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Ethnopharmacological Relevance: Yuanhu Zhitong Prescription (YZP) is a well-known traditional Chinese medicine (TCM) formula for neuropathic pain (NP) therapy with a satisfying clinical efficacy. However, the underlying pharmacological mechanism and its compatibility principle remain unclear., Aim of the Study: This study aims to investigate the analgesic and compatibility mechanisms of YZP on neuropathic pain (NP) at the gene and biological process levels., Materials and Methods: The chronic constriction injury (CCI) rats were intragastrically administrated with extracts of YZP, YH and BZ separately, and then mechanical hypersensitivity were measured to evaluate the analgesic effects between YH and BZ before and after compatibility. Then, RNA-seq and bioinformatics analyses were performed to elucidate the potential mechanisms underlying YZP's analgesia and compatibility. Finally, the expression levels and significant differences of key genes were analyzed., Results: Behaviorally, both YZP and YH effectively alleviated mechanical allodynia in CCI rats, with YZP being superior to YH. In contrast, we did not observe an analgesic effect of BZ. Genetically, YZP, YH, and BZ reversed the expression levels of 52, 34, and 42 aberrant genes in the spinal cord of CCI rats, respectively. Mechanically, YZP was revealed to alleviate NP mainly by modulating the inflammatory response and neuropeptide signaling pathway, which are the dominant effective processes of YH. Interestingly, the effective targets of YZP were especially enriched in leukocyte activation and cytokine-mediated signaling pathways. Moreover, BZ was found to exert an adjunctive effect in enhancing the analgesic effect of YH by promoting skeletal muscle tissue regeneration and modulating calcium ion transport., Conclusions: YH, as the monarch drug, plays a dominant role in the analgesic effect of YZP that effectively relieves NP by inhibiting the spinal inflammation and neuropeptide signaling pathway. BZ, as the minister drug, not only synergistically enhances analgesic processes of YH but also helps to alleviate the accompanying symptoms of NP. Consequently, YZP exerted a more potent analgesic effect than YH and BZ alone. In conclusion, our findings offer new insights into understanding the pharmacological mechanism and compatibility principle of YZP, which may support its clinical application in NP therapy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Gabapentinoids are used worldwide as first-line agents for the treatment of neuropathic pain. Accumulating evidence indicates that one of the antihyperalgesic mechanisms of gabapentinoids is through activation of the noradrenergic pathway of the descending pain inhibition system. However, the involvement of the serotonin pathway is unclear. We investigated the effects of gabapentin (GBP) on the serotonergic pathway of the descending inhibitory system using the spinal nerve ligation (SNL) rat model. As in previous reports, administration of GBP to SNL rats improved paw withdrawal thresholds (PWT). Intrathecally administered serotonin receptor antagonists abolished GBP's amelioration in PWT. GBP did not ameliorate PWT in noradrenaline-depleted SNL rats by DSP-4. However, GBP ameliorated PWT in serotonin-depleted SNL rats by para-chlorophenylalanine, which was not inhibited by intrathecal administration of a serotonin receptor antagonist. Immunohistochemical analysis of serotonin in the spinal dorsal horn revealed a slight, albeit statistically insignificant, increase in 5-HT levels in SNL rats compared to naive rats. However, no apparent changes were observed before or after GBP administration in naive and SNL rats. In conclusion, the involvement of the serotonergic pathway in the antihyperalgesic effects of GBP on the spinal cord is secondary, although it cooperates with the noradrenergic system to produce analgesia., Competing Interests: Conflicts of interest The authors declare no conflicts of interest., (Copyright © 2023 Elsevier Ltd and Japan Neuroscience Society. All rights reserved.)

Ethnopharmacological Relevance: Until now, inflammatory pain, especially ones with central sensitization in the spinal cord, is far from effectively treated. Yu-Xue-Bi Tablets (YXB) is a patented medicine, which has been widely applied for inflammatory pain. However, its therapeutic characteristics and mechanism remain unknown., Aim of the Study: This study is designed to evaluate the analgesic characteristics and explore the underlying mechanism of YXB in the inflammatory pain model induced by Complete Freund's Adjuvant (CFA)., Materials and Methods: The analgesic effects were measured by Von Frey test. The expression of calcitonin gene-related peptide (CGRP) was quantified by immunofluorescence. The expression of immune factors was analyzed via Luminex assay. The further quantifications of C-C Motif chemokine ligand 3 (CCL3) were verified by Enzyme-linked immunosorbent assay (ELISA). The transmigration of macrophage and activation of microglia were evaluated by immunofluorescence. Spinal injections of purified CCL3, CCR1 antagonist (J113863) and CCR5 antagonist (Maraviroc) were used to clarify roles of CCL3 assumed in the pharmacological mechanism of YXB., Results: In CFA mice, YXB ameliorated the mechanical allodynia in dose and time dependent way, suppressed the central sensitization in dose dependent way. In the L5 spinal cord, YXB downregulated the expression of macrophage M1 pro-inflammatory factors TNFRI and CCL3, inhibited the transmigration of circulating macrophage and the activation of microglia. Purified CCL3 led to the transmigration of macrophage, activation of microglia, central sensitization, and mechanical allodynia in the Sham mice. Inhibitors of CCR1 and CCR5 attenuated above symptoms in CFA mice. Purified CCL3 blocked YXB mediated down regulation of CCL3, inhibition of macrophage transmigration, but not activation of microglia., Conclusion: YXB exerts the analgesic effects by inhibiting CCL3-mediated peripheral macrophage transmigrate into spinal cord. This study provided a novel approach for inflammatory pain treatment and new insight into the pharmacological action of YXB., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Pregabalin has been increasingly used in recent years, and becoming a first-line medication for the clinical treatment of neuropathic pain. However, the mechanisms underlying pregabalin-induced neuropathic pain alleviation remain unclear. In this study, we aimed to investigate whether PKC epsilon (PKCε)/ transient receptor potential vanilloid subtype 1(TRPV1) signaling pathway participated in pregabalin-induced analgesia during treatment of neuropathic pain using rat models of spared nerve injury (SNI). The left hind paw withdrawal mechanical thresholds (PWMT) of rats were measured preoperatively one day before and on day 1, 4, 7 and 14 after surgery. On day 7 after SNI surgery, the rats received ligation operation were administrated with pregabalin intraperitoneally and were intrathecally injected with PKC Inhibitor BIM Ⅰ or PKC agonist PMA for seven consecutive days, IL-1β and IL-6 expression levels in the spinal cord of rats were then assessed. Furthermore, we analyzed the PKCε, TRPV1, pTRPV1 and Glial fibrillary acidic protein (GFAP) protein levels and the expression of reactive astrocytes and the PKCε, TRPV1 and pTRPV1 positive cells on day 14 after SNI. Our findings indicated that pregabalin could relieve neuropathic pain to a certain extent by suppressing the PKCε/TRPV1 signaling pathway and inhibiting inflammatory processes in the spinal cord., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Chemotherapy-induced neuropathic pain is a debilitating and common side effect of cancer treatment and so far no effective drug is available for treatment of the serious side effect. Previous studies have demonstrated β2-adrenoreceptor (ADRB2) agonists can attenuate neuropathic pain. However, the role of ADRB2 in paclitaxel -induced neuropathic pain (PINP) remains unclear. In this study, we investigated the effect of formoterol, a long-acting ADRB2 agonist, and related mechanisms in PINP. A rat model of PINP was established by intraperitoneal injection of paclitaxel (2 mg/kg) every other day with a final cumulative dose of 8 mg/kg. Hind paw withdrawal thresholds (PWTs) in response to von Frey filament stimuli were used to evaluate mechanical allodynia. Western blot was used to examine the expression of ADRB2, peroxisome proliferator-activated receptor coactivator-1α (PGC-1α), nuclear respiratory factors 1 (NRF1) and mitochondrial transcription factor A (TFAM) and the immunofluorescence was to detect the cellular localization of ADRB2 and PGC-1α in the spinal cord. Moreover, we measured mitochondrial DNA (mtDNA) copy number by qPCR. In our study, formoterol attenuated established PINP and delayed the onset of PINP. Formoterol restored ADRB2 expression as well as mtDNA copy number and PGC-1α, NRF1, and TFAM protein expression, which are major genes involved in mitochondrial biogenesis, in the spinal cord of PINP rats. Moreover, we found the analgesic effect of formoterol against PINP was partially abolished by PGC-1α inhibitor SR-18292. Collectively, these results demonstrated the activation of ADRB2 with formoterol ameliorates PINP at least partially through induction of mitochondrial biogenesis., (Copyright © 2021 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

The sigma-1 receptor (Sig-1R) plays an important role in spinal pain transmission by increasing phosphorylation of the N-methyl-D-aspartate (NMDA) receptor GluN1 subunit (pGluN1). As a result Sig-1R has been suggested as a novel therapeutic target for prevention of chronic pain. Here we investigated whether interleukin-1β (IL-1β) modulates the expression of the Sig-1R in spinal astrocytes during the early phase of nerve injury, and whether this modulation affects spinal pGluN1 expression and the development of neuropathic pain following chronic constriction injury (CCI) of the sciatic nerve. Repeated intrathecal (i.t.) administration of IL-1β from days 0-3 post-surgery significantly reduced the increased pGluN1 expression at the Ser896 and Ser897 sites in the ipsilateral spinal cord, as well as, the development of mechanical allodynia and thermal hyperalgesia in the ipsilateral hind paw of CCI mice, which were restored by co-administration of IL-1 receptor antagonist with IL-1β. Sciatic nerve injury increased the expression of Sig-1R in astrocytes of the ipsilateral spinal cord, and this increase was suppressed by i.t. administration of IL-1β. Agonistic stimulation of the Sig-1R with PRE084 restored pGluN1 expression and the development of mechanical allodynia that were originally suppressed by IL-1β in CCI mice. Collectively these results demonstrate that IL-1β administration during the induction phase of neuropathic pain produces an analgesic effect on neuropathic pain development by controlling the expression of Sig-1R in spinal astrocytes., (Copyright © 2021 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Background: Neuropathic pain is a debilitating disease with few effective treatments. Emerging evidence indicates the involvement of mitochondrial dysfunction and oxidative stress in neuropathic pain. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a potent regulator of the antioxidant response system. In this study, we investigated whether RTA-408 (RTA, a novel synthetic triterpenoid under clinical investigation) could activate Nrf2 and promote mitochondrial biogenesis (MB) to reverse neuropathic pain and the underlying mechanisms., Methods: Neuropathic pain was induced by chronic constriction injury (CCI) of the sciatic nerve. Pain behaviors were measured via the von Frey test and Hargreaves plantar test. The L4-6 spinal cord was collected to examine the activation of Nrf2 and MB., Results: RTA-408 treatment significantly reversed mechanical allodynia and thermal hyperalgesia in CCI mice in a dose-dependent manner. Furthermore, RTA-408 increased the activity of Nrf2 and significantly restored MB that was impaired in CCI mice in an Nrf2-dependent manner. Peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1 α ) is the key regulator of MB. We found that the PGC-1 α activator also induced a potent analgesic effect in CCI mice. Moreover, the antinociceptive effect of RTA-408 was reversed by the preinjection of the PGC-1 α inhibitor., Conclusions: Nrf2 activation attenuates chronic constriction injury-induced neuropathic pain via induction of PGC-1 α -mediated mitochondrial biogenesis in the spinal cord. Our results indicate that Nrf2 may be a potential therapeutic strategy to ameliorate neuropathic pain and many other disorders with oxidative stress and mitochondrial dysfunction., Competing Interests: The authors have no conflicts of interest to declare., (Copyright © 2021 Jia Sun et al.)

Endomorphin analogs containing unnatural amino acids have demonstrated potent analgesic effects in our previous studies. In the present study, the differences in antinociception and the mechanisms thereof for analogs 1-3 administered intracerebroventricularly and intrathecally were explored. All analogs at different routes of administration produced potent analgesia compared to the parent peptide endomorphin-1. Multiple antagonists and antibodies were used to explore the mechanisms of action of these analogs, and it was inferred that analogs 1-3 stimulated the μ opioid receptor to induce antinociception. Moreover, the antibody data suggested that analog 2 may induce the release of immunoreactive [Leu 5 ]-enkephaline and [Met 5 ]-enkephaline to produce a secondary component of antinociception at the spinal level and analog 3 may stimulate the the release of immunoreactive [Met 5 ]-enkephaline at the spinal level. Finally, analogs 2 and 3 produced no acute tolerance in the spinal cord. We hypothesize that the unique characteristics of the endomorphin analogs result from their capacities to stimulate the release of endogenous antinociceptive substances., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Diabetic neuropathy is one of the most common complications of diabetes mellitus. Excess glutamate release and oxidative stress are hypothesized to be involved in the pathophysiology of diabetes-induced neuropathy. This study was designed to investigate the effect of clavulanic acid (CLAV), a competitive beta-lactamase inhibitor, on the streptozocin (STZ)-induced neuropathic pain and possible mechanisms in the spinal cord of rats. Male Wistar rats were divided into naive group; control group which got a single dose of STZ (50 mg/kg, i.p.), as a model of diabetic neuropathic pain; prophylactic groups: animals received CLAV (10, 20 and 40 mg/kg, i.p.) 1 week after STZ for 10 days; and therapeutic group: animals received 20 mg/kg CLAV, 21 days after STZ for 10 days. Study of pain behaviors was started on days 0, 7, 14, 21, 28, 35 and 42 after STZ. The expression of the glutamate transport 1 (GLT1), genes of oxidative stress including inducible nitric oxide synthase (iNOS), proinflammatory cytokine, tumor necrosis factor alpha (TNF-α), as well as genes involved in the apoptosis including bcl2, bcl2-associated x (bax) were measured in the spinal cord tissue by Real Time PCR, on day 42. On day 21 post injection of STZ, diabetic animals showed significant mechanical allodynia, cold allodynia and thermal hyperalgesia. CLAV in all doses of 10, 20 and 40 mg/kg reduced symptoms of allodynia and hyperalgesia, in both prophylactic and therapeutic regimens. While iNOS, TNF-α, bax/bcl2 were found significantly overexpressed in spinal cord of diabetic animals, their expression in animals received CLAV had been reduced. In contrast, GLT1 that had decreased in the spinal cord of diabetic animals, significantly increased in those received CLAV. CLAV was found a promising candidate for reliving neuropathic pain in diabetes mellitus. Such beneficial effect of CLAV could be, in part, attributed to the increased expression of GLT 1, inhibition of nitrosative stress, anti-inflammation, and inhibition of some apoptotic mediators followed by administration into diabetic animals.

Ethnopharmacological Relevance: Chaiqin chengqi decoction (CQCQD) and its derivatives have been widely used in China for the early management of patients with acute pancreatitis (AP). Numerous studies demonstrate the anti-inflammatory and anti-oxidative effects of CQCQD and derivatives, but whether these effects can be attributed to suppressing neurogenic inflammation, has never been studied., Aim of the Study: To investigate the effects of CQCQD on substance P (SP)-neurokinin 1 receptor (NK1R) based neurogenic inflammation in an experimental AP model., Material and Methods: For AP patients on admission, pain score was accessed by visual analog scale (VAS); the levels of serum SP and expressions of pancreatic SP and NK1R were also determined. For in vivo study, mice received 7 intraperitoneal injections of cerulein (50 μg/kg) at hourly intervals to induce AP, whilst controls received normal saline injections. In the treatment groups, CQCQD (10 g/kg, 200 μl) was intragastrically given at the third, fifth, and seventh of the cerulein injection or the NK1R antagonist CP96345 (5 mg/kg) was intraperitoneally injected 30 min before the first cerulein administration. The von Frey test was performed to evaluate pain behavior. Animals were sacrificed at 12 h from the first cerulein/saline injection for severity assessment. Pharmacology network analysis was used to identify active ingredients of CQCQD for AP and pain. In vitro, freshly isolated pancreatic acinar cells were pre-treated with CQCQD (5 mg/ml), CP96345 (1 μM), or selected active compounds of CQCQD (12.5, 25, and 50 μM) for 30 min, followed by SP incubation for another 30 min., Results: The VAS score as well as the levels of serum SP and expressions of pancreatic SP-NK1R were up-regulated in moderately severe and severe patients compared with those with mild disease. CQCQD, but not CP96345, consistently and significantly ameliorated pain, pancreatic necrosis, and systemic inflammation in cerulein-induced AP as well as inhibited NK1R internalization of pancreatic acinar cells. These effects of CQCQD were associated with reduction of pancreatic SP-NK1R and neuron activity in pancreas, dorsal root ganglia, and spinal cord. Baicalin, emodin, and magnolol, the top 3 active components of CQCQD identified via pharmacology network analysis, suppressed NK1R internalization and NF-κB signal pathway activation in isolated pancreatic acinar cells., Conclusions: CQCQD ameliorated cerulein-induced AP and its associated pain via inhibiting neuron activation-mediated pancreatic acinar cell SP-NK1R signaling pathways and its active compounds baicalin, emodin, and magnolol contributed to this effect., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Treating postoperative (PO) pain is a clinical challenge. Inadequate PO pain management can lead to worse outcomes, for example chronic post-surgical pain. Therefore, acquiring new information on the PO pain mechanism would increase the therapeutic options available. In this paper, we evaluated the role of a natural substance, epigallocatechin-3-gallate (EGCG), on pain and neuroinflammation induced by a surgical procedure in an animal model of PO pain. We performed an incision of the hind paw and EGCG was administered for five days. Mechanical allodynia, thermal hyperalgesia, and motor dysfunction were assessed 24 h, and three and five days after surgery. At the same time points, animals were sacrificed, and sera and lumbar spinal cord tissues were harvested for molecular analysis. EGCG administration significantly alleviated hyperalgesia and allodynia, and reduced motor disfunction. From the molecular point of view, EGCG reduced the activation of the WNT pathway, reducing WNT3a, cysteine-rich domain frizzled (FZ)1 and FZ8 expressions, and both cytosolic and nuclear β-catenin expression, and the noncanonical β-catenin-independent signaling pathways, reducing the activation of the NMDA receptor subtype NR2B (pNR2B), pPKC and cAMP response element-binding protein (pCREB) expressions at all time points. Additionally, EGCG reduced spinal astrocytes and microglia activation, cytokines overexpression and nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB) pathway, downregulating inducible nitric oxide synthase (iNOS) activation, cyclooxygenase 2 (COX-2) expression, and prostaglandin E2 (PGE2) levels. Thus, EGCG administration managing the WNT/β-catenin signaling pathways modulates PO pain related neurochemical and inflammatory alterations.

Neuropathic pain is a debilitating chronic pain condition, and its treatment remains a clinical challenge. Curcumin, a naturally occurring phenolic compound, possesses diverse biological and pharmacological effects but has not yet been approved as a drug due to its low bioavailability. In order to overcome this limitation, we synthesized a potential ester prodrug of curcumin, curcumin diethyl diglutarate (CurDDG). In this study, we evaluated the pharmacological advantages of CurDDG over curcumin in a mouse model of chronic constriction injury (CCI), and the anti-inflammatory effect of CurDDG in LPS-induced RAW 264.7 macrophage cells was accessed to clarify the underline mechanism. Mice were treated with various oral doses of curcumin (25, 50, 100 and 200 mg/kg/day, daily for 14 days) or equimolar doses of CurDDG. CurDDG at all doses tested significantly attenuated CCI-induced thermal hyperalgesia and mechanical allodynia compared with the CCI-control group. CurDDG at 25, 50 and 100 mg/kg demonstrated significantly greater efficacy on both mechanical and thermal hypersensitivities compared to that of curcumin. The effect of CurDDG correlated well with the inhibition of TNF-α and IL-6 levels in both the sciatic nerve and the spinal cord, as compared to its respective control groups. Similarly, in the in vitro study, CurDDG significantly reduced the LPS-induced expression of TNF-α and IL-6. Moreover, CurDDG significantly decreased COX-2 and iNOS levels and attenuated p38, JNK, and ERK1/2 phosphorylation as compared to the curcumin-treated cells. Altogether, this study demonstrated the improved pharmacological effects of curcumin by its diglutarate conjugate, CurDDG., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The recently identified molecule P7C3 has been highlighted in the field of pain research. We examined the effect of intrathecal P7C3 in tissue injury pain evoked by formalin injection and determined the role of the GABA system in the activity of P7C3 at the spinal level. Male Sprague-Dawley rats with intrathecal catheters implanted for experimental drug delivery were studied. The effects of intrathecal P7C3 and nicotinamide phosphoribosyltransferase (NAMPT) administered 10 min before the formalin injection were examined. Animals were pretreated with bicuculline, a GABA-A receptor antagonist; saclofen, a GABA-B receptor antagonist; L-allylglycine, a glutamic acid decarboxylase (GAD) blocker; and CHS 828, an NAMPT inhibitor; to observe involvement in the effects of P7C3. The effects of P7C3 alone and the mixture of P7C3 with GABA receptor antagonists on KCl-induced calcium transients were examined in rat dorsal root ganglion (DRG) neurons. The expression of GAD and the concentration of GABA in the spinal cord were evaluated. Intrathecal P7C3 and NAMPT produced an antinociceptive effect in the formalin test. Intrathecal bicuculline, saclofen, L-allylglycine, and CHS 828 reversed the antinociception of P7C3 in both phases. P7C3 decreased the KCl-induced calcium transients in DRG neurons. Both bicuculline and saclofen reversed the blocking effect of P7C3. The levels of GAD expression and GABA concentration decreased after formalin injection and were increased by P7C3. These results suggest that P7C3 increases GAD activity and then increases the GABA concentration in the spinal cord, which in turn may act on GABA receptors causing the antinociceptive effect against pain evoked by formalin injection., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Overactivation of N-methyl-D-aspartate receptor (NMDAR) in the spinal cord dorsal horn (SDH) in the setting of injury represents a key mechanism of neuropathic pain. However, directly blocking NMDAR or its downstream signaling, interaction between postsynaptic density-95 (PSD-95) and neuronal nitric oxide synthase (nNOS), causes analgesic tolerance, mainly due to GABAergic disinhibition. The aim of this study is to explore the possibility of preventing analgesic tolerance through co-targeting NMDAR downstream signaling and γ-aminobutyric acid type A receptors (GABA A Rs). Methods: Mechanical/thermal hyperalgesia were quantified to assess analgesic effects. Miniature postsynaptic currents were tested by patch-clamp recording to evaluate synaptic transmission in the SDH. GABA-evoked currents were tested on HEK293 cells expressing different subtypes of recombinant GABA A Rs to assess the selectivity of (+)-borneol and ZL006-05. The expression of α2 and α3 subunits of GABA A Rs and BDNF, and nNOS-PSD-95 complex levels were analyzed by western blotting and coimmunoprecipitation respectively. Open field test, rotarod test and Morris water maze task were conducted to evaluate the side-effect of ZL006-05. Results: (+)-Borneol selectively potentiated α2- and α3-containing GABA A Rs and prevented the disinhibition of laminae I excitatory neurons in the SDH and analgesic tolerance caused by chronic use of ZL006, a nNOS-PSD-95 blocker. A dual-target compound ZL006-05 produced by linking ZL006 and (+)-borneol through an ester bond blocked nNOS-PSD-95 interaction and potentiated α2-containing GABA A R selectively. Chronic use of ZL006-05 did not produce analgesic tolerance and unwanted side effects. Conclusion: By targeting nNOS-PSD-95 interaction and α2-containing GABA A R simultaneously, chronic use of ZL006-05 can avoid analgesic tolerance and unwanted side effects. Therefore, we offer a novel candidate drug without analgesic tolerance for treating neuropathic pain., Competing Interests: Competing Interests: A patent on ZL006-05 and its analogues has been authorized in the United States (US9481636B2), China (ZL201210403997.6), Japan (5841672) and European Union (EP2774914B1). Name of inventors: Dong-Ya Zhu, Fei Li and Chun-Xia Luo., (© The author(s).)

D-Amino acid oxidase (DAAO) specifically catalyzes the oxidative deamination of neutral and polar D-amino acids and finally yields byproducts of hydrogen peroxide. Our previous work demonstrated that the spinal astroglial DAAO/hydrogen peroxide (H 2 O 2 ) pathway was involved in the process of pain and morphine antinociceptive tolerance. This study aimed to report mouse strain specificity of DAAO inhibitors on antinociception and explore its possible mechanism. DAAO inhibitors benzoic acid, CBIO, and SUN significantly inhibited formalin-induced tonic pain in Balb/c and Swiss mice, but had no antinociceptive effect in C57 mice. In contrast, morphine and gabapentin inhibited formalin-induced tonic pain by the same degrees among Swiss, Balb/c and C57 mice. Therefore, mouse strain difference in antinociceptive effects was DAAO inhibitors specific. In addition, intrathecal injection of D-serine greatly increased spinal H 2 O 2 levels by 80.0% and 56.9% in Swiss and Balb/c mice respectively, but reduced spinal H 2 O 2 levels by 29.0% in C57 mice. However, there was no remarkable difference in spinal DAAO activities among Swiss, Balb/c and C57 mice. The spinal expression of glutathione (GSH) and glutathione peroxidase (GPx) activity in C57 mice were significantly higher than Swiss and Balb/c mice. Furthermore, the specific GPx inhibitor D-penicillamine distinctly restored SUN antinociception in C57 mice. Our results reported that DAAO inhibitors produced antinociception in a strain-dependent manner in mice and the strain specificity might be associated with the difference in spinal GSH and GPx activity., (© 2021 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.)

Objective: To explore the potential analgesic effect of melatonin and its underlying molecular mechanisms in a neuropathic pain model induced by spinal nerve ligation (SNL)., Methods: The experimental animals were divided into different groups including sham, vehicle, melatonin (MT) treatment, caspase-1 inhibitor (VX-765) treatment and MT2 antagonist (4P-PDOT) treatment. On the first three successive postoperative days, rats were intraperitoneally administered with MT, VX-765 or combination of MT and 4P-PDOT. Hyperalgesic behavior after SNL was evaluated using the paw withdrawal threshold (PWT). We then assessed expression of tumor necrosis factor-α (TNF-α), IL-18, interleukin-1β (IL-1β), NLRP3 inflammasome components, and nuclear factor-κB (NF-κB) activation using enzyme-linked immunosorbent assay kits (ELISA), real-time PCR, immunohistochemistry, and western blot, respectively, in spinal cord horn tissues extracted on postoperative day 7., Results: The results showed that melatonin treatment alleviated SNL-induced allodynia. We observed an SNL-induced upregulation of TNF-α, IL-18, IL-1β, NLRP3, ASC, cleaved caspase-1, and NF-κB in the lumbar spinal cord horn of rats, which was significantly attenuated by intraperitoneal injection of melatonin or VX-765. Additionally, co-treatment of melatonin and 4P-PDOT abrogated the analgesic and anti-inflammatory effect of melatonin., Conclusion: Melatonin had potent analgesic and anti-inflammatory effects in SNL-induced neuropathic pain via NF-κB/NLRP3 inflammasome signaling pathway. Our results therefore suggested that this pathway could represent a novel therapeutic target for the management of neuropathic pain., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Opioids such as morphine are mainstay treatments for clinical pain conditions. Itch is a common side effect of opioids, particularly as a result of epidural or intrathecal administration. Recent progress has advanced our understanding of itch circuits in the spinal cord. However, the mechanisms underlying opioid-induced itch are not fully understood, although an interaction between µ-opioid receptor (MOR) and gastrin-releasing peptide receptor (GRPR) in spinal GRPR-expressing neurons has been implicated. In this study we investigated the cellular mechanisms of intrathecal opioid-induced itch by conditional deletion of MOR-encoding Oprm1 in distinct populations of interneurons and sensory neurons. We found that intrathecal injection of the MOR agonists morphine or DAMGO elicited dose-dependent scratching as well as licking and biting, but this pruritus was totally abolished in mice with a specific Oprm1 deletion in Vgat+ neurons [Oprm1-Vgat (Slc32a1)]. Loss of MOR in somatostatin+ interneurons and TRPV1+ sensory neurons did not affect morphine-induced itch but impaired morphine-induced antinociception. In situ hybridization revealed Oprm1 expression in 30% of inhibitory and 20% of excitatory interneurons in the spinal dorsal horn. Whole-cell recordings from spinal cord slices showed that DAMGO induced outward currents in 9 of 19 Vgat+ interneurons examined. Morphine also inhibited action potentials in Vgat+ interneurons. Furthermore, morphine suppressed evoked inhibitory postsynaptic currents in postsynaptic Vgat- excitatory neurons, suggesting a mechanism of disinhibition by MOR agonists. Notably, morphine-elicited itch was suppressed by intrathecal administration of NPY and abolished by spinal ablation of GRPR+ neurons with intrathecal injection of bombesin-saporin, whereas intrathecal GRP-induced itch response remained intact in mice lacking Oprm1-Vgat. Intrathecal bombesin-saporin treatment reduced the number of GRPR+ neurons by 97% in the lumber spinal cord and 91% in the cervical spinal cord, without changing the number of Oprm1+ neurons. Additionally, chronic itch from DNFB-induced allergic contact dermatitis was decreased by Oprm1-Vgat deletion. Finally, naloxone, but not peripherally restricted naloxone methiodide, inhibited chronic itch in the DNFB model and the CTCL model, indicating a contribution of central MOR signalling to chronic itch. Our findings demonstrate that intrathecal morphine elicits itch via acting on MOR on spinal inhibitory interneurons, leading to disinhibition of the spinal itch circuit. Our data also provide mechanistic insights into the current treatment of chronic itch with opioid receptor antagonist such as naloxone., (© The Author(s) (2020). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Neuropathic pain is still a critical public health problem worldwide. Thereby, the search for novel and more effective strategies against neuropathic pain is urgently considered. It is known that neuroinflammation plays a crucial role in the pathogenesis of neuropathic pain. SedumLineare Thunb. (SLT), a kind of Chinese herb originated from the whole grass of Crassulaceae plant, was reported to possess anti-inflammatory activity. However, whether SLT has anti-nociceptive effect on neuropathic pain and its possible underlying mechanisms remains poorly elucidated. In this study, a rat model of neuropathic pain induced by spared nerve injury (SNI)was applied. SLT (p.o.) was administered to SNI rats once every day lasting for 14 days. Pain-related behaviors were assessed by using paw withdrawal threshold (PWT) and CatWalk gait parameters. Expression levels of inflammatory mediators and pain-related signaling molecules in the spinal cord were detected using western blotting assay. The results revealed that SLT (30, 100, and 300 mg/kg, p.o.) treatment for SNI rats ameliorated mechanical hypersensitivity in a dose-dependent manner. Application of SLT at the most effective dose of 100 mg/kg to SNI rats not only significantly blocked microglial activation, but also markedly reduced the protein levels of spinal HMGB1, TLR4, MyD88, TRAF6, IL-1β, IL-6, and TNF-α, along with an enhancement in gait parameters. Furthermore, SLT treatment dramatically inhibited the phosphorylation levels of both IKK and NF-κB p65 but obviously improved both IκB and IL-10 protein expression in the spinal cord of SNI rats. Altogether, these data suggested that SLT could suppress spinal TLR4/NF-κB signaling pathway in SNI rats, which might at least partly contribute to its anti-nociceptive action, indicating that SLT may serveas a potential therapeutic agent for neuropathic pain., (Copyright © 2021 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Contactin-associated protein-like 2 (CNTNAP2 or CASPR2) is a neuronal transmembrane protein of the neurexin superfamily which is correlated with pain related hypersensitivity. Recent results indicated that the hyperactive phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway may be a promising therapeutic target for pain-related hypersensitivity in patients with dysfunction of CNTNAP2. Resveratrol is one of the most widely studied polyphenols with several beneficial properties. In the present study, we investigated the effects of resveratrol on the pain related hypersensitivity. And we found that the up-regulated phosphorylation of S6 could be suppressed by resveratrol. The nocifensive behavior duration time to heat and chemical algogens stimulation in Cntnap2-deficiency (Cntnap2 -/- ) mice could be attenuated by resveratrol. Our results indicated that resveratrol could rescue the pain related hypersensitivity for Cntnap2 -/- mice may be via mTOR signaling pathway., (Copyright © 2020. Published by Elsevier B.V.)

Agents that modulate the activity of high-voltage gated calcium channels (HVCCs) exhibit experimentally and clinically significant effect by relieving visceral pain. Among these agents, the toxins Phα1β and ω-conotoxin MVIIA effectively reduce chronic pain in rodent models. The molecular mechanisms underlying the chronic pain associated with acute pancreatitis (AP) are poorly understood. Hypercalcemia is a risk factor; the role of cytosolic calcium is considered to be a modulator of pancreatitis. Blockade of Ca 2+ signals may be useful as a prophylactic treatment of pancreatitis. We explored the pathophysiological roles of three peptide toxins: Phα1β and its recombinant form CTK 01512-2-blockers of TRPA1 receptor and HVCCs and ω-conotoxin MVIIA, a specific blocker of N-type calcium channels in cerulein-induced AP. Cerulein injection elicits AP in rats, evidenced by an increase in hyperalgesic pain, inflammatory infiltration, amylase and lipase secretion, and reactive oxygen species, TNF-α, and p65 NF-κB levels. These effects of cerulein-induced AP were abolished by Phα1β and its recombinant form CTK 01512-2, whereas ω-conotoxin MVIIA had no effect on the induced increase in pancreatic enzyme secretion. Our results demonstrate that Phα1β and CTK 01512-2 toxins-antagonists of HVCCs and TRPA1 receptor presented an effective response profile, in the control of nociception and inflammatory process in the AP model in rats, without causing changes in spontaneous locomotion of the rats., (Copyright © 2020. Published by Elsevier B.V.)

Chronic postsurgical pain (CPSP) is a serious issue for many postoperative patients. Though there are numerous treatment options for the prevention of CPSP, none of them is optimal as the mechanisms of the transition from acute to chronic postoperative pain have not been elucidated. Ketamine and opioids have been administered for chronic postoperative pain treatment but induce severe adverse reactions and/or physical dependency. Here, we examined whether pre-administration of the nonselective N-methyl-d-aspartate (NMDA) receptor antagonist magnesium sulfate attenuates CPSP behavior and alters the expression of glutamate ionotropic receptor NMDA type subunit 1a (Grin1 mRNA) in a rat skin/muscle incision and retraction (SMIR) model. We assessed the effects of a single subcutaneous magnesium sulfate injection on nociceptive behaviors including guarding pain, mechanical hyperalgesia, and heat hypersensitivity in rats after SMIR surgery. We used reverse transcription-quantitative PCR (RT-qPCR) to evaluate Grin1 mRNA expression in the dorsal horn of the spinal cord on postoperative day 14. Compared with the vehicle, magnesium sulfate administration before SMIR surgery reduced mechanical hyperalgesia for 17 d Grin1 gene expression was significantly higher on the ipsilateral side than the contralateral side (P = 0.001) on postoperative day 14. The magnesium sulfate injection prevented Grin1 mRNA upregulation in the spinal cord dorsal horn. A single magnesium sulfate injection mitigated SMIR-induced mechanical hyperalgesia possibly by modulating Grin1 expression. Preoperative magnesium sulfate administration could prove to be a simple and safe CPSP treatment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Artemisinin (ART) was initially described for the control of inflammation and pain. However, the mechanisms involved with its antinociceptive effect are still poorly understood. Thus, this present study aimed to investigate the effect of ART in both free and nanocapsulated form on postoperative pain, as well as the participation of the spinal Toll-like receptor 4 (TLR4) in this process. Postoperative pain was induced using the skin/muscle incision retraction (SMIR) model in male Swiss mice. After 3 and 28 days of SMIR, the animals received an intrathecal injection of free or nanocapsulated ART, and the nociceptive threshold was evaluated by von Frey filament test. To evaluate the involvement of the microglia, astrocytes, and TLR4, minocycline (a microglia inhibitor), fluorocitrate (an astrocyte inhibitor), and Lipopolysaccharide Rhodobacter sphaeroides (LPS-RS), a TLR4 antagonist, were intrathecally injected on the third day of SMIR. The levels of spinal TLR4 protein and proinflammatory cytokines tumor necrosis factor-alpha (TNF-α), and interleukin-1-beta (IL-1β) were quantified by western blot and enzyme-linked immunosorbent assay, respectively. The results showed that free ART reduced postoperative pain (P < 0.001, F 5,30  = 7.49, 16.66% for 1000 ng dose; and P < 0.01, F 5,30  = 7.49, 14.58% for 500 ng dose) on the 3rd day of SMIR; while the ART nanocapsule had this effect on both the third (P < 0.001; F 5,30  = 4.94; 43.75, 39.58 and 72.91% for the 250, 500 and 1000 ng doses, respectively) and 28th (P < 0.05; F 5,30  = 7.71; 29.16 and 33.33% for the 500 and 1000 ng doses, respectively) day. The ART nanocapsule had a more potent and longer antinociceptive effect than free ART or morphine. Postoperative pain was also reduced by minocycline and LPS-RS. The ART nanocapsule also reduced the increased levels of TLR4, TNF-α, and IL-1β induced by SMIR. These data suggest that the ART nanocapsule has a potent analgesic effect on postoperative pain at the spinal level, and this response involves the inhibition of TLR4 and the proinflammatory cytokines TNF-α and IL-1β.

Chemotherapy-induced neuropathic pain harms the quality of life patients. Vincristine is an often used chemotherapeutic drug that evokes neuralgia via inflammation. Puerarin (Pue) extracted from Puerariae Lobatae Radix has analgesic and anti-inflammatory effects; however, its possible effect and mechanism in vincristine (Vin)-induced neuropathic pain has not been investigated. The present research aimed to explore whether Pue could relieve chemotherapy-evoked neuropathic pain and the underlying mechanism actions. Rat neuropathic pain was established by intraperitoneal injection of vincristine. Pue was orally administered in two dose levels (25 or 50 mg/kg/d) for three weeks. The paw withdrawal latency and paw withdrawal threshold were performed to evaluate the pain behaviors. Inflammatory cytokines in the spinal cord and dorsal root ganglion were measured by ELISA kits. qRT-PCR, western blot, and immunofluorescence staining were employed to measure the level and expression feature of inflammatory cytokines. Our findings showed that Pue improved hyperalgesia and allodynia. Treatment with Pue restored the levels of tumor necrosis factor-α (TNF-α), and IL-1β and increased the levels of transforming growth factor-β (TGF-β), and interleukin-10 (IL-10). On the molecular level, treatment with Pue down-regulated the protein levels of IL-1β, and NF-κBp65 and up-regulated the protein levels of TGF-β, p-Smad2, and p-Smad3 (TGF-β/Smad) in the spinal cord and DRG. Immunofluorescence staining further demonstrated that Pue decreased the NF-κBp65 protein. Our findings imply that Pue relieved chemotherapy-induced neuropathic pain might be attributable to the suppression of inflammation cytokines. The anti-inflammation action of Pue might be associated with the activation of the TGF-β/Smad pathway, a novel mechanism exploring its prophylactic effect in vincristine-induced neuropathic pain., (Copyright © 2020 Elsevier B.V. All rights reserved.)

In the spinal cord, γ-aminobutyric acid (GABA) interneurons play an essential role in antinociception. However, not all actions of GABA favor antinociception at the supraspinal level. We previously reported that gabaculine, which increases endogenous GABA in the synaptic clefts, induces loss of the righting reflex (LORR) that is one indicator of hypnosis, but not immobility in response to noxious stimulus. A slow pain is transmitted to the spinal cord via C fibers and evokes substance P (SP) release from their terminals. However, the antinociceptive effects of gabaculine are still unknown. Our study examined whether the analgesic effects of the opioid morphine or the α 2 -adrenoceptor agonist dexmedetomidine, whose actions are mediated through facilitation of the descending analgesic pathway, are affected by gabaculine-induced LORR. We also explored the effects of GABA receptor agonists on SP release from cultured dorsal root ganglion (DRG) neurons. All drugs were administered systemically to mice. To assess antinociception, loss of nociceptive response (analgesia) and immobility were evaluated. DRG cells were dissected from rats. Gabaculine produced no analgesia. Either morphine or dexmedetomidine in combination with gabaculine induced immobility; however, the doses of each drug required to induce immobility were much higher than those required to induce analgesia. Capsaicin significantly increased SP release from DRG cells, but a high concentration (1 mM) of the GABA receptor agonist muscimol, propofol, gaboxadol, or baclofen did not inhibit the capsaicin-induced SP release, suggesting that their antinociceptive effects were not through this mechanism. Thus, the gabaculine-induced LORR may inhibit the descending analgesic pathway., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Many people with MS (pwMS) use unregulated cannabis or cannabis products to treat the symptoms associated with the disease. In line with this, Sativex, a synthetic combination of cannabidiol (CBD) and Δ 9 -tetrahydrocannabinol (Δ 9 -THC) has been approved to treat symptoms of spasticity. In animals, CBD is effective in reducing the amounts of T-cell infiltrates in the spinal cord, suggesting CBD has anti-inflammatory properties. By doing this, CBD has shown to delay symptom onset in animal models of multiple sclerosis and slow disease progression. Importantly, combinations of CBD and Δ 9 -THC appear more effective in treating animal models of multiple sclerosis. While CBD reduces the amounts of cell infiltrates in the spinal cord, Δ 9 -THC reduces scores of spasticity. In human studies, the results are less encouraging and conflict with the findings in animals. Drugs which deliver a combination of Δ 9 -THC and CBD in a 1:1 ratio appear to be only moderately effective in reducing spasticity scores, but appear to be almost as effective as current front-line treatments and cause less severe side effects than other treatments, such as baclofen (a GABA-B receptor agonist) and tizanidine (an α2 adrenergic receptor agonist). The findings of the studies reviewed suggest that cannabinoids may help treat neuropathic pain in pwMS as an add-on therapy to already established pain treatments. It is important to note that treatment with cannabinoid compounds may cause significant cognitive dysfunction. Long term double-blind placebo studies are greatly needed to further our understanding of the role of cannabinoids in multiple sclerosis treatment.

Background: These studies were undertaken to investigate whether the ingestion of glycinamide, a precursor of glycine, made more palatable by mixing with a chocolate suspension, improves antinociception in rats., Methods: Two nociception threshold models were employed: the tail-flick latency and vocalization to tail shock, in restricted and freely-moving rats. Glycinamide in a highly palatable commercial chocolate aqueous suspension was provided for ad-lib ingestion after 24 hours of water deprivation. Antinociception threshold testing was performed before and 10, 30, 60, 90, 120, 180, and 240 minutes after the ingestion of the chocolate-glycinamide mixture., Results: Ingestion of the glycinamide-in-chocolate suspension induced antinociception based on the tail shock vocalization and tail-flick latency tests. Ingestion of the glycinamide-in-chocolate suspension induced an 80% elevation in the antinociceptive threshold that persisted for 4 hours., Conclusions: Rats readily ingest the glycine precursor, glycinamide, in an aqueous chocolate mixture, which induces potent and prolonged antinociception., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Paclitaxel, a primary chemotherapeutic agent used to treat numerous solid malignancies, is commonly associated with debilitating peripheral neuropathy. However, a satisfactory gold-standard monotherapy for this neuropathic pain is not currently available. A combination strategy of two or more medications with different properties may achieve more beneficial effects than monotherapy. Thus, we investigated the analgesic efficacies and spinal mechanisms of the combination strategy, including bee venom acupuncture (BVA) and venlafaxine (VLX) against paclitaxel-induced allodynia in mice. Four intraperitoneal infusions of paclitaxel on alternating days (2 mg/kg/day) induced cold and mechanical allodynia for at least 1 week as assessed using acetone and the von Frey hair test, respectively. Co-treatment of BVA (1.0 mg/kg, s.c., ST36) with VLX (40 mg/kg, i.p.) at the medium dose produced a longer-lasting and additive effect than each monotherapy at the highest dose (BVA, 2.5 mg/kg; VLX, 60 mg/kg). Spinal pre-administration of idazoxan (α 2 -adrenergic receptor antagonist, 10 μg), methysergide (mixed 5-HT 1 /5-HT 2 receptor antagonist, 10 μg), or MDL-72222 (5-HT 3 receptor antagonist, 10 μg) abolished this analgesia. These results suggest that the combination therapy with BVA and VLX produces long-lasting and additive analgesic effects on paclitaxel-induced allodynia, via the spinal noradrenergic and serotonergic mechanism, providing a promising clinical strategy.

Objectives: To assess the protective effect of the glucagon-like peptide-1 receptor (GLP-1R) agonist morroniside against neuropathic pain and its downstream mechanisms of activating microglial GLP-1R/interleukin-10 (IL-10)/β-endorphin antinociceptive pathway., Methods: Spinal nerve ligation-induced neuropathic pain rats were intrathecally injected with morroniside, with mechanical paw withdrawal threshold being assessed. The expression of spinal and cultured microglia IL-10 and β-endorphin were detected with qRT-PCR., Key Findings: Morroniside alleviated mechanical allodynia in neuropathic rats, which was blocked by inhibiting or depleting microglia. In addition, neutralizing spinal IL-10 or β-endorphin with specialized antibodies or blocking the μ-opioid receptor was able to fully reverse the morroniside-induced mechanical antiallodynia. Morroniside treatment stimulated the gene expression of IL-10 and β-endorphin in the spinal lumbar enlargements of neuropathic rats as well as in primary cultured microglia. Furthermore, pretreatment with the IL-10 antibody blocked morroniside-stimulated β-endorphin expression in the spinal cords of neuropathic rats and cultured primary microglia, whereas the β-endorphin antibody failed to affect morroniside-stimulated gene expression of IL-10., Conclusions: These results reveal that morroniside produces therapeutic effects in neuropathy through spinal microglial expression of IL-10 and subsequent β-endorphin after activation of GLP-1R., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Paclitaxel-induced neuropathic pain (PINP) is a dose-limiting side effect and is refractory to widely used analgesic drugs. Previous studies have demonstrated a protective role of peroxisome proliferator-activated receptor gama (PPARγ) in neuropathic pain. However, whether PPARγ activation could alleviate PINP remains to be elucidated. Our previous study has validated the analgesic effect of oltipraz, an nuclear factor erythroid-2 related factor 2 (Nrf2) activator, in a rat model of PINP. In this study, we tested the hypothesis that rosiglitazone, a selective agonist of PPARγ, could attenuate PINP through induction of Nrf2/heme oxygenase-1 (HO-1) signaling pathway. Paclitaxel was injected intraperitoneally on four alternate days to induce neuropathic pain. Paw withdrawal threshold was used to evaluate mechanical allodynia. Western blot and immunofluorescence were used to examine the expression and distribution of PPARγ, Nrf2 and HO-1 in the spinal cord. Our results showed that rosiglitazone attenuated established PINP and delayed the onset of PINP via activation of PPARγ, which were reversed by PPARγ antagonist GW9662. Moreover, rosiglitazone inhibited downregulation of PPARγ in the spinal cord of PINP rats. Furthermore, the analgesic effect of rosiglitazone against PINP was abolished by trigonelline, an Nrf2 inhibitor. Finally, rosiglitazone significantly increased expression of Nrf2 and HO-1 in the spinal cord of PINP rats. Collectively, these results indicated that PPARγ activation might mitigate PINP through activating spinal Nrf2/HO-1 signaling pathway. Our results may provide an alternative option for PINP patients., (Copyright © 2020. Published by Elsevier Masson SAS.)

Chronic neuropathic pain poses a significant health problem worldwide, for which effective treatment is lacking. The current work aimed to investigate the potential analgesic effect of isoliquiritin, a flavonoid from Glycyrrhiza uralensis, against neuropathic pain and elucidate mechanisms. Male C57BL/6J mice were subjected to chronic constriction injury (CCI) by loose ligation of their sciatic nerves. Following CCI surgery, the neuropathic mice developed pain-like behaviors, as shown by thermal (heat) hyperalgesia in the Hargreaves test and tactile allodynia in the von Frey test. Repetitive treatment of CCI mice with isoliquiritin (p.o., twice per day for two weeks) ameliorated behavioral hyperalgesia to thermal (heat) stimuli and allodynia to tactile stimuli in a dose-dependent fashion (5, 15 and 45 mg/kg). The isoliquiritin-triggered analgesia seems serotonergically dependent, since its antihyperalgesic and antiallodynic actions were totally abolished by chemical depletion of spinal serotonin by p-chlorophenylalanine, whereas potentiated by 5-HTP (a precursor of 5-HT). Consistently, isoliquiritin-treated neuropathic mice showed escalated levels of spinal monoamines especially 5-HT, with depressed monoamine oxidase activity. Moreover, isoliquiritin-evoked antihyperalgesia and antiallodynia were preferentially counteracted by the 5-HT 1A receptor antagonist WAY-100635 delivered systematically or spinally. Of notable benefit, isoliquiritin was able to correct co-morbid behavioral symptoms of depression and anxiety evoked by neuropathic pain. Collectively, these findings demonstrate, for the first time, the therapeutic efficacy of isoliquiritin on neuropathic hypersensitivity, and this effect is dependent on the spinal serotonergic system and 5-HT 1A receptors., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Neuropathic pain is difficult to treat and remains a major clinical challenge worldwide. While the mechanisms which underlie the development of neuropathic pain are incompletely understood, interferon signaling by the immune system is known to play a role. Here, we demonstrate a role for interferon β (IFNβ) in attenuating mechanical allodynia induced by the spared nerve injury in mice. The results show that intrathecal administration of IFNβ (dosages up to 5,000 U) produces significant, transient, and dose-dependent attenuation of mechanical allodynia without observable effects on motor activity or feeding behavior, as is common with IFN administration. This analgesic effect is mediated by the ubiquitin-like protein interferon-stimulated gene 15 (ISG15), which is potently induced within the spinal cord following intrathecal delivery of IFNβ. Both free and conjugated ISG15 are elevated following IFNβ treatment, and this effect is increased in UBP43 -/- mice lacking a key deconjugating enzyme. The IFNβ-mediated analgesia reduces MAPK signaling activation following nerve injury, and this effect requires induction of ISG15. These findings highlight a new role for IFNβ, ISG15, and MAPK signaling in immunomodulation of neuropathic pain and may lead to new therapeutic possibilities. PERSPECTIVE: Neuropathic pain is frequently intractable in a clinical setting, and new treatment options are needed. Characterizing the antinociceptive potential of IFNβ and the associated downstream signaling pathways in preclinical models may lead to the development of new therapeutic options for debilitating neuropathies., (Copyright © 2019. Published by Elsevier Inc.)

Pain is a percept of critical importance to our daily survival. In most cases, it serves both an adaptive function by helping us respond appropriately in a potentially hostile environment and also a protective role by alerting us to tissue damage. Normally, it is evoked by the activation of peripheral nociceptive nerve endings and the subsequent relay of information to distinct cortical and sub-cortical regions, but under pathological conditions that result in chronic pain, it can become spontaneous. Given that one in three chronic pain patients do not respond to the treatments currently available, the need for more effective analgesics is evident. Two principal obstacles to the development of novel analgesic therapies are our limited understanding of how neuronal circuits that comprise these pain pathways transmit and modulate sensory information under normal circumstances and how these circuits change under pathological conditions leading to chronic pain states. In this review, we focus on the role of inhibitory interneurons in setting pain thresholds and, in particular, how disinhibition in the spinal dorsal horn can lead to aberrant sensory processing associated with chronic pain states.

Oxidative stress induced post-translational protein modifications are associated with the development of inflammatory hypersensitivities. At least 90% of cellular reactive oxygen species (ROS) are produced in the mitochondria, where the mitochondrial antioxidant, manganese superoxide dismutase (MnSOD), is located. MnSOD's ability to reduce ROS is enhanced by the mitochondrial NAD + -dependent deacetylase sirtuin (SIRT3). SIRT3 can reduce ROS levels by deacetylating MnSOD and enhancing its ability to neutralize ROS or by enhancing the transcription of MnSOD and other oxidative stress-responsive genes. SIRT3 can be post-translationally modified through carbonylation which results in loss of activity. The contribution of post-translational SIRT3 modifications in central sensitization is largely unexplored. Our results reveal that SIRT3 carbonylation contributes to spinal MnSOD inactivation during carrageenan-induced thermal hyperalgesia in rats. Moreover, inhibiting ROS with natural and synthetic antioxidants, prevented SIRT3 carbonylation, restored the enzymatic activity of MnSOD, and blocked the development of thermal hyperalgesia. These results suggest that therapeutic strategies aimed at inhibiting post-translational modifications of SIRT3 may provide beneficial outcomes in pain states where ROS have been documented to play an important role in the development of central sensitization., Competing Interests: Declaration of Competing Interest All authors declare no conflict of interest., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

While the acute sensation of pain is protective, signaling the presence of actual or potential bodily harm, its persistence is unpleasant. When pain becomes chronic, it has limited evolutionarily advantage. Despite the differing nature of acute and chronic pain, a common theme is that sufferers seek pain relief. The possibility to medicate pain types as varied as a toothache or postsurgical pain reflects the diverse range of mechanism(s) by which pain-relieving "analgesic" therapies may reduce, eliminate, or prevent pain. Systemic application of an analgesic able to cross the blood-brain barrier can result in pain modulation via interaction with targets at different sites in the central nervous system. A so-called supraspinal mechanism of action indicates manipulation of a brain-defined circuitry. Pre-clinical studies demonstrate that, according to the brain circuitry targeted, varying therapeutic pain-relieving effects may be observed that relate to an impact on, for example, sensory and/or affective qualities of pain. In many cases, this translates to the clinic. Regardless of the brain circuitry manipulated, modulation of brain processing often directly impacts multiple aspects of nociceptive transmission, including spinal neuronal signaling. Consideration of supraspinal mechanisms of analgesia and ensuing pain relief must take into account nonbrain-mediated effects; therefore, in this review, the supraspinally mediated analgesic actions of opioidergic, anti-convulsant, and anti-depressant drugs are discussed. The persistence of poor treatment outcomes and/or side effect profiles of currently used analgesics highlight the need for the development of novel therapeutics or more precise use of available agents. Fully uncovering the complex biology of nociception, as well as currently used analgesic mechanism(s) and site(s) of action, will expedite this process.

Ethenzamide (ETZ), an antipyretic analgesic categorized as a non-steroidal anti-inflammatory drug (NSAID), is widely used as an OTC drug in combination with other NSAIDs. However, its site of action and mechanism underlying its analgesic action have not yet been fully elucidated. In this study, we performed in vitro pharmacological assays to identify the mechanism underlying the analgesic action of ETZ, and also conducted the rat formalin test to investigate its analgesic effect and site of action. Of the 85 receptors, ion channels, transporters and enzymes tested, we found that ETZ binds to the 5-hydroxytryptamine (5HT) 2B receptor in concentration-dependent manner with modest inhibitory effects on monoamine oxidase-A and transient potential vanilloid 1 channel. The 5HT 2B receptor antagonist activity of ETZ was also confirmed in a cellular functional assay. Furthermore, the drug exerted no inhibitory effects on cycrooxygenase-1 and -2. In the rat formalin test, oral administration of ETZ significantly reduced the nociceptive responses of the second phase and also the number of c-Fos-expressing cells in the spinal dorsal horn, in a dose-dependent manner. Moreover, intrathecal administration of ETZ significantly reduced the nociceptive responses. These results suggest that the analgesic effect of ETZ is exerted at least in the spinal cord, and the effect would be attributed to multiple mechanisms of action including 5HT 2B receptor blockade.

The activation of the AMP activated protein kinase (AMPK) exerts antinociceptive effects in acute and neuropathic pain models. Mitochondrial open-reading-frame of the twelve S rRNA-c (MOTS-c), a mitochondrial-derived peptide, regulates many biological activities via activating AMPK. However, the role of MOTS-c in the formalin-induced inflammatory nociception remains unclear. In this study, we investigated the role of MOTS-c in the formalin-induced inflammatory nociception. The antinociceptive effect of MOTS-c was assessed by recording the time spent licking paw. The anti-inflammatory effect of MOTS-c was evaluated by detecting the inflammatory cytokine level changes in the mouse serum. Western blot was used to detect the changes of protein phosphorylation levels in the mouse spinal cord. Changes of c-fos expression in the spinal cord were assessed by immunohistochemistry. Our results showed that the intraperitoneal administration of MOTS-c reduced the time spent on licking in phase 2 in a dose-dependent manner in the formalin test. The antinociceptive effects of MOTS-c (50 mg/kg, i.p.) were attenuated by the AMPK antagonist compound C (10 mg/kg, i.p.). MOTS-c (50 mg/kg, i.p.) significantly reduced pro-inflammatory cytokine levels and elevated the level of anti-inflammatory cytokine in mouse serum. In addition, MOTS-c treatment significantly increased AMPKα phosphorylation level and suppressed formalin-induced extracellular signal-regulated kinase (ERK), c-Jun amino-terminal kinases (JNK), and P38 activation and c-fos expression in the mouse spinal cord. These results suggest that systemic administration of MOTS-c exerts antinociceptive and anti-inflammatory effects, at least partially, through activating AMPK pathway and inhibiting MAP kinases-c-fos signaling pathway in the mouse formalin test., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Bone cancer pain is considered to be mechanistically unique compared with inflammatory or neuropathic pain states. Toll-like receptor 4 (TLR4) is a transmembrane receptor protein which has been reported to be involved in neuropathic pain. However, the role of TLR4 in bone cancer pain is still unclear. Therefore, the aim of this study is to investigate the hypothesis that oxytocin may ameliorate bone cancer pain by suppressing TLR4 in spinal cord. Behavioral analysis and molecular biological experiments were carried out. Our data demonstrated that intrathecally delivery of oxytocin significantly ameliorated the mechanical allodynia and thermal hyperalgesia in bone cancer pain rats. Moreover, oxytocin suppressed the up-regulation of TLR4 and proinflammatory cytokines TNFα and IL-1β in spinal cord of bone cancer pain rats. Therefore, we concluded that intrathecal administration of oxytocin relieves bone cancer pain by suppressing the up-regulation of TLR4, TNFα and IL-1β in spinal cord. Oxytocin possesses analgesic efficacy against bone cancer pain and deserves further to confirm its effectiveness in clinically relevant of cancer pain.

The avulsion of nerve roots of the brachial plexus that is commonly seen in motorcycle accidents is a type of neuropathy due to deafferentation. This type of pain is clinically challenging since therapeutical protocols fail or have severe side effects. Thus, it is proposed to evaluate the antinociceptive activity of the recombinant CTK 01512-2 peptide that is derived from the venom of the Phoneutria nigriventer spider, as a future new therapeutical option. The neuropathic pain was surgically induced by avulsion of the upper brachial plexus trunk in groups of male Wistar rats and after 17 days, they were treated intrathecally with morphine, ziconotide, and CTK 01512-2. Behavioral tests were performed to evaluate mechanical and thermal hyperalgesia, cold allodynia, the functional activity of the front paw, and exploratory locomotion after the treatments. The peripheral blood samples were collected 6 h after the treatments and a comet assay was performed. The spinal cord was removed for the lipoperoxidation dosing of the membranes. The cerebrospinal fluid was analyzed for the dosage of glutamate. The recombinant peptide showed an antinociceptive effect when compared to the other drugs, without affecting the locomotor activity of the animals. Mechanical and thermal hyperalgesia, as well as cold allodynia, were reduced in the first hours of treatment. The levels of glutamate and the damage by membrane lipoperoxidation were shown to be improved, and genotoxicity was not demonstrated. In a scenario of therapeutical failures in the treatment of this type of pain, CTK 01512-2 was shown as a new effective alternative protocol. However, further testing is required to determine pharmacokinetics., (Copyright © 2019. Published by Elsevier Ltd.)

Objective: The phytohormone abscisic acid (ABA) as a signaling molecule exists in various types of organisms from early multicellular to animal cells and tissues. It has been demonstrated that ABA has an antinociceptive effect in rodents. The present study was designed to assess the possible role of PKA and phosphorylated ERK (p-ERK) on the antinociceptive effects of intrathecal (i.t.) ABA in male Wistar rats., Methods: The animals were cannulated intrathecally and divided into different experimental groups (n=6‒7): Control (no surgery), vehicle (received ABA vehicle), ABA-treated groups (received ABA in doses of 10 or 20 µg/rat), ABA plus H.89 (PKA inhibitor)-treated group which received the inhibitor 15 min prior to the ABA injection. Tail-flick and hot-plate tests were used as acute nociceptive stimulators to assess ABA analgesic effects. p-ERK was evaluated in the dorsal portion of the spinal cord using immunoblotting., Results: Data showed that a microinjection of ABA (10 and 20 µg/rat, i.t.) significantly increased the nociceptive threshold in tail flick and hot plate tests. The application of PKA inhibitor (H.89, 100 nM/rat) significantly inhibited ABA-induced analgesic effects. Expression of p-ERK was significantly decreased in ABA-injected animals, which were not observed in the ABA+H.89-treated group., Conclusions: Overall, i.t. administration of ABA (10 µg/rat) induced analgesia and p-ERK down-expression likely by involving the PKA-dependent mechanism.

Eplingiella fruticosa (Lamiaceae), formally known as Hyptis fruticosa, is an important aromatic medicinal herb used in folk medicine in northeastern Brazil. We aimed to evaluate the anti-hyperalgesic effect of essential oil obtained from E. fruticosa (HypEO) complexed with βCD (HypEO-βCD) in a chronic widespread non-inflammatory muscle pain animal model (a mice fibromyalgia-like model, FM). The HypEO was extracted by hydro distillation and its chemical composition was determined by GC-MS/FID. Moreover, Fos protein expression in the spinal cord was assessed by immunofluorescence. (E)-caryophyllene, bicyclogermacrene, 1,8-cineole, α-pinene, β-pinene and 21 other compounds were identified in the HypEO. The treatment with HypEO-βCD produced a longer-lasting anti-hyperalgesic effect compared to HypEO, without alterations in motor coordination or myorelaxant effects. Moreover, HypEO and HypEO-βCD produced a significant anti-hyperalgesic effect over 7 consecutive treatment days. Immunofluorescence assay demonstrated a decrease in Fos protein expression in the spinal cord (p < 0.001). We demonstrated that the anti-hyperalgesic effect produced by HypEO was improved after complexation with β-CD and this seems to be related to the central pain-inhibitory pathway, suggesting the possible use of E. fruticosa for chronic pain management., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Neuropathic pain is highly prevalent in pathological conditions such as diabetes, herpes zoster, trauma, etc. The severity and refractoriness to treatments make neuropathic pain a significant health concern. The transforming growth factor (TGF-β) family of cytokines is involved in pain modulation. Bone morphogenetic proteins (BMPs) constitute the largest subgroup within the TGF-β family. BMP-7 induces the transcription of genes coding endogenous opioid precursors in vitro. However, a nociception modulatory function for this cytokine remains unexplored in vivo. Herein, we show that BMP-7 and its type I receptors were detected in regions of the nervous system involved in pain transmission, processing, and modulation. BMP-7 haploinsufficiency confers to male and female mice a tactile hyperalgesia phenotype to mechanical stimuli, both at baseline and after sciatic nerve injury (SNI). The administration of recombinant BMP-7 (rBMP-7) reduced the severity of the allodynia after SNI in rodents without sexual dimorphism. Central administration of rBMP-7 delayed allodynia development after SNI and reduced the severity of allodynia. The opioid antagonist naloxone antagonized the antinociceptive effect of rBMP-7 in rats. The analgesic effect of morphine was significantly attenuated in BMP-7 +/- mice. The antiallodynic effect of voluntary exercise after SNI, whose mechanism involves the endogenous opioid system, was hampered by BMP-7 deficiency while potentiated by rBMP-7. Our results suggest that BMP-7 may constitute a novel therapeutic target for the treatment of neuropathic pain, which improves the function of the endogenous pain-resolution mechanisms to alleviate chronic pain., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The 'Science of Relief' event, held in Milan on 10-11 May 2019, was aimed at promoting dialog between different stakeholders among scientific associations, pharma industry, healthcare services and related institutions. The goal was to renew interest and attention on the management of pain, sharing new solutions in order to bring the patients and their quality of life to the center of attention. An international group of scientists and clinicians presented and discussed new and known evidence in the field of chronic pain, from physiopathology and diagnosis to the choice of appropriate and timely pharmacological treatments. This paper reports the highlights of those presentations.

The cannabinoid system has been suspected to play a role in the mechanisms of action of dipyrone and paracetamol. Our purpose was to measure the local endocannabinoid and N-acylethanolamide levels in the brain and spinal cord of rats following dipyrone and paracetamol administration. Nociception was assessed 1, 5, and 12 h following drug injections in Wistar rats, using tail-flick and hot-plate tests. The antinociceptive effects of dipyrone (150, 300, and 600 mg/kg, i.p.) and paracetamol (30, 100, and 300 mg/kg, i.p.) were observed. After administration of the highest doses of dipyrone and paracetamol, endocannabinoid (N-arachidonoylethanolamide (AEA), 2-arachidonoylglycerol (2-AG)) and N-acylethanolamide (palmitoylethanolamide (PEA), oleoylethanolamide (OEA)) levels were measured in the periaqueductal gray (PAG), rostral ventromedial medulla (RVM), and spinal cords of rats using tandem mass spectrometry with liquid chromatography. Increased 2-AG levels were observed in the PAG and the RVM 12 h after paracetamol injection; dipyrone exerted no action on 2-AG levels. Analgesic administrations led to a reduction in AEA levels in the RVM and spinal cord; similar decreases in PEA and OEA levels were observed in the RVM and the spinal cord. Dipyrone and paracetamol administrations appear to exert complicated effects on endocannabinoid and N-acylethanolamide levels in rats.

Neuropathic pain is a widespread and debilitating chronic pain and the treatment remains a clinical challenge. Isoorientin (3',4',5,7-tetrahydroxy-6-C-glucopyranosyl flavone) is a natural flavonoid-like compound that exhibits antioxidant and anti-inflammatory activities; however, its effect on neuropathic pain remains unclear. Our study aimed to evaluate the antinociceptive effect of isoorientin in neuropathic pain mouse models induced by chronic constriction injury (CCI). In our study, the mice with CCI were administered with 7.5, 15, and, 30 mg/kg isoorientin for 8 consecutive days. Behavioral parameters were assayed on days 0, 7, 8, 10, 12, and 14 post-CCI surgery. Electrophysiological, histopathological, and biochemical indices were analyzed on day 14. Immunofluorescence was utilized to examine matrix metalloproteinase-9 (MMP-9) and glial cell activation, and proinflammatory cytokine expression levels were detected via Western blot. It is obvious that the treatment of Isoorientin remarkably ameliorated hyperalgesia and allodynia, increased sensory nerve conduction velocities, and restored CCI-induced sciatic nerve damage in mice. Isoorientin treatment significantly increased the total antioxidant capacity (T-AOC), total superoxide dismutase (T-SOD) and catalase (CAT) levels, and decreased the malondialdehyde (MDA) concentrations. Isoorientin also suppressed MMP-9 and glial cell activation, and downregulated tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β) expression levels. Therefore, this study provided a novel approach for neuropathic pain treatment and new insights into the pharmacological action of isoorientin., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Berberine, a chemical found in plants, is used as a supplement for diabetes. This study aimed to investigate the effects and the underlying molecular regulations of berberine in diabetic neuropathic pain in a rat model of diabetes. Rats were injected with streptozotocin (STZ) to induce diabetes and then were treated with berberine. Blood glucose levels and body weight were measured. Thermal and mechanical nociception were assessed by paw pressure test and hot tail immersion test. Oxidative stress was assessed by lipid peroxidation, production of reactive oxygen species (ROS) and catalase activity. Neuroinflammation was assessed by tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) protein levels. Finally, μ-opioid receptor (MOR) protein and mRNA levels were measured. We found that berberine treatment partially suppressed blood glucose levels and restored body weight in diabetic rats. Berberine also suppressed STZ-induced oversensitivity of mechanical and thermal nociception. Additionally, berberine partially suppressed oxidative stress and inflammation of diabetic rats. Finally, berberine significantly enhanced protein and mRNA expression levels of μ-opioid receptor (MOR). Our findings suggest that berberine is a potential therapeutic alleviating diabetes and diabetic neuropathic pain, probably through suppression of oxidative stress and neuroinflammation that may be mediated by MOR.

Celecoxib is a nonsteroidal anti-inflammatory drug (NSAID) commonly used to treat pain conditions in humans. In addition to its blocking activity on cyclooxygenase (COX) enzymes, several other targets could contribute to its analgesic activity. Here we explore the spinal antinociceptive actions of celecoxib and the potential implication of K v 7 channels in mediating its effects. Spinal cord in vitro preparations from hind paw-inflamed animals were used to assess the segmental sensory-motor and the early sensory processing of nociceptive information. Electrophysiological recordings of ventral roots and dorsal horn neurones were obtained, and the effects of celecoxib and K v 7 modulators on responses to repetitive dorsal root stimulation at C-fiber intensity were assessed. Celecoxib applied at clinically relevant concentrations produced depressant effects on responses to dorsal root stimulation recorded from both ventral roots and individual dorsal horn neurones; by contrast, the non-nociceptive monosynaptic reflex was unaffected. The NSAID indomethacin had no effect on spinal reflexes, but further coapplication of celecoxib still produced depressant effects. The depressant actions of celecoxib were abolished after K v 7 channel blockade and mimicked by its structural analog dimethyl-celecoxib, which lacks COX-blocking activity. The present results identify K v 7 channels as novel central targets for celecoxib, which may be relevant to its analgesic effect. This finding contributes to better understand the pharmacology of celecoxib and reinforces both the role of K v 7 channels in modulating the excitability of central pain pathways and its validity as target for the design of analgesics., (Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics.)

Neuropathic pain is a common type of chronic pain caused by trauma or chemotherapy. However, this type of pain is undertreated. TsNTxP is a non-toxic protein isolated from the venom of the scorpion Tityus serrulatus, and it is structurally similar to neurotoxins that interact with voltage-gated sodium channels. However, the antinociceptive properties of this protein have not been characterized. The purpose of this study was to investigate the antinociceptive effects of TsNTxP in acute and neuropathic pain models. Male and female Swiss mice (25-30 g) were exposed to different models of acute pain (tail-flick test and nociception caused by capsaicin intraplantar injection) or neuropathic pain (chronic pain syndrome induced by paclitaxel or chronic constriction injury of the sciatic nerve). Hypersensitivity to mechanical or cold stimuli were evaluated in the models of neuropathic pain. The ability of TsNTxP to alter the release of glutamate in mouse spinal cord synaptosomes was also evaluated. The results showed that TsNTxP exerted antinociceptive effects in the tail-flick test to a thermal stimulus and in the intraplantar capsaicin administration model. Furthermore, TsNTxP was non-toxic and exerted antiallodynic effects in neuropathic pain models induced by chronic constriction injury of the sciatic nerve and administration of paclitaxel. TsNTxP reduced glutamate release from mouse spinal cord synaptosomes following stimulation with potassium chloride (KCl) or capsaicin. Thus, this T. serrulatus protein may be a promising non-toxic drug for the treatment of neuropathic pain., (Copyright © 2019. Published by Elsevier B.V.)

Oxaliplatin is a chemotherapeutic agent used for metastatic colon and other advanced cancers. Most common side effect of oxaliplatin is peripheral neuropathy, manifested in mechanical and cold allodynia. Although the analgesic effect of bee venom has been proven to be effective against oxaliplatin-induced peripheral neuropathy, the effect of its major component; melittin has not been studied yet. Thus, in this study, we investigated whether melittin has an analgesic effect on oxaliplatin-induced allodynia. Intraperitoneal single injection of oxaliplatin (6 mg/kg) induced mechanical and cold allodynia, resulting in increased withdrawal behavior in response to von Frey filaments and acetone drop on hind paw. Subcutaneous melittin injection on acupoint ST36 (0.5 mg/kg) alleviated oxaliplatin-induced mechanical and cold allodynia. In electrophysiological study, using spinal in vivo extracellular recording, it was shown that oxaliplatin-induced hyperexcitation of spinal wide dynamic range neurons in response to peripheral stimuli, and melittin administration inhibited this neuronal activity. In behavioral assessment, analgesic effect of melittin was blocked by intrathecal α1- and α2- adrenergic receptor antagonists administration. Based on these results, we suggest that melittin could be used as an analgesic on oxaliplatin-induced peripheral neuropathy, and that its effect is mediated by activating the spinal α1- and α2-adrenergic receptors.

Inhibition of voltage-gated calcium (CaV) channels is a potential therapy for many neurological diseases including chronic pain. Neuronal CaV1/CaV2 channels are composed of α, β, γ and α2δ subunits. The β subunits of CaV channels are cytoplasmic proteins that increase the surface expression of the pore-forming α subunit of CaV. We targeted the high-affinity protein-protein interface of CaVβ's pocket within the CaVα subunit. Structure-based virtual screening of 50,000 small molecule library docked to the β subunit led to the identification of 2-(3,5-dimethylisoxazol-4-yl)-N-((4-((3-phenylpropyl)amino)quinazolin-2-yl)methyl)acetamide (IPPQ). This small molecule bound to CaVβ and inhibited its coupling with N-type voltage-gated calcium (CaV2.2) channels, leading to a reduction in CaV2.2 currents in rat dorsal root ganglion sensory neurons, decreased presynaptic localization of CaV2.2 in vivo, decreased frequency of spontaneous excitatory postsynaptic potentials and miniature excitatory postsynaptic potentials, and inhibited release of the nociceptive neurotransmitter calcitonin gene-related peptide from spinal cord. IPPQ did not target opioid receptors nor did it engage inhibitory G protein-coupled receptor signaling. IPPQ was antinociceptive in naive animals and reversed allodynia and hyperalgesia in models of acute (postsurgical) and neuropathic (spinal nerve ligation, chemotherapy- and gp120-induced peripheral neuropathy, and genome-edited neuropathy) pain. IPPQ did not cause akinesia or motor impairment, a common adverse effect of CaV2.2 targeting drugs, when injected into the brain. IPPQ, a quinazoline analog, represents a novel class of CaV2.2-targeting compounds that may serve as probes to interrogate CaVα-CaVβ function and ultimately be developed as a nonopioid therapeutic for chronic pain.

Opioids are potent analgesic drugs, but their use has been limited due to their side effects. Antinociceptive effects of D2-like receptor agonists such as quinpirole have been shown at the spinal cord level; however, their efficacy is not as high as that of opioids. Dopaminergic agonists are long-prescribed and well-tolerated drugs that have been useful to treat clinically and experimentally painful conditions. Because current pain treatments are not completely effective, the aim of this work was to determine if a D2-like receptor agonist improves the antinociceptive effects of a μ-opioid receptor agonist. Drugs were intrathecally administered in adult rats; mechanonociceptive and thermonociceptive tests were carried out. Intraplantar injection of complete Freund's adjuvant (CFA) and sciatic loose ligation (SLL) were used for inflammatory and neuropathic models of pain, respectively. In intact animals, D-Ala2, N-MePhe4, Gly-ol-enkephalin (DAMGO; a µ-opioid receptor agonist) increased the paw withdrawal latencies (PWL) in thermal and mechanical nociceptive tests in a dose-dependent manner. Quinpirole (D2-like receptor agonist) increased PWL only in mechanonociception. In the presence of quinpirole (1 nmol), the ED 50 of the mechanical antinociceptive effect of DAMGO was significantly decreased (8-fold). Coadministration of 1 nmol quinpirole and 30 pmol DAMGO completely reversed hyperalgesia in the CFA model, whereas 100 pmol DAMGO plus 1 nmol quinpirole reversed the allodynia in the SLL model. This work offers evidence about a synergistic antinociceptive effect between opioidergic and dopaminergic drugs. This combination may relieve painful conditions resistant to conventional treatments, and it may reduce the adverse effects of chronic opioid administration., (Copyright © 2019 Elsevier B.V. All rights reserved.)