Identification of compounds that cause axonal dieback without cytotoxicity in dorsal root ganglia explants and intervertebral disc cells with potential to treat pain via denervation.

Low back pain, knee osteoarthritis, and cancer patients suffer from chronic pain. Aberrant nerve growth into intervertebral disc, knee, and tumors, are common pathologies that lead to these chronic pain conditions. Axonal dieback induced by capsaicin (Caps) denervation has been FDA-approved to treat painful neuropathies and knee osteoarthritis but with short-term efficacy and discomfort. Herein, we propose to evaluate pyridoxine (Pyr), vincristine sulfate (Vcr) and ionomycin (Imy) as axonal dieback compounds for denervation with potential to alleviate pain. Previous literature suggests Pyr, Vcr, and Imy can cause undesired axonal degeneration, but no previous work has evaluated axonal dieback and cytotoxicity on adult rat dorsal root ganglia (DRG) explants. Thus, we performed axonal dieback screening using adult rat DRG explants in vitro with Caps as a positive control and assessed cytotoxicity. Imy inhibited axonal outgrowth and slowed axonal dieback, while Pyr and Vcr at high concentrations produced significant reduction in axon length and robust axonal dieback within three days. DRGs treated with Caps, Vcr, or Imy had increased DRG cytotoxicity compared to matched controls, but overall cytotoxicity was minimal and at least 88% lower compared to lysed DRGs. Pyr did not lead to any DRG cytotoxicity. Further, neither Pyr nor Vcr triggered intervertebral disc cell death or affected cellular metabolic activity after three days of incubation in vitro. Overall, our findings suggest Pyr and Vcr are not toxic to DRGs and intervertebral disc cells, and there is potential for repurposing these compounds for axonal dieback compounds to cause local denervation and alleviate pain.

Comparative study of susceptibility to methylmercury cytotoxicity in cell types composing rat peripheral nerves: a higher susceptibility of dorsal root ganglion neurons.

Methylmercury is an environmental polluting organometallic compound that exhibits neurotoxicity, as observed in Minamata disease patients. Methylmercury damages peripheral nerves in Minamata patients, causing more damage to sensory nerves than motor nerves. Peripheral nerves are composed of three cell types: dorsal root ganglion (DRG) cells, anterior horn cells (AHCs), and Schwann cells. In this study, we compared cultured these three cell types derived from the rat for susceptibility to methylmercury cytotoxicity, intracellular accumulation of mercury, expression of L-type amino acid transporter 1 (LAT1), which transports methylmercury into cells, and expression of multidrug resistance-associated protein 2 (MRP2), which transports methylmercury-glutathione conjugates into the extracellular space. Of the cells examined, we found that DRG cells were the most susceptible to methylmercury with markedly higher intracellular accumulation of mercury. The constitutive level of LAT1 was higher and that of MRP2 lower in DRG cells compared with those in AHC and Schwann cells. Additionally, decreased cell viability caused by methylmercury was significantly reduced by either the LAT1 inhibitor, JPH203, or siRNA-mediated knockdown of LAT1. On the other hand, an MRP2 inhibitor, MK571, significantly intensified the decrease in the cell viability caused by methylmercury. Our results provide a cellular basis for sensory neve predominant injury in the peripheral nerves of Minamata disease patients.

A phytosphingosine derivative mYG-II-6 inhibits histamine-mediated TRPV1 activation and MRGPRX2-dependent mast cell degranulation.

BACKGROUND: Phytosphingosine and its derivative are known for their skin-protective properties. While mYG-II-6, a phytosphingosine derivative, has shown anti-inflammatory and antipsoriatic effects, its potential antipruritic qualities have yet to be explored. This study aimed to investigate mYG-II-6's antipruritic properties. METHODS: The calcium imaging technique was employed to investigate the activity of ion channels and receptors. Mast cell degranulation was confirmed through the ß-hexosaminidase assay. Additionally, in silico molecular docking and an in vivo mouse scratching behavior test were utilized. RESULTS: Using HEK293T cells transfected with H1R and TRPV1, we examined the impact of mYG-II-6 on histamine-induced intracellular calcium rise, a key signal in itch-mediating sensory neurons. Pretreatment with mYG-II-6 significantly reduced histamine-induced calcium levels and inhibited TRPV1 activity, suggesting its role in blocking the calcium influx channel. Additionally, mYG-II-6 suppressed histamine-induced calcium increase in primary cultures of mouse dorsal root ganglia, indicating its potential antipruritic effect mediated by histamine. Interestingly, mYG-II-6 exhibited inhibitory effects on human MRGPRX2, a G protein-coupled receptor involved in IgE-independent mast cell degranulation. However, it did not inhibit mouse MrgprB2, the ortholog of human MRGPRX2. Molecular docking analysis revealed that mYG-II-6 selectively interacts with the binding pocket of MRGPRX2. Importantly, mYG-II-6 suppressed histamine-induced scratching behaviors in mice. CONCLUSIONS: Our findings show that mYG-II-6 can alleviate histamine-induced itch sensation through dual mechanisms. This underscores its potential as a versatile treatment for various pruritic conditions.

Molecular Aspects Involved in the Mechanisms of Bothrops jararaca Venom-Induced Hyperalgesia: Participation of NK1 Receptor and Glial Cells.

Accidents caused by Bothrops jararaca (Bj) snakes result in several local and systemic manifestations, with pain being a fundamental characteristic. The inflammatory process responsible for hyperalgesia induced by Bj venom (Bjv) has been studied; however, the specific roles played by the peripheral and central nervous systems in this phenomenon remain unclear. To clarify this, we induced hyperalgesia in rats using Bjv and collected tissues from dorsal root ganglia (DRGs) and spinal cord (SC) at 2 and 4 h post-induction. Samples were labeled for Iba-1 (macrophage and microglia), GFAP (satellite cells and astrocytes), EGR1 (neurons), and NK1 receptors. Additionally, we investigated the impact of minocycline, an inhibitor of microglia, and GR82334 antagonist on Bjv-induced hyperalgesia. Our findings reveal an increase in Iba1 in DRG at 2 h and EGR1 at 4 h. In the SC, markers for microglia, astrocytes, neurons, and NK1 receptors exhibited increased expression after 2 h, with EGR1 continuing to rise at 4 h. Minocycline and GR82334 inhibited venom-induced hyperalgesia, highlighting the crucial roles of microglia and NK1 receptors in this phenomenon. Our results suggest that the hyperalgesic effects of Bjv involve the participation of microglial and astrocytic cells, in addition to the activation of NK1 receptors.

NT-3 contributes to chemotherapy-induced neuropathic pain through TrkC-mediated CCL2 elevation in DRG neurons.

Cancer patients undergoing treatment with antineoplastic drugs often experience chemotherapy-induced neuropathic pain (CINP), and the therapeutic options for managing CINP are limited. Here, we show that systemic paclitaxel administration upregulates the expression of neurotrophin-3 (Nt3) mRNA and NT3 protein in the neurons of dorsal root ganglia (DRG), but not in the spinal cord. Blocking NT3 upregulation attenuates paclitaxel-induced mechanical, heat, and cold nociceptive hypersensitivities and spontaneous pain without altering acute pain and locomotor activity in male and female mice. Conversely, mimicking this increase produces enhanced responses to mechanical, heat, and cold stimuli and spontaneous pain in naive male and female mice. Mechanistically, NT3 triggers tropomyosin receptor kinase C (TrkC) activation and participates in the paclitaxel-induced increases of C-C chemokine ligand 2 (Ccl2) mRNA and CCL2 protein in the DRG. Given that CCL2 is an endogenous initiator of CINP and that Nt3 mRNA co-expresses with TrkC and Ccl2 mRNAs in DRG neurons, NT3 likely contributes to CINP through TrkC-mediated activation of the Ccl2 gene in DRG neurons. NT3 may be thus a potential target for CINP treatment.

Paclitaxel-Associated Mechanical Sensitivity and Neuroinflammation Are Sex-, Time-, and Site-Specific and Prevented through Cannabigerol Administration in C57Bl/6 Mice.

Chemotherapy-induced peripheral neuropathy (CIPN) is one of the most prevalent and dose-limiting complications in chemotherapy patients. One identified mechanism underlying CIPN is neuroinflammation. Most of this research has been conducted in only male or female rodent models, making direct comparisons regarding the role of sex differences in the neuroimmune underpinnings of CIPN limited. Moreover, most measurements have focused on the dorsal root ganglia (DRG) and/or spinal cord, while relatively few studies have been aimed at characterizing neuroinflammation in the brain, for example the periaqueductal grey (PAG). The overall goals of the present study were to determine (1) paclitaxel-associated changes in markers of inflammation in the PAG and DRG in male and female C57Bl6 mice and (2) determine the effect of prophylactic administration of an anti-inflammatory cannabinoid, cannabigerol (CBG). In Experiment 1, male and female mice were treated with paclitaxel (8-32 mg/kg/injection, Days 1, 3, 5, and 7) and mechanical sensitivity was measured using Von Frey filaments on Day 7 (Cohort 1) and Day 14 (Cohort 2). Cohorts were euthanized on Day 8 or 15, respectively, and DRG and PAG were harvested for qPCR analysis of the gene expression of markers of pain and inflammation Aig1, Gfap, Ccl2, Cxcl9, Tlr4, Il6, and Calca. In Experiment 2, male and female mice were treated with vehicle or 10 mg/kg CBG i.p. 30 min prior to each paclitaxel injection. Mechanical sensitivity was measured on Day 14. Mice were euthanized on Day 15, and PAG were harvested for qPCR analysis of the gene expression of Aig1, Gfap, Ccl2, Cxcl9, Tlr4, Il6, and Calca. Paclitaxel produced a transient increase in potency to produce mechanical sensitivity in male versus female mice. Regarding neuroinflammation, more gene expression changes were apparent earlier in the DRG and at a later time point in the PAG. Also, more changes were observed in females in the PAG than males. Overall, sex differences were observed for most markers at both time points and regions. Importantly, in both the DRG and PAG, most increases in markers of neuroinflammation and pain occurred at paclitaxel doses higher than those associated with significant changes in the mechanical threshold. Two analytes that demonstrated the most compelling sexual dimorphism and that changed more in males were Cxcl9 and Ccl2, and Tlr4 in females. Lastly, prophylactic administration of CBG protected the male and female mice from increased mechanical sensitivity and female mice from neuroinflammation in the PAG. Future studies are warranted to explore how these sex differences may shed light on the mechanisms of CIPN and how non-psychoactive cannabinoids such as CBG may engage these targets to prevent or attenuate the effects of paclitaxel and other chemotherapeutic agents on the nervous system.

A novel isoquinoline alkaloid HJ-69 isolated from Zanthoxylum bungeanum attenuates inflammatory pain by inhibiting voltage-gated sodium and potassium channels.

ETHNOPHARMACOLOGY RELEVANCE: Zanthoxylum bungeanum Maxim. (Z. bungeanum), a member of the Rutaceae family, has a rich history of traditional use in Asia for treating arthritis and toothache conditions. As characteristic chemical components, numerous kinds of alkaloids have been extracted from plants and their diverse biological activities have been reported. However, research on the isoquinoline alkaloid, a specific type of alkaloids, in Z. bungeanum was scarce. AIM OF THE STUDY: The study aimed to isolate a novel isoquinoline alkaloid from Z. bungeanum and explore its pharmacological activity in vitro and analgesic activity in vivo. MATERIALS AND METHODS: Isoquinoline alkaloid isolation and identification from Z. bungeanum were conducted using chromatographic and spectroscopic methods. The whole-cell patch-clamp technique was applied to assess its impact on neuronal excitability, and endogenous voltage-gated potassium (Kv) and sodium (Nav) currents in acutely isolated mouse small-diameter dorsal root ganglion (DRG) neurons. Its inhibitory impacts on channels were further validated with HEK293 cells stably expressing Nav1.7 and Nav1.8, and Chinese hamster ovary (CHO) cells transiently expressing Kv2.1. The formalin inflammatory pain model was utilized to evaluate the potential analgesic activity in vivo. RESULTS: A novel isoquinoline alkaloid named HJ-69 (N-13-(3-methoxyprop-1-yl)rutaecarpine) was isolated and identified from Z. bungeanum for the first time. HJ-69 significantly suppressed the firing frequency and amplitudes of action potentials in DRG neurons. Consistently, it state-dependently inhibited endogenous Nav currents of DRG neurons, with half maximal inhibitory concentration (IC50) values of 13.06 ± 2.06 µM and 30.19 ± 2.07 µM for the inactivated and resting states, respectively. HJ-69 significantly suppressed potassium currents in DRG neurons, which notably inhibited the delayed rectifier potassium (IK) currents (IC50 = 6.95 ± 1.29 µM) and slightly affected the transient outward potassium (IA) currents (IC50 = 523.50 ± 39.16 µM). Furtherly, HJ-69 exhibited similar potencies on heterologously expressed Nav1.7, Nav1.8, and Kv2.1 channels, which correspondingly represent the main components in neurons. Notably, intraperitoneal administration of 30 mg/kg and 100 mg/kg HJ-69 significantly alleviated pain behaviors in the mouse inflammatory pain model induced by formalin. CONCLUSION: The study concluded that HJ-69 is a novel and active isoquinoline alkaloid, and the inhibition of Nav and Kv channels contributes to its analgesic activity. HJ-69 may be a promising prototype for future analgesic drug discovery based on the isoquinoline alkaloid.

Neurotropin® ameliorates chronic pain associated with scar formation in a mouse model: A gene expression analysis of the inflammatory response.

Background: Scar formation after trauma and surgery involves an inflammatory response and can lead to the development of chronic pain. Neurotropin® (NTP) is a nonprotein extract of inflamed skin of rabbits inoculated with vaccinia virus. It has been widely used for the treatment of chronic pain. However, the in vivo effects of NTP on painful scar formation have not been determined. To investigate the molecular mechanisms underlying the effects of NTP on the inflammatory response, we evaluated gene expression in the scar tissues and dorsal root ganglions (DRGs) of mice administered NTP and control mice. Methods and results: Mice injected with saline or NTP were used as controls; other mice were subjected to surgery on the left hind paw to induce painful scar formation, and then injected with saline or NTP. Hind paw pain was evaluated by measuring the threshold for mechanical stimulation using the von Frey test. The paw withdrawal threshold gradually returned to pre-operative levels over 4 weeks post-operation; NTP-treated mice showed a significantly shortened recovery time of approximately 3 weeks, suggesting that NTP exerted an analgesic effect in this mouse model. Total RNA was extracted from the scarred hind paw tissues and DRGs were collected 1 week post-operation for a microarray analysis. Gene set enrichment analysis revealed that the expression of some gene sets related to inflammatory responses was activated or inhibited following surgery and NTP administration. Quantitative real-time reverse transcription-polymerase chain reaction analysis results for several genes were consistent with the microarray results. Conclusion: The administration of NTP to the hind paws of mice with painful scar formation following surgery diminished nociceptive pain and reduced the inflammatory response. NTP inhibited the expression of some genes involved in the response to surgery-induced inflammation. Therefore, NTP is a potential therapeutic option for painful scar associated with chronic pain.

8-shogaol derived from dietary ginger alleviated acute and inflammatory pain by targeting TRPV1.

Ginger, a well-known spice plant, has been used widely in medicinal preparations for pain relief. However, little is known about its analgesic components and the underlying mechanism. Here, we ascertained, the efficacy of ginger ingredient 8-Shogaol (8S), on inflammatory pain and tolerance induced by morphine, and probed the role of TRPV1 in its analgesic action using genetic and electrophysiology approaches. Results showed that 8S effectively reduced nociceptive behaviors of mice elicited by chemical stimuli, noxious heat as well as inflammation, and antagonized morphine analgesic tolerance independent on opioid receptor function. Genetic deletion of TRPV1 significantly abolished 8S' analgesia action. Further calcium imaging and patch-clamp recording showed that 8S could specifically activate TRPV1 in TRPV1-expressing HEK293T cells and dorsal root ganglion (DRG) neurons. The increase of [Ca2+]i in DRG was primarily mediated through TRPV1. Mutational and computation studies revealed the key binding sites for the interactions between 8S and TRPV1 included Leu515, Leu670, Ile573, Phe587, Tyr511, and Phe591. Further studies showed that TRPV1 activation evoked by 8S resulted in channel desensitization both in vitro and in vivo, as may be attributed to TRPV1 degradation or TRPV1 withdrawal from the cell surface. Collectively, this work provides the first evidence for the attractive analgesia of 8S in inflammatory pain and morphine analgesic tolerance mediated by targeting pain-sensing TRPV1 channel. 8S from dietary ginger has potential as a candidate drug for the treatment of inflammatory pain.

A next generation peripherally restricted Cavα2δ-1 ligand with inhibitory action on Cav2.2 channels and utility in neuropathic pain.

The Voltage-Gated Calcium Channel (VGCC) auxiliary subunit Cavα2δ-1 (CACNA2D1) is the target/receptor of gabapentinoids which are known therapeutics in epilepsy and neuropathic pain. Following damage to the peripheral sensory nervous system, Cavα2δ-1 is upregulated in dorsal root ganglion (DRG) neurons in several animal models of chronic neuropathic pain. Gabapentinoids, such as gabapentin and pregabalin, engage with Cavα2δ-1 via binding an arginine residue (R241) within an RRR motif located at the N-terminus of human Cavα2δ-1. A novel, next generation gabapentinoid, engineered not to penetrate the brain, was able to generate a strong analgesic response in Chronic Constriction Injury animal model of chronic neuropathic pain and showed binding specificity for Cavα2δ-1 versus the Cavα2δ-2 subunit. This novel non-brain penetrant gabapentinoid, binds to R241 and a novel binding site on Cavα2δ-1, which is located within the VGCC_α2 domain, identified as a lysine residue within an IKAK amino acid motif (K634). The overall whole cell current amplitudes were diminished by the compound, with these inhibitory effects being diminished in R241A mutant Cavα2δ-1 subunits. The functional effects occurred at lower concentrations than those needed for inhibition by gabapentin or pregabalin, which apparently bound the Cavα2δ-1 subunit only on the R241 and not on the K634 residue. Our work sets the stage for the identification and characterisation of novel compounds with therapeutic properties in neuropathic pain and possibly in other disorders and conditions which require engagement of the Cavα2δ-1 target.

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

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

Macrophage Infiltration Initiates RIP3/MLKL-Dependent Necroptosis in Paclitaxel-Induced Neuropathic Pain.

Paclitaxel (PTX) is a commonly used antitumor drug. Approximately 80% of all patients receiving PTX chemotherapy develop chemotherapy-induced peripheral neuropathy (CIPN), limiting the use of PTX. Moreover, CIPN responds poorly to conventional analgesics. Experimental evidence suggests that the neuroinflammatory response plays an essential role in paclitaxel-induced peripheral neuropathy (PIPN). Previous studies have confirmed that dorsal root ganglion (DRG) neuron necroptosis and accompanying inflammation are linked with PIPN; however, the potential upstream regulatory mechanisms remain unclear. Preclinical studies have also established that macrophage infiltration in the DRG is associated with PIPN. TNF-α released by activated macrophages is the primary regulatory signal of necroptosis. In this study, we established a rat model of PIPN via quartic PTX administration (accumulated dose: 8 mg/kg, i.p.). The regulatory effect of macrophage infiltration on necroptosis in PIPN was observed using a macrophage scavenging agent (clodronate disodium). The results showed that PTX increased macrophage infiltration and the levels of TNF-α and IL-1ß in the DRG. PTX also upregulated the levels of necroptosis-related proteins, including receptor-interacting protein kinase (RIP3) and mixed-lineage kinase domain-like protein (MLKL) in DRG neurons and promoted MLKL phosphorylation, resulting in neuronal necrosis and hyperalgesia. In contrast, clodronate disodium effectively removed macrophages, reduced the levels of RIP3, MLKL, and pMLKL, and decreased the number of necrotic cells in the DRG of PIPN rats, alleviating the behavioral pain abnormalities. These results suggest that PTX promotes macrophage infiltration, which results in the release of TNF-α and IL-1ß in the DRG and the initiation of neuronal necroptosis via the RIP3/MLKL pathway, ultimately leading to neuropathic pain.

CEBPß regulation of endogenous IGF-1 in adult sensory neurons can be mobilized to overcome diabetes-induced deficits in bioenergetics and axonal outgrowth.

Aberrant insulin-like growth factor 1 (IGF-1) signaling has been proposed as a contributing factor to the development of neurodegenerative disorders including diabetic neuropathy, and delivery of exogenous IGF-1 has been explored as a treatment for Alzheimer's disease and amyotrophic lateral sclerosis. However, the role of autocrine/paracrine IGF-1 in neuroprotection has not been well established. We therefore used in vitro cell culture systems and animal models of diabetic neuropathy to characterize endogenous IGF-1 in sensory neurons and determine the factors regulating IGF-1 expression and/or affecting neuronal health. Single-cell RNA sequencing (scRNA-Seq) and in situ hybridization analyses revealed high expression of endogenous IGF-1 in non-peptidergic neurons and satellite glial cells (SGCs) of dorsal root ganglia (DRG). Brain cortex and DRG had higher IGF-1 gene expression than sciatic nerve. Bidirectional transport of IGF-1 along sensory nerves was observed. Despite no difference in IGF-1 receptor levels, IGF-1 gene expression was significantly (P < 0.05) reduced in liver and DRG from streptozotocin (STZ)-induced type 1 diabetic rats, Zucker diabetic fatty (ZDF) rats, mice on a high-fat/ high-sugar diet and db/db type 2 diabetic mice. Hyperglycemia suppressed IGF-1 gene expression in cultured DRG neurons and this was reversed by exogenous IGF-1 or the aldose reductase inhibitor sorbinil. Transcription factors, such as NFAT1 and CEBPß, were also less enriched at the IGF-1 promoter in DRG from diabetic rats vs control rats. CEBPß overexpression promoted neurite outgrowth and mitochondrial respiration, both of which were blunted by knocking down or blocking IGF-1. Suppression of endogenous IGF-1 in diabetes may contribute to neuropathy and its upregulation at the transcriptional level by CEBPß can be a promising therapeutic approach.

Chondroitin and glucosamine sulphate reduced proinflammatory molecules in the DRG and improved axonal function of injured sciatic nerve of rats.

Neuropathic pain (NP) is an abnormality resulting from lesion or damage to parts of the somatosensory nervous system. It is linked to defective quality of life and often poorly managed. Due to the limited number of approved drugs, limited efficacy and side effects associated with the approved drugs, drugs or drug combinations with great efficacy and very minimal or no side effects will be of great advantage in managing NP. This study aimed at investigating the synergistic antinociceptive effects of the combination of glucosamine sulphate (GS) (240 mg/kg) and chondroitin sulphate (CS) (900 mg/kg) in chronic constriction injury (CCI)-induced neuropathy in rats. Forty-two Wistar rats were randomly distributed into seven groups (n = 6). Sciatic nerve was ligated with four loose ligatures to induce NP. Effects of drugs were examined on stimulus and non-stimulus evoked potentials, expression of dorsal root ganglia (DRG) pain modulators and structural architecture of DRG. Oral administration of GS and CS for 21 days reduced hyperalgesia, allodynia, sciatic nerve functional aberration and DRG pain modulators. Histopathology and immunohistochemistry revealed restoration of structural integrity of DRG. Our result showed that the combination of GS and CS produced antinociceptive effects by attenuating hyperalgesia, allodynia and downregulation of NP mediators. GS and CS additionally produced synergistic analgesic effect over its individual components.

Effect of Shixiao San on inflammatory factors and pain in rats with endometriosis.

ETHNOPHARMACOLOGICAL RELEVANCE: In the practice of traditional Chinese medicine, endometriosis is believed to be caused by blood stasis and is characterised by dysmenorrhea, which is difficult to control. Shixiao San (SXS) has a long history of use in the treatment of gynaecological diseases. The prescriptions composed of SXS include Typhae Pollen and Faeces Trogopterori, both of which have anti-inflammatory activity. In addition, Typhae Pollen can be used to treat many kinds of blood stasis diseases. AIM OF THE STUDY: The purpose of the present study was to investigate the effect of SXS on pain relief in rats with endometriosis and to preliminarily explore its mechanism of action in alleviating pain. MATERIAL AND METHODS: Ten rats received sham operation as the Sham group, and 30 endometriosis model rats were randomly divided into three groups: the Model, Shixiao San-Low (SXS-L), and Shixiao San-High (SXS-H) groups. The rats were administered the appropriate treatment via intragastric gavage for 4 weeks. The thermal radiation pain and mechanical pain thresholds of the rats were measured every 7 days after treatment. Finally, the distribution density of nerve fibres in endometrial tissue, the inflammatory infiltration of the dorsal root ganglion (DRG), the expression of TRPV1 in the DRG, and the expression of IL-1ß, TNF-α, and IL-6 in ectopic tissue were measured. RESULTS: After SXS treatment, the growth of ectopic tissue in rats with endometriosis was significantly suppressed, their thermal radiation pain and mechanical pain thresholds increased, the density of nerve fibres and the expression of inflammatory factors in ectopic tissues reduced, and inflammatory cells infiltration in the DRG of the animals alleviated. Meanwhile, the expression of TRPV1 in the DRG was downregulated in rats with endometriosis. CONCLUSIONS: SXS could possibly inhibit the development of endometriosis and relieve pain in patients with endometriosis by reducing inflammatory responses in ectopic tissue and the DRG.

IQM-PC332, a Novel DREAM Ligand with Antinociceptive Effect on Peripheral Nerve Injury-Induced Pain.

Neuropathic pain is a form of chronic pain arising from damage of the neural cells that sense, transmit or process sensory information. Given its growing prevalence and common refractoriness to conventional analgesics, the development of new drugs with pain relief effects constitutes a prominent clinical need. In this respect, drugs that reduce activity of sensory neurons by modulating ion channels hold the promise to become effective analgesics. Here, we evaluated the mechanical antinociceptive effect of IQM-PC332, a novel ligand of the multifunctional protein downstream regulatory element antagonist modulator (DREAM) in rats subjected to chronic constriction injury of the sciatic nerve as a model of neuropathic pain. IQM-PC332 administered by intraplantar (0.01-10 µg) or intraperitoneal (0.02-1 µg/kg) injection reduced mechanical sensitivity by ≈100% of the maximum possible effect, with ED50 of 0.27 ± 0.05 µg and 0.09 ± 0.01 µg/kg, respectively. Perforated-patch whole-cell recordings in isolated dorsal root ganglion (DRG) neurons showed that IQM-PC332 (1 and 10 µM) reduced ionic currents through voltage-gated K+ channels responsible for A-type potassium currents, low, T-type, and high voltage-activated Ca2+ channels, and transient receptor potential vanilloid-1 (TRPV1) channels. Furthermore, IQM-PC332 (1 µM) reduced electrically evoked action potentials in DRG neurons from neuropathic animals. It is suggested that by modulating multiple DREAM-ion channel signaling complexes, IQM-PC332 may serve a lead compound of novel multimodal analgesics.

An experimental study of tangzhouling on morphological changes of nissl bodies in the dorsal root ganglion of DM rats / Un estudio experimental de tangzhouling sobre los cambios morfológicos de los cuerpos de nissl en el ganglio de la raíz dorsal de ratas DM

SUMMARY: This study aims to investigate the effect of Tangzhouling on the morphological changes of Nissl bodies in the dorsal root ganglion of DM Rats. In this study, 69 rats were randomly divided into a control group (n = 10) and a model group (n = 59). The rats in the model group were randomly divided into a diabetic group (n = 11), a vitamin C group (n = 12), a low dose Tangzhouling group (n = 12), a medium dose Tangzhouling group (n = 12) and a high dose Tangzhouling group (n = 12). The dose of Tangzhouling in the low dose group was 5 times that of the adult dose, being 0.44g/kg/d. The dose of Tangzhouling in the medium dose group was 10 times that of the adult dose, being 0.88g/kg/d. The dose of Tangzhouling in the high dose group was 20 times that of the adult dose, being 1.75g/kg/d. All doses above are crude drug dosages. Rats in the vitamin C group were given 10 times the dose of an adult, being, 0.05 g/ kg/d. The diabetic group and the control group were given the same amount of distilled water. Drug delivery time is 16 weeks. The dorsal root ganglion was placed in a freezing tube at the end of the experiment. The morphological changes of Nissl bodies in the dorsal root ganglion were detected by HE and Nissl staining. The study results showed that vitamin C had no significant effect on the quantity, size and nucleolus. Tangzhouling can improvee the morphology, quantity and nucleolus of Nissl bodies to a certain extent, and the high dose is better than the lower dose. Tangzhouling capsules can improve the nerve function of DM rats through Nissl bodies.

RESUMEN: Este estudio tuvo como objetivo investigar el efecto de Tangzhouling en los cambios morfológicos de los cuerpos de Nissl en el ganglio de la raíz dorsal de las ratas DM. En este estudio, 69 ratas se dividieron aleatoriamente en un grupo control (n = 10) y un grupo modelo (n = 59). Las ratas del grupo modelo se dividieron aleatoriamente en un grupo diabéticos (n = 11), un grupo vitamina C (n = 12), un grupo de dosis baja de Tangzhouling (n = 12), un grupo de dosis media de Tangzhouling (n = 12) y un grupo de dosis alta de Tangzhouling (n = 12). La dosis de Tangzhouling en el grupo de dosis baja fue 5 veces mayor que la dosis del adulto, siendo 0,44 g/kg/d. La dosis de Tangzhouling en el grupo de dosis media fue 10 veces mayor que la dosis del adulto, siendo 0,88 g/kg/d. La dosis de Tangzhouling en el grupo de dosis alta fue 20 veces mayor que la dosis del adulto, siendo 1,75 g/kg/d. Todas las dosis anteriores son dosis de fármaco crudo. Se les administró 10 veces la dosis de un adulto a las ratas del grupo vitamina C, siendo 0,05 g/kg/d. El grupo de diabéticos y el grupo de control recibieron la misma cantidad de agua destilada. El tiempo de entrega del fármaco fue de 16 semanas. El ganglio de la raíz dorsal se colocó en un tubo de congelación al final del experimento. Los cambios morfológicos de los cuerpos de Nissl en el ganglio de la raíz dorsal se detectaron mediante tinción de HE y Nissl. Los resultados del estudio mostraron que la vitamina C no tuvo un efecto significativo sobre la cantidad, el tamaño y el nucléolo. Tangzhouling puede mejorar la morfología, la cantidad y el nucléolo de los cuerpos de Nissl hasta cierto punto, y es mejor la dosis alta que la dosis baja. Las cápsulas de Tangzhouling pueden mejorar la función nerviosa de las ratas DM a través de los cuerpos de Nissl.

Pentobarbital may protect against neurogenic inflammation after surgery via inhibition of substance P release from peripheral nerves of rats.

The inflammatory response related to surgery is considered surgical inflammation. Most anesthetic agents directly or indirectly suppress the immune response. However, the intravenous anesthetics pentobarbital and ketamine were reported to inhibit the lipopolysaccharide-induced inflammatory response such as cytokines formation. Neurogenic inflammation is inflammation originating from the local release of inflammatory mediators, such as substance P (SP), by primary afferent neurons after noxious stimuli like surgery. Thus, in this study, we examined whether pentobarbital and ketamine suppress SP release from cultured dorsal root ganglion (DRG) neurons. DRG cells were dissected from male Wistar rats. Released SP was measured by radioimmunoassay. We demonstrated that higher concentrations of pentobarbital (100-1,000 µM) significantly inhibited capsaicin (100 nM)-induced, but not high K+ (50 mM)-induced, SP release from DRG cells, although a high concentration of ketamine (1 mM) did not. This study revealed that pentobarbital functions between the activation of vanilloid receptor subtype 1 (TRPV1) receptors, to which capsaicin selectively binds, and the opening of voltage-operated Ca2+ channels (VOCC) in the nerve endings. Therefore, the anti-inflammatory action of pentobarbital is mediated through different mechanisms than those of ketamine. Thus, the inhibitory effect of pentobarbital on SP release from peripheral terminals may protect against neurogenic inflammation after surgery.

Makatoxin-3, a thermostable Nav1.7 agonist from Buthus martensii Karsch (BmK) scorpion elicits non-narcotic analgesia in inflammatory pain models.

ETHNOPHARMACOLOGICAL RELEVANCE: Chronic pain management represents a serious healthcare problem worldwide. The use of opioid analgesics for pain has always been hampered by their side effects; in particular, the addictive liability associated with chronic use. Finding a morphine replacement has been a long-standing goal in the field of analgesia. In traditional Chinese medicine, processed Buthus martensii Karsch (BmK) scorpion has been used as a painkiller to treat chronic inflammatory arthritis and spondylitis, so called "Scorpio-analgesia". However, the molecular basis and the underline mechanism for the Scorpio-analgesia are still unclear. AIM OF THE STUDY: The study aims to investigate the molecular basis of "Scorpio analgesia" and identify novel analgesics from BmK scorpion. MATERIALS AND METHODS: In this study, the analgesic abilities were determined using formalin-, acetic acid- and complete Freund's adjuvant-induced pain models. The effect of BmK venom and processed BmK venom on Nav1.7 were detected by whole-cell voltage-clamp recordings on HEK293-hNav1.7 stable cell line. Action potentials in Dorsal root ganglion (DRG) neurons induced by Makatoxin-3-R58A were recorded in current-clamp mode. The content of Makatoxin-3 was detected using competitive enzyme-linked immunosorbent assay based on the Makatoxin-3 antibody. High performance liquid chromatography, western blot and circular dichroism spectroscopy were used to analysis the stability of Makatoxin-3. RESULTS: Here we demonstrate that Makatoxin-3, an α-like toxin in BmK scorpion venom targeting Nav1.7 is the critical component in Scorpio-analgesia. The analgesic effect of Makatoxin-3 could not be reversed by naloxone and is more potent than Nav1.7-selective inhibitors and non-steroidal anti-inflammatory drugs in inflammatory models. Moreover, a R58A mutant of Makatoxin-3 is capable of eliciting analgesia effect without inducing pain response. CONCLUSIONS: This study advances ion channel biology and proposes Nav1.7 agonists, rather than the presumed Nav1.7-only blockers, for non-narcotic relief of chronic pain.

Multitarget nociceptor sensitization by a promiscuous peptide from the venom of the King Baboon spider.

The King Baboon spider, Pelinobius muticus, is a burrowing African tarantula. Its impressive size and appealing coloration are tempered by reports describing severe localized pain, swelling, itchiness, and muscle cramping after accidental envenomation. Hyperalgesia is the most prominent symptom after bites from P. muticus, but the molecular basis by which the venom induces pain is unknown. Proteotranscriptomic analysis of P. muticus venom uncovered a cysteine-rich peptide, δ/κ-theraphotoxin-Pm1a (δ/κ-TRTX-Pm1a), that elicited nocifensive behavior when injected into mice. In small dorsal root ganglion neurons, synthetic δ/κ-TRTX-Pm1a (sPm1a) induced hyperexcitability by enhancing tetrodotoxin-resistant sodium currents, impairing repolarization and lowering the threshold of action potential firing, consistent with the severe pain associated with envenomation. The molecular mechanism of nociceptor sensitization by sPm1a involves multimodal actions over several ion channel targets, including NaV1.8, KV2.1, and tetrodotoxin-sensitive NaV channels. The promiscuous targeting of peptides like δ/κ-TRTX-Pm1a may be an evolutionary adaptation in pain-inducing defensive venoms.