Sida cordifolia L. attenuates behavioral hypersensitivity by interfering with KIF17-NR2B signaling in rat model of neuropathic pain.

ETHNOPHARMACOLOGICAL RELEVANCE: Sida cordifolia L., a perennial subshrub belonging to the Malvaceae family, holds noteworthy significance in the Indian Ayurvedic System and global texts. Roots of this plant are reported to be useful in neurodegenerative disorders, facial paralysis, and treating several neuropathic pain conditions such as neuralgia, and sciatica. However, despite these claims, there remains a dearth of experimental evidence showcasing the effectiveness of Sida cordifolia L. roots in mitigating neuropathic pain. AIM OF THE STUDY: The primary objective of this study was to assess the analgesic properties of the whole extract (SCE) obtained from the roots of Sida cordifolia L., as well as its aqueous fraction (SAF) in rat model of chronic constriction injury (CCI)-induced neuropathic pain. Furthermore, in-depth phytochemical and molecular biology studies were conducted to identify the potential phytoconstituents and unveil the underlying mechanisms of action. MATERIAL AND METHODS: DCM: Methanol (1:1) was used to extract the roots of Sida cordifolia L. to get whole extract (SCE) and was subjected to phytochemical investigations including LC-MS analysis. Analgesic potential of SCE was evaluated in chronic constriction injury (CCI) model of neuropathic pain in rats followed by its bioactivity guided fractionation using in-vitro anti-inflammatory assay and assessment of most potent fraction (SAF) in in-vivo pain model. We have also performed the detailed phytochemical and molecular biology investigations to delineate the mechanism of action of Sida cordifolia root extract. RESULTS: Chronic constriction injury leads to significant decrease in paw withdrawal threshold and paw withdrawal latency indicating development of hypersensitivity in rodents. Treatment with SCE and its most potent aqueous fraction (SAF) leads to significant and dose-dependent reduction in pain-like behavior of nerve injured rats. Pro-inflammatory cytokines (TNF-α, IL-1ß), glia cell markers (Iba1, ICAM1), neuropeptides (CGRP and Substance P), KIF-17 and NR2B expressions were found to be significantly upregulated in DRG and spinal cord of nerve injured rats. Treatment with SCE and SAF suppressed oxido-inflammatory cascade along with attenuation of KIF-17 mediated NR2B trafficking and neuroinflammation in DRG and spinal tissues of neuropathic rats. HPTLC and HR-MS analysis suggest betaine as major constituent in SAF which along with other phytoconstituents. CONCLUSIONS: Both the whole extract (SCE) and the aqueous fraction (SAF) demonstrate a significant reduction in mechanical and thermal hypersensitivity by inhibiting KIF-17 mediated NR2B signaling in nerve injured rats and may be used as a potential alternative for the treatment of chronic pain. Our findings support the use of roots of Sida cordifolia L. in neuropathic pain conditions as acclaimed by its traditional use.

Based on spinal central sensitization creating analgesic screening approach to excavate anti-neuropathic pain ingredients of Corydalis yanhusuo W.T.Wang.

ETHNOPHARMACOLOGICAL RELEVANCE: Corydalis Rhizome (RC) as a traditional analgesic Chinese medicine is the dried tuber of Corydalis yanhusuo W.T.Wang. Many efforts have revealed that RC could effectively alleviate neuropathic pain, while its active ingredients in neuropathic pain are still not clear. AIM OF THE STUDY: Spinal central sensitization contributes greatly to neuropathic pain, and neuron, astrocyte and microglia play important roles in spinal central sensitization. The aim of the present study is to excavate active compounds in RC regulating spinal central sensitization to inhibit neuropathic pain. MATERIALS AND METHODS: Immunofluorescence and western blotting were used to determine protein expression levels. Gene expression levels were detected by RT-PCR. PC12 neuronal cells, C6 astrocyte cells, and BV2 microglia cells were cultured for in vitro studies. Targeting multi types of cells extraction combined with HPLC-Q-TOF-MS/MS was established to identify components binding to above cells. Animal studies were used to verify the analgesic activities of components. RESULTS: Total alkaloids of RC (RC-TA) significantly relieved neuropathic pain in chronic constriction injury (CCI) rats and repressed spinal central sensitization. Eight components of RC-TA were found to bind to PC12, C6, or BV2 cells. They could respectively suppress the activation of cells in vitro and alleviate CCI-induced neuropathic pain, among which glaucine and dehydrocorydaline induced antinociception was stronger than l-THP. Meanwhile, glaucine had no effect on acute or chronic inflammatory pain, and its antinociception in neuropathic pain could be abolished by dopamine D1 receptor agonist. CONCLUSIONS: Employing multi types of cells based on spinal central sensitization rather than single cell may allow for more thorough excavation of active substances. Glaucine was firstly found could attenuate neuropathic pain but not other types of pain which indicated that different alkaloids in RC exert distinct analgesic effects on different pain models, and gluacine has the potential to be developed as an analgesic drug specifically for neuropathic pain relieving.

Intranasal oxytocin alleviates comorbid depressive symptoms in neuropathic pain via elevating hippocampal BDNF production in both female and male mice.

The comorbidity of pain and depression is frequently observed in patients suffering from chronic pain and depression. However, the comorbid mechanism is not well elucidated and the therapeutic medication is still inadequate. Oxytocin is a neuropeptide synthesized in the hypothalamus. It has been reported to relieve chronic pain and depressive symptoms. However, the analgesic action and mechanisms of oxytocin have mainly been investigated using peripheral or spinal administration. Because of the advantage of intranasal delivery of oxytocin in crossing the blood-brain barrier, we investigated the effect of intranasal application of oxytocin on neuropathic pain and comorbid depressive symptoms in both female and male mice. In female and male mice receiving spared nerve injury (SNI) surgery, intranasal oxytocin (2.4 µg, daily for 28 days) attenuated depression-like behavior, but did not alleviate mechanical hyperalgesia. Intranasal oxytocin not only inhibited the activation of microglia and astrocytes, but also increased the downregulated oxytocin receptor (OTR) expression, reversed the elevated GluN2A, and restored the decreased BDNF expression in the hippocampus. SNI also decreased OTR expression in the spinal cord and increased spinal GluN2A and BDNF. However, intranasal oxytocin treatment did not change the expression levels of OTR, GluN2A, or BDNF in the spinal cord of neuropathic mice. The results suggest that the oxytocin signaling in the hippocampus is involved in the comorbidity of pain and depression, and intranasal oxytocin may have the potential to treat depressive symptoms in neuropathic pain patients.

[Possible involvement of FFAR1 signaling in mouse emotional behaviors through the regulation of brain monoamine releases].

The free fatty acid receptor 1 (FFAR1) is suggested to function as a G protein-coupled receptor for medium- to long-chain free fatty acids. We have previously shown that FFAR1 signaling pathway plays an important suppressive role in spinal nociceptive processing after peripheral inflammation and nerve injury, and that FFAR1 agonists might serve as a new class of analgesics for treating inflammatory and neuropathic pain. To further pursue the functional significance of central FFAR1 signaling, we investigated the possible involvement of FFAR1 in endogenous pain modulation, depressive-like behavior, and aberrant behavior induced by addictive drugs using FFAR1 agonist (GW9508), FFAR1 antagonist (GW1100), and FFAR1 gene-deficient mice. As a result, FFAR1-deficient mice were found to exhibit stronger inflammatory and peripheral neuropathic pain-like behavior as well as depressive-like behavior. In particular, we noticed that peripheral nerve injury-induced depressive-like behavior was insensitive to imipramine. Next, we employed in vivo microdialysis to investigate whether FFAR1 is actually involved in the regulation of brain monoamines (dopamine and serotonin) releases. Our findings suggest that FFAR1 indirectly regulates dopamine release by promoting serotonin release. Thus, we are currently investigating how FFAR1 is involved in behavioral changes induced by addictive drugs such as cocaine and morphine. In this review, we briefly discuss about the possible involvement of FFAR1 in cocaine-induced locomotor hyperactivity.

Integration of microbiota and metabolomics reveals the analgesic mechanisms of emodin against neuropathic pain.

BACKGROUND AND OBJECTIVE: Neuropathic pain (NeP) induced dysbiosis of intestinal microbiota in chronic constriction injury (CCI) rats. Emodin has analgesic effect but the detailed mechanism is not clear at the present time. This study aims to explore the underling mechanism of action of emodin against NeP with in CCI model. METHODS: Male SD rats (180-220 g) were randomly divided into three groups: sham group, CCI group, and emodin group. Behavioral tests were performed to evaluate the therapeutic effects of emodin on CCI model. Feces and spinal cords of all rats were collected 15 days after surgery. 16S rDNA sequencing, untargeted metabolomics, qPCR and ELISA were performed. RESULTS: Mechanical withdrawal thresholds (MWT), thermal withdrawal latency (TWL) and Sciatic functional index (SFI) in emodin group were significantly higher than CCI group (P < 0.05). Emodin not only inhibited the expression of pro-inflammatory cytokines in the spinal cords and colonic tissue, but also increased the expression of tight junction protein in colonic tissue. 16S rDNA sequencing showed that emodin treatment changed the community structure of intestinal microbiota in CCI rats. Untargeted metabolomics analysis showed that 33 differential metabolites were screened out between CCI group and emodin group. After verification, we found that emodin increased the level of S-adenosylmethionine (SAM) and Histamine in the spinal cord of CCI rats. CONCLUSION: Emodin was effective in relieving neuropathic pain, which is linked to inhibition inflammatory response, increasing the proportion of beneficial bacteria and beneficial metabolites.

Electroacupuncture exerts prolonged analgesic and neuroprotective effects in a persistent dental pain model induced by multiple dental pulp injuries: GABAergic interneurons-astrocytes interaction.

Pain within the trigeminal system, particularly dental pain, is poorly understood. This study aimed to determine whether single or multiple dental pulp injuries induce persistent pain, its association with trigeminal central nociceptive pathways and whether electroacupuncture (EA) provides prolonged analgesic and neuroprotective effects in a persistent dental pain model. Models of single dental pulp injury (SDPI) and multiple dental pulp injuries (MDPI) were used to induce trigeminal neuropathic pain. The signs of dental pain-related behavior were assessed using the mechanical head withdrawal threshold (HWT). Immunofluorescence and western blot protocols were used to monitor astrocyte activation, changes in apoptosis-related proteins, and GABAergic interneuron plasticity. SDPI mice exhibited an initial marked decrease in HWT from days one to 14, followed by progressive recovery from days 21 to 42. From days 49 to 70, the HWT increased and returned to the control values. In contrast, MDPI mice showed a persistent decrease in HWT from days one to 70. MDPI increased glial fibrillary acidic protein (GFAP) and decreased glutamine synthetase (GS) and glutamate transporter-1 (GLT1) expression in the Vi/Vc transition zone of the brainstem on day 70, whereas no changes in astrocytic markers were observed on day 70 after SDPI. Increased expression of cleaved cysteine-aspartic protease-3 (cleaved caspase-3) and Bcl-2-associated X protein (Bax), along with decreased B-cell lymphoma/leukemia 2 (Bcl-2), were observed at day 70 after MDPI but not after SDPI. The downregulation of glutamic acid decarboxylase (GAD65) expression was observed on day 70 only after MDPI. The effects of MDPI-induced lower HWT from days one to 70 were attenuated by 12 sessions of EA treatment (days one to 21 after MDPI). Changes in astrocytic GFAP, GS, and GLT-1, along with cleaved caspase-3, Bax, Bcl-2, and GAD65 expression observed 70 days after MDPI, were reversed by EA treatment. The results suggest that persistent dental pain in mice was induced by MDPI but not by SDPI. This effect was associated with trigeminal GABAergic interneuron plasticity along with morphological and functional changes in astrocytes. EA exerts prolonged analgesic and neuroprotective effects that might be associated with the modulation of neuron-glia crosstalk mechanisms.

Pregabalin inhibits purinergic P2Y2 receptor and TRPV4 to suppress astrocyte activation and to relieve neuropathic pain.

BACKGROUNDS: Transient receptor potential vanilloid 4 (TRPV4)-mediated astrocyte activation is critical to neuropathic pain. Pregabalin, a widely used drug to treat chronic pain, is reported to lower the intracellular calcium level. However, the molecular mechanism by which pregabalin decreases the intracellular calcium level remains unknown. Purinergic P2Y2 receptor-a member of the G protein-coupled receptor (GPCR) family-regulates calcium-related signal transduction in astrocyte activation. We investigated whether P2Y2 receptor is involved in the pharmacological effects of pregabalin on neuropathic pain. METHODS: Neuropathic pain was induced by chronic compression of the dorsal root ganglion (CCD) in rats. Paw withdrawal mechanical threshold (PWMT) was used for behavioral testing. Intracellular calcium concentration was measured using a fluorescent calcium indicator (Fluo-4 AM). RESULTS: We found that P2Y2 receptor protein was upregulated and astrocytes were activated in the experimental rats after CCD surgery. Lipopolysaccharide (LPS) increased the intracellular calcium concentration and induced astrocyte activation in cultured astrocytes but was prevented via P2Y2 receptor inhibitor AR-C118925 or pregabalin. Furthermore, plasmid-mediated P2Y2 receptor overexpression induced an elevation of the intracellular calcium levels and inflammation in astrocytes, which was abolished by the TRPV4 inhibitor HC-067047. AR-C118925, HC-067047, and pregabalin relieved neuropathic pain and inflammation in rats after CCD surgery. Finally, plasmid-mediated P2Y2 receptor overexpression induced neuropathic pain in rats, which was abolished by pregabalin administration. CONCLUSIONS: Pathophysiological variables that upregulated the P2Y2 receptor/TRPV4/calcium axis contribute to astrocyte activation in neuropathic pain. Pregabalin exerts an analgesic effect by inhibiting this pathway.

Epigenetic regulation of beta-endorphin synthesis in hypothalamic arcuate nucleus neurons modulates neuropathic pain in a rodent pain model.

Although beta-endorphinergic neurons in the hypothalamic arcuate nucleus (ARC) synthesize beta-endorphin (ß-EP) to alleviate nociceptive behaviors, the underlying regulatory mechanisms remain unknown. Here, we elucidated an epigenetic pathway driven by microRNA regulation of ß-EP synthesis in ARC neurons to control neuropathic pain. In pain-injured rats miR-203a-3p was the most highly upregulated miRNA in the ARC. A similar increase was identified in the cerebrospinal fluid of trigeminal neuralgia patients. Mechanistically, we found histone deacetylase 9 was downregulated following nerve injury, which decreased deacetylation of histone H3 lysine-18, facilitating the binding of NR4A2 transcription factor to the miR-203a-3p gene promoter, thereby upregulating miR-203a-3p expression. Further, increased miR-203a-3p was found to maintain neuropathic pain by targeting proprotein convertase 1, an endopeptidase necessary for the cleavage of proopiomelanocortin, the precursor of ß-EP. The identified mechanism may provide an avenue for the development of new therapeutic targets for neuropathic pain treatment.

Traditional Chinese medicine alleviating neuropathic pain targeting purinergic receptor P2 in purinergic signaling: A review.

Past studies have suggested that Chinese herbal may alleviate neuropathic pain, and the mechanism might target the inhibition of purinergic receptor P2. This review discusses whether traditional Chinese medicine target P2 receptors in neuropathic pain and its mechanism in order to provide references for future clinical drug development. The related literatures were searched from Pubmed, Embase, Sinomed, and CNKI databases before June 2023. The search terms included"neuropathic pain", "purinergic receptor P2", "P2", "traditional Chinese medicine", "Chinese herbal medicine", and "herb". We described the traditional Chinese medicine alleviating neuropathic pain via purinergic receptor P2 signaling pathway including P2X2/3 R, P2X3R, P2X4R, P2X7R, P2Y1R. Inhibition of activating glial cells, changing synaptic transmission, increasing painful postsynaptic potential, and activating inflammatory signaling pathways maybe the mechanism. Purine receptor P2 can mediate the occurrence of neuropathic pain. And many of traditional Chinese medicines can target P2 receptors to relieve neuropathic pain, which provides reasonable evidences for the future development of drugs. Also, the safety and efficacy and mechanism need more in-depth experimental research.

Characterisation of GFAP-Expressing Glial Cells in the Dorsal Root Ganglion after Spared Nerve Injury.

Satellite glial cells (SGCs), enveloping primary sensory neurons' somas in the dorsal root ganglion (DRG), contribute to neuropathic pain upon nerve injury. Glial fibrillary acidic protein (GFAP) serves as an SGC activation marker, though its DRG satellite cell specificity is debated. We employed the hGFAP-CFP transgenic mouse line, designed for astrocyte studies, to explore its expression within the peripheral nervous system (PNS) after spared nerve injury (SNI). We used diverse immunostaining techniques, Western blot analysis, and electrophysiology to evaluate GFAP+ cell changes. Post-SNI, GFAP+ cell numbers increased without proliferation, and were found near injured ATF3+ neurons. GFAP+ FABP7+ SGCs increased, yet 75.5% of DRG GFAP+ cells lacked FABP7 expression. This suggests a significant subset of GFAP+ cells are non-myelinating Schwann cells (nmSC), indicated by their presence in the dorsal root but not in the ventral root which lacks unmyelinated fibres. Additionally, patch clamp recordings from GFAP+ FABP7-cells lacked SGC-specific Kir4.1 currents, instead displaying outward Kv currents expressing Kv1.1 and Kv1.6 channels specific to nmSCs. In conclusion, this study demonstrates increased GFAP expression in two DRG glial cell subpopulations post-SNI: GFAP+ FABP7+ SGCs and GFAP+ FABP7- nmSCs, shedding light on GFAP's specificity as an SGC marker after SNI.

A Single Injection of rAAV-shmTOR in Peripheral Nerve Persistently Attenuates Nerve Injury-Induced Mechanical Allodynia.

Activation of mammalian target of rapamycin (mTOR) has been known as one of the contributing factors in nociceptive sensitization after peripheral injury. Its activation followed by the phosphorylation of downstream effectors causes hyperexcitability of primary sensory neurons in the dorsal root ganglion. We investigated whether a single injection of rAAV-shmTOR would effectively downregulate both complexes of mTOR in the long-term and glial activation as well. Male SD rats were categorized into shmTOR (n = 29), shCON (n = 23), SNI (n = 13), and Normal (n = 8) groups. Treatment groups were injected with rAAV-shmTOR or rAAV-shCON, respectively. DRG tissues and sciatic nerve were harvested for Western blot and immunohistochemical analyses. Peripheral sensitization was gradually attenuated in the shmTOR group, and it reached a peak on PID 21. Western blot analysis showed that both p-mTORC1 and p-mTORC2 were downregulated in the DRG compared to shCON and SNI groups. We also found decreased expression of phosphorylated p38 and microglial activation in the DRG. We first attempted a therapeutic strategy for neuropathic pain with a low dose of AAV injection by interfering with the mTOR signaling pathway, suggesting its potential application in pain treatment.

Evaluated periodontal tissues and oxidative stress in rats with neuropathic pain-like behavior.

BACKGROUND: Oxidative stress has a critical effect on both persistent pain states and periodontal disease. Voltage-gated sodium NaV1.7 (SCN9A), and transient receptor potential ankyrin 1 (TRPA1) are pain genes. The goal of this study was to investigate oxidative stress markers, periodontal status, SCN9A, and TRPA1 channel expression in periodontal tissues of rats with paclitaxel-induced neuropathic pain-like behavior (NPLB). METHODS AND RESULTS: Totally 16 male Sprague Dawley rats were used: control (n = 8) and paclitaxel-induced pain (PTX) (n = 8). The alveolar bone loss and 8-hydroxy-2-deoxyguanosine (8-OHdG) levels were analyzed histometrically and immunohistochemically. Gingival superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) activities (spectrophotometric assay) were measured. The relative TRPA1 and SCN9A genes expression levels were evaluated using quantitative real-time PCR (qPCR) in the tissues of gingiva and brain. The PTX group had significantly higher alveolar bone loss and 8-OHdG compared to the control. The PTX group had significantly lower gingival SOD, GPx and CAT activity than the control groups. The PTX group had significantly higher relative gene expression of SCN9A (p = 0.0002) and TRPA1 (p = 0.0002) than the control in gingival tissues. Increased nociceptive susceptibility may affect the increase in oxidative stress and periodontal destruction. CONCLUSIONS: Chronic pain conditions may increase TRPA1 and SCN9A gene expression in the periodontium. The data of the current study may help develop novel approaches both to maintain periodontal health and alleviate pain in patients suffering from orofacial pain.

IL-6/JAK2/STAT3 axis mediates neuropathic pain by regulating astrocyte and microglia activation after spinal cord injury.

After spinal cord injury (SCI), the control of activated glial cells such as microglia and astrocytes has emerged as a promising strategy for neuropathic pain management. However, signaling mechanism involved in glial activation in the process of neuropathic pain development and maintenance after SCI is not well elucidated. In this study, we investigated the potential role and mechanism of the JAK2/STAT3 pathway associated with glial cell activation in chronic neuropathic pain development and maintenance after SCI. One month after contusive SCI, the activation of JAK2/STAT3 pathway was markedly upregulated in both microglia and astrocyte in nociceptive processing regions of the lumbar spinal cord. In addition, both mechanical allodynia and thermal hyperalgesia was significantly inhibited by a JAK2 inhibitor, AG490. In particular, AG490 treatment inhibited both microglial and astrocyte activation in the lumbar (L) 4-5 dorsal horn and significantly decreased levels of p-p38MAPK, p-ERK and p-JNK, which are known to be activated in microglia (p-p38MAPK and p-ERK) and astrocyte (p-JNK). Experiments using primary cell cultures also revealed that the JAK2/STAT3 pathway promoted microglia and astrocyte activation after lipopolysaccharide stimulation. Furthermore, JAK2/STAT3 signaling and pain behaviors were significantly attenuated when the rats were treated with anti-IL-6 antibody. Finally, minocycline, a tetracycline antibiotic, inhibited IL-6/JAK2/STAT3 signaling pathway in activated glial cells and restored nociceptive thresholds and the hyperresponsiveness of dorsal neurons. These results suggest an important role of the IL-6/JAK2/STAT3 pathway in the activation of microglia and astrocytes and in the maintenance of chronic below-level pain after SCI.

A humanized chemogenetic system inhibits murine pain-related behavior and hyperactivity in human sensory neurons.

Hyperexcitability in sensory neurons is known to underlie many of the maladaptive changes associated with persistent pain. Chemogenetics has shown promise as a means to suppress such excitability, yet chemogenetic approaches suitable for human applications are needed. PSAM4-GlyR is a modular system based on the human α7 nicotinic acetylcholine and glycine receptors, which responds to inert chemical ligands and the clinically approved drug varenicline. Here, we demonstrated the efficacy of this channel in silencing both mouse and human sensory neurons by the activation of large shunting conductances after agonist administration. Virally mediated expression of PSAM4-GlyR in mouse sensory neurons produced behavioral hyposensitivity upon agonist administration, which was recovered upon agonist washout. Stable expression of the channel led to similar reversible suppression of pain-related behavior even after 10 months of viral delivery. Mechanical and spontaneous pain readouts were also ameliorated by PSAM4-GlyR activation in acute and joint pain inflammation mouse models. Furthermore, suppression of mechanical hypersensitivity generated by a spared nerve injury model of neuropathic pain was also observed upon activation of the channel. Effective silencing of behavioral hypersensitivity was reproduced in a human model of hyperexcitability and clinical pain: PSAM4-GlyR activation decreased the excitability of human-induced pluripotent stem cell-derived sensory neurons and spontaneous activity due to a gain-of-function NaV1.7 mutation causing inherited erythromelalgia. Our results demonstrate the contribution of sensory neuron hyperexcitability to neuropathic pain and the translational potential of an effective, stable, and reversible humanized chemogenetic system for the treatment of pain.

Mitochondrial transplantation attenuates traumatic neuropathic pain, neuroinflammation, and apoptosis in rats with nerve root ligation.

Traumatic neuropathic pain (TNP) is caused by traumatic damage to the somatosensory system and induces the presentation of allodynia and hyperalgesia. Mitochondrial dysfunction, neuroinflammation, and apoptosis are hallmarks in the pathogenesis of TNP. Recently, mitochondria-based therapy has emerged as a potential therapeutic intervention for diseases related to mitochondrial dysfunction. However, the therapeutic effectiveness of mitochondrial transplantation (MT) on TNP has rarely been investigated. Here, we validated the efficacy of MT in treating TNP. Both in vivo and in vitro TNP models by conducting an L5 spinal nerve ligation in rats and exposing the primary dorsal root ganglion (DRG) neurons to capsaicin, respectively, were applied in this study. The MT was operated by administrating 100 µg of soleus-derived allogeneic mitochondria into the ipsilateral L5 DRG in vivo and the culture medium in vitro. Results showed that the viable transplanted mitochondria migrated into the rats' spinal cord and sciatic nerve. MT alleviated the nerve ligation-induced mechanical and thermal pain hypersensitivity. The nerve ligation-induced glial activation and the expression of pro-inflammatory cytokines and apoptotic markers in the spinal cord were also repressed by MT. Consistently, exogenous mitochondria reversed the capsaicin-induced reduction of mitochondrial membrane potential and expression of pro-inflammatory cytokines and apoptotic markers in the primary DRG neurons in vitro. Our findings suggest that MT mitigates the spinal nerve ligation-induced apoptosis and neuroinflammation, potentially playing a role in providing neuroprotection against TNP.

Alleviation of neuropathic pain with neuropeptide Y requires spinal Npy1r interneurons that coexpress Grp.

Neuropeptide Y targets the Y1 receptor (Y1) in the spinal dorsal horn (DH) to produce endogenous and exogenous analgesia. DH interneurons that express Y1 (Y1-INs; encoded by Npy1r) are necessary and sufficient for neuropathic hypersensitivity after peripheral nerve injury. However, as Y1-INs are heterogenous in composition in terms of morphology, neurophysiological characteristics, and gene expression, we hypothesized that a more precisely defined subpopulation mediates neuropathic hypersensitivity. Using fluorescence in situ hybridization, we found that Y1-INs segregate into 3 largely nonoverlapping subpopulations defined by the coexpression of Npy1r with gastrin-releasing peptide (Grp/Npy1r), neuropeptide FF (Npff/Npy1r), and cholecystokinin (Cck/Npy1r) in the superficial DH of mice, nonhuman primates, and humans. Next, we analyzed the functional significance of Grp/Npy1r, Npff/Npy1r, and Cck/Npy1r INs to neuropathic pain using a mouse model of peripheral nerve injury. We found that chemogenetic inhibition of Npff/Npy1r-INs did not change the behavioral signs of neuropathic pain. Further, inhibition of Y1-INs with an intrathecal Y1 agonist, [Leu31, Pro34]-NPY, reduced neuropathic hypersensitivity in mice with conditional deletion of Npy1r from CCK-INs and NPFF-INs but not from GRP-INs. We conclude that Grp/Npy1r-INs are conserved in higher order mammalian species and represent a promising and precise pharmacotherapeutic target for the treatment of neuropathic pain.

Melatonin Improves Mitochondrial Dysfunction and Attenuates Neuropathic Pain by Regulating SIRT1 in Dorsal Root Ganglions.

Neuropathic pain is a debilitating chronic pain condition and is refractory to the currently available treatments. Emerging evidence suggests that melatonin exerts analgesic effects in rodent models of neuropathic pain. Nevertheless, the exact underlying mechanisms of the analgesic effects of melatonin on neuropathic pain are largely unknown. Here, we observed that spinal nerve ligation (SNL) in rats L5 and L6 induced an obvious decrease in the 50% paw withdrawal threshold (PWT) and paw withdrawal latency (PWL), indicating the induction of mechanical allodynia and the hyperalgesia, and melatonin prevented the genesis and maintenance of mechanical allodynia and the hyperalgesia. Notably, the inhibitory action of melatonin on SNL-induced mechanical allodynia and heat hypersensitivity was inhibited by a SIRT1 inhibitor (EX527). Melatonin treatment increased the expression of neuronal sirtuin1 (SIRT1) in DRGs following nerve injury. Furthermore, melatonin treatment restored the injury-dependent decrease in mitochondrial membrane potential and peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) and reduced the injury-dependent increase in hydrogen peroxide and 8-hydroxy-2-deoxyguanosine (8-OHdG), which was inhibited by EX527. In addition, we found that EX527 impeded the inhibitory effects of melatonin on the SNL-induced increased expression of cytokines tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1ß). In conclusion, the above data demonstrated that melatonin alleviated mechanical allodynia and hyperalgesia induced by peripheral nerve injury via SIRT1 activation. Melatonin resolved mitochondrial dysfunction-oxidative stress-dependent and neuroinflammation mechanisms that were driven by SIRT1 after nerve injury.

LncRNA FTX ameliorates neuropathic pain by targeting miR-320a in a rat model of chronic constriction injury.

INTRODUCTION: Long non-coding RNAs (lncRNAs) participate in the process of neuropathic pain (NP). Herein, the goal of this research was to examine the roles of lncRNA five prime to XIST (FTX) in influencing chronic constriction injury (CCI)-induced NP. MATERIAL AND METHODS: We have established a rat CCI model to simulate NP in vivo. Reverse transcription-quantitative PCR (RT-qPCR) was used to detect mRNA levels of FTX, microRNA (miR)-320a, and runt-related transcription factor 2 (RUNX2) in the spinal cord. This was followed by subsequent regulation of FTX or miR-320a levels in vivo by intrathecal injection of overexpression FTX or miR-320a mimic lentivirus. The behaviour of rat NP the paw withdrawal threshold (PWT) and paw withdrawal latency (PWL). Enzyme-linked immunosorbent assay (ELISA) was used to assess the secretion of pro-inflammatory and anti-inflammatory factors in the spinal cord tissue. A correlation between FTX and miR-320a, and RUNX2 was validated by luciferase reporter. RESULTS: FTX levels were reduced in CCI rats ( p < 0.05), and miR-320a was a direct target of FTX. Overexpression of FTX typically reduced PWL and PWT as well as neuroinflammation thus alleviating NP ( p < 0.05). However, increasing miR-320a reversed the alleviation of FTX on NP, increased PWL and PWT, and promoted neuroinflammation ( p < 0.05). Additionally, RUNX2, which is a miR-320a target gene, was significantly repressed in CCI rats and its expression was increased by FTX, however, this increase was attenuated by elevated miR-320a ( p < 0.05). CONCLUSIONS: In the CCI-induced NP rat model, FTX attenuates NP and neuroinflammation by regulating the miR-320a/RUNX2 axis. This provides a new vision for NP treatment.

Activation of the TNF-α-Necroptosis Pathway in Parvalbumin-Expressing Interneurons of the Anterior Cingulate Cortex Contributes to Neuropathic Pain.

The hyperexcitability of the anterior cingulate cortex (ACC) has been implicated in the development of chronic pain. As one of the key causes of ACC hyperexcitation, disinhibition of the ACC may be closely related to the dysfunction of inhibitory parvalbumin (PV)-expressing interneurons (PV-INs). However, the molecular mechanism underlying the ACC PV-INs injury remains unclear. The present study demonstrates that spared sciatic nerve injury (SNI) induces an imbalance in the excitation and inhibition (E/I) of the ACC. To test whether tumor necrosis factor-α (TNF-α) upregulation in the ACC after SNI activates necroptosis and participates in PV-INs damage, we performed a differential analysis of transcriptome sequencing using data from neuropathic pain models and found that the expression of genes key to the TNF-α-necroptosis pathway were upregulated. TNF-α immunoreactivity (IR) signals in the ACCs of SNI rats were co-located with p-RIP3- and PV-IR, or p-MLKL- and PV-IR signals. We then systematically detected the expression and cell localization of necroptosis-related proteins, including kinase RIP1, RIP3, MLKL, and their phosphorylated states, in the ACC of SNI rats. Except for RIP1 and MLKL, the levels of these proteins were significantly elevated in the contralateral ACC and mainly expressed in PV-INs. Blocking the ACC TNF-α-necroptosis pathway by microinjecting TNF-α neutralizing antibody or using an siRNA knockdown to block expression of MLKL in the ACC alleviated SNI-induced pain hypersensitivity and inhibited the upregulation of TNF-α and p-MLKL. Targeting TNF-α-triggered necroptosis within ACC PV-INs may help to correct PV-INs injury and E/I imbalance in the ACC in neuropathic pain.

[Mechanism of Mongolian drug Naru-3 in initiation of neuroinflammation of neuropathic pain from MMP9/IL-1ß signaling pathway].

Neuropathic pain(NP) has similar phenotypes but different sequential neuroinflammatory mechanisms in the pathological process. It is of great significance to inhibit the initiation of neuroinflammation, which has become a new direction of NP treatment and drug development in recent years. Mongolian drug Naru-3 is clinically effective in the treatment of trigeminal neuralgia, sciatica, and other NPs in a short time, but its pharmacodynamic characteristics and mechanism of analgesia are still unclear. In this study, a spinal nerve ligation(SNL) model simulating clinical peripheral nerve injury was established and the efficacy and mechanism of Naru-3 in the treatment of NPs was discussed by means of behavioral detection, side effect evaluation, network analysis, and experimental verification. Pharmacodynamic results showed that Naru-3 increased the basic pain sensitivity threshold(mechanical hyperalgesia and thermal radiation hyperalgesia) in the initiation of SNL in animals and relieved spontaneous pain, however, there was no significant effect on the basic pain sensitivity threshold and motor coordination function of normal animals under physiological and pathological conditions. Meanwhile, the results of primary screening of target tissues showed that Naru-3 inhibited the second phase of injury-induced nociceptive response of formalin test in mice and reduced the expression of inflammatory factors in the spinal cord. Network analysis discovered that Naru-3 had synergy in the treatment of NP, and its mechanism was associated with core targets such as matrix metalloproteinase-9(MMP9) and interleukin-1ß(IL-1ß). The experiment further took the dorsal root ganglion(DRG) and the stage of patho-logical spinal cord as the research objects, focusing on the core targets of inducing microglial neuroinflammation. By means of Western blot, immunofluorescence, agonists, antagonists, behavior, etc., the mechanism of Naru-3 in exerting NP analgesia may be related to the negative regulation of the MMP9/IL-1ß signaling pathway-mediated microglia p38/IL-1ß inflammatory loop in the activation phase. The relevant research enriches the biological connotation of Naru-3 in the treatment of NP and provides references for clinical rational drug use.