was used to detect the concentration of β-EP according to the instructions. Five animals were used in each experimental group.

High-Performance Liquid Chromatography

The concentrations of norepinephrine (NE) in the nucleus ceruleus, dopamine (DA), and 5-hydroxytryptamine (5-HT) in the hypothalamus were detected by the Eicom HTEC 500 high-performance liquid chromatography system. The standard solutions of NE (MW 337.3), DA (MW 158.17), 5-HT (MW 212.68), and isoproterenol (internal standard; MW 247.7) with 1 ng/mL concentration were prepared by serial dilution. Hippocampus tissue was homogenized with 0.2 M perchloric acid including 100 uM EDTA-2Na at 4°C. The following working conditions were maintained in the HPLC system described by Wilson. ¹⁴ The levels of neurotransmitters were represented as ng/mg of wet tissue. Five animals were used in each experimental group.

Western Blot

N-methyl-D-aspartic acid receptor (NMDAR1) and c-fos expression levels in the rostral ventromedial medulla (RVM) and spinal dorsal horn sections (T13-L2 and L6-S2) were analyzed by Western blot according to standard protocols. In brief, RVM tissue was washed with ice-cold phosphate buffered saline and scraped in lysis buffer. The insoluble material was removed by centrifugation at 12,000×g for 20 minutes. Twenty micrograms proteins were processed bysodium salt-polyacrylamide gelelectrophoresis (SDS-PAGE) separation in 12.5% gel and transferred to a 0.45 μm nitrocellulose membrane. Nonspecific binding sites were blocked with tris buffer saline (TBS) containing 5% nonfat dry milk for 1 hour at 37°C. The membrane was then incubated with primary antibodies (1:1000 dilution) against NMDAR1 receptor and c-fos, or against β-actin as controls, followed by incubation with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies (1:10,000 dilution). Immunoreactive proteins were detected by enhanced chemiluminescence according to the instructions of the manufacturer (Pierce, Rockford, Illinois, USA). Three animals were used in each experimental group.

Statistical Analysis

Data are represented as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was used to determine statistically significant differences among the groups. A P value of < .05 was considered to be statistically significant.

Results

The Antinociceptive Effects of HJBDO in Acetic Acid-Induced Writhing

Initially, we displayed the antinociceptive effects of HJBDO in mice. We observed that for analgesia, the number of writhings (Figure 1A) and the inhibitory rate (Figure 1B) in HJBDO-L, HJBDO-M, and HJBDO-H treated groups showed dose-dependent alteration (P < .05, P < .01, P < .001, respectively) during the 30-minute acetic acid writhing test, with the HJBDO-H treated group having the fewest writhings. Figure 1C shows that the latency time after treatment with HJBDO differed significantly from the 7.5 mL/kg•d (229.5 ± 57.84 seconds) group to the 30 mL/kg•d (404 ± 70.34 seconds) group, compared to the model group.

Figure 1.

(A) The number of writhings treated by Hua-Jian-Ba-Du Ointment (HJBDO) with low (7.5 mL/kg•d), moderate (15 mL/kg•d), and high (30 mL/kg•d) dosages. (B) The antinociceptive effects (%Es) produced by HJBDO. (C) Changes in the latency of acetic acid-induced writhing in mice treated with different doses of HJBDO. All data are presented as mean ± standard error. N = 9. The experiment was independently repeated 3 times. *P < .05 versus model group; **P < .01 versus model group; ***P < .001 versus model group.

HJBDO Regulated IL-2, TNF-α, IL-6, and PGE₂ Levels in Serum or Peritoneal Fluid

Inflammatory and anti-inflammatory factors in serum or peritoneal fluid were detected to evaluate the mechanism of antinociceptive effects of HJBDO in vivo. As shown in Figure 2, acetic acid significantly decreased the secretion of IL-2 in serum (Figure 2A: 0.62-fold, P < .05) and obviously increased the contents of TNF-α, IL-6, and PGE₂ in peritoneal fluid above basal levels (Figure 2B: TNF-α, 3.4-fold, P < .001; Figure 2C: IL-6, 2.63-fold, P < .001; Figure 2D: PGE₂, 1.58-fold, P < .01). The low dose of HJBDO could not reverse the levels of IL-2 and TNF-α, with no significant difference compared with the model group, whereas the moderate or high dose of HJBDO suppressed the levels of IL-2 and TNF-α significantly (P < .05 or P < .01). For IL-6 and PGE₂, only high dose HJBDO could remarkably inhibit their secretion (P < .05), with a reduction of 73.1% and 74%, respectively.

Figure 2.

After acetic acid injection for 30 minutes, blood was taken from the mice and serum was separated for the detection of (A) interleukin-2 (IL-2) level, and the peritoneal fluid was collected for the examination of (B) tumor necrosis factor-α level, (C) IL-2 level, and (D) prostaglandin E₂ level in different doses of Hua-Jian-Ba-Du Ointment by enzyme-linked immunosorbent assay. All data are presented as mean ± standard error. N = 5. The experiment was independently repeated 3 times. #P < .05 versus control group; ##P < .001 versus control group; ###P < .001 versus model group; *P < .05 versus model group; **P < .01 versus model group.

HJBDO Modulated Monoamine Neurotransmitters in Brain Tissues

β-EP has also been reported to be involved in visceral pain. Therefore, we investigated its concentration in dorsal raphe nucleus tissue. The concentration of β-EP was dramatically decreased after acetic acid induction (P < .05), which was improved by HJBDO with a moderate or high dose other than low dose (Figure 3A). For the neurotransmitters in the nucleus ceruleus and hypothalamus of the model group, NE (Figure 3C) and DA (Figure 3B) levels were notably increased (P < .001), whereas the 5-HT level in the hypothalamus (Figure 3D) was decreased (P < .01). The concentrations of the above 3 neurotransmitters were reversed considerably by HJBDO with a moderate (P < .01, P < .001, respectively) or high dose (P < .05, P < .001, respectively); this did not appear in the low dose group.

Figure 3.

The dorsal raphe nucleus was separated from the brain tissue for the confirmation of β-endorphin (A) by radioimmunoassay assay. The hypothalamus or nucleus ceruleus section was collected for the determination of (B) dopamine, (C) norepinephrine, and (D) 5-hydroxytryptamine by high performance liquid chromatography. All data are presented as mean ± standard error. N = 5. The experiment was independently repeated 3 times. #P < .05 versus control group; ##P < .001 versus control group; ###P < .001 versus model group; *P < .05 versus model group; **P < .01 versus model group.

HJBDO Down-regulated the NMDAR1 and c-fos Expressions in Both RVM and Spinal Dorsal Horn

Protein levels of NMDAR1 and c-fos in both RVM and spinal dorsal horn sections were investigated. As shown in Figures 4B and 4C, levels of NMDAR1 in both RVM and spinal dorsal horn sections were both up-regulated in the model group by 2.29-fold (P < .01) and 3.65-fold (P < .01), respectively, compared with the control in response to acetic acid. Compared with the model group, HJBDO did not down-regulate the level of NMDAR1 at low doses, whereas a high or moderate dose HJBDO reduced the NMDAR1 level in both RVM and spinal dorsal horn significantly (Figures 4A-4C) (P < .05). Data from immunostaining and quantification of c-fos are shown in Figures 4D to 4F. For the c-fos expression in the spinal dorsal horn sections, c-fos expression treated by HJBDO declined obviously from 7.5 mL/kg•d to 30 mL/kg•d (P < .05) in a dose-dependent manner. For c-fos expression in the RVM, few differences were observed between the model and the low or moderate dose groups, indicating that HJBDO at these doses had no inhibitory effect on c-fos. Quantitative analysis revealed statistically significant down-regulation of c-fos in the high dose HJBDO treated groups compared with the model group (P < .05).

Figure 4.

Immunoblots of both rostral ventromedial medulla (RVM) and spinal dorsal horn were probed with anti-N-methyl-D-aspartic acid receptor (anti-NMDAR1) (A), anti-c-fos (D) antibodies, whereas the anti-β-actin antibody served as the loading controls. Bar graphs showed the densities of NMDAR1 bands in spinal dorsal horn (B) and RVM (C), as well as the c-fos bands in spinal dorsal horn (E) and RVM (F). Values represent the mean relative density relative to the loading control. All data are presented as mean ± standard error. N = 3. The experiment was independently repeated 3 times. ##P < .001 versus control group; *P < .05 versus model group; **P < .01 versus model group.

Discussion

The prevalence of pain among patients with cancer has brought great suffering and mental stress to patients. Adequate analgesia was achieved by 45% to 100% of the patients with cancer pain who were evaluated using the World Health Organization ladder. ¹⁵ It has been suggested that effective cancer pain management should be multidisciplinary and multimodal, use combination therapies, and be individualized with the aim of optimizing pain relief with minimization of adverse effects.^16-18 Complementary and alternative medicine, such as TCM, is usually used as combined therapies in pain relief.^19,20

We first demonstrated the analgesic effect of HJBDO in a mouse model induced by acetic acid. In agreement with the outcome of clinical observation in our hospital, we found that administration of a clinically equivalent dose of HJBDO before the onset of acetic-acid induction significantly decreased the number of writhings, increased the inhibitory rate, and prolonged the latency time, which suggested that this formula was an effective and safe strategy for visceral pain. Hua-Jian-Ba-Du Ointment, a formula that has been used for visceral pain in cancer patients, is composed against the pathological changes of cancer pain including poor blood circulation, occlusion, and condensation. Separately, Turmeric Rhizoma (Curcumae Longae) and Scolopendra subspinipes mutilans L Koch resolve hard lumps, promote blood circulation, and remove blood stasis; Radix et Rhizoma Rhei eliminates toxins; Herba Asari and Borneol Borneolum, characterized as pungent and fragrant, smooth meridians and facilitate transdermal absorption. Common Monkshood Mother Root (Radix Aconiti) eliminates cold to stop pain, and Amur Corktree Bark (Cortex Phellodendri) clears away heat and toxic material. As a bicyclic monoterpene, Bingpian has been evidenced to possess significant central and peripheral antinociceptive activity due to its anti-inflammatory activity. ²¹ In addition, the pharmacological effect of the major components of these herbs also supports its application for analgesia. Asarum volatile oil, a major component of Herba Asari, has been reported to have analgesic effects mediated by modulating the content of NO, PGE₂, and MDA. ²² Atisine alkaloids, the major constituents of Common Monkshood Mother Root, carried out a naloxone-dependent antinociceptive effect through opioid receptor modulation. ²³ It is most likely that desirable effects of the 4 herbs are synergistic in HJBDO. The relative contribution of each herb to the outcome needs to be clarified.

Visceral pain transmission is characterized by afferent fibers that are normally unresponsive to stimuli but become activated in the inflammatory process. ²⁴ Intraperitoneal injection of acetic acid represents a mixed model and both activates visceral pain and induces inflammation in the subcutaneous and muscular layers of the abdominal wall and the subdiaphragmatic visceral organs. ²⁵ Many inflammatory cytokines such as TNF-α and IL-6 are involved in the process of pain.^26,27 To further elucidate the inflammatory mechanism of HJBDO at different intervention doses, proinflammatory cytokines and anti-inflammatory cytokines in the serum and peritoneal fluid were examined. It was observed that secretion of proinflammatory cytokines TNF-α, IL-6, and PEG₂ increased above basal levels, whereas the anti-inflammatory cytokine IL-2 was restricted to different degrees in response to acetic acid damage. A low dose of HJBDO could not reverse inflammatory cytokine changes. Per contra, moderate to high doses of HJBDO could regulate inflammatory cytokines to different levels in both serum and peritoneal fluid, indicating a possible mechanism of analgesic effect of HJBDO.

Furthermore, we investigated the central mechanism for the analgesic effect of HJBDO. β-endorphin, the major endogenous opioid peptide, is an agonist of the opioid receptors and is therefore considered to be the main transmitter for the analgesic system.^28,29 As an analgesic substance, β-EP in dorsal raphe nucleus tissue could be improved by HJBDO at a moderate or high dose, which indicated that HJBDO may mimic the analgesic substance. It is noteworthy that the abnormal secretion of neurotransmitters (including 5-HT and NE) weakens the descending inhibitory effect of the modulation of nociceptive transmission. Meanwhile, the sensitivity of the dopamine system plays the same role in pain modulation. ³⁰ These abnormal phenomena were also observed in an animal model with pain. ³¹ 5-hydroxytryptamine in the central nervous system has analgesic effects, in which downward conductive fiber has synaptic connections with β-endorphin neurons at the spinal cord dorsal horn. The results obtained in the present study show that HJBDO improves the 5-HT content, which is beneficial to exert its analgesic effect. Combined with the result of β-EP, we can speculate that HJBDO-induced 5-HT may lead to β-EP secretion to the opium receptor and form presynaptic inhibition, followed by blocking the excitatory conduction substance P, thus reducing the pain impulses. Norepinephrine in the nucleus ceruleus was also considerably decreased by HJBDO, which may be due to blocking NE uptake and inhibiting NE reuptake as well as to interaction with the NE receptor. The increased DA cannot be reversed by HJBDO, suggesting that the nociceptive modulation of HJBDO is rarely associated with the dopamine system.

Another neurotransmitter involved in the analgesic effects on visceral pain is glutamate. The concentration of the excitatory amino acid glutamate in acetic acid-induced writhing was increased in the spine. ³² Over the past few years, the involvement of NMDA in the transmission of nociceptive information, including the development of central sensitization and wind-up phenomena, has increased interest in NMDA receptor antagonists as antinociceptive drugs. ³³ C-fos protein is activated considerably in the spinal dorsal horn by noxious stimulation in the peripheral somatic and visceral and has been widely used to study the level of spinal cord nociceptive transmission and its pharmacological and physiological mechanisms. ³⁴ The RVM has been well documented to contribute both inhibitory and facilitatory influences on spinal nociceptive transmission. ³⁵ In our present article, the NMDAR1 and c-fos expression after acetic acid injury were both up-regulated in both spinal dorsal horn and RVM, indicating a facilitatory influence on spinal nociceptive transmission. Hua-Jian-Ba-Du Ointment at a moderate or high dose could remarkably suppress the NMDAR1 expression, suggesting that the enhanced signal of the NMDA receptor at spinal dorsal horn neurons may be inhibited by HJBDO. According to this result, we speculate that HJBDO has a similar mechanism of analgesic effect to NMDA receptor antagonists. Furthermore, c-fos in the RVM could be inhibited by HJBDO from low to high doses, indicating that HJBDO partially alleviated downward nociceptive information.

In conclusion, HJBDO exhibited an analgesic effect on visceral pain in mice, which is in line with our clinical observation. The observed beneficial role of HJBDO and this effect might be attributable to the regulation of neurotransmitters and c-fos in the brain. Further investigations are required to identify the mechanisms behind the HJBDO analgesia.