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Cynandione A attenuates neuropathic pain through p38β MAPK-mediated spinal microglial expression of β-endorphin

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Highlights

  • Cynandione A is the principle acetophenone of Cynanchi Wilfordii Radix.

  • Intrathecal injection of cynandione A produced antinociception in neuropathic rats.

  • Cynandione A stimulated β-endorphin expression in spinal microglia.

  • p38β MAPK mediated cynandione A-induced β-endorphin release and antiallodynia.

Abstract

Cynanchi Wilfordii Radix (baishouwu), a medicinal herb, has been widely used in Asia to treat a variety of diseases or illnesses. Cynandione A isolated from C. Wilfordii is the principle acetophenone and exhibits neuroprotective and anti-inflammatory activities. This study aims to evaluate the antihypersensitivity activities of cynandione A in neuropathy and explored its mechanisms of action. Intrathecal injection of cynandione A dose-dependently attenuated spinal nerve ligation-induced mechanical allodynia and thermal hyperalgesia, with maximal possible effects of 57% and 59%, ED50s of 14.9 μg and 6.5 μg, respectively. Intrathecal injection of cynandione A significantly increased β-endorphin levels in spinal cords of neuropathic rats and its treatment concentration-dependently induced β-endorphin expression in cultured primary microglia (but not in neurons or astrocytes), with EC50s of 38.8 and 20.0 μM, respectively. Cynandione A also non-selectively upregulated phosphorylation of mitogen-activated protein kinases (MAPKs), including p38, extracellular signal regulated kinase (ERK1/2), and extracellular signal regulated kinase (JNK) in primary microglial culture; however, cynandione A-stimulated β-endorphin expression was completely inhibited by the specific p38 activation inhibitor SB203580, but not by the ERK1/2 or JNK activation inhibitors. Knockdown of spinal p38β but not p38α using siRNA also completely blocked cynandione A-induced β-endorphin expression in cultured microglial cells. Furthermore, cynandione A-induced antiallodynia in neuropathy was totally inhibited by the microglial inhibitor minocycline, SB203580, anti-β-endorphin antibody, and μ-opioid receptor antagonist CTAP (but not the κ- or δ-opioid receptor antagonist). These results suggest that cynandione A attenuates neuropathic pain through upregulation of spinal microglial expression β-endorphin via p38β MAPK activation.

Introduction

Cynanchi Wilfordii Radix (baishouwu) is an appellative name for the root tubers of Cynanchum wilfordii and Cynanchum auriculatum (The Health Department and National Chinese Medicine Office, 1999). C. Wilfordii has been widely used as a traditional herbal medicine in Asia countries to treat a variety of medical conditions, such as insomnia, anxiety, anemia, senescence, and various geriatric diseases (Koo et al., 2015). In animal experiments, the C. Wilfordii extracts and fractions exhibited various biological functions, including blocking free radicals production (Song and Ding, 1997), enhancing immunity (Gu et al., 1987), reducing serum cholesterols (Niu et al., 1988), and exhibiting anti-tumor (Shan et al., 2005) and gastroprotection activities (Shan et al., 2006). Cynandione A, the principle acetophenone isolated from C. Wilfordii, has shown to attenuate glutamate-induced cytotoxicity and markedly improve neurological deficit scores in the rat model of cerebral ischemia (Yue et al., 2012). Cynandione A also significantly improved survival of mice with lethal endotoxemia (Kim et al., 2015). However, to date, there are no scientific reports published on its antinociceptive property particularly in neuropathy.

Mitogen-activated protein kinases (MAPKs) are a family of evolutionally conserved proteins and include three major members, i.e., p38, extracellular signal regulated kinase (ERK1/2), and c-Jun N-terminal kinase (JNK) for three different signaling cascades (Ji and Suter, 2007, Pyo et al., 1999, Pyo et al., 1998). After being activated via phosphorylation, MAPKs play a critical role in cell signaling and gene expression, particularly in relation to microglial activation (Ledeboer et al., 2005, Sorge et al., 2015, Taves et al., 2013, Tsuda et al., 2004). Lipopolysaccharides (LPS), the main contributing factor to inflammation, can activate several signaling cascades, including MAPKs and the transcription factor NF-κB (Lu et al., 2008, Weinstein et al., 1992, Yang et al., 2000). Accumulated evidence indicates that spinal microglia play a crucial role in initiation and development of neuropathic pain (Jin et al., 2003, Raghavendra et al., 2004, Taves et al., 2013, Wang et al., 2014, Wang et al., 2012) and activated microglia in chronic pain states induce production of different proinflammatory factors (tumor necrosis factor-α (TNF-α), interleukin (IL)-6, IL-1β, and brain-derived neurotrophic factor (BDNF)) (Chauvet et al., 2001, Taves et al., 2013). These cytokines released from activated microglia consequently sensitize the neurons in the spinal dorsal horn by altering the excitatory or inhibitory synaptic transmission, contributing to pain facilitation (Kawasaki et al., 2008). Cynandione A and the extracts of C. Wilfordii were recently reported to reduce LPS-increased plasma levels of proinflammatory cytokines (TNF-α, IL-6 and IL-1β) in septic mice, and nitric oxide and prostaglandin E2 in microglia and macrophages (Kim et al., 2015, Yang et al., 2014). Cynandione A can also significantly inhibit ERK1/2 and p38 phosphorylation (but barely changing JNK activation) and NF-κB activation (Kim et al., 2015). It is thus possible that cynandione A produced antinociception by blockade of proinflammatory cytokine expression via inhibition of MAPK and NF-κB activation.

Microglia are the key cells in overall brain maintenance and act as the main form of active immune defense in the central nervous system. They also exhibit a “protective state” that involves the anti-inflammatory cascades or tissue repair, in addition to their well-known “destructive state” that induces inflammation (Taves et al., 2013). We have recently illustrated that microglia-released opioid peptide transmitters were actively involved in the protective state of microglia. Specifically, glucagon-like peptide-1 (GLP-1) receptors expressed in microglia were a potential molecular target for treatment of chronic pain (Gong et al., 2014c) and ischemia (Jia et al., 2015). Their agonists, including the peptidic agonists GLP-1(7-36) and exenatide, non-peptidic agonist WB4-24, and herbal agonists shanzhiside methylester, geniposide, loganin, morroniside and catalpol, stimulated microglia in the spinal dorsal horn and hippocampus to express and release β-endorphin, which subsequently activated post-synaptic neuronal μ-opioid receptors, leading to antinociception and neuroprotection (Fan et al., 2015, Fan et al., 2016, Gong et al., 2014a, Gong et al., 2014c, Jia et al., 2015, Xu et al., 2017, Zhu et al., 2014). Furthermore, the herb aconitum-derived bulleyaconitine A and bullatine A exhibited antihypersensitivity activity through a direct stimulation of dynorphin A expression in spinal microglia (Huang et al., 2016, Li et al., 2016). Our preliminary data also showed that cynandione A antinociception in neuropathy was blocked by intrathecal injection of the specific opioid receptor antagonist naloxone, suggesting that cynandione A produces antinociception possibly through expression and secretion of opioid peptides from spinal microglia.

In this study, we aimed to investigate whether cynandione A possesses antinociceptive effects in the rat neuropathic pain model induced by tight ligation of L5/L6 spinal nerves. We further explored the mechanisms underlying cynandione A antinociception, particularly the involvement of expression of spinal microglial opioid peptides and proinflammatory cytokines. Our data revealed that cynandione A produced antinociception in neuropathy via β-endorphin overexpression in spinal microglia and subsequent activation of post-synaptic neuronal μ-opioid receptors.

Section snippets

Drugs and reagents

Cynandione A was extracted and purified by Shandong Academy of Sciences and the purity was more than 95% determined using the high performance liquid chromatography. Its molecular weight was verified by a high resolution mass spectrum (Waters Corporation, Milford, MA, USA). Lidocaine and minocycline were purchased from the First Chengdu Pharmaceuticals Group (Chengdu, China) and Yuanye Biotech (Shanghai, China). 5′-Guanidinonaltrindole (GNTI) and SP600125 were from Sigma Aldrich (St. Louis, MO,

Cynandione A attenuated pain hypersensitivity in neuropathy

The antinociceptive effects of spinal cynandione A on mechanical allodynia and thermal hyperalgesia were assessed in L5/L6 spinal nerve-ligated rats. Six groups of neuropathic rats received a single intrathecal injection of 10 μL of the vehicle (10% DMSO and 20% PEG400 in saline) or cynandione A at a dose of 3, 10, 30, 100 or 300 μg. The paw withdrawal thresholds and withdrawal latencies were subsequently (with 10-min interval) measured before, and 0.5, 1, 2, and 4 h after drug injection. After

Discussion

Our current study characterized the antinociceptive effects of cynandione A in spinal nerve ligation-induced neuropathy, which was well developed 2–3 weeks after surgery. Intrathecal injection of cynandione A did not alter normal nociceptive responses in the contralateral paws, whereas it was dose-dependent to attenuate mechanical allodynia and thermal hyperalgesia in the ipsilateral paws, with Emax values of 57% and 59% MPE, ED50 values of 14.9 μg and 6.5 μg, respectively. Our results, for the

Author contributions

Conceived and designed the experiments: YXW, QH, and XW; performed the experiments: QH, XFM, HYW, HL and MLS; provided cynandione A: XW; analyzed the data: YXW and QH; and preparation of the paper: YXW and QH.

Conflict of interest

The authors declare no competing financial interests in this work.

Acknowledgments

This study was supported in part by a grant from the National Natural Science Foundation of China (No. 81673403).

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