(+)-Catechin attenuates CCI-induced neuropathic pain in male rats by promoting the Nrf2 antioxidant pathway to inhibit ROS/TLR4/NF-κB-mediated activation of the NLRP3 inflammasome.

The objective of this study was to investigate the potential mechanisms by which (+)-catechin alleviates neuropathic pain. Thirty-two male Sprague-Dawley rats were divided into four groups: the sham group, the chronic constriction injury (CCI)group, the CCI+ ibuprofen group, and the CCI+ (+)-catechin group. CCI surgery induces thermal hyperalgesia in rats and (+)-catechin ameliorated CCI-induced thermal hyperalgesia and repaired damaged sciatic nerve in rats. CCI decreased SOD levels in male rat spinal cord dorsal horn and promoted MDA production, induced oxidative stress by increasing NOX4 levels and decreasing antioxidant enzyme HO-1 levels, and also increased protein levels of TLR4, p-NF-κB, NLRP3 inflammasome components, and IL-1ß. In contrast, (+)-catechin reversed the above results. In i vitro experiments, (+)-catechin reduced the generation of reactive oxygen species (ROS) in GMI-R1 cells after LPS stimulation and attenuated the co-expression of IBA-1 and NLRP3. It also showed significant inhibition of the NF-κB and NLRP3 inflammatory pathways and activation of the Nrf2-mediated antioxidant system. Overall, these findings suggest that (+)-catechin inhibits the activation of the NLRP3 inflammasome through the triggering of the Nrf2-induced antioxidant system, the inhibition of the TLR4/NF-κB pathway, and the production of ROS to alleviate CCI-induced neuropathic pain in male rats.

TRAF6 promotes spinal microglial M1 polarization to aggravate neuropathic pain by activating the c-JUN/NF-kB signaling pathway.

BACKGROUND: The neuropathic pain with complex networks of neuroinflammatory activation severely limits clinical therapeutic research. TNF receptor-associated factor 6 (TRAF6) is associated with multiple inflammatory diseases. However, there remains confusion about the effects and mechanisms of TRAF6 in neuropathic pain. METHODS: A chronic constriction injury (CCI) model was developed to simulate neuralgia in vivo. We overexpressed or knocked down TRAF6 in CCI mice, respectively. Activation of microglia by TRAF6, the inflammatory response, and disease progression were inspected using WB, qRT-PCR, immunofluorescence, flow cytometry, and ELISA assays. Moreover, the mechanism of M1/M2 polarization activation of microglia by TRAF6 was elaborated in BV-2 cells. RESULTS: TRAF6 was enhanced in the spinal neurons and microglia of the CCI mice model compared with the sham operation group.. Down-regulation of TRAF6 rescued the expression of Iba-1. In response to mechanical and thermal stimulation, PWT and PWL were improved after the knockdown of TRAF6. Decreased levels of pro-inflammatory factors were observed in TRAF6 knockdown groups. Meanwhile, increased microglial M1 markers induced by CCI were limited in mice with TRAF6 knockdown. In addition, TRAF6 overexpression has the precise opposite effect on CCI mice or microglia polarization. We also identifed that TRAF6 activated the c-JUN/NF-kB pathway signaling; the inhibitor of c-JUN/NF-kB could effectively alleviate the neuropathic pain induced by upregulated TRAF6 in the CCI mice model. CONCLUSION: In summary, this study indicated that TRAF6 was concerned with neuropathic pain, and targeting the TRAF6/c-JUN/NF-kB pathway may be a prospective target for treating neuropathic pain.

Bioinformatics and systems biology approach to identify the pathogenetic link of neurological pain and major depressive disorder.

Neurological pain (NP) is always accompanied by symptoms of depression, which seriously affects physical and mental health. In this study, we identified the common hub genes (Co-hub genes) and related immune cells of NP and major depressive disorder (MDD) to determine whether they have common pathological and molecular mechanisms. NP and MDD expression data was downloaded from the Gene Expression Omnibus (GEO) database. Common differentially expressed genes (Co-DEGs) for NP and MDD were extracted and the hub genes and hub nodes were mined. Co-DEGs, hub genes, and hub nodes were analyzed for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment. Finally, the hub nodes, and genes were analyzed to obtain Co-hub genes. We plotted Receiver operating characteristic (ROC) curves to evaluate the diagnostic impact of the Co-hub genes on MDD and NP. We also identified the immune-infiltrating cell component by ssGSEA and analyzed the relationship. For the GO and KEGG enrichment analyses, 93 Co-DEGs were associated with biological processes (BP), such as fibrinolysis, cell composition (CC), such as tertiary granules, and pathways, such as complement, and coagulation cascades. A differential gene expression analysis revealed significant differences between the Co-hub genes ANGPT2, MMP9, PLAU, and TIMP2. There was some accuracy in the diagnosis of NP based on the expression of ANGPT2 and MMP9. Analysis of differences in the immune cell components indicated an abundance of activated dendritic cells, effector memory CD8+ T cells, memory B cells, and regulatory T cells in both groups, which were statistically significant. In summary, we identified 6 Co-hub genes and 4 immune cell types related to NP and MDD. Further studies are needed to determine the role of these genes and immune cells as potential diagnostic markers or therapeutic targets in NP and MDD.

Sevoflurane impedes neuropathic pain by maintaining endoplasmic reticulum stress and oxidative stress homeostasis through inhibiting the activation of the PLCγ/CaMKII/IP3R signaling pathway.

OBJECTIVE: To investigate the effect of sevoflurane on neuropathic pain induced by chronic constriction injury (CCI) of sciatic nerve in mice, and to elucidate its mechanism by animal experiments. METHODS AND RESULTS: Thirty-two C57BL/6 mice were randomly divided into four groups: Sham group, Model group, Control group and Sevoflurane group. First, a mouse model of neuropathic pain was established. Then, the mice in each group were killed on Day 14 after operation to harvest the enlarged lumbosacral spinal cord. In contrast with the Model group, the Sevoflurane group displayed a significantly increased paw withdrawal mechanical threshold (PWMT) and significantly prolonged paw withdrawal thermal latency (PWTL) from Day 5 after operation. The morphological changes of lumbosacral spinal cord were observed by hematoxylin-eosin (HE) staining and transmission electron microscopy. Pathological results showed that sevoflurane reduced nuclear pyknosis in lumbosacral spinal cord tissue, with a large number of mitochondrial crista disappearance and mitochondrial swelling. The results of Western blotting showed that sevoflurane significantly decreased the protein expressions of phosphorylated phospholipase Cγ (p-PLCγ), phosphorylated calcium/calmodulin-dependent protein kinase II (p-CaMKII) and phosphorylated inositol 1,4,5-triphosphate receptor (p-IP3R), and reduced the protein expressions of endoplasmic reticulum (ER) stress proteins glucose-regulated protein 78 (GRP78) and GRP94, oxidative stress-related proteins P22 and P47 and inflammatory factors nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), interleukin-1 ß (IL-1ß), and tumor necrosis factor-α (TNF-α). CONCLUSIONS: Sevoflurane inhibits neuropathic pain by maintaining ER stress and oxidative stress homeostasis through inhibiting the activation of the PLCγ/CaMKII/IP3R signaling pathway.

[Mongolian medicine Naru-3 reduces neuroinflammation in maintenance stage of neuropathic pain by inhibiting astrocyte activation].

Neuropathic pain(NP) is difficult to be treated since it has similar phenotypes but different pathogenesis in different pathological stages. Targeted intervention of the core regulatory elements at different pathological stages of NP has become a new direction of drug research and development in recent years and provides the possibility for the treatment of NP. The Mongolian medicine Naru-3(NR-3) is effective in the treatment of sciatica and trigeminal neuralgia, the mechanisms of which remain unknown. On the basis of the previous study of the priming stage, this study established the mouse model of spinal nerve ligation(SNL) and measured the changes of pain thresholds by behavioral tests. The network analysis, Western blot, immunofluorescence assay, ELISA, and agonist/antagonist were employed to decipher the mechanism of NR-3 in the treatment of NP in the maintenance stage. The results showed that NR-3 increased the mechanical and thermal pain thresholds of SNL mice, while it had no significant effect on the basal pain threshold of normal mice. NR-3 may relieve the pain in the maintenance stage of NP by blocking the matrix metalloproteinase 2(MMP2)/interleukin-1ß(IL-1ß) pathway in the astrocytes of the dorsal root ganglion(DRG) and spinal cord. The findings have enriched the biological connotation of NR-3 in the treatment of the maintenance stage of NP and provide reference for the rational use of this medicine in clinical practice.

Silencing of FTO inhibits oxidative stress to relieve neuropathic pain by m6A modification of GPR177.

BACKGROUND: Neuropathic pain (NP) is a challenging health condition owing to its complex nature and associated multiple etiologies. The occurrence of NP involves the abnormal activity of neurons mediated by oxidative stress (OS). Previous research has demonstrated that m6A methylation plays a role in the regulatory pathway of NP. This study aimed to investigate the specific molecular pathways through which m6A methylation modifiers alleviate NP. METHODS: For this purpose, an NO rat model was developed via spared nerve injury (SNI), followed by quantifying the animal's pain assessment via paw withdrawal threshold (PWT) and paw withdrawal latency (PWL). The OS in SNI rats was evaluated by measuring reactive oxygen species, superoxide dismutase, and catalase (CAT) in spinal cord tissues. Moreover, quantitative-real-time polymerase chain reaction and western blot analysis were employed for detecting fat mass and obesity-associated (FTO) and GPR177 levels, while m6A levels of GPR117 were analyzed via MeRIP. RESULTS: The results indicated an enhanced OS with highly expressed FTO in spinal cord tissue samples, where knocking down Fto effectively relieved NP and OS in SNI rats. Mechanistic investigations revealed that Fto-mediated reduction of Grp177 m6A modification was involved in the WNT5a/TRPV1 axis-mediated OS remission of NP. Moreover, in vitro experiment results indicated that YTHDF2 was an important m6A methylated reading protein for this process. CONCLUSIONS: Fto silencing leads to increased m6A methylation of Grp177 through a YTHDF2-dependent mechanism, resulting in decreased Grp177 stability and ultimately reducing NP in rats by OS suppression.

Tuina alleviates neuropathic pain through regulate the activation of microglia and the secretion of inflammatory cytokine in spinal cord.

OBJECTIVE: To observe the analgesic effects of Tuina on neuropathic pain (NPP) and the underlying mechanisms. METHODS: Forty-eight Sprague-Dawley (SD) rats were assigned by random into three treatment groups: sham, chronic constriction injury (CCI), and Tuina. Each group contained sixteen rats. CCI model was generated by ligating the right sciatic nerve. Behavioral changes of CCI were assessed by the paw withdrawal threshold (PWT) and paw withdrawal latency (PWL). In addition, biochemical techniques such as immunofluorescence staining, enzyme-linked immunosorbent assay (ELISA) and Western blotting were used to profile levels of microglia activation and inflammatory factors in the spinal dorsal horn (SDH) of rats. Tuina (clockwise pressing and rubbing) was performed at Chengshan (BL57) to observe the analgesic effects on CCI rats and the underlying mechanisms. RESULTS: Rats with CCI experienced significant reduction in the PWT and PWL of the right hind paw relative to CCI group at day 3. Tuina treatment rescued this situation significantly on days 10 and 14. Besides, Iba-1, microglia M1 receptor CD68, tumor necrosis factor-α (TNF-α), and interleukin-1ß (IL-1ß) were higher in the right SDH for CCI group compared to the sham group on day 14. As expected, Tuina partially downregulated the CCI-induced overexpressed Iba-1, CD68, TNF-α, and IL-1ß in the SDH of CCI model. CONCLUSION: Tuina induces a time-dependent cumulative analgesic effect in CCI rats by inhibiting the activation of microglia and the secretion of IL-1ß and TNF-α in SDH.

Morin Regulates M1/M2 Microglial Polarization via NF-κB p65 to Alleviate Vincristine-Induced Neuropathic Pain.

Background: Morin can alleviate vincristine-induced neuropathic pain via inhibiting neuroinflammation. Microglial cells play an important role in initiating and maintenance of pain and neuroinflammation. It remains unclear whether morin exerts antinociceptive properties through the regulation of microglial cells. This study aimed to elucidate the mechanisms of morin against neuropathic pain focusing on microglial cells. Methods: The thermal withdrawal latency and mechanical withdrawal threshold were used as measures of pain behaviours. Histological abnormalities of the sciatic nerve were observed with transmission electron microscopy. The sciatic functional index and the sciatic nerve conduction velocity were used as measures of the functional deficits of the sciatic nerve. Inflammatory factors were detected using ELISA. The expression of M1/M2 polarization markers of microglia and nuclear factor κB (NF-κB) p65 were measured by immunofluorescence, real-time quantitative PCR and Western blotting. Results: Morin alleviated vincristine-induced abnormal pain, sciatic nerve injury, and neuroinflammatory response in rats. Furthermore, morin decreased the expression of NF-κB P65 and M1 activation markers, increased the expression of M2 activation markers. Additionally, phorbol 12-myristate 13-acetate reversed the effects of morin on microglial polarization, the production of inflammatory factors and neuropathic pain, while ammonium pyrrolidine dithiocarbamate showed the opposite effects. Conclusion: Our results demonstrate that morin inhibits neuroinflammation to alleviate vincristine-induced neuropathic pain via inhibiting the NF-κB signalling pathway to regulate M1/M2 microglial polarization.

HCN channels in the lateral habenula regulate pain and comorbid depressive-like behaviors in mice.

AIMS: Comorbid anxiodepressive-like symptoms (CADS) in chronic pain are closely related to the overactivation of the lateral habenula (LHb). Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels have been implicated to play a key role in regulating neuronal excitability. However, the role of HCN channels in the LHb during CADS has not yet been characterized. This study aimed to investigate the effect of HCN channels in the LHb on CADS during chronic pain. METHODS: After chronic neuropathic pain induction by spared nerve injury (SNI), mice underwent a sucrose preference test, forced swimming test, tail suspension test, open-field test, and elevated plus maze test to evaluate their anxiodepressive-like behaviors. Electrophysiological recordings, immunohistochemistry, Western blotting, pharmacological experiments, and virus knockdown strategies were used to investigate the underlying mechanisms. RESULTS: Evident anxiodepressive-like behaviors were observed 6w after the SNI surgery, accompanied by increased neuronal excitability, enhanced HCN channel function, and increased expression of HCN2 isoforms in the LHb. Either pharmacological inhibition or virus knockdown of HCN2 channels significantly reduced LHb neuronal excitability and ameliorated both pain and depressive-like behaviors. CONCLUSION: Our results indicated that the LHb neurons were hyperactive under CADS in chronic pain, and this hyperactivation possibly resulted from the enhanced function of HCN channels and up-regulation of HCN2 isoforms.

CBP Expression Contributes to Neuropathic Pain via CREB and MeCP2 Regulation in the Spared Nerve Injury Rat Model.

Background and Objectives: This study aimed to investigate the relationship between neuropathic pain and CREB-binding protein (CBP) and methyl-CpG-binding protein 2 (MeCP2) expression levels in a rat model with spared nerve injury (SNI). Materials and Methods: Rat (male Sprague-Dawley white rats) models with surgical SNI (n = 6) were prepared, and naive rats (n = 5) were used as controls. The expression levels of CBP and MeCP2 in the spinal cord and dorsal root ganglion (DRG) were compared through immunohistochemistry at 7 and 14 days after surgery. The relationship between neuropathic pain and CBP/MeCP2 was also analyzed through intrathecal siRNA administration. Results: SNI induced a significant increase in the number of CBPs in L4 compared with contralateral DRG as well as with naive rats. The number of MeCP2 cells in the dorsal horn on the ipsilateral side decreased significantly compared with the contralateral dorsal horn and the control group. SNI induced a significant decrease in the number of MeCP2 neurons in the L4 ipsilateral DRG compared with the contralateral DRG and naive rats. The intrathecal injection of CBP siRNA significantly inhibited mechanical allodynia induced by SNI compared with non-targeting siRNA treatment. MeCP2 siRNA injection showed no significant effect on mechanical allodynia. Conclusions: The results suggest that CBP and MeCP2 may play an important role in the generation of neuropathic pain following peripheral nerve injury.

New Insight into Neuropathic Pain: The Relationship between α7nAChR, Ferroptosis, and Neuroinflammation.

Neuropathic pain, which refers to pain caused by a lesion or disease of the somatosensory system, represents a wide variety of peripheral or central disorders. Treating neuropathic pain is quite demanding, primarily because of its intricate underlying etiological mechanisms. The central nervous system relies on microglia to maintain balance, as they are associated with serving primary immune responses in the brain next to cell communication. Ferroptosis, driven by phospholipid peroxidation and regulated by iron, is a vital mechanism of cell death regulation. Neuroinflammation can be triggered by ferroptosis in microglia, which contributes to the release of inflammatory cytokines. Conversely, neuroinflammation can induce iron accumulation in microglia, resulting in microglial ferroptosis. Accumulating evidence suggests that neuroinflammation, characterized by glial cell activation and the release of inflammatory substances, significantly exacerbates the development of neuropathic pain. By inhibiting microglial ferroptosis, it may be possible to prevent neuroinflammation and subsequently alleviate neuropathic pain. The activation of the homopentameric α7 subtype of the neuronal nicotinic acetylcholine receptor (α7nAChR) has the potential to suppress microglial activation, transitioning M1 microglia to an M2 phenotype, facilitating the release of anti-inflammatory factors, and ultimately reducing neuropathic pain. Recent years have witnessed a growing recognition of the regulatory role of α7nAChR in ferroptosis, which could be a potential target for treating neuropathic pain. This review summarizes the mechanisms related to α7nAChR and the progress of ferroptosis in neuropathic pain according to recent research. Such an exploration will help to elucidate the relationship between α7nAChR, ferroptosis, and neuroinflammation and provide new insights into neuropathic pain management.

Jinmaitong alleviates diabetic neuropathic pain by inhibiting JAK2/STAT3 signaling in microglia of diabetic rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Jinmaitong (JMT) is a prescription of Traditional Chinese Medicine that is composed of 12 crude drugs. It has been used in the treatment of diabetic neuropathic pain (DNP) for more than 30 years. AIM OF STUDY: Microglia are thought to play an important role in neuropathic pain. This study aimed to evaluate the protective effect of JMT against DNP and to investigate the underlying mechanisms in which the microglia and JAK2/STAT3 signaling pathway were mainly involved. MATERIALS AND METHODS: The chemical composition of JMT was analyzed using liquid chromatography tandem mass spectrometry. The diabetes model was constructed using 11 to 12-week-old male Zucker diabetic fatty (ZDF) rat (fa/fa). The model rats were divided into 5 groups and were given JMT at three dosages (11.6, 23.2, and 46.4 g/kg, respectively, calculated as the crude drug materials), JAK inhibitor AG490 (positive drug, 10 µg/day), and placebo (deionized water), respectively, for eight weeks (n = 6). Meanwhile, Zucker lean controls (fa/+) were given a placebo (n = 6). Body weight was tested weekly and blood glucose was monitored every 2 weeks. The mechanical allodynia and heat hyperalgesia were assessed using mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) tests. After treatment, the microglia activation marker Iba-1, CD11B, CD68, neuroinflammatory mediators, and mediators of the JAK2/STAT3 signaling pathway were compared between different groups. The mRNA and protein levels of target genes were assessed by quantitative real-time PCR and Western Blot, respectively. RESULTS: We found that JMT significantly inhibited the overactivation of microglia in spinal cords, and suppressed neuroinflammation of DNP model rats, thereby ameliorating neurological dysfunction and injuries. Furthermore, these effects of JMT could be attributed to the inhibition of the JAK2/STAT3 signaling pathway. CONCLUSIONS: Our findings suggested that JMT effectively ameliorated DNP by modulating microglia activation via inhibition of the JAK2/STAT3 signaling pathway. The present study provided a basis for further research on the therapeutic strategies of DNP.

Minocycline Abrogates Individual Differences in Nerve Injury-Evoked Affective Disturbances in Male Rats and Prevents Associated Supraspinal Neuroinflammation.

Chronic neuropathic pain precipitates a complex range of affective and behavioural disturbances that differ markedly between individuals. While the reasons for differences in pain-related disability are not well understood, supraspinal neuroimmune interactions are implicated. Minocycline has antidepressant effects in humans and attenuates affective disturbances in rodent models of pain, and acts by reducing neuroinflammation in both the spinal cord and brain. Previous studies, however, tend not to investigate how minocycline modulates individual affective responses to nerve injury, or rely on non-naturalistic behavioural paradigms that fail to capture the complexity of rodent behaviour. We investigated the development and resolution of pain-related affective disturbances in nerve-injured male rats by measuring multiple spontaneous ethological endpoints on a longitudinal naturalistic foraging paradigm, and the effect of chronic oral minocycline administration on these changes. Disrupted foraging behaviours appeared in 22% of nerve-injured rats - termed 'affected' rats - and were present at day 14 but partially resolved by day 21 post-injury. Minocycline completely prevented the emergence of an affected subgroup while only partly attenuating mechanical allodynia, dissociating the relationship between pain and affect. This was associated with a lasting downregulation of ΔFosB expression in ventral hippocampal neurons at day 21 post-injury. Markers of microglia-mediated neuroinflammation were not present by day 21, however proinflammatory microglial polarisation was apparent in the medial prefrontal cortex of affected rats and not in CCI minocycline rats. Individual differences in affective disturbances following nerve injury are therefore temporally related to altered microglial morphology and hippocampal neuronal activation, and are abrogated by minocycline.

ESRRG-controlled downregulation of KCNN1 in primary sensory neurons is required for neuropathic pain.

Peripheral nerve injury-induced neuronal hyperactivity in the dorsal root ganglion (DRG) participates in neuropathic pain. The calcium-activated potassium channel subfamily N member 1 (KCNN1) mediates action potential afterhyperpolarization (AHP) and gates neuronal excitability. However, the specific contribution of DRG KCNN1 to neuropathic pain is not yet clear. We report that chronic constriction injury (CCI) of the unilateral sciatic nerve or unilateral ligation of the fourth lumbar nerve produced the downregulation of Kcnn1 mRNA and KCNN1 protein in the injured DRG. This downregulation was partially attributed to a decrease in DRG estrogen-related receptor gamma (ESRRG), a transcription factor, which led to reduced binding to the Kcnn1 promoter. Rescuing this downregulation prevented CCI-induced decreases in total potassium voltage currents and AHP currents, reduced excitability in the injured DRG neurons, and alleviated CCI-induced development and maintenance of nociceptive hypersensitivities, without affecting locomotor function and acute pain. Mimicking the CCI-induced DRG KCNN1 downregulation resulted in augmented responses to mechanical, heat, and cold stimuli in naive mice. Our findings indicate that ESRRG-controlled downregulation of DRG KCNN1 is likely essential for the development and maintenance of neuropathic pain. Thus, KCNN1 may serve as a potential target for managing this disorder.

Discovery of a CCR2-targeting pepducin therapy for chronic pain.

Targeting the CCL2/CCR2 chemokine axis has been shown to be effective at relieving pain in rodent models of inflammatory and neuropathic pain, therefore representing a promising avenue for the development of non-opioid analgesics. However, clinical trials targeting this receptor for inflammatory conditions and painful neuropathies have failed to meet expectations and have all been discontinued due to lack of efficacy. To overcome the poor selectivity of CCR2 chemokine receptor antagonists, we generated and characterized the function of intracellular cell-penetrating allosteric modulators targeting CCR2, namely pepducins. In vivo, chronic intrathecal administration of the CCR2-selective pepducin PP101 was effective in alleviating neuropathic and bone cancer pain. In the setting of bone metastases, we found that T cells infiltrate dorsal root ganglia (DRG) and induce long-lasting pain hypersensitivity. By acting on CCR2-expressing DRG neurons, PP101 attenuated the altered phenotype of sensory neurons as well as the neuroinflammatory milieu of DRGs, and reduced bone cancer pain by blocking CD4+ and CD8+ T cell infiltration. Notably, PP101 demonstrated its efficacy in targeting the neuropathic component of bone cancer pain, as evidenced by its anti-nociceptive effects in a model of chronic constriction injury of the sciatic nerve. Importantly, PP101-induced reduction of CCR2 signaling in DRGs did not result in deleterious tumor progression or adverse behavioral effects. Thus, targeting neuroimmune crosstalk through allosteric inhibition of CCR2 could represent an effective and safe avenue for the management of chronic pain.

SIRT1 mediates the excitability of spinal CaMKIIα-positive neurons and participates in neuropathic pain by controlling Nav1.3.

AIMS: Neuropathic pain is a common chronic pain disorder, which is largely attributed to spinal central sensitization. Calcium/calmodulin-dependent protein kinase II alpha (CaMKIIα) activation in the spinal dorsal horn (SDH) is a major contributor to spinal sensitization. However, the exact way that CaMKIIα-positive (CaMKIIα+) neurons in the SDH induce neuropathic pain is still unclear. This study aimed to explore the role of spinal CaMKIIα+ neurons in neuropathic pain caused by chronic constriction injury (CCI) and investigate the potential epigenetic mechanisms involved in CaMKIIα+ neuron activation. METHODS: CCI-induced neuropathic pain mice model, Sirt1loxP/loxP mice, and chemogenetic virus were used to investigate whether the activation of spinal CaMKIIα+ neurons is involved in neuropathic pain and its involved mechanism. Transcriptome sequence, western blotting, qRT-PCR, and immunofluorescence analysis were performed to assay the expression of related molecules and activation of neurons. Co-immunoprecipitation was used to observe the binding relationship of protein. Chromatin immunoprecipitation (ChIP)-PCR was applied to analyze the acetylation of histone H3 in the Scn3a promoter region. RESULTS: The expression of sodium channel Nav1.3 was increased and the expression of SIRT1 was decreased in the spinal CaMKIIα+ neurons of CCI mice. CaMKIIα neurons became overactive after CCI, and inhibiting their activation relieved CCI-induced pain. Overexpression of SIRT1 reversed the increase of Nav1.3 and alleviated pain, while knockdown of SIRT1 or overexpression of Nav1.3 promoted CaMKIIα+ neuron activation and induced pain. By knocking down spinal SIRT1, the acetylation of histone H3 in the Scn3a (encoding Nav1.3) promoter region was increased, leading to an increased expression of Nav1.3. CONCLUSION: The findings suggest that an aberrant reduction of spinal SIRT1 after nerve injury epigenetically increases Nav1.3, subsequently activating CaMKIIα+ neurons and causing neuropathic pain.

Cannabinoid CB2 receptors in primary sensory neurons are implicated in CB2 agonist-mediated suppression of paclitaxel-induced neuropathic nociception and sexually-dimorphic sparing of morphine tolerance.

Cannabinoid CB2 agonists show therapeutic efficacy without unwanted CB1-mediated side effects. The G protein-biased CB2 receptor agonist LY2828360 attenuates the maintenance of chemotherapy-induced neuropathic nociception in male mice and blocks development of morphine tolerance in this model. However, the cell types involved in this phenomenon are unknown and whether this therapeutic profile is observed in female mice has never been investigated. We used conditional deletion of CB2 receptors to determine the cell population(s) mediating the anti-allodynic and morphine-sparing effects of CB2 agonists. Anti-allodynic effects of structurally distinct CB2 agonists (LY2828360 and AM1710) were present in paclitaxel-treated CB2f/f mice and in mice lacking CB2 receptors in CX3CR1 expressing microglia/macrophages (CX3CR1CRE/+; CB2f/f), but were absent in mice lacking CB2 receptors in peripheral sensory neurons (AdvillinCRE/+; CB2f/f). The morphine-sparing effect of LY28282360 occurred in a sexually-dimorphic manner, being present in male, but not female, mice. LY2828360 treatment (3 mg/kg per day i.p. x 12 days) blocked the development of morphine tolerance in male CB2f/f and CX3CR1CRE/+; CB2f/f mice with established paclitaxel-induced neuropathy but was absent in male (or female) AdvillinCRE/+; CB2f/f mice. Co-administration of morphine with a low dose of LY2828360 (0.1 mg/kg per day i.p. x 6 days) reversed morphine tolerance in paclitaxel-treated male CB2f/f mice, but not AdvillinCRE/+; CB2f/f mice of either sex. LY2828360 (3 mg/kg per day i.p. x 8 days) delayed, but did not prevent, the development of paclitaxel-induced mechanical or cold allodynia in either CB2f/f or CX3CR1CRE/+; CB2f/f mice of either sex. Our findings have potential clinical implications.

Roles of Thermosensitive Transient Receptor Channels TRPV1 and TRPM8 in Paclitaxel-Induced Peripheral Neuropathic Pain.

Paclitaxel, a microtubule-stabilizing chemotherapy drug, can cause severe paclitaxel-induced peripheral neuropathic pain (PIPNP). The roles of transient receptor potential (TRP) ion channel vanilloid 1 (TRPV1, a nociceptor and heat sensor) and melastatin 8 (TRPM8, a cold sensor) in PIPNP remain controversial. In this study, Western blotting, immunofluorescence staining, and calcium imaging revealed that the expression and functional activity of TRPV1 were upregulated in rat dorsal root ganglion (DRG) neurons in PIPNP. Behavioral assessments using the von Frey and brush tests demonstrated that mechanical hyperalgesia in PIPNP was significantly inhibited by intraperitoneal or intrathecal administration of the TRPV1 antagonist capsazepine, indicating that TRPV1 played a key role in PIPNP. Conversely, the expression of TRPM8 protein decreased and its channel activity was reduced in DRG neurons. Furthermore, activation of TRPM8 via topical application of menthol or intrathecal injection of WS-12 attenuated the mechanical pain. Mechanistically, the TRPV1 activity triggered by capsaicin (a TRPV1 agonist) was reduced after menthol application in cultured DRG neurons, especially in the paclitaxel-treated group. These findings showed that upregulation of TRPV1 and inhibition of TRPM8 are involved in the generation of PIPNP, and they suggested that inhibition of TRPV1 function in DRG neurons via activation of TRPM8 might underlie the analgesic effects of menthol.

Dysfunction of Small-Conductance Ca2+-Activated Potassium (SK) Channels Drives Amygdala Hyperexcitability and Neuropathic Pain Behaviors: Involvement of Epigenetic Mechanisms.

Neuroplasticity in the amygdala and its central nucleus (CeA) is linked to pain modulation and pain behaviors, but cellular mechanisms are not well understood. Here, we addressed the role of small-conductance Ca2+-activated potassium (SK) channels in pain-related amygdala plasticity. The facilitatory effects of the intra-CeA application of an SK channel blocker (apamin) on the pain behaviors of control rats were lost in a neuropathic pain model, whereas an SK channel activator (NS309) inhibited pain behaviors in neuropathic rats but not in sham controls, suggesting the loss of the inhibitory behavioral effects of amygdala SK channels. Brain slice electrophysiology found hyperexcitability of CeA neurons in the neuropathic pain condition due to the loss of SK channel-mediated medium afterhyperpolarization (mAHP), which was accompanied by decreased SK2 channel protein and mRNA expression, consistent with a pretranscriptional mechanisms. The underlying mechanisms involved the epigenetic silencing of the SK2 gene due to the increased DNA methylation of the CpG island of the SK2 promoter region and the change in methylated CpG sites in the CeA in neuropathic pain. This study identified the epigenetic dysregulation of SK channels in the amygdala (CeA) as a novel mechanism of neuropathic pain-related plasticity and behavior that could be targeted to control abnormally enhanced amygdala activity and chronic neuropathic pain.

Purpurin ameliorated neuropathic allodynia and hyperalgesia by modulating neuronal mitochondrial bioenergetics and redox status in type 1 diabetic mice.

A substantial proportion of diabetic patients suffer a debilitating and persistent pain state, known as peripheral painful neuropathy that necessitates improved therapy or antidote. Purpurin, a natural anthraquinone compound from Rubia tinctorum L., has been reported to possess antidepressant activity in preclinical studies. As antidepressants have been typically used as standard agents against persistent neuropathic pain, this study aimed to probe the effect of purpurin on neuropathic pain associated with streptozotocin-induced type 1 diabetes in male C57BL6J mice. The Hargreaves test and the von Frey test were used to assess the pain-like behaviors, shown as heat hyperalgesia and mechanical allodynia respectively. Chronic treatment of diabetic mice with purpurin not only ameliorated the established symptoms of heat hyperalgesia and mechanical allodynia, but also arrested the development of these pain states given preemptively at low doses. Although purpurin treatment hardly impacted on metabolic disturbance in diabetic mice, it ameliorated exacerbated oxidative stress in pain-associated tissues, improved mitochondrial bioenergetics in dorsal root ganglion neurons and restored nerve conduction velocity in sciatic nerves. Notably, the analgesic actions of purpurin were modified by pharmacologically manipulating redox status and mitochondrial bioenergetics. These findings unveil the analgesic activity of purpurin, an effect that is causally associated with its bioenergetics-enhancing and antioxidant effects, in mice with type 1 diabetes.