S1P/S1PR1 signaling is involved in the development of nociceptive pain.

Background: Pain is a complex perception involving unpleasant somatosensory and emotional experiences. However, the underlying mechanisms that mediate its different components remain unclear. Sphingosine-1-phosphate (S1P), a metabolite of sphingomyelin and a potent lipid mediator, initiates signaling via G protein-coupled receptors (S1PRs) on cell surfaces. It serves as a second messenger in cellular processes such as proliferation and apoptosis. Nevertheless, the neuropharmacology of sphingolipid signaling in pain conditions within the central nervous system remains largely unexplored and controversial. Methods: Chronic nociceptive pain models were induced in vivo by intraplantar injection of 20 µL complete Freund's adjuvant (CFA) into the left hind paws. We assessed S1P and S1PR1 expression in the spinal cords of CFA model mice. Functional antagonists of S1PR1 or S1PR1-specific siRNA were administered daily following CFA model establishment. Paw withdrawal response frequency (PWF) and paw withdrawal latency (PWL) were measured to evaluate mechanical allodynia and thermal hyperalgesia, respectively. RT-PCR assessed interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α levels. Western blotting and immunofluorescence were used to analyze glial fibrillary acidic protein (GFAP), ionized calcium-binding adapter molecule (Iba1), STAT3, ERK, and p38 MAPK protein expression. Results: In the chronic nociceptive pain model induced by CFA, S1P and S1PR1 expression levels were significantly elevated, leading to activation of spinal cord glial cells. S1PR1 activation also promoted MMP2-mediated cleavage of mature IL-1ß. Additionally, S1PR1 activation upregulated phosphorylation of STAT3, ERK, and p38 MAPK in glial cells, profoundly impacting downstream signaling pathways and contributing to chronic nociceptive pain. Conclusion: The S1P/S1PR1 axis plays a pivotal role in the cellular and molecular mechanisms underlying nociceptive pain. This signaling pathway modulates glial cell activation and the expression of pain-related genes (STAT3, ERK, p38 MAPK) and inflammatory factors in the spinal dorsal horn. These findings underscore the potential of targeting the S1P system for developing novel analgesic therapies.

Astaxanthin alleviates fibromyalgia pain and depression via NLRP3 inflammasome inhibition.

Fibromyalgia is characterised by widespread chronic pain and is often accompanied by comorbidities such as sleep disorders, anxiety, and depression. Because it is often accompanied by many adverse symptoms and lack of effective treatment, it is important to search for the pathogenesis and treatment of fibromyalgia. Astaxanthin, a carotenoid pigment known for its anti-inflammatory and antioxidant properties, has demonstrated effective analgesic effects in neuropathic pain. However, its impact on fibromyalgia remains unclear. Therefore, in this study, we constructed a mouse model of fibromyalgia and investigated the effect of astaxanthin on chronic pain and associated symptoms through multiple intragastrical injections. We conducted behavioural assessments to detect pain and depression-like states in mice, recorded electroencephalograms to monitor sleep stages, examined c-Fos activation in the anterior cingulate cortex, measured activation of spinal glial cells, and assessed levels of inflammatory factors in the brain and spinal cord, including interleukin (IL)-1ß, IL-6, and tumour necrosis factor- α(TNF-α).Additionally, we analysed the expression levels of IL-6, IL-10, NOD-like receptor thermal protein domain associated protein 3 (NLRP3), Apoptosis-associated speck-like protein containing CARD, and Caspase-1 proteins. The findings revealed that astaxanthin significantly ameliorated mechanical and thermal pain in mice with fibromyalgia and mitigated sleep disorders and depressive-like symptoms induced by pain. A potential mechanism underlying these effects is the anti-inflammatory action of astaxanthin, likely mediated through the inhibition of the NLRP3 inflammasome, which could be one of the pathways through which astaxanthin alleviates fibromyalgia. In conclusion, our study suggests that astaxanthin holds promise as a potential analgesic medication for managing fibromyalgia and its associated symptoms.

Diosmetin alleviates neuropathic pain by regulating the Keap1/Nrf2/NF-κB signaling pathway.

BACKGROUND: Neuropathic pain, a chronic condition with a high incidence, imposes psychological burdens on both patients and society. It is urgent to improve pain management and develop new analgesic drugs. Traditional Chinese medicine has gained popularity as a method for pain relief. Diosmetin (Dio) is mainly found in Chinese herbal medicines with effective antioxidant, anti-cancer, and anti-inflammatory properties. There are few known mechanisms underlying the effectiveness of Dio in treating neuropathic pain. However, the complete understanding of its therapeutic effect is missing. PURPOSE: This study aimed to evaluate Dio's therapeutic effects on neuropathic pain models and determine its possible mechanism of action. We hypothesized that Dio may activate antioxidants and reduce inflammation, inhibit the activation of Kelch-like epichlorohydrin-associated protein 1 (Keap1) and nuclear factor-k-gene binding (NF-κB), promote the metastasis of nuclear factor erythroid 2-related factor 2 (Nrf2) and the expression of heme oxygenase 1 (HO-1), thus alleviating the neuropathic pain caused by spinal nerve ligation. METHODS: Chronic nociceptive pain mouse models were established in vivo by L4 spinal nerve ligation (SNL). Different dosages of Dio (10, 50, 100 mg/kg) were intragastrically administered daily from the third day after the establishment of the SNL model. Allodynia, caused by mechanical stimuli, and hyperalgesia, caused by heat, were assessed using the paw withdrawal response frequency (PWF) and paw withdrawal latency (PWL), respectively. Cold allodynia were assessd by acetone test. RT-PCR was used to detect the content of interleukin-(IL)- 1ß, IL-6 and tumor necrosis factor (TNF)-a. Immunofluorescence and western blotting were employed to assess the expression levels of Glial fibrillary acidic protein (GFAP), ionized calcium-binding adapter molecule (Iba1), Keap1, Nrf2, HO-1, and NF-κB p-p65 protein. RESULTS: Dio administration relieved SNL-induced transient mechanical and thermal allodynia in mice. The protective effect of Dio in the SNL model was associated with its anti-inflammatory and anti-glial responses in the spinal cord. Dio inhibited both inflammatory factors and macrophage activation in the DRG. Furthermore, Dio regulated the Keap1/Nrf2/NF-κB signaling pathway. HO-1 and Nrf2 were upregulated following Dio administration, which also decreased the levels of Keap1 and NF-κB p65 protein. CONCLUSION: Mice with SNL-induced neuropathic pain were therapeutically treated with Dio. Dio may protect against pain by inhibiting inflammatory responses and improved Keap1/Nrf2/NF-κB pathway. These results highlight the potential therapeutic effect of Dio for the development of new analgesic drugs.

Sirtuin 2 Alleviates Chronic Neuropathic Pain by Suppressing Ferroptosis in Rats.

Neuropathic pain (NP) is chronic and associated with poor effects of general analgesia. It affects patients' health and quality of life. The apoptotic process of lipid peroxidation caused by iron overload is called ferroptosis, which may be associated with nervous system disease. A recent study has found that sirtuin 2 (SIRT2) achieves a neuroprotective effect by suppressing ferroptosis. Herein, we aimed to examine whether SIRT2 regulated spared nerve injury (SNI)-induced NP by suppressing ferroptosis in rats. A rat model of NP was induced in adult male Sprague-Dawley rats weighing 200-250 g. Mechanical allodynia was observed from the first day after SNI and continued for 14 days. Compared with age-matched control rats, the expression of SIRT2 and ferroportin 1 (FPN1) decreased in the L4-6 spinal cord of the SNI-induced NP rats. In addition, we observed that the levels of both iron and anti-acyl-coenzyme A synthetase long-chain family member 4 (ACSL4) were significantly increased in the spinal cord after SNI, while the expression of glutathione peroxidase 4 (GPX4) was decreased. Furthermore, an intrathecal injection of SIRT2 overexpressed recombinant adenovirus, which upregulated the expression of SIRT2, attenuated mechanical allodynia, enhanced the level of FPN1, inhibited intracellular iron accumulation, and reduced oxidant stress levels, thereby reversing the changes to ACSL4 and GPX4 expression in the SNI rats. This evidence suggests that SIRT2-targeted therapeutics may help relieve the symptoms of chronic NP.

Astaxanthin alleviates neuropathic pain by inhibiting the MAPKs and NF-κB pathways.

Neuropathic pain is a complex condition that usually lasts a lifetime and has a major negative impact on life after injury. Improving pain management is an important and unmet need. Astaxanthin (AST) is a natural marine medicine with effective antioxidant and anti-inflammatory properties and neuroprotective effects. However, few mechanisms can explain the role of AST in the treatment of neuropathic pain. In the present study, we examined its potential to eliminate spinal nerve ligation (SNL) damage by inhibiting the phosphorylation of extracellular signal-regulated kinase (ERK)1/2, phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK), nuclear factor-κB (NF-κB) p65 and the inflammatory response. The results of behavior tests indicated the promising role of AST in analgesic effect in SNL mice. AST decreased the neuronal and non-neuronal activation, the levels of the inflammatory signaling mediators (p-ERK1/2 p-p38 MAPK and NF-κB p65) and inflammatory cytokine expression (interleukin [IL]-1, IL-17, IL-6, and tumor necrosis factor-α [TNF-α]. These results suggest that AST is a promising candidate to reduce nociceptive hypersensitization after SNL.

IL-23/IL-17A/TRPV1 axis produces mechanical pain via macrophage-sensory neuron crosstalk in female mice.

Although sex dimorphism is increasingly recognized as an important factor in pain, female-specific pain signaling is not well studied. Here we report that administration of IL-23 produces mechanical pain (mechanical allodynia) in female but not male mice, and chemotherapy-induced mechanical pain is selectively impaired in female mice lacking Il23 or Il23r. IL-23-induced pain is promoted by estrogen but suppressed by androgen, suggesting an involvement of sex hormones. IL-23 requires C-fiber nociceptors and TRPV1 to produce pain but does not directly activate nociceptor neurons. Notably, IL-23 requires IL-17A release from macrophages to evoke mechanical pain in females. Low-dose IL-17A directly activates nociceptors and induces mechanical pain only in females. Finally, deletion of estrogen receptor subunit α (ERα) in TRPV1+ nociceptors abolishes IL-23- and IL-17-induced pain in females. These findings demonstrate that the IL-23/IL-17A/TRPV1 axis regulates female-specific mechanical pain via neuro-immune interactions. Our study also reveals sex dimorphism at both immune and neuronal levels.

STING suppresses bone cancer pain via immune and neuronal modulation.

Patients with advanced stage cancers frequently suffer from severe pain as a result of bone metastasis and bone destruction, for which there is no efficacious treatment. Here, using multiple mouse models of bone cancer, we report that agonists of the immune regulator STING (stimulator of interferon genes) confer remarkable protection against cancer pain, bone destruction, and local tumor burden. Repeated systemic administration of STING agonists robustly attenuates bone cancer-induced pain and improves locomotor function. Interestingly, STING agonists produce acute pain relief through direct neuronal modulation. Additionally, STING agonists protect against local bone destruction and reduce local tumor burden through modulation of osteoclast and immune cell function in the tumor microenvironment, providing long-term cancer pain relief. Finally, these in vivo effects are dependent on host-intrinsic STING and IFN-I signaling. Overall, STING activation provides unique advantages in controlling bone cancer pain through distinct and synergistic actions on nociceptors, immune cells, and osteoclasts.

Sirt2 in the Spinal Cord Regulates Chronic Neuropathic Pain Through Nrf2-Mediated Oxidative Stress Pathway in Rats.

Reduction in Nrf2-mediated antioxidant response in the central nervous system plays an important role in the development and maintenance of neuropathic pain (NP). However, the mechanisms regulating Nrf2 activity in NP remain unclear. A recent in vitro study revealed that Sirt2, a member of the sirtuin family of proteins, affects antioxidant capacity by modulating Nrf2 activity. Here we examined whether central Sirt2 regulates NP through Nrf2-mediated oxidative stress pathway. In a rat model of spared nerve injury (SNI)-induced NP, mechanical allodynia and thermal hyperalgesia were observed on day 1 and up to day 14 post-SNI. The expression of Sirt2, Nrf2 and its target gene NQO1 in the spinal cord in SNI rats, compared with sham rats, was significantly decreased from day 7 and remained lower until the end of the experiment (day 14). The mechanical allodynia and thermal hyperalgesia in SNI rats were ameliorated by intrathecal injection of Nrf2 agonist tBHQ, which normalized expression of Nrf2 and NQO1 and reversed SNI-induced decrease in antioxidant enzyme superoxide dismutase (SOD) and increase in oxidative stress marker 8-hydroxy-2'-deoxyguanosine (8-OHdG) in the spinal cord. Moreover, intrathecal injection of a recombinant adenovirus expressing Sirt2 (Ad-Sirt2) that upregulated expression of Sirt2, restored expression of Nrf2 and NQO1 and attenuated oxidative stress in the spinal cord, leading to improvement of thermal hyperalgesia and mechanical allodynia in SNI rats. These findings suggest that peripheral nerve injury downregulates Sirt2 expression in the spinal cord, which inhibits Nrf2 activity, leading to increased oxidative stress and the development of chronic NP.

Activation of GPR37 in macrophages confers protection against infection-induced sepsis and pain-like behaviour in mice.

GPR37 was discovered more than two decades ago, but its biological functions remain poorly understood. Here we report a protective role of GPR37 in multiple models of infection and sepsis. Mice lacking Gpr37 exhibited increased death and/or hypothermia following challenge by lipopolysaccharide (LPS), Listeria bacteria, and the mouse malaria parasite Plasmodium berghei. Sepsis induced by LPS and Listeria in wild-type mice is protected by artesunate (ARU) and neuroprotectin D1 (NPD1), but the protective actions of these agents are lost in Gpr37-/- mice. Notably, we found that ARU binds to GPR37 in macrophages and promotes phagocytosis and clearance of pathogens. Moreover, ablation of macrophages potentiated infection, sepsis, and their sequelae, whereas adoptive transfer of NPD1- or ARU-primed macrophages reduced infection, sepsis, and pain-like behaviors. Our findings reveal physiological actions of ARU in host cells by activating macrophages and suggest that GPR37 agonists may help to treat sepsis, bacterial infections, and malaria.

Computer-aided Discovery of a New Nav1.7 Inhibitor for Treatment of Pain and Itch.

BACKGROUND: Voltage-gated sodium channel Nav1.7 has been validated as a perspective target for selective inhibitors with analgesic and anti-itch activity. The objective of this study was to discover new candidate compounds with Nav1.7 inhibitor properties. The authors hypothesized that their approach would yield at least one new compound that inhibits sodium currents in vitro and exerts analgesic and anti-itch effects in mice. METHODS: In silico structure-based similarity search of 1.5 million compounds followed by docking to the Nav1.7 voltage sensor of Domain 4 and molecular dynamics simulation was performed. Patch clamp experiments in Nav1.7-expressing human embryonic kidney 293 cells and in mouse and human dorsal root ganglion neurons were conducted to test sodium current inhibition. Formalin-induced inflammatory pain model, paclitaxel-induced neuropathic pain model, histamine-induced itch model, and mouse lymphoma model of chronic itch were used to confirm in vivo activity of the selected compound. RESULTS: After in silico screening, nine compounds were selected for experimental assessment in vitro. Of those, four compounds inhibited sodium currents in Nav1.7-expressing human embryonic kidney 293 cells by 29% or greater (P < 0.05). Compound 9 (3-(1-benzyl-1H-indol-3-yl)-3-(3-phenoxyphenyl)-N-(2-(pyrrolidin-1-yl)ethyl)propanamide, referred to as DA-0218) reduced sodium current by 80% with a 50% inhibition concentration of 0.74 µM (95% CI, 0.35 to 1.56 µM), but had no effects on Nav1.5-expressing human embryonic kidney 293 cells. In mouse and human dorsal root ganglion neurons, DA-0218 reduced sodium currents by 17% (95% CI, 6 to 28%) and 22% (95% CI, 9 to 35%), respectively. The inhibition was greatly potentiated in paclitaxel-treated mouse neurons. Intraperitoneal and intrathecal administration of the compound reduced formalin-induced phase II inflammatory pain behavior in mice by 76% (95% CI, 48 to 100%) and 80% (95% CI, 68 to 92%), respectively. Intrathecal administration of DA-0218 produced acute reduction in paclitaxel-induced mechanical allodynia, and inhibited histamine-induced acute itch and lymphoma-induced chronic itch. CONCLUSIONS: This study's computer-aided drug discovery approach yielded a new Nav1.7 inhibitor that shows analgesic and anti-pruritic activity in mouse models.

Neuromodulation, Specialized Proresolving Mediators, and Resolution of Pain.

The current crises in opioid abuse and chronic pain call for the development of nonopioid and nonpharmacological therapeutics for pain relief. Neuromodulation-based approaches, such as spinal cord stimulation, dorsal root ganglion simulation, and nerve stimulation including vagus nerve stimulation, have shown efficacy in achieving pain control in preclinical and clinical studies. However, the mechanisms by which neuromodulation alleviates pain are not fully understood. Accumulating evidence suggests that neuromodulation regulates inflammation and neuroinflammation-a localized inflammation in peripheral nerves, dorsal root ganglia/trigeminal ganglia, and spinal cord/brain-through neuro-immune interactions. Specialized proresolving mediators (SPMs) such as resolvins, protectins, maresins, and lipoxins are lipid molecules produced during the resolution phase of inflammation and exhibit multiple beneficial effects in resolving inflammation in various animal models. Recent studies suggest that SPMs inhibit inflammatory pain, postoperative pain, neuropathic pain, and cancer pain in rodent models via immune, glial, and neuronal modulations. It is noteworthy that sham surgery is sufficient to elevate resolvin levels and may serve as a model of resolution. Interestingly, it has been shown that the vagus nerve produces SPMs and vagus nerve stimulation (VNS) induces SPM production in vitro. In this review, we discuss how neuromodulation such as VNS controls pain via immunomodulation and neuro-immune interactions and highlight possible involvement of SPMs. In particular, we demonstrate that VNS via auricular electroacupuncture effectively attenuates chemotherapy-induced neuropathic pain. Furthermore, auricular stimulation is able to increase resolvin levels in mice. Thus, we propose that neuromodulation may control pain and inflammation/neuroinflammatioin via SPMs. Finally, we discuss key questions that remain unanswered in our understanding of how neuromodulation-based therapies provide short-term and long-term pain relief.

PD-1 blockade inhibits osteoclast formation and murine bone cancer pain.

Emerging immune therapy, such as with the anti-programmed cell death-1 (anti-PD-1) monoclonal antibody nivolumab, has shown efficacy in tumor suppression. Patients with terminal cancer suffer from cancer pain as a result of bone metastasis and bone destruction, but how PD-1 blockade affects bone cancer pain remains unknown. Here, we report that mice lacking Pdcd1 (Pd1-/-) demonstrated remarkable protection against bone destruction induced by femoral inoculation of Lewis lung cancer cells. Compared with WT mice, Pd1-/- mice exhibited increased baseline pain sensitivity, but the development of bone cancer pain was compromised in Pd1-/- mice. Consistently, these beneficial effects in Pd1-/- mice were recapitulated by repeated i.v. applications of nivolumab in WT mice, even though nivolumab initially increased mechanical and thermal pain. Notably, PD-1 deficiency or nivolumab treatment inhibited osteoclastogenesis without altering tumor burden. PD-L1 and CCL2 are upregulated within the local tumor microenvironment, and PD-L1 promoted RANKL-induced osteoclastogenesis through JNK activation and CCL2 secretion. Bone cancer upregulated CCR2 in primary sensory neurons, and CCR2 antagonism effectively reduced bone cancer pain. Our findings suggest that, despite a transient increase in pain sensitivity following each treatment, anti-PD-1 immunotherapy could produce long-term benefits in preventing bone destruction and alleviating bone cancer pain by suppressing osteoclastogenesis.

Resolvin D5 Inhibits Neuropathic and Inflammatory Pain in Male But Not Female Mice: Distinct Actions of D-Series Resolvins in Chemotherapy-Induced Peripheral Neuropathy.

Earlier studies have demonstrated that essential fatty acid-derived specialized pro-resolving mediators (SPMs) promote the resolution of inflammation and pain. However, the potential analgesic actions of SPMs in chemotherapy-induced peripheral neuropathy (CIPN) are not known. Recent results also showed sex dimorphism in immune cell signaling in neuropathic pain. Here, we evaluated the analgesic actions of D-series resolvins (RvD1, RvD2, RvD3, RvD4, and RvD5) on a CIPN in male and female mice. Paclitaxel (PTX, 2 mg/kg), given on days 0, 2, 4, and 6, produced robust mechanical allodynia in both sexes at 2 weeks. Intrathecal injection of RvD1 and RvD2 (100 ng, i.t.) at 2 weeks reversed PTX-induced mechanical allodynia in both sexes, whereas RvD3 and RvD4 (100 ng, i.t.) had no apparent effects on either sex. Interestingly, RvD5 (100 ng, i.t.) only reduced mechanical allodynia in male mice but not in female mice. Notably, PTX-induced mechanical allodynia was fully developed in Trpv1 or Trpa1 knockout mice, showing no sex differences. Also, intrathecal RvD5 reduced mechanical allodynia in male mice lacking Trpv1 or Trpa1, whereas female mice with Trpv1 or Trpa1 deficiency had no response to RvD5. Finally, RvD5-induced male-specific analgesia was also confirmed in an inflammatory pain condition. Formalin-induced second phase pain (licking and flinching) was reduced by intrathecal RvD5 in male but not female mice. These findings identified RvD5 as the first SPM that shows sex dimorphism in pain regulation. Moreover, these results suggest that specific resolvins may be used to treat CIPN, a rising health concern in cancer survivors.