Mother root of Aconitum carmichaelii Debeaux exerts antinociceptive effect in Complet Freund’s Adjuvant-induced mice: roles of dynorpin/kappa-opioid system and transient receptor potential vanilloid type-1 ion channel

Background: Processed Chuanwu (PCW), the mother root of Aconitum carmichaelii Debeauxv, has been widely used as a classic Traditional Chinese Medicine for pain relieve for over two millennia clinically. However, its action on chronic inflammatory pain has not been clarified. Here, we investigated the antinociceptive effect of PCW in complete freund’s adjuvant (CFA)-induced mice and its possible mechanisms associated with opioid system and TRPV1 ion channel. Methods: Male ICR mice were intraplantarly injected with CFA. PCW (0.34, 0.68 and 1.35 g/kg) was orally given to mice once a day for 7 days. Von frey hairs and planter test were assessed to evaluate the antinociceptive effect of PCW. To investigate the participation of dynorphin/opioid system in PCW antinociception, subtype-specific opioid receptor antagonists or anti-dynorphin A antiserum were used. To eliminate other central mechanisms that contribute to PCW antinociception, hot plate (50 °C) test were performed. Further, involvements of TRPV1 in PCW antinociception were evaluated in CFA-induced TRPV1−/− and TRPV1+/+ C57BL/6 male mice, and in capsaicin-induced nociception ICR naive mice pretreated with nor-BNI. Meanwhile, calcium imaging was performed in HEK293T-TRPV1 cells. Finally, rotarod, open-field tests and body temperature measurement were carried out to assess side effects of PCW. Results: PCW dose-dependently attenuated mechanical and heat hypersensitivities with no tolerance, which could be partially attenuated by coadministration of k-opioid receptor antagonist nor-binaltorphimine (nor-BNI) or antidynorphin A (1–13) antiserum. And PCW antinociception was totally erased by pretreatment with nor-BNI in the hot plate test. In addition, PCW antinociception was decreased in TRPV1−/− mice compared to TRPV1+/+ group. And PCW still manifested inhibitory effects in capsaicin-induced nociception with nor-BNI pretreatment. PCW significantly inhibited capsaicin-induced calcium influx in HEK293T-TRPV1 cells. Finally, no detectable side effects were found in naive mice treated with PCW. © 2015 Wang et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Open Access *Correspondence: [email protected]; [email protected] Chao Wang and Danni Sun contributed equally to this article 1 Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, No. 16, Nanxiaojie, Dongzhimennei, Beijing 100700, China Full list of author information is available at the end of the article Page 2 of 13 Wang et al. J Transl Med (2015) 13:284 Background Inflammatory pain after tissue injury results in peripheral and central sensitization in peripheral tissues or spinal cord [1]. Indeed, hypersensitivities to thermal and mechanical stimuli are well documented characteristic symptom of chronic inflammatory pain [1, 2]. This chronic process causes patients to live with disability and continues to impart high health cost, economic loss to society. The mechanisms underlying the inflammatory pain remain to be elucidated. It is well-known that activation of opioid receptors result in the inhibition of chronic inflammatory pain [3]. On the other hand, transient receptor potential (TRP) ion channels in nociceptor peripheral terminals and dorsal root ganglias (DRG), especially TRPV1, contribute to the initiation and maintenance of hypersensitivity under inflammatory pain condition [4], and the deleting or inhibiting activity of TRPV1 leads to reduced inflammatory hyperalgesia [4–6]. Recently, several reports suggested that the activation of opioid receptors via inhibition of adenylyl cyclase suppresses TRPV1 and other nonselective cation currents stimulated by inflammatory agents to induce antinociception [7–9]. Current analgesics aimed to modulate pain transduction and transmission in neurons has limited success [1]. There are two main classes of analgesic drugs: opioids and non-steroidal anti-inflammatory drugs (NSAIDs), whereas opioid analgesics are limited by tolerance, somnolence, respiratory depression, confusion, constipation and addiction [10, 11], NSAIDs are restricted by side effects such as gastrointestinal ulcers, bleeding, myocardial infarction and stroke [3, 12, 13]. And the induction of hyperthermia by TRPV1 antagonists has hampered the development of new drugs as analgesics [14]. Therefore, novel and efficient agents that are lack of central side effects and of adverse effects typical of NSAIDs are needed [3]. There is a growing interest in the utilization of medicinal plants for prevention and treatment of pain. The mother and lateral root of Aconitum carmichaelii Debx, called as “Chuanwu” (CW) and “Fuzi” in Chinese respectively, have been widely used to relieve pain and treat rheumatic arthritis and other inflammatory conditions for over 2000 years in clinic. Previous studies have shown that processed Fuzi, obtained after autoclaving crude Aconiti tuber (Ranunculaceae Aconitum carmichaeli Debeaux; Iwate, Japan) to minimize its toxicity, are efficient in relieving pain in repeated cold stress mice, chronic constriction injury (CCI) neuropathic pain as well as adjuvant articular inflammation in rats [15, 16]. And the analgesic effect of processed Fuzi is mediated by spinal κand μ-opioid receptors in CCI rats and in tailflick test [17–20]. On the other hand, it is found that CW has an anti-arthritic effect in complete Freund’s adjuvant (CFA)-induced arthritis rats [21], and methanol extracts of crude Aconitum roots have anti-inflammatory effects in inhibiting acid-induced vascular permeability and carrageen-induced hind paw edema in mice [22]. However, the action of processed CW (PCW) on chronic inflammatory pain remains unclear. In the present study, we investigated the antinociceptive effect of PCW in CFAinduced cutaneous inflammation and its possible mechanisms associated with opioid system and TRPV1 ion channel were also explored. Methods This study was supported by the Research Ethics Committee of China Academy of Chinese Medical Sciences, Beijing, China (Permit Number: 2015–2028). 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 and International Association for Suicide Prevention (IASP). All efforts were made to demonstrate consistent effects of the drug treatments and minimize the suffering of animals. PCW preparation and UPLC–MS and UPLC‐MS2 analysis PCW preparation PCW was purchased from Beijing Huamiao Chinese medicine Engineering Development Center (Beijing, China) and authenticated by Professor Shilin Hu, China Academy of Chinese Medical Sciences. Hypaconitine and mesaconitine were purchased from the Chinese Authenticating Institute of Material and Biological Products (Beijing, China). Benzoylmesaconine, benzoylhypacoitine, aconitine, and benzoylaconitine were purchased from Lan Yuan Biological technology Co., Ltd. (Shanghai, China). Reserpine as the internal standard was purchased from Sigma (USA). Conclusions: This study shows PCW’s potent antinociceptive effect in inflammatory conditions without obvious side effects. This effect may result from the activation of κ-opioid receptor via dynorpin release and the inhibition of TRPV1. These findings indicate that PCW might be a potential agent for the management of chronic inflammatory pain.