Elsevier

Journal of Ethnopharmacology

Volume 195, 4 January 2017, Pages 81-88
Journal of Ethnopharmacology

Analgesic effect of coumarins from Radix angelicae pubescentis is mediated by inflammatory factors and TRPV1 in a spared nerve injury model of neuropathic pain

https://doi.org/10.1016/j.jep.2016.11.046Get rights and content

Abstract

Ethnopharmacological relevance

Coumarins from Radix angelicae pubescentis (CRAP) are a major active component that are isolated from dried roots of Angelica biserrata Yuan et Shan, which has been used clinically to cure headaches for a long period of time, and it is an effective treatment for pain. The aim of the present study was to investigate the analgesic effect of CRAP on a spared nerve injury (SNI) model of neuropathy.

Materials and methods

Antinociceptive effects of CRAP were assessed in Sprague-Dawley male rats using a spared nerve injury model of neuropathic pain. Inflammatory factors were determined by Enzyme-linked immunosorbent assay (ELISA). Transient receptor potential cation channel 1 (TRPV1) and Phosphorylated extracellular regulated protein kinases (pERK) were detected by Immunofluorescence and Western blotting, respectively.

Results

The high performance liquid chromatography (HPLC) analysis showed the presence of osthole and columbianadin in Radix angelicae pubescentis. CRAP induced the dose-dependent effect of on attenuating the development of mechanical hypersensitivity. Molecular profiling revealed that CRAP reduced the levels of pro-inflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) and significantly attenuated the expression of TRPV1 and pERK in damaged DRG neurons.

Conclusion

This results demonstrate that CRAP possess remarkable antinociceptive activities which may be due to osthole and columbianadin at least in part, supporting the folkloric usage of the plant to treat various pain diseases.

Introduction

Primary nervous system damage or neurological dysfunction may cause neuropathic pain, which is a devastating neurological disease that seriously affects patients’ quality of life (Costigan et al., 2009, Treede et al., 2008, Dworkin et al., 2010). Current clinical medications for neuropathic pain relief primarily include antiepileptics, opioid analgesics, antidepressants, and topical lidocaine (Xu et al., 2012, Xu et al., 2012, Dworkin et al., 2007, Rice and Hill, 2006). The treatment options for neuropathic pain syndromes are not entirely effective and produce a myriad of side effects, including drug addiction, constipation, orthostatic hypotension, misuse, somnolence and hyperalgesia (Stacey and Swift, 2006, Dharmshaktu et al., 2012, Chou et al., 2009). Therefore, it is necessary to explore new pharmacological tools for the treatment of neuropathic pain.

Radix angelicae pubescentis isolated from the dried roots of Angelica biserrata Yuan et Shan belong to the Umbelliferae family. Radix angelicae pubescentis was initially described in Shennong Ben Cao Jing as a traditional Chinese medicine to cure rheumatism via the elimination of inflammation and the alleviation of pain with a long history (Li et al., 2015). Radix angelicae pubescentis is used clinically in China to cure headache caused by cold weather, rheumatism and lassitude (Chang et al., 2014). Previous studies revealed that the ethanol extracts of Radix angelicae pubescentis possess potent analgesic activity. These extracts extend the incubation period of mouse writhing induced by acetic acid and prolong the tail-curl latency in mice tail immersion tests compared to aspirin (Li et al., 2013). Courmarin, which is the main active ingredient of Radix angelicae pubescentis, has been proved to be effective on pain (Park et al., 2013). CRAP are primarily composed of osthole, columbianadin, columbianedin, columbianetin, columbianetin acetate, xanthotoxin and bergapten. However, whether CRAP exerts protective effects and alleviates neuropathic pain is not known.

Pain is classically transmitted from sensory nerve endings to the dorsal root ganglion (DRG), spinal cord and cerebrum (Chizh and Illes, 2001, Glombiewski et al., 2010). Neuropathic pain may arise from discharge at the site of axonal injury and spontaneous activity in DRG neurons (Ma et al., 2003, Thakor et al., 2009, Wu et al., 2001). Numerous inflammatory cytokines initially gather in the injured tissue, lower activation thresholds in nociceptors produces neuropathic pain (Xu et al., 2012, Xu et al., 2012Xu et al., 2012, Xu et al., 2012). Numerous studies demonstrated that inflammation and the immune response play important roles in neuropathic pain. Pro-inflammatory cytokines, such as TNF-α, IL-1β and IL-6, exhibit temporal up-regulation, which leads to a series of changes in DRG neurons that contribute to the development of neuropathic pain after nerve injury (Cao et al., 2015, Hwang et al., 2005). TRPV1 is a member of the TRP family proteins, and it is widely distributed in DRG neurons. TRPV1 is involved in pain transduction, transmission, perception and modulation (Hwang et al., 2005, Jara-Oseguera et al., 2008, Starowicz et al., 2008). Activated TRPV1 facilitates cation flux from the extracellular to intracellular space, which leads to the release of substance P and calcitonin-gene related peptide (CGRP) and pain generation (Szallasi and Sheta, 2012). TRPV1 expression is down-regulated in damaged DRGs after peripheral nerve injury, but it is up-regulated in the undamaged population (Fukuoka et al., 2002, Hudson et al., 2001). Numerous endogenous and exogenous signals regulate TRPV1 activity and promote pain generation or sensitization, such as G protein-coupled receptors, tyrosine kinase receptors, protein kinase C (PKC), and mitogen-activated protein kinases (MAPKs), which are associated with TRPV1 sensitization (Ferrari et al., 2014, Ji et al., 2002, Palazzo et al., 2012). ERK modulation of TRPV1 activity plays a critical role in pain perception, and it has been widely studied in various pain models (Jin et al., 2003, Svensson et al., 2003). ERK phosphorylation is necessary to maintain the cytokine-mediated TRPV1 sensitization in sensory neurons (Hensellek et al., 2007). It is reported that coumarin derivative specifically activate the nociceptor TRPV1 and reverse the inflammatory pain in mice through channel desensitization (Wei et al., 2016). However, how CRAP act on pain control is still not clear.

This study examined the relationships between CRAP and pain processing in a SNI model of neuropathic pain. We also measured the expression of kinases that influence TRPV1 sensitization and correlated changes in TNF-α, IL-1β and IL-6 expression during the development of allodynia. These studies provide a mechanistic framework for further studies of the use of CRAP as an effective treatment for neuropathic pain.

Section snippets

Drugs and agent

Radix angelicae pubescentis was purchased from the Xijing hospital pharmacy. CRAP was prepared in our laboratory. Briefly, Radix angelicae pubescentis was ground into a powder and 10 g powder was extracted with 500 mL ethanol twice for 2 h under ultrasonic oscillation at room temperature (25 °C). The ethanol was retrieved, and the extract was concentrated in a Rotary evaporation instrument for approximately 0.5 h at 60 °C and transferred to an evaporating dish. The dish was placed in a water bath at

HPLC analysis of CRAP

In the present study, firstly we carried out the qualitative and quantitative analysis of CRAP. With RP-HPLC, peaks of CRAP were compared with chemical marks, osthole (1, Rt: 19.812 min), and columbianadin (2, Rt: 23.476 min) were proved as the main components of CRAP (Fig. 1). Quantitative results of two constituents and method validation were seen in Table 1, Table 2, Table 3.

Linearity and calibration curves. Linearity of four compounds calibration curves was established by calculating the

Discussion

This study reports the anti-nociceptive effects and molecular mechanisms of action of CRAP. Behavioral testing demonstrated that CRAP administration alleviated mechanical allodynia in neuropathic rats. CRAP also suppressed the production of pro-inflammatory cytokines. We also demonstrated that the increased abundance of TRPV1 in damaged DRG neurons was involved in the anti-nociceptive effect of CRAP. CRAP significantly attenuated pERK expression, which is a known downstream effector in the MAPK

Conclusions

CRAP significantly prevented neuropathic pain and attenuated the development of mechanical hypersensitivity induced by SNI. This study suggests that the anti-nociceptive activities of CRAP are associated with pro-inflammatory cytokines (TNF-α, IL-1β and IL-6), TRPV1 and pERK in peripheral nervous pain systems. Therefore, CRAP is a promising candidate as a therapeutic anti-nociceptive agent.

Conflict of interest

The authors declare that there are no conflicts of interest in this paper.

Acknowledgments

This work was supported by a grant from the National New Drug “R&D” Project (No. 2011ZXJ09302). The authors are grateful for the proofreading of this manuscript by Mr. Zongtao Lin in the Department of Pharmaceutical Sciences, University of Tennessee Health Science Center and Mr. Bo Wang in the Department of Developmental Neurobiology, St. Jude Children's Research Hospital.

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