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Inflammatory pain such as arthritic pain is typically treated with opioids and COX-2 inhibitors with well-known side effects. Transient receptor potential subtype V1 (TRPV1) and A1 (TRPA1) contribute importantly to the genesis of inflammatory pain via both peripheral mechanisms (peripheral sensitization) and spinal cord mechanisms (central sensitization). Although these TRP channels have been intensively studied, little is known about their endogenous inhibitors. Recent studies have demonstrated that the endogenous lipid mediators resolvins (RvE1 and RvD1), derived from omega-3 unsaturated fatty acids, are potent inhibitors for inflammatory pain, without noticeable side effects. However, the molecular mechanisms underlying resolvins’ distinct analgesic actions in mice are unclear. Resolvin D2 (RvD2) is a novel family member of resolvins. Here we report that RvD2 is a remarkably potent inhibitor of TRPV1 (IC50=0.1 nM) and TRPA1 (IC50= 2 nM) in primary sensory neurons, whereas RvE1 and RvD1 selectively inhibited TRPV1 (IC50=1 nM) and TRPA1 (IC50=9 nM), respectively. Accordingly, RvD2, RvE1, and RvD1 differentially regulated TRPV1 and TRPA1 agonist-elicited acute pain and spinal cord synaptic plasticity (sEPSC frequency increase). RvD2 also abolished inflammation-induced sEPSC increases (frequency and amplitude), without affecting basal synaptic transmission. Intrathecal administration of RvD2 at very low doses (0.01-1 ng) prevented formalin-induced spontaneous pain. Intrathecal RvD2 also reversed adjuvant-induced inflammatory pain without altering baseline pain and motor function. Finally, intrathecal RvD2 reversed C-fiber stimulation-evoked long-term potentiation in the spinal cord. Our findings suggest distinct roles of resolvins in regulating TRP channels and identify RvD2 as a potent endogenous inhibitor for TRPV1/A1 and inflammatory pain.
Tissue injury is known to produce inflammation and inflammatory pain. It is generally believed that inflammatory pain is driven by peripheral sensitization in primary sensory neurons following stimulation of inflammatory mediators (Hucho and Levine, 2007;Basbaum et al., 2009). Tissue injury-induced spinal cord synaptic plasticity, i.e. central sensitization, also contributes importantly to the development and maintenance of inflammatory pain (Ji et al., 2003;Kuner, 2010). Long-term potentiation (LTP) in the spinal cord (Ruscheweyh et al., 2011) is a unique form of central sensitization for persistent pain development. Transient receptor potential vanilloid 1 (TRPV1) and ankyryn 1 (TRPA1) are two critical types of TRP channels that are strongly implicated in the genesis of inflammatory pain (Caterina et al., 2000; McMahon and Wood, 2006). Activation of TRPV1 and TRPA1 can enhance inflammatory pain not only via well-demonstrated peripheral sensitization (Caterina et al., 2000;Dai et al., 2007;Bautista et al., 2006;Ji et al., 2002) but also via central sensitization, by increasing glutamate release from primary afferent terminals to enhance synaptic transmission (Kosugi et al., 2007). Great pharmaceutical effort has been devoted to developing small molecule inhibitors of TRPV1 and TRPA1. Interestingly, several lipid mediators have been identified as endogenous activators of TRPV1 (Hwang et al., 2000;Patwardhan et al., 2009). However, little is known about the endogenous inhibitors of TRP channels.
Resolvins are endogenous lipid mediators generated during the resolution phase of acute inflammation from omega-3 polyunsaturated fatty acids and display potent pro-resolving and anti-inflammatory actions (Serhan et al., 2008;Serhan et al., 2002). Resolvin D1 (RvD1) and Resolvin E1 (RvE1) are derived from docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), respectively (Serhan et al., 2008). Recent studies have shown that both peripheral (intraplantar) and central (intrathecal) administration of RvE1 and RvD1 could effectively reduced inflammatory and postoperative pain (Xu et al., 2010;Huang et al., 2011;Bang et al., 2010). Interestingly, resolvins also modulate the activity of TRP channels (Ji et al., 2011). RvE1 blocked capsaicin-induced spontaneous pain and spinal cord synaptic plasticity (Xu et al., 2010), whereas RvD1 inhibited TRPA1 in dorsal root ganglion (DRG) neurons (Bang et al., 2010). However, the efficacy (IC50 value) of RvE1 and RvD1 in inhibiting TRPV1/A1 in primary sensory neurons has not been evaluated in the previous studies.
Resolvin D2 (RvD2), derived from DHA, is a novel family member of resolvin. The complete structure of RvD2 was recently established (Spite et al., 2009). RvD2 is a potent regulator of leukocytes and controls microbial sepsis (Spite et al., 2009). Hence the role of RvD2 in regulating TRPV1/A1 and pain control was of interest. We investigated whether different resolvins play distinct roles in regulating inflammatory pain, TRPV1/A1, and spinal cord synaptic plasticity.
Adult CD1 mice (male, 8-10 weeks) were used for behavioral and pharmacological studies. Young CD1 mice (4-6 weeks) were used for electrophysiological studies in spinal cord slices and DRG neurons. To produce acute and persistent inflammatory pain, diluted formalin (5%, 20 μl), carrageenan (1%, 20 μl), or complete Freund's adjuvant (CFA, 20 μl, 1 mg/ml, Sigma) was injected into the plantar surface of a hindpaw. All the animal procedures were approved by the Animal Care Committee of Harvard Medical School.
Capsaicin, carrageenan, allyl isothiocyanate (AITC), and GDPβS were obtained from Sigma. AMG9810 and pertussis toxin were purchased from Tocris. RvD1, RvD2, DHA, and EPA were purchased from Cayman Chemical. RvE1 was a kind gift from Resolvyx Pharmaceutical Inc (Cambridge, MA). Resolvins were initially isolated in exudates from the resolution phase of self-limited acute inflammation (Serhan et al., 2002). After the full structural elucidation, resolvin's physical and anti-inflammatory properties were confirmed by total organic synthesis (Serhan et al., 2008). All resolvins were prepared in PBS. For intrathecal injection, spinal cord puncture was made with a 30G needle between the L5 and L6 level to deliver reagents (10 μl) to the cerebral spinal fluid.
DRG neuron cultures were prepared as previously reported (Park et al., 2011). DRG cells were plated on glass cover slips and cultured in a neurobasal defined medium (with 2% B27 supplement, Invitrogen) for 24 hours before use. Whole-cell current-clamp recordings were performed at room temperature (28°C) to measure inward currents with Axopatch-200B amplifier (Axon Instruments). The pipette solution for voltage-clamp contains (in mM): 126 K-gluconate, 10 NaCl, 1 MgCl2, 10 EGTA, 2 NaATP, and 0.1 MgGTP, adjusted to pH 7.4 (with KOH) and osmolarity 295 - 300 mOsm. In some cases, GDPβS (2.5 mM) was included in intracellular solution to block GPCRs. Extracellular solution was prepared by adding 0 mM CaCl2 and 2 mM EGTA for Ca2+ chelation. It contains (in mM): 140 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, 10 glucose, adjusted to pH 7.4. The recording chamber (300 μl) was continuously superfused (2-3 ml/min). Series resistance was compensated for (> 80%), and leak subtraction was performed. Data were low-pass-filtered at 2 KHz, sampled at 10 KHz. The pClamp8 (Axon Instruments) software was used during experiments and analysis. Voltage clamp was performed at a holding potential of -60 mV.
A portion of the lumbar spinal cord (L4-L5) was removed from mice, as previously reported (Xu et al., 2010) Transverse slices (400-600 μm) were cut on a vibrating microslicer. The slices were perfused with Kreb's solution (8-10 ml/min) saturated with 95% O2 and 5% CO2 at 37°C. The whole cell patch-clamp recordings were made from lamina IIo neurons in voltage clamp mode at 37°C. After establishing the whole-cell configuration, neurons were held at the potential of -70 mV to record sEPSCs. The internal solution contains (in mM): 135 K-gluconate, 5 KCl, 0.5 CaCl2, 2 MgCl2, 5 EGTA, 5 HEPES, 5 ATP-Mg, adjusted to pH 7.4. The perfusion solution contains (in mM): 117 NaCl, 3.6 KCl, 2.5 CaCl2, 1.2 MgCl2, 1.2 NaH2PO4, 25 NaHCO3, and 11 glucose. The resistance of a typical patch pipette is 5-10 MΩ. Membrane currents were amplified with an Axopatch 200B amplifier (Axon Instruments) in voltage-clamp mode. Signals were filtered at 2 kHz and digitized at 5 kHz. Data were stored with a personal computer using pCLAMP 10 software and analyzed with Mini Analysis (Synaptosoft Inc.).
Spinal cord LTP was recorded in vivo as we recently demonstrated (Park et al., 2011). Mice were anesthetized with urethane (1.5 g/kg, IP). A laminectomy was performed at vertebrae T13-L1 to expose the lumbar enlargement, and the left sciatic nerve was exposed for bipolar electrical stimulation. The vertebral column was firmly suspended by rostral and caudal clamps on the stereotaxic frame. Following electrical stimulation of the sciatic nerve, the field potentials were recorded in the ipsilateral L4-5 spinal cord segments with glass microelectrodes, 100-300 μm from the surface of the cord. After recording stable responses following test stimuli (2x C-fiber threshold, 0.5 ms, 1 min interval, every 5 min) for > 40 min, conditioning tetanic stimulation (5 times of C-fiber threshold, 100 Hz, 1 s, 4 trains, 10 s interval) was delivered to the sciatic nerve for inducing LTP of C-fiber-evoked field potentials. For intrathecal drug delivery, a PE5 catheter was inserted at L5-L6 level via lumbar puncture.
Animals were habituated to the testing environment for two days and were kept in plastic boxes for 30 min habituation before testing. Heat sensitivity was tested by radiant heat using Hargreaves apparatus (Hargreaves et al., 1988). For testing capsaicin and mustard oil (AITC)-induced spontaneous pain (nocifensive behavior), capsaicin or AITC was intraplantarly injected, and the time spent on nocifensive behavior (flinching and licking) was recorded for 5 min. For formalin test, the time spent in licking and flinching the affected paws was recorded every 5 min for 45 min. For testing mechanical sensitivity, mice were put in boxes on an elevated metal mesh floor, and the plantar surface of each hind paw was stimulated with a series of von Frey hairs with logarithmically incrementing stiffness (0.02-2.56 g, Stoelting), presented perpendicular to the plantar surface. The 50% paw withdrawal threshold was determined using Dixon's up-down method. For Rota-Rod test, mice were put in the treadmill with accelerating speed and the seconds spent before the first fall were recorded. For all the behavioral tests, the experimenters were blinded to the treatments.
All data were expressed as mean ± s.e.m. Differences between groups were compared using student t-test (two groups), One-Way ANOVA (multiple groups) followed by posthoc Bonnferroni test, or Two-Way ANOVA for analyzing the LTP data. The criterion for statistical significance was P<0.05.
First, we used patch clamp recordings in dissociated mouse DRG neurons to test whether RvD2 would modulate TRPV1 and TRPA1 activity. Perfusion of DRG neurons with capsaicin (100 nM) elicited a marked TRPV1 current, and this current was dose-dependently inhibited by RvD2 (Fig. 1A,B). Strikingly, RvD2 inhibited TRPV1 currents with an extremely low IC50, 0.1 nM (i.e. 0.04 ng/ml, Figure 1B and Table-1). For comparison we also tested the efficacy of RvE1 and RvD1 in inhibiting TRPV1 current. Of interest RvE1 inhibited TRPV1 currents with IC50=1.0 nM, which is 10 times higher than that of RvD2, whereas RvD1 had no effect even at high concentrations (Figure 1C, Table-1).
Next, we examined whether RvD2 would also modulate TRPA1 current, induced by mustard oil allyl isothiocyanate (AITC, 300 μM). RvD2 potently inhibited TRPA1 currents with IC50=2.1 nM (Figure 1D,E and Table-1). In sharp contrast, RvD1 inhibited TRPA1 currents with IC50=8.5 nM, which is 4 times higher than that of RvD2, whereas RvE1 did not inhibit TRPA1 currents even at high concentrations (Figure 1F and Table-1).
Notably, the molecular weights of RvE1 (350.4), RvD1 (376.5), and RvD2 (376.5), as well as their precursors DHA (328.5) and EPA (302.5) are comparable (Table-1). Further comparison revealed that DHA and EPA inhibited TRPV1 currents with IC50 of 1200 and 224 nM, respectively, without affecting TRPA1 currents (Table-1). In addition, AMG9810, a commonly used TRPV1 antagonist, inhibited TRPV1 currents with IC50=163 nM (Park et al., 2011), which is ≈ 1600 times higher than that of RvD2. Collectively, there results indicate that resolvins and their precursors differentially regulate TRPV1 and TRPA1 currents.
Pro-resolving mediators such as LXA4, RvE1 and RvD1 each signal via specific GPCRs (Serhan et al., 2008). To determine the involvement of GPCRs in resolvins’ actions, we pretreated DRG cultures with selective Gαi-coupled GPCR inhibitor, pertussis toxin (PTX, 0.5 μg/ml) for 18 hours. After PTX treatment, RvD2 could not alter TRPV1 and TRPA1 currents (Figure 1G, P>0.05, n=6 neurons). We also blocked the action of G proteins by intracellular delivery of GDPβS (2.5 mM, 8 min) via recording electrode. GDPβS treatment completely blocked RvD2's inhibition on TRPV1 and TRPA1 currents (Fig. 1H). Taken together, these data suggest that 1) RvD2 does not act directly on TRPV1/A1, and 2) RvD2's effects are mediated by specific Gαi-coupled GPCRs in DRG neurons.
We further examined whether specific resolvins could differentially regulate TRPV1 and TRPA1-mediated pain in mice. Intraplantar injection of capsaicin (1 μg) or AITC (1 μg) elicited marked spontaneous pain behaviors (flinching/licking) in mice. Both capsaicin and AITC-induced nocifensive behaviors were significantly reduced by intraplantar RvD2 (10 ng, Figure 2A). In contrast, intraplantar RvE1 and RvD1 (10 ng) each selectively inhibited capsaicin and AITC-induced pain. At much higher doses (10 μg), the precursors EPA and DHA also inhibited capsaicin- but not AITC-induced pain. Of note, RvD2 was more potent than RvE1 in inhibiting capsaicin-induced spontaneous pain (Figure 2A). Therefore, both electrophysiological and behavioral data suggest distinct roles of resolvins in modulating TRPV1/A1-elicited inward currents and pain behaviors.
Intraplantar injection of formalin, carrageenan (CRG), and complete Freund's adjuvant (CFA) is known to elicit acute, sub-acute, and chronic inflammatory pain, respectively (Xu et al., 2010). Peripheral (intraplantar) administration of RvD2 (10 ng) largely prevented CRG-induced heat hyperalgesia (Fig. 2B) and mechanical allodynia (Fig. 2C), and reduced CRG-induced paw edema (data not shown). Spinal (intrathecal, i.t.) preemptive treatment of RvD2 (0.01-1 ng) also dose-dependently inhibited formalin-induced spontaneous pain in the 2nd phase but not in the 1st phase (Fig. 2D), indicating a specific inhibition of central sensitization. Strikingly, even 0.01 ng of RvD2 had significant antinociceptive action (Fig. 2D).
In addition to preventing the development of inflammatory pain, RvD2 also inhibited persistent inflammatory pain after post-treatment. Intrathecal administration of RvD2 (10 ng), 3 days after CFA-induced inflammation, effectively reduced CFA-induced heat hyperalgesia and mechanical allodynia for > 3 hours (Fig. 2E and data not shown).
We further tested the effects of RvD2 on baseline pain and motor function. RvD2 (10 ng, i.t.) did not alter baseline pain sensitivity to thermal and mechanical stimulation (Fig. 2F, and data not shown). Neither did this treatment affect the animal's motor function in the Rota-rod test (Fig. 2G). Thus, RvD2 can potently attenuate inflammatory pain in acute, sub-acute, and persistent/chronic phase, without affecting normal pain perception and motor function.
Given an important role of synaptic plasticity in generating inflammatory pain (Ji et al., 2003), we assessed the different roles of RvD2, RvE1, and RvD2 in spinal cord synaptic plasticity induced by TRPV1/A1 agonist. It is generally believed that changes in the frequency and amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) in spinal cord neurons are caused by altered glutamate neurotransmission via respective presynaptic and postsynaptic mechanisms (Xu et al., 2010). Patch clamp recording in lamina II neurons of spinal cord slices showed that RvD2 alone (1 ng/ml) altered neither the sEPSC frequency (Fig. 3A,B) nor sEPSC amplitude (P>0.05, n=5 neurons). Capsaicin (1 μM) and AITC (300 μM) substantially increased the frequency of sEPSCs (Figure 3A,B) but not the amplitude of sEPSCs (P>0.05, n=6), indicating that TRPV1/A1 agonist potentiated glutamate release from presynaptic terminals of primary afferents. Notably, the capsaicin- and AITC-induced increases in sEPSC frequency were does-dependently blocked by RvD2 (0.1 and 1 ng/ml, Fig 3A, B). Instead, RvE1 and RvD1 only selectively inhibited capsaicin- and AITC-induced sEPSC frequency increase, respectively (Fig. 3A, B). Notably, the concentrations required for inhibiting TRPV1/A1-evoked synaptic transmission is: RvD2<RvE1<RvD1.
CFA elicited significant increases both in sEPSC frequency (68%) and amplitude (23%) in spinal cord slices prepared from inflamed mice (one day). Strikingly, these increases in sEPSC frequency and amplitude were abolished by RvD2 at a low concentration (1 ng/ml) (Fig. 3C-E), indicating that RvD2 could modulate synaptic plasticity via both presynaptic (sEPSC frequency) and post-synaptic (sEPSC amplitude) mechanisms.
C-fiber-induced spinal long-term potentiation (LTP) is a persistent form of synaptic plasticity in the spinal cord (Ruscheweyh et al., 2011). Tetanic stimulation of the C-fibers in the sciatic nerve evoked marked LTP of C-fiber-evoked field potentials in the dorsal horn (Fig. 3F), which was completely reversed by RvD2 (10 ng), intrathecally administrated 2 h after LTP induction (Figure 3F, P < 0.05, Two-way ANOVA).
Although great effort in pain research and drug development has been devoted to developing TRPV1 and TRPA1 antagonists, little is known about the endogenous inhibitors of TRPV1/A1. Resolvins are endogenous anti-inflammatory and proresolving lipid mediators produced during the resolution phase of inflammation (Serhan et al., 2008). The present study has identified resolvins as highly potent endogenous inhibitors for TRPV1/A1. In particular, we demonstrated distinct roles of RvD1, RvD2, and RvE1 in regulating TRPV1 and TRPA1 activity. Although previous studies indicated different inhibition of TRPV1 and TRPA1 by RvE1 (Xu et al., 2010) and RvD1 (Bang et al., 2010), respectively, the present study further calculated the IC50 for RvE1 inhibition of TRPV1 (IC50=1.0 nM) and RvD1 inhibition of TRPA1 (IC50=8.5 nM). The most striking finding of this study is RvD2, a novel member of resolving family, inhibited both TRPV1 and TRPA1 with superior potency: IC50=0.1 nM for TRPV1 and IC50=2.1 nM for TRPA1. To the best our knowledge, RvD2 is probably the most potent inhibitor of TRPV1, among all the published compounds. Importantly, RvD2's inhibitory actions are mediated by specific GPCRs, which can clearly separate this endogenous inhibitor from classic TRPV1/A1 antagonists that act directly on the channels. In parallel, RvD2 also inhibited TRPV1/A1-induced spontaneous pain, as well as TRPV1/A1 and inflammation-evoked spinal cord synaptic plasticity (sEPSC increases).
Our findings demonstrated that peripheral and central administration of RvD2, at very low doses (0.01-10 ng), reduced inflammatory pain in acute, sub-acute, and persistent conditions. RvD2 also reduced different modalities of inflammatory pain including spontaneous pain and evoked pain (heat hyperalgesia and mechanical allodynia), as a result of inhibiting TRPV1/A1 and inflammation-induced synaptic plasticity. In addition, RvD2 may also attenuate inflammatory pain via TRPV1/A1-independent mechanisms. For example, RvD2 but not the TRPV1 antagonist AMG9810 reversed spinal cord LTP (Fig. 3F, Park et al., 2011).
Inflammatory pain such as arthritic pain is typically treated with opioids and COX-2 inhibitors with well-known side effects (Sommer and Birklein, 2010;Ji et al., 2011). Given its high potency and wide safety range, RvD2 and its related compounds are promising new therapeutics for treating pain associated with inflammation. However, the detailed signaling mechanisms of RvD2 in pain relief remain unclear. It is important to understand these molecular means which endow RvD2 to be so potent for pain relief. Elucidation of the RvD2 signaling mechanisms is also critical in future clinical development for targeting the RvD2 receptors and signaling pathways with small molecule agonists.
The work was supported in part by US National Institutes of Health grants R01-DE17794, NS54932 to RRJ, and R01-GM38765, P01-GM095467 to CNS, and R01-NS67686 to both RRJ and CNS. We thank the Resolvyx Pharmaceuticals Inc. for providing resolvins and Dr. Philip J. Vickers for helpful discussion.
C.N.S. is an inventor on patents [resolvins] assigned to BWH and licensed to Resolvyx Pharmaceuticals. C.N.S. is a scientific founder of Resolvyx Pharmaceuticals and owns equity in the company. C.N.S.’ interests were reviewed and are managed by the Brigham and Women1s Hospital and Partners HealthCare in accordance with their conflict of interest policies.