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Inhibitory GABA-ergic neurotransmission is fundamental for the adult vertebrate central nervous system and requires low chloride concentration in neurons, maintained by KCC2, a neuroprotective ion transporter that extrudes intracellular neuronal chloride. To identify Kcc2 gene expression‑enhancing compounds, we screened 1057 cell growth-regulating compounds in cultured primary cortical neurons. We identified kenpaullone (KP), which enhanced Kcc2/KCC2 expression and function in cultured rodent and human neurons by inhibiting GSK3ß. KP effectively reduced pathologic pain-like behavior in mouse models of nerve injury and bone cancer. In a nerve-injury pain model, KP restored Kcc2 expression and GABA-evoked chloride reversal potential in the spinal cord dorsal horn. Delta-catenin, a phosphorylation-target of GSK3ß in neurons, activated the Kcc2 promoter via KAISO transcription factor. Transient spinal over-expression of delta-catenin mimicked KP analgesia. Our findings of a newly repurposed compound and a novel, genetically-encoded mechanism that each enhance Kcc2 gene expression enable us to re-normalize disrupted inhibitory neurotransmission through genetic re-programming., Lack of expression and function of chloride ion-extruding transporter KCC2 in central neurons, a consequence of various forms of neural injury, is strongly suggested to contribute to chronic pain. Here the authors identify from a screen of cancer drugs a kinase-inhibitor, kenpaullone, as an enhancer of Kcc2/KCC2 gene expression and show that it (i) alleviates pain like behaviour in animal models, (ii) repairs neural-circuit disrupting elevated chloride in pain relay neurons in the dorsal spinal cord.

Chemotherapy-induced peripheral neuropathy (CIPN) is the de facto clinical side effect that limits the administration of anti-cancer treatments. Recently, we reported that intrathecally injected bone marrow stromal cells (BMSCs) reduced nerve trauma-induced neuropathic pain in male mice via TGF-β1 signaling. In this study, we examined sex-dependent pain relief mediated by intrathecally delivered BMSCs and TGF-β1 in paclitaxel (PTX)-induced CIPN. BMSCs were prepared from primary cultures of male or female mice separately. A single intrathecal injection of BMSCs, prepared from male donors, completely prevented the development of PTX-evoked mechanical allodynia in male mice. However, female mice showed no analgesic response to either male or female BMSCs. Additionally, male mice did not demonstrate an analgesic response to BMSCs from female donors. Intrathecal injection of TGF-β1 neutralizing antibody reversed the analgesic action of BMSCs. Interestingly, spinal administration of TGF-β1 reduced mechanical allodynia in male mice but not in female mice. Ex vivo patch-clamp recordings in spinal cord slices revealed that TGF-β1 inhibited PTX-induced synaptic plasticity, i.e. increase in spontaneous excitatory synaptic currents (sEPsCs), in spinal cord neurons from male mice only. Intrathecal TGF-β1 increased the paw withdrawal threshold in von Frey testing in naïve mice of males but not females, and the antinociceptive effect of TGF-β1 in males was blocked by orchiectomy-induced androgen deficiency. Together, these findings reveal sex dimorphism in BMSC control of mechanical pain through spinal TGF-β1 signaling.

Objective. Herpetic-related neuralgia (HN) caused by varicella-zoster virus (VZV) infection is one of the most typical and common neuropathic pain in the clinic. However, due to the lack of an animal model to simulate HN, the potential mechanisms and therapeutic approaches for the prevention and treatment of HN are still unclear. This study aims to provide a comprehensive understanding of the molecular mechanisms and potential therapeutic targets of HN. Methods. We used an HSV-1 infection-induced HN mouse model and screened the differentially expressed genes (DEGs) in the DRG and spinal cord using an RNAseq technique. Moreover, the bioinformatics methods were used to figure out the signaling pathways and expression regulation patterns of the DEGs enriched. In addition, quantitative real-time RT-PCR and western blot was carried out to further confirm the expression of DEGs. Results. HSV-1 inoculation in mice resulted in mechanical allodynia, thermal hyperalgesia, and cold allodynia, following the infection of HSV-1 in both DRG and spinal cord. Besides, HSV-1 inoculation induced an up-regulation of ATF3, CGRP, and GAL in DRG and activation of astrocytes and microglia in the spinal cord. Moreover, 639 genes were up-regulated, 249 genes were down-regulated in DRG, whereas 534 genes were up-regulated, and 12 genes were down-regulated in the spinal cord of mice 7 days after HSV-1 inoculation. GO and KEGG enrichment analysis suggested that immune responses and cytokine-cytokine receptor interaction are involved in DRG and spinal cord neurons in mice after HSV-1 infection. In addition, CCL5 and its receptor CCR5 were significantly up-regulated in DRG and spinal cord upon HSV-1 infection in mice. And blockade of CCR5 exhibited a significant analgesic effect and suppressed the up-regulation of inflammatory cytokines in DRG and spinal cord induced by HSV-1 infection in mice. Conclusions. We reported a reliable HN model with HSV-1 infection-induced allodynia and hyperalgesia in mice through dysregulation of immune response and cytokine-cytokine receptor interaction mechanism.

Background and purpose The μ-opioid receptor (μ receptor) is the primary target for opioid analgesics. The 7-transmembrane (TM) and 6TM μ receptor isoforms mediate inhibitory and excitatory cellular effects. Here, we developed compounds selective for 6TM- or 7TM-μ receptors to further our understanding of the pharmacodynamic properties of μ receptors. Experimental approach We performed virtual screening of the ZINC Drug Now library of compounds using in silico 7TM- and 6TM-μ receptor structural models and identified potential compounds that are selective for 6TM- and/or 7TM-μ receptors. Subsequently, we characterized the most promising candidate compounds in functional in vitro studies using Be2C neuroblastoma transfected cells, behavioural in vivo pain assays using various knockout mice and in ex vivo electrophysiology studies. Key results Our virtual screen identified 30 potential candidate compounds. Subsequent functional in vitro cellular assays shortlisted four compounds (#5, 10, 11 and 25) that demonstrated 6TM- or 7TM-μ receptor-dependent NO release. In in vivo pain assays these compounds also produced dose-dependent hyperalgesic responses. Studies using mice that lack specific opioid receptors further established the μ receptor-dependent nature of identified novel ligands. Ex vivo electrophysiological studies on spontaneous excitatory postsynaptic currents in isolated spinal cord slices also validated the hyperalgesic properties of the most potent 6TM- (#10) and 7TM-μ receptor (#5) ligands. Conclusion and implications Our novel compounds represent a new class of ligands for μ receptors and will serve as valuable research tools to facilitate the development of opioids with significant analgesic efficacy and fewer side-effects.

The innate immune regulator STING is a critical sensor of self- and pathogen-derived DNA. DNA sensing by STING leads to the induction of type-I interferons (IFN-I) and other cytokines, which promote immune-cell-mediated eradication of pathogens and neoplastic cells1,2. STING is also a robust driver of antitumour immunity, which has led to the development of STING activators and small-molecule agonists as adjuvants for cancer immunotherapy3. Pain, transmitted by peripheral nociceptive sensory neurons (nociceptors), also aids in host defence by alerting organisms to the presence of potentially damaging stimuli, including pathogens and cancer cells4,5. Here we demonstrate that STING is a critical regulator of nociception through IFN-I signalling in peripheral nociceptors. We show that mice lacking STING or IFN-I signalling exhibit hypersensitivity to nociceptive stimuli and heightened nociceptor excitability. Conversely, intrathecal activation of STING produces robust antinociception in mice and non-human primates. STING-mediated antinociception is governed by IFN-Is, which rapidly suppress excitability of mouse, monkey and human nociceptors. Our findings establish the STING-IFN-I signalling axis as a critical regulator of physiological nociception and a promising new target for treating chronic pain.

Interactions between central glial cells and neurons in the pain circuitry are critical contributors to the pathogenesis of chronic pain. In the central nervous system (CNS), two major glial cell types predominate: astrocytes and microglia. Injuries or pathological conditions which evoke pain are concurrently associated with the presence of a reactive microglia or astrocyte state, which is characterized by a variety of changes in the morphological, molecular, and functional properties of these cells. In this review, we highlight the changes that reactive microglia and astrocytes undergo following painful injuries and insults and discuss the critical and interactive role these two cell types play in the initiation and maintenance of chronic pain. Additionally, we focus on several crucial mechanisms by which microglia and astrocytes contribute to chronic pain and provide commentary on the therapeutic promise of targeting these pathways. In particular, we discuss how the inflammasome in activated microglia drives maturation and release of key pro-inflammatory cytokines, which drive pain through neuronal- and glial regulations. Moreover, we highlight several potentially-druggable hemichannels and proteases produced by reactive microglia and astrocytes in pain states and discuss how these pathways regulate distinct phases during pain pathogenesis. We also review two emerging areas in chronic pain research: 1) sexually dimorphic glial cell signaling and 2) the role of oligodendrocytes. Finally, we highlight important considerations for potential pain therapeutics targeting glial cell mediators as well as questions that remain in our conceptual understanding of glial cell activation in pain states. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13311-020-00905-7) contains supplementary material, which is available to authorized users.

Background: Currently, chemotherapy-induced peripheral neuropathic pain (CIPNP) has no effective treatment. Oxytocin (OXT) and the oxytocin receptor (OXTR) play roles in central analgesia. However, the expression and role of OXTR in the peripheral nervous system in CIPNP remain unclear. Here, we studied the peripheral analgesic profiles of OXTR and related mechanisms in CIPNP pain models.Methods: The chemotherapeutic agent paclitaxel (PTX) was used to establish CIPNP. qRT-PCR, fluorescent in situ hybridization, western blotting, and immunocytochemistry were used to observe the properties of OXTR expression in dorsal root ganglion (DRG). The analgesic effects were assessed by the hot plate and the Von Frey tests. The whole-cell patch-clamp was performed to record the effects of the OXTR activation on the excitability of DRG neurons and the excitatory transmissions from the primary afferents to the spinal cord.Results: The expression of OXTR in DRG neurons was boosted significantly after PTX treatment, which is rarely expressed in the vehicle group without sex difference. The activation of OXTR exhibited analgesic effects and decreased the excitability of DRG neurons. Additionally, the OXTR activation impaired the voltage-gated sodium current through the PLC/PKC pathway. Oxytocin also suppressed the excitatory transmission in the spinal dorsal horn and the excitatory inputs from the primary afferents in PTX-treated mice.Conclusions: Paclitaxel upregulates the expression of OXTR in DRG neurons. This upregulation leads to effective analgesic effects of peripheral oxytocin which is mediated by the reduction of the excitability of DRG neurons and the excitatory transmission in the spinal dorsal horn. Our results suggested that OXTR on peripheral sensory neurons can be utilized to relieve CIPNP.

Background. Serum chitinase-3-like protein 1 (CHI3L1) is a promising marker for diagnosing liver fibrosis. This meta-analysis was carried out to assess the diagnostic performance of serum CHI3L1 for the estimation of liver fibrosis. Methods. Systematic searches were performed on PubMed, Embase, Web of Science, Scopus, the Cochrane Library, Google Scholar, Sinomed, the China National Knowledge Infrastructure (CNKI), the Chinese Medical Journal Database, and the Wanfang databases for available studies. The primary studies were screened strictly according to inclusion and exclusion criteria, and sensitivity, specificity, and other measures of accuracy of serum CHI3L1 for evaluating liver fibrosis were pooled with 95% confidence intervals. I 2 was calculated to assess heterogeneity, and sources of heterogeneity were explored by subgroup analysis. Deeks’ test was used to assess for publication bias, and likelihood ratio was used to determine posttest probability. Results. Our research integrated 11 articles, accounting for 1897 patients older than 18 years old. The pooled sensitivity and specificity for significant fibrosis, advanced fibrosis, and cirrhosis were 0.79 and 0.82 with an area under the receiver operating characteristic curve (AUC) of 0.85, 0.81 and 0.83 with an AUC of 0.91, and 0.72 and 0.74 with an AUC of 0.85, respectively. Random-effects models were used to assess for significant heterogeneity, and subgroup analysis showed that age and aetiology of included patients were likely sources of heterogeneity. No potential publication bias was found for serum CHI3L1 in the diagnosis of significant fibrosis, advanced fibrosis, or cirrhosis, and posttest probability was moderate. Conclusion. Measurement of serum CHI3L1 is a feasible diagnostic tool for liver fibrosis.

Oxytocin (OT) and its receptor are promising targets for the treatment and prevention of the neuropathic pain. In the present study, we compared the effects of a single and continuous intrathecal infusion of OT on nerve injury-induced neuropathic pain behaviours in mice and further explore the mechanisms underlying their analgesic properties. We found that three days of continuous intrathecal OT infusion alleviated subsequent pain behaviours for 14 days, whereas a single OT injection induced a transient analgesia for 30 min, suggesting that only continuous intrathecal OT attenuated the establishment and development of neuropathic pain behaviours. Supporting this behavioural finding, continuous intrathecal infusion, but not short-term incubation of OT, reversed the nerve injury-induced depolarizing shift in Cl

The license plate detection and recognition (LPDR) system is one of the practical applications of optical character recognition (OCR) technology in the field of automobile transportation. This paper investigates several state-of-the-art machine and deep learning algorithms for the Chinese license plate recognition based on convolutional neural networks (CNN), long short term memory (LSTM), and k-nearest neighbors (KNN) models. Comparing the performance of these models on the Chinese City Parking Dataset (CCPD) demonstrates that the convolutional recurrent neural network (CRNN) model with an accuracy of 95% is the most accurate and performs better than other models to detect the license plates.

Patients with advanced stage cancers frequently suffer from severe pain as a result of bone metastasis and bone destruction, for which there is no efficacious treatment. Here, using multiple mouse models of bone cancer, we report that agonists of the immune regulator STING (stimulator of interferon genes) confer remarkable protection against cancer pain, bone destruction, and local tumor burden. Repeated systemic administration of STING agonists robustly attenuates bone cancer-induced pain and improves locomotor function. Interestingly, STING agonists produce acute pain relief through direct neuronal modulation. Additionally, STING agonists protect against local bone destruction and reduce local tumor burden through modulation of osteoclast and immune cell function in the tumor microenvironment, providing long-term cancer pain relief. Finally, these in vivo effects are dependent on host-intrinsic STING and IFN-I signaling. Overall, STING activation provides unique advantages in controlling bone cancer pain through distinct and synergistic actions on nociceptors, immune cells, and osteoclasts., There is an unmet clinical need to develop therapies to alleviate metastatic bone pain, frequently observed in patients with advanced cancers. Here, using mouse models of bone cancer pain, the authors show that STING agonists not only suppress bone cancer tumor burden, but also attenuate bone pain and reduce cancer-induced bone destruction.

The aim of this work is to provide a comprehensive and unbiased understanding at the molecular correlates of peripheral nerve injury. In this study, we screened the differentially expressed genes (DEGs) in the DRG from rats using RNA-seq technique. Moreover, the bioinformatics methods were used to figure out the signaling pathways and expression regulation pattern of the DEGs enriched in. In addition, quantitative real-time RT-PCR was carried out to further confirm the expression of DEGs. 414 genes were upregulated, while 184 genes were downregulated in the DRG of rats 7 days after partial sciatic nerve ligation (pSNL) surgery. Moreover, GO and KEGG enrichment analysis suggested that most of the altered genes were involved in inflammatory responses and signaling transduction. In addition, our results state that they shared similar characters in the DRG among four types of neuropathic pain models. Eighteen genes have been altered (17 of them were upregulated) in the DRG of all four types of neuropathic pain models, in which Vgf, Atf3, Cd74, Gal, Jun, Npy, Serpina3n, and Hspb1 have been reported to be involved in neuropathic pain. Quantitative real-time RT-PCR results further confirmed the mRNA expression levels of Vgf, Atf3, Cd74, Gal, Jun, Npy, Serpina3n, and Hspb1 in the DRG of rats with pSNL surgery. The present study suggested that these eight genes may play important roles in neuropathic pain, revealing that these genes might serve as therapeutic targets for neuropathic pain. Moreover, anti-inflammatory therapy might be an effective approach for neuropathic pain treatment and prevention.

Haematococcus pluvialis is a unicellular green alga with great commercial value, due to its synthesis of powerful antioxidant astaxanthin. H. pluvialis was mainly distributed in small water bodies but was also observed in eutrophicated lakes, and even coexisted with Microcystis. However, Haematococcus cells never prevail in eutrophicated water bodies. Phosphorus is the main limiting factor in most aquatic ecosystems and may have a role in the distribution of H. pluvialis. Here, we focused on the physiological responses of H. pluvialis to various phosphorus conditions (0.002, 0.02, 0.2, and 2 mM), and compared with a bloom-forming cyanobacterium Microcystis wesenbergii. Growth determination suggested that high phosphorus conditions (0.2 mM and 2 mM) favor the growth of H. pluvialis cells, but H. pluvialis cells have a shorter duration of log phase than M. wesenbergii cells. Growth determination also indicated H. pluvialis cells had lower tolerability to low phosphorus (0.002 mM). Qualitative comparisons from long-term and short-term phosphorus uptake experiments, polyphosphate accumulation and extracellular alkaline phosphatase expression analysis suggested two different phosphorus utilization strategies in the two species. H. pluvialis cells were characterized with the induction of extracellular alkaline phosphatase to survive phosphorus-deficient condition, while M. wesenbergii cells were characterized with quick uptake of phosphorus and accumulation more of polyphosphate in phosphorus-replete conditions. To our knowledge, this is the first study to demonstrate features of phosphorus uptake and utilization in H. pluvialis, which will increase our understanding in the distribution of H. pluvialis.

Chemotherapy-induced peripheral neuropathic pain (CIPNP) is a major dose- and therapy-limiting side effect that is particularly difficult to treat. Huachansu, an aqueous extract from toad skin, is a widely used anti-cancer natural product in China. Clinical findings have established the safety and effectiveness of Huachansu in combination with chemotherapy to promote the therapeutic efficacy while alleviate the side effects, especially cancer-related pain symptoms. Unfortunately, experimental data on the effects and mechanisms of Huachansu in combination with chemotherapy is not available. In this study, the effects of Huachansu were tested in vivo on a rat model of oxaliplatin-induced CIPNP. The results show, a single injection of Huachansu 2.5 g/kg produced a short-term analgesic effect on pre-established oxaliplatin-induced CIPNP after 60 min, as indicated by decreased mechanical and thermal hypersensitivity in comparison to oxaliplatin-treated rats. Repeated doses of Huachansu, given during CIPNP induction, prevented the development of oxaliplatin-induced CIPNP. This prophylactic effect of Huachansu was associated with suppressed oxaliplatin-induced TRPV1 up-regulation in the dorsal root ganglia and spinal astrocyte activation. These findings reveal Huachansu therapeutic potential in treating and preventing CIPNP.

Background Nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2)-induced oxidative stress, including the production of reactive oxygen species (ROS) and hydrogen peroxide, plays a pivotal role in neuropathic pain. Although the activation and plasma membrane translocation of protein kinase C (PKC) isoforms in dorsal root ganglion (DRG) neurons have been implicated in multiple pain models, the interactions between NOX2-induced oxidative stress and PKC remain unknown. Methods A spared nerve injury (SNI) model was established in adult male rats. Pharmacologic intervention and AAV-shRNA were applied locally to DRGs. Pain behavior was evaluated by Von Frey tests. Western blotting and immunohistochemistry were performed to examine the underlying mechanisms. The excitability of DRG neurons was recorded by whole-cell patch clamping. Results SNI induced persistent NOX2 upregulation in DRGs for up to 2 weeks and increased the excitability of DRG neurons, and these effects were suppressed by local application of gp91-tat (a NOX2-blocking peptide) or NOX2-shRNA to DRGs. Of note, the SNI-induced upregulated expression of PKCε but not PKC was decreased by gp91-tat in DRGs. Mechanical allodynia and DRG excitability were increased by ψεRACK (a PKCε activator) and reduced by εV1-2 (a PKCε-specific inhibitor). Importantly, εV1-2 failed to inhibit SNI-induced NOX2 upregulation. Moreover, the SNI-induced increase in PKCε protein expression in both the plasma membrane and cytosol in DRGs was attenuated by gp91-tat pretreatment, and the enhanced translocation of PKCε was recapitulated by H2O2 administration. SNI-induced upregulation of PKCε was blunted by phenyl-N-tert-butylnitrone (PBN, an ROS scavenger) and the hydrogen peroxide catalyst catalase. Furthermore, εV1-2 attenuated the mechanical allodynia induced by H2O2 Conclusions NOX2-induced oxidative stress promotes the sensitization of DRGs and persistent pain by increasing the plasma membrane translocation of PKCε.

Agonists of the innate immune regulator stimulator of interferon genes (STING) have shown great efficacy in promoting antitumor immunity in preclinical models, leading to their exploration in cancer immunotherapy trials. Patients with advanced stage cancers frequently suffer from severe pain as a result of bone metastasis and bone destruction, for which there is no efficacious treatment. Here, using multiple mouse models of metastatic bone cancer, we report that STING agonists confer remarkable protection against cancer pain, bone destruction, and local tumor burden. Repeated systemic administration of STING agonists robustly attenuated bone cancer-induced pain symptoms and improved locomotor function. Interestingly, STING agonists provided acute pain relief through direct neuronal modulation, as ex vivo incubation of STING agonists reduced excitability of pain-sensing nociceptive neurons from tumor-bearing mice. In addition, STING agonists protected local bone destruction and reduced local tumor burden through modulation of osteoclast and immune cell function in the tumor microenvironment, providing long-term cancer pain relief. Finally, these in vivo effects were dependent on host-intrinsic STING and Ifnar1. Overall, STING activation provides unique advantages in controlling metastatic bone cancer pain through distinct and synergistic actions on nociceptors, immune cells, and osteoclasts.

The protease activated receptor (PAR) family is a group of G-protein coupled receptors (GPCRs) activated by proteolytic cleavage of the extracellular domain. PARs are expressed in a variety of cell types with crucial roles in homeostasis, immune responses, inflammation, and pain. PAR3 is the least researched of the four PARs, with little known about its expression and function. We sought to better understand its potential function in the peripheral sensory nervous system. Mouse single-cell RNA sequencing data demonstrates that PAR3 is widely expressed in dorsal root ganglion (DRG) neurons. Co-expression of PAR3 mRNA with other PARs was identified in various DRG neuron subpopulations, consistent with its proposed role as a coreceptor of other PARs. We developed a lipid tethered PAR3 agonist, C660, that selectively activates PAR3 by eliciting a Ca2+ response in DRG and trigeminal neurons. In vivo, C660 induces mechanical hypersensitivity and facial grimacing in WT but not PAR3-/- mice. We characterized other nociceptive phenotypes in PAR3-/- mice and found a loss of hyperalgesic priming in response to IL-6, carrageenan, and a PAR2 agonist, suggesting that PAR3 contributes to long-lasting nociceptor plasticity in some contexts. To examine the potential role of PAR3 in regulating the activity of other PARs in sensory neurons, we administered PAR1, PAR2, and PAR4 agonists and assessed mechanical and affective pain behaviors in WT and PAR3-/- mice. We observed that the nociceptive effects of PAR1 agonists were potentiated in the absence of PAR3. Our findings suggest a complex role of PAR3 in the physiology and plasticity of nociceptors. PERSPECTIVE: We evaluated the role of PAR3, a G-protein coupled receptor, in nociception by developing a selective peptide agonist. Our findings suggest that PAR3 contributes to nociception in various contexts and plays a role in modulating the activity of other PARs.

Background Obesity is characterized by increased adipose tissue mass that results from increased fat cell size (hypertrophy) and number (hyperplasia). The molecular mechanisms that govern the regulation and differentiation of adipocytes play a critical role for better understanding of the pathological mechanism of obesity. However, the mechanism of adipocyte differentiation is still unclear. Objective The present study aims to compare the gene expression changes during adipocyte differentiation in the transcriptomic level, which may help to better understand the mechanism of adipocyte differentiation. Methods RNA sequencing (RNA-seq) technology, GO and KEGG analysis, quantitative RT-PCR, and oil red O staining methods were used in this study. Results A lot of genes were up- or down-regulated between each two differentiation stages of 3T3-L1 cells. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that lipid metabolism and oxidation-reduction reaction were mainly involved in the whole process of adipocyte differentiation. Decreased immune response and cell cycle adhesion occurred in the late phase of adipocyte differentiation, which was demonstrated by divergent expression pattern analysis. Moreover, quantitative RT-PCR results showed that the mRNA expression levels of Trpv4, Trpm4, Trpm5, and Trpm7 were significantly decreased in the differentiated adipocytes. On the other hand, the mRNA expression levels of Trpv1, Trpv2, Trpv6, and Trpc1 were significantly increased in the differentiated adipocytes. Besides, the mRNA expressions of TRPV2 and TRPM7 were also significantly increased in subcutaneous white adipose tissue from diet-induced mice. In addition, the activation of TRPM7, TRPV1, and TRPV2 suppressed the differentiation of adipocytes. Conclusion These data present the description of transcription profile changes during adipocyte differentiation and provides an in-depth analysis of the possible mechanisms of adipocyte differentiation. These data offer new insight into the understanding of the mechanisms of adipocyte differentiation.

Oxytocin (OT), a neuropeptide involved in the regulation of complex social and sexual behavior in mammals, has been proposed as a treatment for a number of psychiatric disorders including pain. It has been well documented that central administration of OT elicits strong scratching and grooming behaviors in rodents. However, these behaviors were only described as symptoms, few studies have investigated their underlying neural mechanisms. Thus, we readdressed this question and undertook an analysis of spinal circuits underlying OT-induced scratching behavior in the present study. We demonstrated that intrathecal OT induced robust but transient hindpaw scratching behaviors by activating spinal OT receptors (OTRs). Combining the pre-clinical and clinical evidence, we speculated that OT-induced scratching may be an itch symptom. Further RNAscope studies revealed that near 80% spinal GRP neurons expressed OTRs. OT activated the expression of c-fos mRNA in spinal GRP neurons. Chemical ablation of GRPR neurons significantly reduced intrathecal OT-induced scratching behaviors. Given GRP/GRPR pathway plays an important role in spinal itch transmission, we proposed that OT binds to the OTRs expressed on the GRP neurons, and activates GRP/GRPR pathway to trigger itch-scratching behaviors in mice. These findings provide novel evidence relevant for advancing understanding of OT-induced behavioral changes, which will be important for the development of OT-based drugs to treat a variety of psychiatric disorders.

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 (i.t.) 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 (i.t.) opioid-induced itch by conditional deletion of MOR-encodingOprm1in distinct populations of interneurons and sensory neurons. We found that i.t. injection of the MOR agonists morphine or DAMGO elicited dose-dependent scratching, but this pruritus was totally abolished in mice with a specificOprm1deletion in Vgat+neurons (Oprm1-Vgat). Loss of MOR in somatostatin+interneurons and TRPV1+sensory neurons did not affect morphine-induced itch but impaired morphine-induced antinociception.In situhybridization revealedOprm1expression 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 out of 19 Vgat+interneurons examined. Morphine also inhibited action potentials in Vgat+interneurons and suppressed evoked IPSCs in postsynaptic Vgat-excitatory neurons, suggesting a mechanism of disinhibition by MOR agonists. Notably, morphine-elicited itch was suppressed by i.t. administration of NPY and abolished by spinal ablation of GRPR+neurons, whereas i.t. GRP-induced itch response remained intact in mice lackingOprm1-Vgat. Additionally, chronic itch from DNFB-induced allergic contact dermatitis was decreased byOprm1-Vgatdeletion. Finally, naloxone, but not peripherally restricted naloxone methiodide, inhibited chronic itch in the DNFB model and the cutaneous T-cell lymphoma (CTCL) model, indicating a contribution of central MOR signaling to chronic itch. Our findings demonstrate that i.t. morphine elicits itch via acting on MOR on spinal inhibitory interneurons, leading to disinhibition of the spinal itch circuit. Our data also suggest that chronic itch could be effectively treated with CNS-targeted naloxone.

Oxaliplatin, a platinum-based chemotherapeutic drug, which is used as first-line treatment for some types of colorectal carcinoma, causes peripheral neuropathic pain in patients. In addition, an acute peripheral pain syndrome develop in almost 90% of patients immediately after oxaliplatin treatment, which is poorly understood mechanistically but correlates with incidence and severity of the later-occurring neuropathy. Here we investigated the effects of acute oxaliplatin treatment in a murine model, showing that male and female mice develop mechanical hypersensitivity 24 h after oxaliplatin treatment. Interestingly, we found that the levels of several lipids were significantly altered in nervous tissue during oxaliplatin-induced acute pain. Specifically, the linoleic acid metabolite 9,10-EpOME (epoxide of linoleic acid) as well as the lysophospholipids lysophosphatidylcholine (LPC) 18:1 and LPC 16:0 were significantly increased 24 h after oxaliplatin treatment in sciatic nerve, DRGs, or spinal cord tissue as revealed by untargeted and targeted lipidomics. In contrast, inflammatory markers including cytokines and chemokines, ROS markers, and growth factors are unchanged in the respective nervous system tissues. Importantly, LPC 18:1 and LPC 16:0 can induce Ca2+transients in primary sensory neurons, and we identify LPC 18:1 as a previously unknown endogenous activator of the ligand-gated calcium channels transient receptor potential V1 and M8 (transient receptor potential vanilloid 1 and transient receptor potential melastatin 8) in primary sensory neurons using both pharmacological inhibition and genetic knockout. Additionally, a peripheral LPC 18:1 injection was sufficient to induce mechanical hypersensitivity in naive mice. Hence, targeting signaling lipid pathways may ameliorate oxaliplatin-induced acute peripheral pain and the subsequent long-lasting neuropathy.SIGNIFICANCE STATEMENTThe first-line cytostatic drug oxaliplatin can cause acute peripheral pain and chronic neuropathic pain. The former is causally connected with the chronic neuropathic pain, but its mechanisms are poorly understood. Here, we performed a broad unbiased analysis of cytokines, chemokines, growth factors, and ∼200 lipids in nervous system tissues 24 h after oxaliplatin treatment, which revealed a crucial role of lysophospholipids lysophosphatidylcholine (LPC) 18:1, LPC 16:0, and 9,10-EpOME in oxaliplatin-induced acute pain. We demonstrate for the first time that LPC 18:1 contributes to the activation of the ion channels transient receptor potential vanilloid 1 and transient receptor potential melastatin 8 in sensory neurons and causes mechanical hypersensitivity after peripheral injectionin vivo. These findings suggest that the LPC-mediated lipid signaling is involved in oxaliplatin-induced acute peripheral pain.

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.

Emerging immunotherapies with monoclonal antibodies against programmed cell death protein–1 (PD-1) have shown success in treating cancers. However, PD-1 signaling in neurons is largely unknown. We recently reported that dorsal root ganglion (DRG) primary sensory neurons express PD-1 and activation of PD-1 inhibits neuronal excitability and pain. Opioids are mainstay treatments for cancer pain, and morphine produces antinociception via mu opioid receptor (MOR). Here, we report that morphine antinociception and MOR signaling require neuronal PD-1. Morphine-induced antinociception after systemic or intrathecal injection was compromised in Pd1−/− mice. Morphine antinociception was also diminished in wild-type mice after intravenous or intrathecal administration of nivolumab, a clinically used anti–PD-1 monoclonal antibody. In mouse models of inflammatory, neuropathic, and cancer pain, spinal morphine antinociception was compromised in Pd1−/− mice. MOR and PD-1 are coexpressed in sensory neurons and their axons in mouse and human DRG tissues. Morphine produced antinociception by (i) suppressing calcium currents in DRG neurons, (ii) suppressing excitatory synaptic transmission, and (iii) inducing outward currents in spinal cord neurons; all of these actions were impaired by PD-1 blockade in mice. Loss of PD-1 also enhanced opioid-induced hyperalgesia and tolerance and potentiates opioid-induced microgliosis and long-term potentiation in the spinal cord in mice. Last, intrathecal infusion of nivolumab inhibited intrathecal morphine-induced antinociception in nonhuman primates. Our findings demonstrate that PD-1 regulates opioid receptor signaling in nociceptive neurons, leading to altered opioid-induced antinociception in rodents and nonhuman primates.

Summary: Chronic allergic itch is a common symptom affecting millions of people and animals, but its pathogenesis is not fully explained. Herein, we show that periostin, abundantly expressed in the skin of patients with atopic dermatitis (AD), induces itch in mice, dogs, and monkeys. We identify the integrin αVβ3 expressed on a subset of sensory neurons as the periostin receptor. Using pharmacological and genetic approaches, we inhibited the function of neuronal integrin αVβ3, which significantly reduces periostin-induced itch in mice. Furthermore, we show that the cytokine TSLP, the application of AD-causing MC903 (calcipotriol), and house dust mites all induce periostin secretion. Finally, we establish that the JAK/STAT pathway is a key regulator of periostin secretion in keratinocytes. Altogether, our results identify a TSLP-periostin reciprocal activation loop that links the skin to the spinal cord via peripheral sensory neurons, and we characterize the non-canonical functional role of an integrin in itch. : Mishra et al. demonstrate periostin-induced itch in mice, dogs, and monkeys and identify the integrin αVβ3 as the periostin neuronal receptor. They find that keratinocytes release periostin in response to TSLP, thus identifying a possible reciprocal vicious circle implicating the cytokine TSLP and periostin in chronic allergic itch. Keywords: atopic dermatitis, AD, chronic allergic itch, integrin, JAK/STAT, keratinocytes, NPPB, periostin, sensory neuron, TRPV1, TRPA1, TSLP

It has been reported that nicotinamide-overload induces oxidative stress associated with insulin resistance, the key feature of type 2 diabetes mellitus (T2DM). This study aimed to investigate the effects of B vitamins in T2DM. Glucose tolerance tests (GTT) were carried out in adult Sprague-Dawley rats treated with or without cumulative doses of B vitamins. More specifically, insulin tolerance tests (ITT) were also carried out in adult Sprague-Dawley rats treated with or without cumulative doses of Vitamin B3. We found that cumulative Vitamin B1 and Vitamin B3 administration significantly increased the plasma H_2O_2 levels associated with high insulin levels. Only Vitamin B3 reduced muscular and hepatic glycogen contents. Cumulative administration of nicotinic acid, another form of Vitamin B3, also significantly increased plasma insulin level and H_2O_2 generation. Moreover, cumulative administration of nicotinic acid or nicotinamide impaired glucose metabolism. This study suggested that excess Vitamin B1 and Vitamin B3 caused oxidative stress and insulin resistance.

Adipose tissues play key roles in energy homeostasis. Brown adipocytes and beige adipocytes in white adipose tissue (WAT) share the similar characters of thermogenesis, both of them could be potential targets for obesity management. Several thermo-sensitive transient receptor potential channels (thermoTRPs) are shown to be involved in adipocyte biology. However, the expression pattern of thermoTRPs in adipose tissues from obese mice is still unknown. The mRNA expression of thermoTRPs in subcutaneous WAT (sWAT) and interscapular brown adipose tissue (iBAT) from lean and obese mice were measured using reverse transcriptase-quantitative PCRs (RT-qPCR). The results demonstrated that all 10 thermoTRPs are expressed in both iBAT and sWAT, and without significant difference in the mRNA expression level of thermoTRPs between these two tissues. Moreover, Trpv1 and Trpv3 mRNA expression levels in both iBAT and sWAT were significantly decreased in high fat diet (HFD)-induced obese mice and db/db (leptin receptor deficient) mice. Trpm2 mRNA expression level was significantly decreased only in sWAT from HFD-induced obese mice and db/db mice. On the other hand, Trpv2 and Trpv4 mRNA expression levels in iBAT and sWAT were significantly increased in HFD-induced obese mice and db/db mice. Taken together, we conclude that all 10 thermoTRPs are expressed in iBAT and sWAT. And several thermoTRPs differentially expressed in adipose tissues from HFD-induced obese mice and db/db mice, suggesting a potential involvement in anti-obesity regulations.

Paclitaxel-induced acute pain syndrome (P-APS), characterized by deep muscle aches and arthralgia, occurs in more than 70% of patients who receive paclitaxel. P-APS can be debilitating for patients and lead to reductions and discontinuation of potentially curable therapy. Despite being relatively common in clinical practice, no clear treatment exists for P-APS and the underlying mechanisms remain poorly defined. Regulation of glutamatergic transmission by metabotropic glutamate receptors (mGluRs) has received growing attention with respect to its role in neuropathic pain. To our knowledge, no study has been conducted on alterations and functions of group III mGluR7 signaling in P-APS. In the present study, we determined whether a single administration of paclitaxel induces glutamatergic alterations and whether mGluR7 activation blocks paclitaxel-induced neuropathic pain by suppressing glial reactivity in the spinal cord. A single paclitaxel injection dose-dependently induced acute mechanical and thermal hypersensitivity, and was associated with increased glutamate level accompanied by reduction in mGluR7 expression in the spinal cord. Selective activation of mGluR7 by its positive allosteric modulator, AMN082, blocked the development of paclitaxel-induced acute mechanical and thermal hypersensitivity, without affecting the normal pain behavior of control rats. Moreover, activation of mGluR7 by AMN082 inhibited glial reactivity and decreased pro-inflammatory cytokine release during P-APS. Abortion of spinal glial reaction to paclitaxel alleviated paclitaxel-induced acute mechanical and thermal hypersensitivity. There results support the hypothesis that spinal mGluR7 signaling plays an important role in P-APS; Selective activation of mGluR7 by its positive allosteric modulator, AMN082, blocks P-APS in part by reducing spinal glial reactivity and neuroinflammatory process.

(−)-menthol, a major form of menthol, is one of the most commonly used chemicals. Many studies have demonstrated that (−)-menthol produces analgesic action through peripheral mechanisms which are mainly mediated by activation of TRPM8. Moreover, intrathecal injection of menthol induces analgesia as well. However, the central actions and mechanisms of (−)-menthol remain unclear. Here, we have investigated the action of (−)-menthol on excitatory synaptic transmission in spinal lamina II layer which plays a pivotal role in modulating nociceptive transmission from the periphery by using patch-clamp technique in mice spinal cord. We found that (−)-menthol increased miniature excitatory postsynaptic current frequency. The frequency increases which (−)-menthol induced were in a dose-dependent manner (EC50: 0.1079 mM). However, neither genetic knockout nor pharmacological inhibition of TRPM8 could block (−)-menthol-induced effects entirely. Furthermore, this increase was also impaired by TRPA1 antagonist HC030031, but abolished utterly by co-application of TRPM8 and TRPA1 antagonist. Our results indicate that (−)-menthol increases the excitatory synaptic transmission by activating either TRPA1 or TRPM8 channels in spinal lamina II layer.

The development of multitarget opioid drugs has emerged as an attractive therapeutic strategy to eliminate opioid-related side effects. Our previous study developed a series of opioid and neuropeptide FF pharmacophore-containing chimeric peptides, including DN-9 (Tyr-D.Ala-Gly-NMe.Phe-Gly-Pro-Gln-Arg-Phe-NH2), which produced potent nontolerance forming analgesia at the supraspinal level. In the present study, the antinociceptive effects of DN-9 in a series of preclinical pain models and the potential side-effects were investigated at the spinal level in mice. In the tail-flick test, intrathecal injection of DN-9 produced potent analgesia with an ED50 value at 1.33 pmol, and the spinal antinociception of DN-9 was mainly mediated by μ- and κ-opioid receptors. In addition, DN-9-induced spinal antinociception was augmented by the neuropeptide FF receptors antagonist. Furthermore, DN-9 could decrease both the frequency and amplitude of sEPSCs in lamina IIo neurons of the spinal cord, which were mediated by opioid receptors. In contrast to morphine, chronic intrathecal treatments with DN-9 did not induce analgesic tolerance, c-Fos expression or microglial activation. Intrathecal injection of DN-9 showed potent analgesia with antinociceptive ED50 values between .66 and 55.04 pmol in different pain models, including the formalin test, acetic acid-induced writhing test, carrageenan-induced inflammatory pain and neuropathic pain. Moreover, DN-9 did not show side effects in locomotor function and coordination, gastrointestinal transit inhibition, the cardiovascular system, and body temperature regulation at antinociceptive doses. Taken together, the present study showed DN-9 produced effective, nontolerance forming analgesia with reduced side effects at the spinal level. DN-9 might be a promising compound for developing multifunctional opioid analgesics with limited adverse effects. PERSPECTIVE: This article presents the potent and nontolerance forming analgesia effects of DN-9 in a series of preclinical pain models with less opioid related adverse effects at the spinal level in mice. This study also demonstrates that DN-9 has translational potential into an intrathecal analgesic.

Cav3 channels play an important role in modulating chronic pain. However, less is known about the functional changes of Cav3 channels in superficial spinal dorsal horn in neuropathic pain states. Here, we examined the effect of partial sciatic nerve ligation (PSNL) on either expression or electrophysiological properties of Cav3 channels in superficial spinal dorsal horn. Our in vivo studies showed that the blockers of Cav3 channels robustly alleviated PSNL-induced mechanical allodynia and thermal hyperalgesia, which lasted at least 14 days following PSNL. Meanwhile, PSNL triggered an increase in both mRNA and protein levels of Cav3.2 but not Cav3.1 or Cav3.3 in rats. However, in Cav3.2 knockout mice, PSNL predominantly attenuated mechanical allodynia but not thermal hyperalgesia. In addition, the results of whole-cell patch-clamp recordings showed that both the overall proportion of Cav3 current-expressing neurons and the Cav3 current density in individual neurons were elevated in spinal lamina II neurons from PSNL rats, which could not be recapitulated in Cav3.2 knockout mice. Altogether, our findings reveal that the elevated functional Cav3.2 channels in superficial spinal dorsal horn may contribute to the mechanical allodynia in PSNL-induced neuropathic pain model.

Chronic allergic itch is a major symptom that affects millions of people and animals, but its pathogenesis is not fully explained. Here, we show that one of the mediators abundantly present in the skin of patients with atopic dermatitis, periostin, induces itch via sensory neurons in mice, dogs, and monkeys. We identified the periostin receptor, the integrin αvβ3 expressed on a subset of sensory neurons. Using pharmacological and genetic approaches, we inhibited the function of this neuronal integrin αvβ3, which significantly reduced periostin-mediated itch in mice. Furthermore, we showed that the cytokine TSLP, and the application of the AD-causing MC903 (calcipotriol) and house dust mites induced periostin secretion. Finally, we established that the JAK/STAT pathway is a key regulator of periostin secretion in keratinocytes. Overall, our results identified a TSLP-periostin reciprocal activation that links the skin to the central nervous system via peripheral sensory neurons, and we characterized the non-canonical functional role of a neuronal integrin in itch behavior.

We have previously reported that oxytocin produces an inward current at a holding potential of −70 mV without a change in glutamatergic excitatory transmission in adult male rat spinal lamina II (substantia gelatinosa; SG) neurons that play a pivotal role in regulating nociceptive transmission. Oxytocin also enhanced GABAergic and glycinergic spontaneous inhibitory transmissions in a manner sensitive to a voltage-gated Na+-channel blocker tetrodotoxin. These actions were mediated by oxytocin-receptor activation. Such a result was different from that obtained by other investigators in young male rat superficial dorsal horn neurons in which an oxytocin-receptor agonist enhanced glutamatergic and GABAergic but not glycinergic spontaneous transmissions. In order to know a developmental change and also sexual difference in the actions of oxytocin, we examined its effect on spontaneous synaptic transmission in adult female and young male rat SG neurons by using the whole-cell patch-clamp technique in spinal cord slices. In adult female rats, oxytocin produced an inward current at −70 mV without a change in excitatory transmission. GABAergic and glycinergic transmissions were enhanced by oxytocin, the duration of which enhancement was much shorter than in adult male rats. In young (11–21 postnatal days) male rats, oxytocin produced not only an inward but also outward current at −70 mV, and presynaptically inhibited or facilitated excitatory transmission, depending on the neurons tested; both GABAergic and glycinergic transmissions were enhanced by oxytocin. The inhibitory transmission enhancements in adult female and young male rats were sensitive to tetrodotoxin. Although the data may not be enough to be estimated, it is suggested that synaptic modulation by oxytocin in SG neurons, i.e., cellular mechanism for its antinociceptive action, exhibits a developmental change and sexual difference., Highlights • Oxytocin modulated synaptic transmission in spinal dorsal horn neurons. • The synaptic modulation produced by oxytocin exhibited a developmental change. • The synaptic modulation produced by oxytocin exhibited a sexual difference.