Sinomenine produces peripheral analgesic effects via inhibition of voltage-gated sodium currents

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SINOMENINE PRODUCES PERIPHERAL ANALGESIC EFFECTS VIA INHIBITION OF VOLTAGE-GATED SODIUM CURRENTS

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JEONG-YUN LEE, ay SEO-YEON YOON, a,by JONGHWA WON, a HAN-BYUL KIM, a YOUNGNAM KANG b,c AND SEOG BAE OH a,b*

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Department of Brain and Cognitive Sciences, College of Natural Sciences, Seoul National University, Seoul, Republic of Korea b

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Dental Research Institute and Department of Neurobiology & Physiology, School of Dentistry, Seoul National University, Seoul, Republic of Korea

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Department of Neuroscience and Oral Physiology, Osaka University Graduate School of Dentistry, Osaka, Japan

Abstract—Sinomenium acutum has been used in traditional medicine to treat a painful disease such as rheumatic arthritis and neuralgia. Sinomenine, which is a main bioactive ingredient in Sinomenium acutum, has been reported to have an analgesic effect in diverse pain animal models. However little is known about the detailed mechanisms underlying peripheral analgesic effect of sinomenine. In the present study, we aimed to elucidate its cellular mechanism by using formalin-induced acute inflammatory pain model in mice. We found that intraperitoneal (i.p.) administration of sinomenine (50 mg/kg) suppressed formalin-induced paw licking behavior in both the first and the second phase. Formalin-induced c-Fos protein expression was also suppressed by sinomenine (50 mg/kg i.p.) in the superficial dorsal horn of spinal cord. Whole-cell patch-clamp recordings from small-sized dorsal root ganglion (DRG) neurons revealed that sinomenine reversibly increased the spike threshold and the threshold current intensity for evoking a single spike and decreased firing frequency of action potentials evoked in response to a long current pulse. Voltagegated sodium currents (INa) were also significantly reduced by sinomenine in a dose-dependent manner (IC50 = 2.3 ± 0.2 mM). Finally, we confirmed that intraplantar application of sinomenine suppressed formalin-induced pain behavior only in the first phase, but not the second phase. Taken together, our results suggest that sinomenine has a peripheral analgesic effect by inhibiting INa. Ó 2017 IBRO. Published by Elsevier Ltd. All rights reserved.

Key words: pain, analgesia, sinomenine, formalin test, c-Fos, voltage-gated sodium channel. *Correspondence to, S.B. Oh: Department of Neurobiology & Physiology, School of Dentistry, Seoul National University, 101 Daehak-ro, Jongno-gu, Seoul 03080, Republic of Korea. E-mail address: [email protected] (S. B. Oh). y These authors contributed equally to this work. Abbreviations: APs, action potentials; DRG, dorsal root ganglion; INa, voltage-gated sodium currents; VGSCs, voltage-gated sodium channels. http://dx.doi.org/10.1016/j.neuroscience.2017.06.024 0306-4522/Ó 2017 IBRO. Published by Elsevier Ltd. All rights reserved. 1

INTRODUCTION

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Sinomenium acutum has been traditionally used as a herbal medicine to treat rheumatic arthritis, arrhythmia and neuralgia (Zhao et al., 2012). Sinomenine is its main bioactive ingredient in Sinomenium acutum, which is well known to have immunosuppressive and anti-inflammatory effects (Wang and Li, 2011). Interestingly, recent studies have also demonstrated analgesic effects of sinomenine. Sinomenine has been reported to have analgesic effects on neuropathic pain models as well as inflammatory pain models (Gao et al., 2013). Sinomenine-induced analgesia is reversed by a GABAA receptor antagonist in neuropathic pain models (Zhu et al., 2014). Apart from the pathological animal pain models, tail flick test shows that sinomenine has analgesic effects by activating opioid lreceptor (Wang et al., 2008). While these studies have found potential cellular targets for sinomenine in the central nervous system (CNS) (Zhu et al., 2016), little is known about the cellular mechanism or sinomenineinduced peripheral analgesia. Primary afferent nociceptive neurons such as Ad and C-fibers convey pain signals from peripheral injury sites to the CNS. Voltage-gated sodium channels (VGSCs) are the main ion channels for generating action potentials, thereby responsible for transmitting pain signals in the nociceptive neurons (Mathie, 2010; Waxman and Zamponi, 2014; McEntire et al., 2016). Thus, VGSCs in primary afferent neurons are the key molecular target for diverse analgesics including local anesthetics such as lidocaine (Inan et al., 2009). In line with this, we previously showed that eugenol, the main component in clove plant, has local anesthetic actions like lidocaine in periphery by inhibiting voltage-gated sodium currents (INa), thereby providing a pharmacological mode of action for their wide use in dental clinic to alleviate dental pain (Park et al., 2009). It is also possible to selectively block pain signals by blocking VGSCs with the permanently charged lidocaine derivative QX-314 that enters through large-pore ion channels selectively expressed in nociceptive neurons (Binshtok et al., 2009; Kim et al., 2010; Puopolo et al., 2013). In this study, we sought to explore the analgesic effect of sinomenine by using formalin-induced acute pain model and further focused its peripheral mechanisms by examining its effects on the excitability of nociceptive neurons. We found that sinomenine can produce peripheral analgesic effect by reducing cellular excitability of nociceptive neurons and the inhibition of INa is likely to contribute to its action.

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EXPERIMENTAL PROCEDURES

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Animals

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Male C57BL/6 mice weighing 20–28 g were used for the experiment. They were housed 4–6 per cage at a temperature-controlled room (23 ± 1 °C, 12 h/12 h light/dark cycle) and maintained with pellet diet and tap water ad libitum. All surgical and experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at Seoul National University.

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Formalin test

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Mice were acclimated in cage at least for a week and then adapted in an acrylic observation chamber (size ranges 12  12  12 cm) before the experiment at least three times. A mirror was located at 45° angle below the chamber to observe the paws. Formalin test was performed by intraplantar (i.pl.) injection of 1% formalin as previously described (Cho et al., 2006). On the day of test, mice were acclimatized for 30 minutes in an acrylic chamber and then 20 ll of 1% formalin was injected subcutaneously into the plantar surface of the right hind paw with a 31-gauge needle of 0.3-ml insulin syringe. Following formalin injection, the animals were immediately placed in a test chamber and recorded using a video camera for a period of 40 min. The time mice spent licking or biting was measured during each 5 min by an observer who was blinded to the treatment. Formalin-induced pain behaviors during 0–10 min after formalin injection represented the first phase and during 10–40 min after formalin injection represented the second phase.

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c-Fos immunohistochemistry

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All procedures were prepared as previously described (Badral et al., 2013). Animals were sacrificed 2 h after formalin injection for immunohistochemical analysis of c-Fos proteins. Animals were anesthetized by intraperitoneal (i. p.) injection of pentobarbital (60 mg/kg) and transcardially perfused with 0.01 M phosphate-buffered saline (PBS) including 500 U/L heparin followed by 4% paraformaldehyde (PFA) in 0.01 M PBS. The spinal cord was postfixed by 4% PFA overnight and transferred to 30% sucrose in 0.01 M PBS for 2–3 weeks. Frozen specimen was transversely sectioned into 30 lm with cryotome and sections were stored in cryoprotectant at 20 °C. Free floating sections were washed with 0.01 M PBS and incubated in 0.3% H2O2 (in distilled water) for 30 min at room temperature (RT). After elimination of endogenous peroxidase, sections were washed with 0.01 M PBS and Pre blocked with 5% normal goat serum (NGS) in PBS with 0.3% triton  (PBST) for 1 h at RT. Sections were incubated in 1st antibody (PC38, Calbiochem, USA; 1:1000 in 1% NGS (in 0.3% PBST)) for 48 h at 4 °C, washed with 0.01 M PBS to remove 1st antibody and incubated in biotinylated goat anti rabbit (BA1000, Vector laboratories, USA; 1:400 in 0.01 M PBS) for 2 h at RT. Sections were processed with ABC kit (PK-6100, Vectastain ABC kit, Vector laboratories, USA), visualized with DAB kit (DAB substrate kit for per-

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oxidase, Vector laboratories, USA) and then mounted on slide glass. After air drying, all sections were cover slipped with mountant (H-1000, Vectashield mounting medium, Vector laboratories, USA).

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For the quantification of c-Fos expression, we confirmed that most c-Fos expression induced by injection of 1% formalin (i.pl.) localized in Lumbar (L) 4–5 segment. Four sections with highest expression of c-Fos per animal were chosen and superficial dorsal horn (lamina I-II) of L4-5 segments was selected for analysis. The number of c-Fos-positive neurons was counted blindedly and the mean value was used as representative counts. Using image J, the image of selected sections was converted to gray scale, background subtracted, enhanced and sharpened. Intensity threshold was adjusted and then analyzed.

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Dorsal root ganglion (DRG) culture

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DRG neurons were prepared as previously described (Kim et al., 2009). Mice were sacrificed by exposure to isoflurane and decapitated. DRG were isolated in cold HBSS (Welgene, Korea) and incubated in 3-ml HBSS containing 1 mg/ml collagenase (Roche, USA) / 2.4 U/ml dispase II (Roche, USA) for 1 h at 37 °C. Then DRG were digested by 0.25% trypsin in PBS for 7 min at 37 °C, inactivated by 2-ml 0.25% trypsin inhibitor (Sigma, USA) and 2-ml DMEM containing 10% fetal bovine serum (Gibco, USA)/1% pen strep (Gibco, USA) and washed by 4-ml DMEM (FBS/pen strep). DRG neurons were triturated using fire-polished pasteur pipette to separate cells. The cells were resuspended in neurobasal medium (Gibco, USA) containing B-27 supplement (Gibco, USA), 1% pen strep, L-glutamine (1 mM), placed on cover slips coated with poly-D-lysine (Sigma, USA). After 1 h cells were fed with fresh neurobasal medium (B-27 supplement / pen strep / L-glutamine) and maintained in a 5% CO2 – 95% O2 incubator at 37 °C.

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Electrophysiological recordings

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Whole-cell current- and voltage-clamp recordings were performed as previously described (Park et al., 2009) in small-sized DRG neurons (<20 lm diameter) at RT with an Axopatch 200B amplifier to record action potentials (APs) and INa. Data were sampled at 10 kHz. The patch pipettes were pulled from borosilicate capillaries. When filled with following pipette solutions, their resistances were 2–6 MO. A 10 mV liquid junction potential was corrected. The extracellular solution was driven by gravity and continuously perfused (1–2 ml/min). The pipette solution for current clamp contained (in mM): K-gluconate 140, CaCl2 1, MgCl2 2, EGTA 10, K2ATP 5, HEPES 10, adjusted to pH 7.4 with KOH, osmolarity 300 mOsm. Extracellular solution for current clamp contained (in mM): NaCl 140, HEPES 10, CaCl2 2, MgCl2 1, glucose 10, KCl 5, adjusted to pH 7.4 with NaOH, osmolarity 300 mOsm. Single APs were evoked by 5-ms depolarizing current pulses. The spike threshold was measured

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Rota-rod test

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Mice were placed on the horizontal bar rotating at a speed of 4 r.p.m using rota-rod apparatus (DJ-4009, Dae-Jong Engineering &Clean Technology, Seoul, Korea). Twenty-four hours before the actual rota-rod test all mice were tested and those that were able to remain on the rod for at least 120 s were included in the study. Performance time on the bar (in sec) and number of falls over 2 min were measured. The scores were then compared and analyzed 5 min (PRE) before and 30 min (POST) after treatment with vehicle or sinomenine. The test was repeated 3 times and the mean value for each animal was recorded.

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Administration of sinomenine

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Sinomenine hydrochloride was purchased from TCI chemicals (Tokyo, Japan). In the formalin test, to examine the systemic effects of sinomenine, sinomenine was diluted in normal saline and intraperitoneally injected at a dose of 25 mg/kg, 50 mg/kg or 75 mg/kg in a volume of 10 ml/kg body weight 30 min prior to the formalin injection. Control animals received (i.p.) an equivalent volume of vehicle. To evaluate the peripheral effects of sinomenine, formalin test was performed after i.pl. injection of sinomenine (500 lg per mouse) which was diluted in 1% formalin (20 ll/ saline). For electrophysiological A recordings, sinomenine was 150 dissolved in the extracellular solutions to their concentration. The solution was driven to cell by gravity and continuously perfused (1–2 ml/ 100 min).

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Statistical analysis

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Statistical analysis was performed using GraphPad Prism version 6.0 (GraphPad Software, USA). Comparison between two groups was made using the unpaired or paired Student’s t-test to analyze the expression of c-Fos, electrophysiological recordings of APs, formalin test and rota-rod test. For multiple comparisons, data were analyzed using the one-way ANOVA

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followed by Tukey’s test to determine formalin test and electrophysiological recordings of INa. Data are presented as mean ± SEM. Differences with p < 0.05 were considered significant.

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RESULTS

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The effect of sinomenine on formalin-induced pain in mice

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We investigated the analgesic effect of sinomenine using formalin model as it is a reliable model for testing the analgesic effect and mechanism of a drug (Hunskaar and Hole, 1987; Tjolsen et al., 1992; Cho et al., 2006). Injection of 1% formalin (i.pl.) elicited typical biphasic pain behavior (Fig. 1A). As compared with the vehicle group, 25 mg/kg (i.p.) sinomenine did not induce statistically significant decrease in formalin-induced pain behavior (Fig. 1B). However, sinomenine-treated group (i.p.) had a significant analgesic effect in both the first phase and second phase, at the dose of 50 mg/kg and 75 mg/kg (Fig. 1B). In the first phase, formalin-induced pain behavior was reduced by 45 ± 9.8% at the dose of 50 mg/kg (*p < 0.05) and 81.4 ± 5.1% at the dose of 75 mg/kg, respectively (***p < 0.001). In the second phase, formalin-induced pain behavior was reduced by 38.5 ± 6.8% at the dose of 50 mg/kg (**p < 0.01) and 96.3 ± 2% at the dose of 75 mg/kg, respectively (***p < 0.001). The expression of c-Fos protein is a measure of acute nociception following noxious stimulation (Harris, 1998). Thus, it is commonly used to show the analgesic property of the drug (Badral et al., 2013). Injection of 1% formalin (i.pl.) caused high c-Fos expression in superficial dorsal horn innervated by L4-5 DRG neurons (Fig. 2A). As compared with the vehicle group, sinomenine (50 mg/kg i.p.) reduced the number of c-Fos-positive neurons in superficial dorsal horn by 32.1 ± 10.1% (*p = 0.0292) (Fig. 2B and C).

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at the onset of the positive deflection of the first differentiation of the spike potential. A series of APs were evoked by 500-ms depolarizing current pulses. The pipette solution for voltage clamp contained (in mM): CsCl 100, sodium L glutamic acid 5, TEACl 30, CaCl2 0.1, MgCl2 2, EGTA 11, HEPES 10 adjusted to pH 7.4 with CsOH osmolarity 300 mOsm. Extracellular solution for voltage clamp contained (in mM): NaCl 140, HEPES 10, CaCl2 2, MgCl2 1, glucose 10, KCl 5, adjusted to pH 7.4 with NaOH, osmolarity 300 mOsm. The INa was evoked by a test pulse stepped to 10 mV from a holding potential of 90 mV every 20 s.

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Fig. 1. Intraperitoneal administration of sinomenine on formalin-induced pain behavior (A) Effects of sinomenine (SIN) on the time course curves of formalin-induced pain behavior. (B) Formalininduced pain behavior was divided into two phases and analyzed. The bar graph represents first phase (0–10 min) and second phase (10–40 min) of formalin-induced pain behavior. Intraperitoneal injection of sinomenine inhibited formalin-induced pain behavior in both first phase and second phase at the dose of 50 mg/kg or 75 mg/kg. *p < 0.05, **p < 0.01 and ***p < 0.001 (oneway ANOVA followed by Tukey’s test).

Please cite this article in press as: Lee J-Y et al. Sinomenine produces peripheral analgesic effects via inhibition of voltage-gated sodium currents. Neuroscience (2017), http://dx.doi.org/10.1016/j.neuroscience.2017.06.024

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Fig. 2. Effects of sinomenine on formalin-induced c-Fos expression at the Lumbar 4–5 segments (A) Intraplantar injection of 1% formalin induced cFos expression in superficial dorsal horn of Lumbar 4–5 (L4-5) segments. (B) Intraperitoneal injection of sinomenine (SIN, 50 mg/kg) decreased formalin-induced c-Fos expression in superficial dorsal horn of L4-5. (C) The quantification of c-Fos expression. 50 mg/kg sinomenine suppressed formalin-induced c-Fos expression in superficial dorsal horn of L4-5 as compared with vehicle. *p < 0.05 (unpaired t test, two-tailed), Scale bar = 200 lm, SDH; superficial dorsal horn.

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Collectively, our results showed that sinomenine produces an analgesic effect in acute inflammatory pain, suggesting that peripheral nociceptive neurons might be targeted by sinomenine. The effect of sinomenine on APs and VGSCs in smallsized DRG neurons We next investigated the effects of sinomenine on the excitability of small-sized DRG neurons (<20 lm diameter). 1 mM sinomenine reversibly increased the spike threshold from –31 ± 1.6 mV to –26.6 ± 2.1 mV (**p = 0.0023) and the current threshold by 21.5 ± 5% (**p = 0.0032) (Fig. 3A), and also significantly decreased AP amplitude by 18.1 ± 3.2% (**p = 0.0013) and AP overshoot level by 28.1 ± 4.9% (**p = 0.0021), when single APs were evoked in response to injection of short current pulses (Fig. 3A). Concomitantly, halfwidth of AP was increased by 89.3 ± 29.9% (**p = 0.0057) (Fig. 3A). We next examined the effects of sinomenine on the firing frequency measured during a series of APs evoked in response to long current pulses with supra-threshold intensities. Although we examined the effect of 1 mM sinomenine on a series of APs, 1 mM sinomenine was not sufficient to show the effect on firing frequencies. We found that AP firing frequency was also decreased by 3 mM sinomenine and restored considerably after washout (Fig. 3B). Compared with the control, 3 mM sinomenine reduced AP firing frequency by 39.5 ± 6% (**p = 0.007). Because the VGSCs are responsible for the generation of the APs, we tested the effect of sinomenine on INa in small-sized DRG neurons by a command pulse stepped from 90 to 10 mV. As compared with the control, 3 mM sinomenine

significantly inhibited INa in small-sized DRG neurons by 63.3 ± 3.2% (***p < 0.001) (Fig. 4A). INa was decreased by sinomenine in a dose-dependent manner with 0.1 mM by 7.7 ± 1%, 0.3 mM by 13 ± 2%, 1 mM by 29.9 ± 2.3%, 3 mM by 63.3 ± 3.2% and 10 mM by 83.3 ± 3.2%. (Fig. 4B, IC50 = 2.3 ± 0.2 mM). These results suggest peripheral action of sinomenine on VGSCs in nociceptive neurons, which might be associated with analgesic effect of sinomenine on acute inflammatory pain.

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Intraplantar application of sinomenine on formalininduced pain behavior

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To clarify the peripheral action of sinomenine, we then examined whether peripheral injection of sinomenine indeed produces analgesic effects. The dose of 500-lg sinomenine was determined based on our behavior result (Fig. 1) which was expected to show no analgesic effect when administered systemically. We found that injection of 500-lg sinomenine (i.pl.), co-administrated with 1% formalin, indeed reduced the first phase of formalin-induced pain behavior by 38.2 ± 5.9% (**p = 0.0057), but not the second phase of formalininduced pain behavior (Fig. 5).

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The effect of sinomenine on motor function in rotarod test

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Because drug-induced motor function impairment may result in false-positive results in nocifensive pain behavior, we carried out rota-rod test to examine the effect of sinomenine on motor function. We did not observe motor activity impairment at the dose of 25 mg/ kg and 50 mg/kg, whereas there was a significant motor

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Fig. 3. Effects of sinomenine on action potentials in small sized dorsal root ganglion neurons (A) The inhibitory effect of sinomenine on action potentials (APs). A representative APs trace (a, b). 1 mM sinomenine (SIN) increased the spike threshold (a, c) and the current threshold (b, d), and inhibited AP amplitude (e) and peak (f). After application of 1 mM sinomenine, half-width of AP was increased (g). (B) Sinomenine decreased AP firing frequency. Series of APs were recorded during 500 ms depolarizing current step (a). 3 mM sinomenine inhibited AP firing frequency (n = 4) (b). Series of APs returned partially after washout (c). **p < 0.01 (paired t test, two-tailed), Asterisk = spike threshold.

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(*p = 0.0267) and decreased (***p = 0.0002) in rota-rod test.

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tion of sinomenine significantly decreased formalin-induced pain a b behavior in both first phase and sec-10 mV ond phase. However, i.pl. injection of 3 mM sinomenine -90 mV 8 sinomenine decreased formalininduced pain behavior only in the first phase. These results suggest that 6 1) sinomenine has peripheral analgesic 3) mechanism. 4 The analgesic effects of 2) 1 nA sinomenine via i.p. route on the second phase might result from its 2 2 ms 2) central mechanisms. Recent studies showed that analgesic effects of 0 sinomenine are related to a GABAA 3) 1 2 3 4 5 6 receptor and opioid l-receptor, both 1) of them are cellular targets for time (min) sinomenine in the CNS. Furthermore, in a previous study, systemic administration (i.p.) of B n.s. c VGSC blockers attenuates formalin100 1.0 induced pain behavior in the second 80 phase (Blackburn-Munro et al., 0.8 2002). However, peripherally adminis60 tered VGSC blocker such as lidocaine 0.6 suppresses formalin-induced pain *** 40 behavior only in the first phase (Inan 0.4 et al., 2009). Thus, it seems that sino20 0.2 menine has peripheral action, in addi0 tion to its central actions previously 0.1 1 10 0.0 demonstrated by other groups. log[Agonist], mM The gene c-Fos encodes nuclear sinomenine protein Fos in response to neuronal activation and has been used as a Fig. 4. Effects of sinomenine on voltage-gated sodium channel in small sized dorsal root ganglion marker for neuronal activity (Harris, neurons. (A) 3 mM sinomenine (SIN) inhibited voltage-gated sodium current (INa). Time course of 1998). Following the noxious stimulaeffects of 3 mM sinomenine on INa (1) before, 2) during, 3) after the application of 3 mM tion, primary afferent nociceptive neusinomenine) (a). INa trace at the points indicated (b). Normalized INa. As compared with control rons (Ad and C-fiber) transmit pain 3 mM sinomenine significantly decreased INa (n = 6) (c). ***p < 0.001 (repeated measures ANOVA followed by Tukey’s test) (B) INa was decreased by sinomenine in a dose dependent signal to second order neurons in manner with IC50 = 2.3 ± 0.2 mM. (0.1 mM n = 4, 0.3 mM n = 5, 1 mM n = 8, 3 mM n = 6, the superficial dorsal horn of spinal 10 mM n = 5). cord. Thus, c-Fos are expressed in the superficial dorsal horn of spinal cord after noxious stimulation DISCUSSION (Catherine et al., 1997) and the reduction of c-Fos expresIn the present study, we show peripherally mediatedsion is commonly used to show peripheral analgesic analgesic action of sinomenine. We found that not only effects (Badral et al., 2013). In this study, we found that i.p application, but also i.pl. application, of sinomenine sinomenine (50 mg/kg i.p.) decreased formalin-induced produces analgesic effect on formalin-induced pain c-Fos expression in the superficial dorsal horn. This result model. Sinomenine inhibited formalin-induced c-Fos further supports a peripheral action of sinomenine. expression in the superficial dorsal horn of spinal cord. The activity of primary nociceptive neurons (Ad and CUsing whole-cell patch-clamp recordings, we also found fiber) is regulated by ion channels and is important for that sinomenine reduces cellular excitability by inhibiting pain pathway (Waxman and Zamponi, 2014). Because INa in small-sized DRG neurons. pain threshold and intensity are directly correlated with The formalin-induced pain model in rodents typically AP threshold and AP frequency, respectively, in such shows biphasic pain behavior, each caused by different afferent, we examined whether sinomenine modulate mechanisms (Hunskaar and Hole, 1987; Shibata et al., the excitability of small-sized DRG neurons. Using patch 1989; Tjolsen et al., 1992). The first phase occurs due clamp recordings, we first showed that sinomenine to direct stimulation of nociceptor in C-fiber and the secincreased AP threshold and decreased AP firing freond phase is a pain response caused by peripheral quency. This strongly suggests that sinomenine has an inflammation and functional changes in central processanalgesic effect. Consistent with an involvement of ing (Shibata et al., 1989). Our study showed that i.p. injecVGSCs in the generation of AP, sinomenine affected Please cite this article in press as: Lee J-Y et al. Sinomenine produces peripheral analgesic effects via inhibition of voltage-gated sodium currents. Neuroscience (2017), http://dx.doi.org/10.1016/j.neuroscience.2017.06.024

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Sinomenine has pharmacological actions on diverse ion channels at a 150 2 phase formalin + SIN 500 µg (n=7) 500 wide range of concentrations. formalin (n=6) n.s. Sinomenine inhibits voltage-gated 400 calcium currents in cultured rat 100 cortical neurons at concentration of 300 0.05–1 lm (Wu et al., 2011). 10 lm sinomenine inhibited ATP-activated 200 1 phase current in HEK293 cells expressing ** 50 the P2X3 receptor (Rao et al., 2017). 100 In this study, we observed that sino0 menine was effective at a concentration range of mM on the cellular 0 excitability and VGSCs in small0 10 20 30 40 time (min) sized DRG neurons. Thus, VGSCs may exhibit less sensitivity to sinomeFig. 5. Intraplantar administration of sinomenine on formalin-induced pain behavior (A) Effects of nine compared to VGCCs and P2X3 sinomenine (SIN) on the time course curves of formalin-induced pain behavior. (B) Formalinchannels. Likewise, we previously induced pain behavior was divided into two phases and analyzed. The bar graph represented first showed that diverse ion channels phase (0–10 min) and second phase (10–40 min) of formalin-induced pain behavior. Intraplantar injection of sinomenine (500 lg) inhibited formalin-induced pain behavior only in the first phase. expressed in sensory neurons are tar** p < 0.01 (unpaired t test, two-tailed). geted by eugenol with different sensitivity (Lee et al., 2005; Park et al., 2006; Yeon et al., 2011). Given A B VGCCs and P2X3 channels are also PRE expressed in DRG neurons, it *** n.s. n.s. n.s. * POST 6 remains to be determined whether 120 VGCCs and P2X3 channels in noci100 ceptive neurons are indeed targeted 4 80 by sinomenine. The effective analgesic dose of 60 n.s. n.s. n.s. sinomenine (50 mg/kg i.p.) does not 2 40 elicit motor activity impairment. 20 When we investigated the effect of 0 0 sinomenine on motor function in 0 25 50 75 0 25 50 75 rota-rod test, the dose of 50 mg/kg (i. Sinomenine (mg/kg, i.p.) Sinomenine (mg/kg, i.p.) p.) were analgesic without motor Fig. 6. Effects of sinomenine in rota-rod test. The performance time on rota-rod (A) and the activity impairment (Fig. 6). number of falls (B) was measured 5 min (PRE) before and 30 min (POST) after intraperitoneal (i. However, we observed motor p.) injection with vehicle or sinomenine. Sinomenine had no effect on rota-rod test at the dose of dysfunction and some sedative effect 25 mg/kg (n = 6) or 50 mg/kg (n = 7). 75 mg/kg sinomenine showed significant difference in rotaat high dose of sinomenine (75 mg/ rod score (n = 6). *p < 0.05 and ***p < 0.0001 (paired t test, two-tailed). kg i.p.). Because systemic administration of sinomenine has a VGSCs. Furthermore, it is well known that VGSCs conside effect such as sedation, gastric tribute to AP overshoot phase (Renganathan et al., intestine and kidney damage at high dose, it is 2001) and, sinomenine inhibited AP overshoot phase in important to use sinomenine in low concentrations. In our experiments. So, the cause of widening of the AP many analgesics as well as sinomenine, the side effects width could be the reduction of potassium channel activaof systemic administration are an important issue. Thus, tion due to the sinomenine-induced decrease in VGSC various drug administration methods have been applied activation. In the present study, INa was decreased by to reduce drug concentration and side effects. For sinomenine in a dose dependent manner. Sinomenine example, patch form of lidocaine was used to reduce has been demonstrated to inhibit L-type calcium channel systemic adverse effects and effective in neuropathic and ASIC channel in cortical neurons (Wu et al., 2011). patients in clinical practice (Gammaitoni et al., 2003). In addition, vasodilatory action of sinomenine is involved Sinomenine also showed anti-inflammatory effects when with inhibition of calcium channels (Nishida and Satoh, topically administrated via patch and spray formulations 2006). These studies show the possibility of ion channel (Li et al., 2010). In our study, we found that i.pl. adminisregulation by sinomenine in various cells. On the other tration of sinomenine show analgesic effect at the low hand, our study proposes that sinomenine regulate concentration that did not show analgesic effects when VGSCs in primary sensory neurons, which can produce administered systemically (2025 mg/kg i.pl. versus peripheral analgesic effect by reducing cellular excitability 25 mg/kg i.p.), implying its potential therapeutic use as a of nociceptive neurons. local analgesic in the periphery. number of Falls

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Based on our findings, we propose that the peripheral analgesic mechanisms of sinomenine are mediated by reduction of cellular excitability in small-sized DRG neurons, with VGSC as one of the molecular targets of sinomenine. Therefore, sinomenine could be a potential pharmacological therapeutic agent as local anesthetic and analgesics.

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CONLFICT OF INTEREST

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All authors declare no conflict of interest.

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AUTHOR CONTRIBUTIONS

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S.B.O. conceived the idea, obtained funding for the study and guided the project; J.Y.L and S.Y.Y performed formalin test and c-Fos staining; J.Y.L and J.W performed whole cell patch clamp; S.Y.Y performed rota-rod test; J.Y.L and S.Y.Y, performed behavior and histological data analysis; J.Y.L, J.W and Y.K performed whole cell patch clamp data analysis; J.Y.L, S.Y.Y, H.B. K, Y.K and S.B.O wrote the manuscript. All authors gave final approval and agree to be accountable for all aspects of the work.

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Acknowledgments—This research was supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF) funded by the Ministry of Science, ICT & Future Planning (No. 2016M3A9B6021209 and No. 2015R1D1A1A01059208). Y.K. was supported by Brain Pool Program through the Korean Federation of Science and Technology Societies (KOFST) funded by the Ministry of Science, ICT and Future Planning.

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REFERENCES

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Badral B, Davies AJ, Kim YH, Ahn JS, Hong SD, Chung G, Kim JS, Oh SB (2013) Pain fiber anesthetic reduces brainstem Fos after tooth extraction. J Dent Res 92:1005–1010. Binshtok AM, Gernerm P, Oh SB, Puopolo M, Suzuki S, Roberson DP, Herbert T, Wang C-F, Kim D, Chung G, Mitani AA, Wang GK, Bean BP, Woolf CJ (2009) Coapplication of Lidocaine and the Permanently Charged Sodium Channel locker QX-314 Produces a Long-lasting Nociceptive Blockade in Rodents. Anesthesiology 111:127–137. Blackburn-Munro G, Ibsen N, Erichsen HK (2002) A comparison of the anti-nociceptive effects of voltage-activated Na+ channel blockers in the formalin test. Eur J Pharmacol 445:231–238. Catherine A, K. TB, Alex PM, I. BA (1997) Differential contribution of the two phases of the formalin test to the pattern of c-fos expression in the rat spinal cord: studies with remifentanil and lidocaine. pain 69:101–110. Cho IH, Chung YM, Park CK, Park SH, Lee H, Kim D, Piao ZG, Choi SY, Lee SJ, Park K, Kim JS, Jung SJ, Oh SB (2006) Systemic administration of minocycline inhibits formalin-induced inflammatory pain in rat. Brain Res 1072:208–214. Gammaitoni AR, Alvarez NA, Galer BS (2003) Safety and tolerability of the lidocaine patch 5%, a targeted peripheral analgesic: a review of the literature. J Clin Pharmacol 43:111–117. Gao T, Hao J, Wiesenfeld-Hallin Z, Wang DQ, Xu XJ (2013) Analgesic effect of sinomenine in rodents after inflammation and nerve injury. Eur J Pharmacol 721:5–11. Harris JA (1998) Using c-fos as a neural marker of pain. Brain Res Bull 45:1–8. Hunskaar S, Hole K (1987) The formalin test in mice dissociation between non inflammatory inflammatory pain. Pain 30:103–114.

516 517 518 519 520 521 522

Inan S, Dun NJ, Cowan A (2009) Inhibitory effect of lidocaine on pain and itch using formalin-induced nociception and 5’guanidinonaltrindole-induced scratching models in mice: behavioral and neuroanatomical evidence. Eur J Pharmacol 616:141–146. Kim HY, Kim K, Li HY, Chung G, Park CK, Kim JS, Jung SJ, Lee MK, Ahn DK, Hwang SJ, Kang Y, Binshtok AM, Bean BP, Woolf CJ, Oh SB (2010) Selectively targeting pain in the trigeminal system. Pain 150:29–40. Kim YH, Park CK, Back SK, Lee CJ, Hwang SJ, Bae YC, Na HS, Kim JS, Jung SJ, Oh SB (2009) Membrane-delimited coupling of TRPV1 and mGluR5 on presynaptic terminals of nociceptive neurons. J Neurosci 29:10000–10009. Lee MH, Yeon KY, Park CK, Li HY, Fang Z, Kim MS, Choi SY, Lee SJ, Lee S, Park K, Lee JH, Kim JS, Oh SB (2005) Eugenol inhibits calcium currents in dental afferent neurons. J Dent Res 84:848–851. Li X, Li X, Zhou Y, Liu Y, Guo M, Zhu Q, Xie Y, Fan Z (2010) Development of patch and spray formulations for enhancing topical delivery of sinomenine hydrochloride. J Pharm Sci 99:1790–1799. Mathie A (2010) Ion channels as novel therapeutic targets in the treatment of pain. J Pharm Pharmacol 62:1089–1095. McEntire DM, Kirkpatrick DR, Dueck NP, Kerfeld MJ, Smith TA, Nelson TJ, Reisbig MD, Agrawal DK (2016) Pain transduction: a pharmacologic perspective. Expert Rev Clin Pharmacol 9:1069–1080. Nishida S, Satoh H (2006) In vitro pharmacological actions of sinomenine on the smooth muscle and the endothelial cell activity in rat aorta. Life Sci 79:1203–1206. Park CK, Kim K, Jung SJ, Kim MJ, Ahn DK, Hong SD, Kim JS, Oh SB (2009) Molecular mechanism for local anesthetic action of eugenol in the rat trigeminal system. Pain 144:84–94. Park CK, Li HY, Yeon KY, Jung SJ, Choi SY, Lee SJ, Lee S, Park K, Kim JS, Oh SB (2006) Eugenol inhibits sodium currents in dental afferent neurons. J Dent Res 85:900–904. Puopolo M, Binshtok AM, Yao GL, Oh SB, Woolf CJ, Bean BP (2013) Permeation and block of TRPV1 channels by the cationic lidocaine derivative QX-314. J Neurophysiol 109:1704–1712. Rao S, Liu S, Zou L, Jia T, Zhao S, Wu B, Yi Z, Wang S, Xue Y, Gao Y, Xu C, Li G, Xu H, Zhang C, Liang S (2017) The effect of sinomenine in diabetic neuropathic pain mediated by the P2X3 receptor in dorsal root ganglia. Purinergic Signal. Renganathan M, Cummins TR, Waxman SG (2001) Contribution of Nav1.8 Sodium channels to Action Potential Electrogenesis in DRG Neurons. J Neurophysiol 86. Shibata M, Ohkubo T, Takahashi H, Inoki R (1989) Modified formalin test characteristic biphasic pain response. Pain 38:347–352. Tjolsen A, Berge OG, Hunskaar S, Rosland JH, Hole K (1992) The formalin test: an evaluation of the method. Pain 51:5–17. Wang MH, Chang CK, Cheng JH, Wu HT, Li YX, Cheng JT (2008) Activation of opioid mu-receptor by sinomenine in cell and mice. Neurosci Lett 443:209–212. Wang Q, Li XK (2011) Immunosuppressive and anti-inflammatory activities of sinomenine. Int Immunopharmacol 11:373–376. Waxman SG, Zamponi GW (2014) Regulating excitability of peripheral afferents: emerging ion channel targets. Nat Neurosci 17:153–163. Wu WN, Wu PF, Chen XL, Zhang Z, Gu J, Yang YJ, Xiong QJ, Ni L, Wang F, Chen JG (2011) Sinomenine protects against ischaemic brain injury: involvement of co-inhibition of acid-sensing ion channel 1a and L-type calcium channels. Br J Pharmacol 164:1445–1459. Yeon KY, Chung G, Kim YH, Hwang JH, Davies AJ, Park MK, Ahn DK, Kim JS, Jung SJ, Oh SB (2011) Eugenol reverses mechanical allodynia after peripheral nerve injury by inhibiting hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. Pain 152:2108–2116.

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Zhao XX, Peng C, Zhang H, Qin LP (2012) Sinomenium acutum: a review of chemistry, pharmacology, pharmacokinetics, and clinical use. Pharm Biol 50:1053–1061. Zhu Q, Sun Y, Mao L, Liu C, Jiang B, Zhang W, Li JX (2016) Antinociceptive effects of sinomenine in a rat model of postoperative pain. Br J Pharmacol 173:1693–1702.

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Zhu Q, Sun Y, Zhu J, Fang T, Zhang W, Li JX (2014) Antinociceptive effects of sinomenine in a rat model of neuropathic pain. Sci Rep 4:7270.

(Received 4 May 2017, Accepted 19 June 2017) (Available online xxxx)

Please cite this article in press as: Lee J-Y et al. Sinomenine produces peripheral analgesic effects via inhibition of voltage-gated sodium currents. Neuroscience (2017), http://dx.doi.org/10.1016/j.neuroscience.2017.06.024

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