Characterizing the opioidergic mechanisms of repetitive transcranial magnetic stimulation-induced analgesia: a randomized controlled trial.

ABSTRACT: Repetitive transcranial magnetic stimulation (rTMS) is a promising technology to reduce chronic pain. Investigating the mechanisms of rTMS analgesia holds the potential to improve treatment efficacy. Using a double-blind and placebo-controlled design at both stimulation and pharmacologic ends, this study investigated the opioidergic mechanisms of rTMS analgesia by abolishing and recovering analgesia in 2 separate stages across brain regions and TMS doses. A group of 45 healthy participants were equally randomized to the primary motor cortex (M1), the dorsolateral prefrontal cortex (DLPFC), and the Sham group. In each session, participants received an intravenous infusion of naloxone or saline before the first rTMS session. Participants then received a second dose of rTMS session after the drugs were metabolized at 90 minutes. M1-rTMS-induced analgesia was abolished by naloxone compared with saline and was recovered by the second rTMS run when naloxone was metabolized. In the DLPFC, double but not the first TMS session induced significant pain reduction in the saline condition, resulting in less pain compared with the naloxone condition. In addition, TMS over the M1 or DLPFC selectively increased plasma concentrations of ß-endorphin or encephalin, respectively. Overall, we present causal evidence that opioidergic mechanisms are involved in both M1-induced and DLPFC-rTMS-induced analgesia; however, these are shaped by rTMS dosage and the release of different endogenous opioids.

NAMPT/NAD+/PARP1 Pathway Regulates CFA-Induced Inflammatory Pain via NF-κB Signaling in Rodents.

Emerging evidence has implicated nicotinamide adenine dinucleotide (NAD+) metabolism in various inflammatory diseases. In the study, the role of NAD+ metabolism in Complete Freund's Adjuvant (CFA)-evoked inflammatory pain and the underlying mechanisms are investigated. The study demonstrated that CFA induced upregulation of nicotinamide phosphoribosyltransferase (NAMPT) in dorsal root ganglia (DRG) without significant changes in the spinal cord. Inhibition of NAMPT expression by intrathecal injection of NAMPT siRNA alleviated CFA-induced pain-like behavior, decreased NAD+ contents in DRG, and lowered poly-(ADP-ribose) polymerase 1 (PARP1) activity levels. These effects are all reversed by the supplement of nicotinamide mononucleotide (NMN). Inhibition of PARP1 expression by intrathecal injection of PARP1 siRNA alleviated CFA-induced pain-like behavior, while elevated NAD+ levels of DRG. The analgesic effect of inhibiting NAMPT/NAD+/PARP1 axis can be attributed to the downregulation of the NF-κB/IL-1ß inflammatory pathway. Double immunofluorescence staining showed that the expression of NAMPT/NAD+/PARP1 axis is restricted to DRG neurons. In conclusion, PARP1 activation in response to CFA stimulation, fueled by NAMPT-derived NAD+, mediates CFA-induced inflammatory pain through NF-κB/IL-1ß inflammatory pathway.

A2Hgx(SeO3)y (A = K, Rb, Cs): Three Alkali Metal Mercury Selenites Featuring Unique 1D [HgOm(SeO3)n]∞ Chains.

Herein, three alkali metal mercury selenites, K2Hg2(SeO3)3, Rb2Hg2(SeO3)3, and Cs2Hg3(SeO3)4, were successfully obtained by a hydrothermal method. The three compounds featured same one-dimensional (1D) [HgOm(SeO3)n]∞ chain structure that consisting of distorted Hg-O polyhedra and SeO3 triangular pyramids with stereochemically active lone pair (SCALP) electrons. Interestingly, the rich coordination environment of Hg atoms and the size difference of alkali metal cations lead to diverse arrangement of SeO3 groups, which makes them exhibit different birefringence. The band gaps of the three compounds indicate that they are potential ultraviolet (UV) optical materials. Detailed theoretical calculations demonstrate that the combined effects of SeO3 triangular pyramids and Hg-O polyhedra are responsible for the optical characteristics of the reported compounds.

Spinal CBX2 contributes to neuropathic pain by activating ERK signaling pathway in male mice.

The deregulated spinal cord proteins induced by nerve injury are the key to neuropathic pain. Integrated transcriptome and translatome analyses can screen out deregulated proteins controlled by only post-transcriptional regulation. By comparing RNA sequencing (RNA-seq) and ribosome profiling sequencing (Ribo-seq) data, we identified an upregulated protein, chromobox 2 (CBX2), with its mRNA level unchanged in the spinal cord after peripheral nerve injury. CBX2 was mainly distributed in the spinal cord neurons. Blocking the SNL-induced increase of spinal CBX2 attenuated the neuronal and astrocytes hyperactivities and pain hypersensitivities in both the development and maintenance phases. Conversely, mimicking the upregulation of CBX2 in the spinal cord facilitated the activities of neurons and astrocytes and produced evoked nociceptive hypersensitivity and spontaneous pain. Our results also revealed that activating the ERK pathway, upregulating CXCL13 in neurons, and CXCL13 further inducing astrocyte activation were possible downstream signaling mechanisms of CBX2 in pain processing. In conclusion, upregulation of CBX2 after nerve injury leads to nociceptive hyperalgesia by promoting neuronal and astrocyte hyperactivities through the ERK pathway. Inhibiting CBX2 upregulation may be therapeutically beneficial.

Hg3(SeO3)2(SO4): A UV Nonlinear Optical Mercury Selenite Sulfate Constructed by Neat [Hg6O8(SeO3)4]∞ Layers and SO4 Tetrahedra.

A novel mercury selenite sulfate named Hg3(SeO3)2(SO4) has been successfully synthesized under a mild hydrothermal method. Hg3(SeO3)2(SO4) crystallizes in a monoclinic space group P21 and features a unique three-dimensional (3D) frame structure formed by [Hg6O8(SeO3)4]∞ layers and SO4 tetrahedra, which enables it to exhibit a comprehensive performance of a moderate second-harmonic generation (SHG) response of approximately 1.3 times that of baseline KH2PO4 (KDP), a moderate birefringence (0.118@546 nm), and a wide band gap (4.70 eV), which indicates that it has potential for application as an ultraviolet (UV) nonlinear optical material. Detailed theoretical calculations show that the Hg2+-based polyhedra with large polarizability and deformability and the SeO3 groups with stereochemically active lone pair (SCALP) electrons are the main contributors to moderate optical properties.

NAD+ metabolism in peripheral neuropathic pain.

Nicotinamide adenine dinucleotide (NAD+) is an omnipresent metabolite that participates in redox reactions. Multiple NAD+-consuming enzymes are implicated in numerous biological processes, including transcription, signaling, and cell survival. Multiple pieces of evidence have demonstrated that NAD+-consuming enzymes, including poly(ADP-ribose) polymerases (PARPs), sirtuins (SIRTs), and sterile alpha and TIR motif-containing 1 (SARM1), play major roles in peripheral neuropathic pain of various etiologies. These NAD+ consumers primarily participate in peripheral neuropathic pain via mechanisms such as mitochondrial dysfunction, oxidative stress, and inflammation. Furthermore, NAD+ synthase and nicotinamide phosphoribosyltransferase (NAMPT) have recently been found to contribute to the regulation of pain. Here, we review the evidence indicating the involvement of NAD+ metabolism in the pathological mechanisms of peripheral neuropathic pain. Advanced understanding of the molecular and cellular mechanisms associated with NAD+ in peripheral neuropathic pain will facilitate the development of novel treatment options for diverse types of peripheral neuropathic pain.

Lysophosphatidylcholine: Potential Target for the Treatment of Chronic Pain.

The bioactive lipid lysophosphatidylcholine (LPC), a major phospholipid component of oxidized low-density lipoprotein (Ox-LDL), originates from the cleavage of phosphatidylcholine by phospholipase A2 (PLA2) and is catabolized to other substances by different enzymatic pathways. LPC exerts pleiotropic effects mediated by its receptors, G protein-coupled signaling receptors, Toll-like receptors, and ion channels to activate several second messengers. Lysophosphatidylcholine (LPC) is increasingly considered a key marker/factor positively in pathological states, especially inflammation and atherosclerosis development. Current studies have indicated that the injury of nervous tissues promotes oxidative stress and lipid peroxidation, as well as excessive accumulation of LPC, enhancing the membrane hyperexcitability to induce chronic pain, which may be recognized as one of the hallmarks of chronic pain. However, findings from lipidomic studies of LPC have been lacking in the context of chronic pain. In this review, we focus in some detail on LPC sources, biochemical pathways, and the signal-transduction system. Moreover, we outline the detection methods of LPC for accurate analysis of each individual LPC species and reveal the pathophysiological implication of LPC in chronic pain, which makes it an interesting target for biomarkers and the development of medicine regarding chronic pain.

Dimethyl Itaconate Attenuates CFA-Induced Inflammatory Pain via the NLRP3/ IL-1ß Signaling Pathway.

Itaconate plays a prominent role in anti-inflammatory effects and has gradually been ushered as a promising drug candidate for treating inflammatory diseases. However, its significance and underlying mechanism for inflammatory pain remain unexplored. In the current study, we investigated the effects and mechanisms of Dimethyl Itaconate (DI, a derivative of itaconate) on Complete Freund's adjuvant (CFA)-induced inflammatory pain in a rodent model. Here, we demonstrated that DI significantly reduced mechanical allodynia and thermal hyperalgesia. The DI-attenuated neuroinflammation was evident with the amelioration of infiltrative macrophages in peripheral sites of the hind paw and the dorsal root ganglion. Concurrently, DI hindered the central microglia activation in the spinal cord. Mechanistically, DI inhibited the expression of pro-inflammatory factors interleukin (IL)-1ß and tumor necrosis factor alpha (TNF-α) and upregulated anti-inflammatory factor IL-10. The analgesic mechanism of DI was related to the downregulation of the nod-like receptor protein 3 (NLRP3) inflammasome complex and IL-1ß secretion. This study suggested possible novel evidence for prospective itaconate utilization in the management of inflammatory pain.

Dimethyl itaconate inhibits neuroinflammation to alleviate chronic pain in mice.

The metabolite itaconate has both anti-inflammatory and immunomodulatory effects. However, its influence on chronic pain is unclear. Here, we demonstrated that intraperitoneal injection of the itaconate derivative dimethyl itaconate (DI) alleviated chronic pain symptoms, such as allodynia and hyperalgesia, in spinal nerve ligation (SNL) and inflammatory pain models. Moreover, intraperitoneal DI reduced the secretion of inflammatory cytokines (i.e., interleukin-1ß, tumour necrosis factor-alpha) in dorsal root ganglion (DRG), spinal cord and hind paw tissues, suppressed the activation of macrophages in the DRG and glial cells in the spinal dorsal horn and decreased the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) in the DRG and spinal cord. DI boosted nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) levels in the DRG and spinal cord of SNL mice. Intraperitoneal administration of the Nrf2 inhibitor ML385 abolished the analgesic effect of DI and decreased the expression of Nrf2 in the DRG and spinal cord. Similarly, administration of DI potently reversed the lipopolysaccharide (LPS)-induced inflammatory effect in microglia. Reduction of endogenous itaconate levels by pretreatment with immune-responsive gene 1 (IRG1) siRNA blocked Nrf2 expression, which impaired the analgesic and anti-inflammatory effects of DI in vitro. Therefore, our findings revealed for the first time that intraperitoneal DI elicited anti-inflammatory effect and sustained chronic pain relief, which may be regarded as a promising therapeutic agent for chronic pain treatment.

MeCP2 Epigenetic Silencing of Oprm1 Gene in Primary Sensory Neurons Under Neuropathic Pain Conditions.

Opioids are the last option for the pharmacological treatment of neuropathic pain, but their antinociceptive effects are limited. Decreased mu opioid receptor (MOR) expression in the peripheral nervous system may contribute to this. Here, we showed that nerve injury induced hypermethylation of the Oprm1 gene promoter and an increased expression of methyl-CpG binding protein 2 (MeCP2) in injured dorsal root ganglion (DRG). The downregulation of MOR in the DRG is closely related to the augmentation of MeCP2, an epigenetic repressor, which could recruit HDAC1 and bind to the methylated regions of the Oprm1 gene promoter. MeCP2 knockdown restored the expression of MOR in injured DRG and enhanced the analgesic effect of morphine, while the mimicking of this increase via the intrathecal infusion of viral vector-mediated MeCP2 was sufficient to reduce MOR in the DRG. Moreover, HDAC1 inhibition with suberoylanilide hydroxamic acid, an HDAC inhibitor, also prevented MOR reduction in the DRG of neuropathic pain mice, contributing to the augmentation of morphine analgesia effects. Mechanistically, upregulated MeCP2 promotes the binding of a high level of HDCA1 to hypermethylated regions of the Oprm1 gene promoter, reduces the acetylation of histone H3 (acH3) levels of the Oprm1 gene promoter, and attenuates Oprm1 transcription in injured DRG. Thus, upregulated MeCP2 and HDAC1 in Oprm1 gene promoter sites, negatively regulates MOR expression in injured DRG, mitigating the analgesic effect of the opioids. Targeting MeCP2/HDAC1 may thus provide a new solution for improving the therapeutic effect of opioids in a clinical setting.

Sevoflurane preconditioning promotes mesenchymal stem cells to relieve myocardial ischemia/reperfusion injury via TRPC6-induced angiogenesis.

BACKGROUND: Ischemic heart diseases is one of the leading causes of death worldwide. Although revascularization timely is an effective therapeutic intervention to salvage the ischemic myocardium, reperfusion itself causes additional myocardial injury called ischemia/reperfusion (I/R) injury. Bone marrow-derived mesenchymal stem cells (MSCs) is one of the promising cells to alleviate ischemic myocardial injury. However, this cell therapy is limited by poor MSCs survival after transplantation. Here, we investigated whether sevoflurane preconditioning could promote MSCs to attenuate myocardial I/R injury via transient receptor potential canonical channel 6 (TRPC6)-induced angiogenesis. METHODS: The anti-apoptotic effect of sevoflurane preconditioning on MSCs was determined by Annexin V-FITC/propidium iodide staining. TRPC6, hypoxia-inducible factor-1α (HIF-1α), Chemokine receptor 4 (CXCR4) and vascular endothelial growth factor (VEGF) protein expressions and VEGF release from MSCs were determined after hypoxia and reoxygenation (H/R). Small interfering RNA (siRNA) was used to knock down TRPC6 gene expression in MSCs. The angiogenesis of human umbilical vein endothelial cells (HUVECs) co-cultured with MSCs was determined by Matrigel tube formation. Myocardial I/R mouse model was induced by occluding left anterior descending coronary artery for 30 min and then reperfusion. MSCs or sevoflurane preconditioned MSCs were injected around the ligature border zone 5 min before reperfusion. Left ventricle systolic function, infarction size, serum LDH, cTnI and inflammatory cytokines were determined after reperfusion. RESULTS: Sevoflurane preconditioning up-regulated TRPC6, HIF-1α, CXCR4 and VEGF expressions in MSCs and VEGF release from MSCs under H/R, which were reversed by knockdown of TRPC6 gene using siRNA in MSCs. Furthermore, sevoflurane preconditioning promoted the angiogenic and anti-inflammatory effect of HUVECs co-cultured with MSCs. Sevoflurane preconditioned MSCs improved left ventricle systolic function and alleviated myocardial infarction and inflammation in mice subjected to I/R insult. CONCLUSION: The current findings reveal that sevoflurane preconditioned MSCs boost angiogenesis in HUVECs subjected to H/R insult and attenuate myocardial I/R injury, which may be mediated by TRPC6 up-regulated HIF-1α, CXCR4 and VEGF.

ZNF382 controls mouse neuropathic pain via silencer-based epigenetic inhibition of Cxcl13 in DRG neurons.

Nerve injury-induced changes of gene expression in dorsal root ganglion (DRG) are critical for neuropathic pain genesis. However, how these changes occur remains elusive. Here we report the down-regulation of zinc finger protein 382 (ZNF382) in injured DRG neurons after nerve injury. Rescuing this down-regulation attenuates nociceptive hypersensitivity. Conversely, mimicking this down-regulation produces neuropathic pain symptoms, which are alleviated by C-X-C motif chemokine 13 (CXCL13) knockdown or its receptor CXCR5 knockout. Mechanistically, an identified cis-acting silencer at distal upstream of the Cxcl13 promoter suppresses Cxcl13 transcription via binding to ZNF382. Blocking this binding or genetically deleting this silencer abolishes the ZNF382 suppression on Cxcl13 transcription and impairs ZNF382-induced antinociception. Moreover, ZNF382 down-regulation disrupts the repressive epigenetic complex containing histone deacetylase 1 and SET domain bifurcated 1 at the silencer-promoter loop, resulting in Cxcl13 transcriptional activation. Thus, ZNF382 down-regulation is required for neuropathic pain likely through silencer-based epigenetic disinhibition of CXCL13, a key neuropathic pain player, in DRG neurons.

Cholinergic-Induced Specific Oscillations in the Medial Prefrontal Cortex to Reverse Propofol Anesthesia.

General anesthesia is a drug-induced reversible state comprised of altered states of consciousness, amnesia, analgesia, and immobility. The medial frontal cortex (mPFC) has been discovered to modulate the level of consciousness through cholinergic and glutamatergic pathways. The optogenetic tools combined with in vivo electrophysiological recording were used to study the neural oscillatory modulation mechanisms in mPFC underlying the loss of consciousness (LOC) and emergence. We found that optogenetic activation of both cholinergic and glutamatergic neurons in the basal forebrain (BF) reversed the hypnotic effect of propofol and accelerated the emergence from propofol-induced unconsciousness. The cholinergic light-activation during propofol anesthesia increased the power in the ß (12-20 Hz) and low γ (20-30 Hz) bands. Conversely, glutamatergic activation increased the power at less specific broad (1-150 Hz) bands. The cholinergic-induced alteration to specific power bands after LOC had opposite effects to that of propofol. These results suggested that the cholinergic system might act on more specific cortical neural circuits related to propofol anesthesia.

The Emerging Application of Itaconate: Promising Molecular Targets and Therapeutic Opportunities.

Metabolites have recently been found to be involved in significant biological regulation and changes. Itaconate, an important intermediate metabolite isolated from the tricarboxylic acid cycle, is derived from cis-aconitate decarboxylation mediated by immune response gene 1 in mitochondrial matrix. Itaconate has emerged as a key autocrine regulatory component involved in the development and progression of inflammation and immunity. It could directly modify cysteine sites on functional substrate proteins which related to inflammasome, signal transduction, transcription, and cell death. Itaconate can be a connector among immunity, metabolism, and inflammation, which is of great significance for further understanding the mechanism of cellular immune metabolism. And it could be the potential choice for the treatment of inflammation and immune-related diseases. This study is a systematic review of the potential mechanisms of metabolite associated with different pathology conditions. We briefly summarize the structural characteristics and classical pathways of itaconate and its derivatives, with special emphasis on its promising role in future clinical application, in order to provide theoretical basis for future research and treatment intervention.

MMP24 Contributes to Neuropathic Pain in an FTO-Dependent Manner in the Spinal Cord Neurons.

Nerve injury-induced gene expression change in the spinal cord is critical for neuropathic pain genesis. RNA N6-methyladenosine (m6A) modification represents an additional layer of gene regulation. We showed that spinal nerve ligation (SNL) upregulated the expression of matrix metallopeptidase 24 (MMP24) protein, but not Mmp24 mRNA, in the spinal cord neurons. Blocking the SNL-induced upregulation of spinal MMP24 attenuated local neuron sensitization, neuropathic pain development and maintenance. Conversely, mimicking MMP24 increase promoted the spinal ERK activation and produced evoked nociceptive hypersensitivity. Methylated RNA Immunoprecipitation Sequencing (MeRIP-seq) and RNA Immunoprecipitation (RIP) assay indicated the decreased m6A enrichment in the Mmp24 mRNA under neuropathic pain condition. Moreover, fat-mass and obesity-associated protein (FTO) was colocalized with MMP24 in spinal neurons and shown increased binding to the Mmp24 mRNA in the spinal cord after SNL. Overexpression or suppression of FTO correlates with promotion or inhibition of MMP24 expression in cultured spinal cord neurons. In conclusion, SNL promoted the m6A eraser FTO binding to the Mmp24 mRNA, which subsequently facilitated the translation of MMP24 in the spinal cord, and ultimately contributed to neuropathic pain genesis.

Pretreatment with AM1241 Enhances the Analgesic Effect of Intrathecally Administrated Mesenchymal Stem Cells.

Mesenchymal stem cells have cannabinoid (CB) receptors type 1 and type 2 and can alleviate a variety of neuropathic pains, including chronic constriction injury (CCI). A selective CB2 receptor agonist is AM1241. In the present study, it was found that mice with CCI displayed a longer duration of mechanical and thermal analgesia when intrathecally (i.t.) injected with AM1241-treated mesenchymal stem cells, compared to those injected with untreated mesenchymal stem cells or AM1241 alone. Moreover, CCI-induced upregulation of the phosphorylated extracellular signal-regulated kinase (ERK) 1/2 (p-ERK1/2) was inhibited following i.t. injection of AM1241-treated mesenchymal stem cells and this inhibition was noticeably higher compared to injection with untreated mesenchymal stem cells. The expression of transforming growth factor-ß1 (TGF-ß1) was also analyzed in the dorsal root ganglion (DRGs) and spinal cord of CCI mice. In untreated CCI mice, expression of TGF-ß1 was increased, whereas pretreatment with AM1241-treated mesenchymal stem cells regulated the expression of TGF-ß1 on 10 days and 19 days after surgery. In addition, i.t. injection of exogenous TGF-ß1 slightly alleviated neuropathic pain whilst neutralization of TGF-ß1 potently blocked the effect of AM1241-treated mesenchymal stem cells on thermal hyperalgesia and mechanical allodynia of CCI mice. In an in vitro experiment, AM1241 could enhance the release of TGF-ß1 in the supernatant of BMSCs after lipopolysaccharide (LPS) simulation. Taken together, the findings of the current study show that i.t. administration of AM1241-treated mesenchymal stem cells has a positive effect on analgesia and that TGF-ß1 and p-ERK1/2 may be the molecular signaling pathway involved in this process.

Mesenchymal Stem Cells and their Exosomes: Promising Therapeutics for Chronic Pain.

Chronic pain is a common condition that seriously affects the quality of human life with variable etiology and complicated symptoms; people who suffer from chronic pain may experience anxiety, depression, insomnia, and other harmful emotions. Currently, chronic pain treatments are nonsteroidal anti-inflammatory drugs and opioids; these drugs are demonstrated to be insufficient and cause severe side effects. Therefore, research into new therapeutic strategies for chronic pain is a top priority. In recent years, stem cell transplantation has been demonstrated to be a potent alternative for the treatment of chronic pain. Mesenchymal stem cells (MSCs), a type of pluripotent stem cell, exhibit multi-directional differentiation, promotion of stem cell implantation, and immune regulation; they have also been shown to exert analgesic effects in several chronic pain models. Exosomes produced by MSCs have been demonstrated to relieve painful symptoms with fewer side effects. In this review, we summarize the therapeutic use of MSCs in various chronic pain studies. We also discuss ways to enhance the treatment effect of MSCs. We predict in the future, cell-free therapies for chronic pain will develop from exosomes secreted by MSCs.

Bioinformatics Analysis Identifies p53 as a Candidate Prognostic Biomarker for Neuropathic Pain.

Neuropathic pain (NP) is a type of chronic pain that is different from the common type of pain. The mechanisms of NP are still poorly understood. Exploring the key genes and neurobiological changes in NP could provide important diagnostic and treatment tools for clinicians. GSE24982 is an mRNA-seq dataset that we downloaded from the Gene Expression Omnibus database to identify key genes in NP. Differentially expressed genes (DEGs) were identified using the BRB-ArrayTools software and R. Functional and pathway enrichment analyses of the DEGs were performed using Metascape. A protein-protein interaction network was created and visualized using Cytoscape. A total of 123 upregulated DEGs were obtained. Among these genes, p53 was the node with the highest degree; hence, we validated it experimentally using a chronic constriction injury mouse model. Our results showed that overexpression of the p53 gene, and the subsequent increase in caspase-3 expression, in dorsal root ganglion neurons led to increased apoptotic changes in these neurons. p53 may therefore be partly responsible for the development of chronic constriction injury-induced NP.