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Alpha 7 nicotinic receptor-targeted cinobufagin induces antinociception and inhibits NF-#B signaling pathway in DRG neurons Longsheng Xu, Xiaoping Zhang, Qingli Feng, Ying Zheng, Huadong Ni, Hui Shen, and Ming Yao ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00369 • Publication Date (Web): 24 Sep 2018 Downloaded from http://pubs.acs.org on September 26, 2018
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ACS Chemical Neuroscience
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Alpha 7 nicotinic receptor-targeted cinobufagin induces
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antinociception and inhibits NF-κB signaling pathway in
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DRG neurons
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Longsheng Xu1, Xiaoping Zhang1, Qingli Feng1, Ying Zheng1, Huadong Ni1, Hui Shen1, Ming Yao1*
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Jiaxing University, Jiaxing 314001, China
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* Correspondence and requests for materials should be addressed to M.Y. (email:
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Department of Anesthesiology and Pain Medicine, The First Affiliated Hospital of
[email protected])
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Abstract
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Cinobufagin (CBG) has been shown to have antinociceptive properties. Nevertheless,
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the antinociceptive effect and mechanism of CBG are still unclear. The present study
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was designed to investigate the antinociceptive effect of CBG in the thermal and
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chemical pain models, and further to explore the molecular target and potential signal
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pathway. As shown in the hot-plate test, formalin test, and acetic acid writhing test in
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mice, administration of CBG produced significant antinociceptive activity in a
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dose-dependent manner, and the antinociceptive effect was blocked by intraperitoneal
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pretreatment of methyllycaconitine citrate (an α7 nicotinic receptor antagonist) and
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intrathecal delivery of an α7 nicotinic receptor antagonist siRNA (α7-siRNA).
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Immunofluorescence demonstrated that the α7 nicotinic receptor and IκB/NF-κB were
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co-expressed in primary cultured lumbar DRG neurons. In the chemical pain models
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and primary cultured DRG neurons, Western blot analysis showed that the formation
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of p-IκB and p-NF-κB was regulated by CBG, and the effect of CBG was inhibited by
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α7-siRNA, and ELISA analysis indicated that CBG also regulated the expression of
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inflammatory cytokines through the α7 nicotinic receptor in DRG. These results
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suggest that CBG may activate a7 nicotinic receptor, thereby triggering the inhibition
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of DRG NF-κB signaling pathway, resulting in antinociceptive effect in mice.
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Keywords
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Cinobufagin; antinociceptive; α7 nicotinic receptor; NF-κB signaling pathway; DRG
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neurons; inflammatory cytokines 1
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Introduction
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Prevention and treatment of pain has become an important public health topic. About
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20-30% of the world's population has long been plagued by various types of pain (1).
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Among them, chronic pain in middle-aged and elderly patients accounts for 50-75%
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of the elderly. Sustained pain easily leads to limited mobility, decreased immunity,
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weakened psychological and social function, and more susceptible to other diseases
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(2). Therefore, pain has become a public health problem that seriously affects people's
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quality of life and a social problem that needs to be solved urgently.
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At present, the drugs used for clinical treatment of pain are mainly opioid
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analgesics, non-steroidal anti-inflammatory drugs, anticonvulsants, antidepressants,
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and local anesthetics (3). Although many patients received benefits from these
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treatment, less than 50% of painful symptoms were effectively controlled (4).
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Because of the limitations of the mechanisms of these drugs, existing drugs have
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serious addictiveness, tolerability, side effects, and long-term safety problems (5, 6).
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Therefore, the development of new drugs with non-addictive, mild adverse reactions,
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and strong analgesic effects has become very urgent.
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The development of high-effective and low-toxic analgesics is closely related to
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their molecular targets. The research involving acetylcholine (ACh) receptors
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(including nicotinic receptors and muscarinic receptors) is a new field of interest in
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the development of new drugs. Studies have shown that analgesic-related central
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nicotinic receptor subtypes are mainly α4β2 nicotinic receptors, α7 nicotinic receptors
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(α7nAChR), and α3 nicotinic receptors (7). Studies have shown that intraventricular
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administration of choline can significantly inhibit a variety of acute pain in rats and
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mice and that its effect was mainly mediated by α7nAChR (8). Topical application of
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α7nAChR agonists in peripheral tissues reduced inflammatory pain in animals by
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reducing the release of proinflammatory cytokines, such as anti-tumor necrosis
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factor-α (TNF-α), thereby reducing the inflammatory response (9). Therefore,
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targeting the high-specificity cholinergic receptor subtypes will be a breakthrough in
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the discovery of non-addictive, highly effective, and low-toxic analgesic drug.
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Cinobufagin (CBG), a major bioactive ingredient of the bufanolide steroid
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compounds, extracted from the skin of the toad Bufo bufo gargarizans Cantor (10). It
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has effects of reducing swelling and relieving a variety of pain. Chen et al. found that
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CBG significantly relieved foot cancer pain in a mouse model of plantar pain (11). 2
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ACS Chemical Neuroscience
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There are research reports that CBG plays an important role in relief of cancer pain
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with minor side effects. It can be used for a long-term therapy or in a combination
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with other analgesics in order to reinforce the duration effects. However, its potential
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mechanisms underlying pain relief remain unclear. This severely restricts the further
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development and use of CBG (12, 13).
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Hence, in the present study, a behavioral approach was used to assess the
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antinociceptive effect of CBG in the thermal and chemical pain models. Its possible
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molecular targets and signaling pathways of the analgesic effects were studied. Our
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data revealed that CBG exerted significant antinociceptive effects in the thermal and
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chemical pain models possibly via activating 7nAChR thus thereby triggering the
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inhibition of NF-κB signaling pathway. This paper elucidated the role of 7nAChR and
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NF-κB signaling pathway in CBG-mediated analgesia.
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RESULTS AND DISCUSSION
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Antinociceptive tests
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Hot-plate test
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CBG (0.5-2.0 mg/kg) significantly induced an increase of the latency time of
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licking or jumping compared to the control in the hot-plate test. The antinociceptive
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action maintained 15-75 min and reached a maximum at 30 min. The peak PTEs were
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5.1%, 23.8%, and 41.3% at 0.5, 1.0, and 2.0 mg/kg CBG at 30 min, respectively. The
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antinociceptive action of morphine (3 mg/kg) peaked at 30 min and the PTE was
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37.0%. Repeated-measures ANOVA showed significant differences between the
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groups (p < 0.05, n = 11; Fig. 1A), and the inhibitory effect of CBG showed both
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dose- and time-dependent manner.
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Acetic acid writhing test
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Injection of 0.6% acetic acid (10 ml/kg) induced abdominal constrictions in
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vehicle group. Administration with CBG (0.5-2.0 mg/kg) significantly reduced the
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number of abdominal constrictions by 18.9%, 40.7%, and 55.2%, respectively.
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Aspirin (300 mg/kg) reduced of the number abdominal constrictions by 74.6%. The
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difference was statistically significant (p < 0.05, n = 11; Fig. 1B) when compared to
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the control, and the inhibitory effect of CBG showed dose-dependent manner.
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Formalin test
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As shown in Fig. 2C, 2.5% formalin (20 µl) induced the increased frequency of
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licking or biting in the right hind paw in vehicle group. CBG (0.5-2.0 mg/kg)
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significantly reduced the frequency of licking or biting in the second phase by 28.2%,
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44.1%, and 59.0%, respectively. Aspirin decreased the number by 60.1%. The
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difference was statistically significant (p < 0.05, n = 11; Fig. 1C) when compared to
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the control, and the inhibitory effect of CBG showed dose-dependent manner. In the
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first phase, CBC at 2 mg/kg dose showed a significant effect (Fig.1C) and no effects
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was found in other CBG groups.
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CBG has also been used as an effective traditional Chinese medicine to treat
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conditions like swelling, pain, and heart failure (14). Zhang et al. confirmed that six
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active toad fat-soluble ingredients had analgesic effects, of which CBG was the most
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potent ingredient in the analgesic effect in the acetic acid writhing and hot-plate test
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(15). Jin et al. and Zhou et al. found that CBG could significantly reduce the number
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of writhing in mice and increased the heat threshold in mice (16, 17). In the aspect of
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cancerous analgesia, CBG was the first to be proven to significantly relieve foot pain
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in experimental mice (11). The antinociceptive effect of CBG was detected in the
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current study using the hot-plate test, formalin test, and acetic acid writhing test. The
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antinociceptive effect of CBG was not only consistent with the previous study but
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also dose-dependent.
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Currently, there are a variety of toads or its main components for the compound
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drug for clinical use. Although it has been explained from the perspective of
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traditional Chinese medicine theory, such as clearing away heat and toxic materials,
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endometriosis, ventilation, and analgesia, but molecular pharmacological mechanism
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of its antinociceptive effect is still unknown (18). The present study attempted to
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reveal molecular mechanism of its analgesia.
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Silencing of α7nAChR by siRNA in DRG
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Cell bodies in DRG are often used as in vitro models to study nociception and
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other forms of sensory processing (19). In the current study, DRG neurons were
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selected because the majority of them are involved in nociceptive processing (20). In
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order to select a siRNA oligonucleotide for an efficient knockdown of α7nAChR,
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three siRNA oligonucleotides targeting mouse α7nAChR were co-transfected to
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mouse DRG neurons (Fig.2A) and α7nAChR expression was evaluated by RT-qPCR.
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It was found that 536-siRNA (p < 0.05, n = 3; Fig. 2B) and 731-siRNA (p < 0.01, n = 4
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ACS Chemical Neuroscience
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3; Fig. 2B) had effective inhibition with 731-siRNA of the higher potency (Fig. 2B).
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Therefore, 731-siRNA was selected to knockdown α7nAChR.
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The effects of intrathecal delivery of α7-siRNA on the protein levels of
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α7nAChR in mouse DRG were also analyzed by Western blot. As shown in Fig. 2C,
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24 h after intrathecal injection of α7-siRNA, the α7nAChR protein level in DRG was
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decreased when compared to MC-siRNA control (p < 0.05, n = 3; Fig. 2C).
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There were references suggested that the alpha 7 nAChR agonists, such as
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nicotine, inhibited alpha 7 nAChR-mediated pain in rats and mice acute pain model
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(21, 22). In vitro experiments showed that ACh (a cholinergic neurotransmitter)
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significantly inhibited endotoxin-induced TNF release from peripheral macrophages.
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Nicotine had the same effect (23). In 2000, Borovikova et al. first proposed the
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concept of "cholinergic anti-inflammatory pathway", which controlled the
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inflammatory response through a series of changes in cholinergic neurons (24). This
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regimen is rapid onset, precise positioning, and short-term effect. It is not only an
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important physiological mechanism, but also has been a potential target of therapeutic
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intervention for inflammatory diseases (25).
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Blocking tests for α7nAChR
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Mice were pretreated with normal saline (i.p.), MLA (3 mmol/kg, i.p.) or
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α7-siRNA (10 nmol, i.t.), respectively. 15 min later, (24h for the group of α7-siRNA)
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animals were gavaged with CBG solution or 2.0 mg/kg CBG. The antinociceptive
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effects of CBG were measured in the hot-plate test, acetic acid writhing test, and
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formalin test. CBG had strong antinociceptive activity The PTE was 27.9% at 45 min
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in the hot-plate test, and the inhibitions were 55.2% and 62.4% in the acetic acid
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writhing test and formalin test, respectively. After pretreatment with MLA or
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α7-siRNA, the antinociceptive actions of CBG were significantly reduced. During the
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period from 15 to 60 min after administration, the differences were statistically
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significant between α7-siRNA/CBG, MLA/CBG and control groups in the hot-plate
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test, and the PTE was reduced to 11.4% and 14.9% at 45 min (p < 0.05, n = 11; Fig.
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3A). The antinociceptive effects were reduced to 18.9% and 21.8% in the acetic acid
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writhing test, one-way ANOVA showed significant differences among the groups (p
