Neuronal Sodium Channels Research Articles

Cardiac dysfunction associated with lacosamide in a premature infant with Hypoxic Ischemic Encephalopathy: A case report.

Lacosamide [Vimpat® Harris FRC Corporation. © 2022 UCB, Inc. Smyrna, GA 30080] is an antiseizure medication which acts through blockage of voltage-gated neuronal sodium channels. Its recent implementation in the neonatal population has been extrapolated from adult and pediatric data suggesting a favorable safety profile. (1) Of note, preterm infants have unique developmental characteristics that may predispose them to increased risk of adverse reactions. We present a case of a preterm neonate who developed left ventricular (LV) dysfunction coinciding with the initiation of lacosamide.

Unraveling the dynamics of firing patterns for neurons with impairment of sodium channels.

Various factors such as mechanical trauma, chemical trauma, local ischemia, and inflammation can impair voltage-gated sodium channels (Nav) in neurons. These impairments lead to a distinctive leftward shift in the activation and inactivation curves of voltage-gated sodium channels. The resulting sodium channel impairments in neurons are known to affect firing patterns, which play a significant role in neuronal activities within the nervous system. However, the underlying dynamic mechanism for the emergence of these firing patterns remains unclear. In this study, we systematically investigated the effects of sodium channel dysfunction on individual neuronal dynamics and firing patterns. By employing codimension-1 bifurcation analysis, we revealed the underlying dynamical mechanism responsible for the generation of different firing patterns. Additionally, through codimension-2 bifurcation analysis, we theoretically determined the distribution of firing patterns on different parameter planes. Our results indicate that the firing patterns of impaired neurons are regulated by multiple parameters, with firing pattern transitions caused by the degree of sodium channel impairment being more diverse than those caused by the ratio of impaired sodium channel and current. Furthermore, we observed that the firing pattern of tonic firing is more likely to be the norm in impaired sodium channel neurons, providing valuable insights into the signaling of impaired neurons. Overall, our findings highlight the intricate relationships among sodium channel impairments, neuronal dynamics, and firing patterns, shedding light on the impact of disruptions in ion concentration gradients on neuronal function.

Neurological Aspects of Sarcopenia: A Comprehensive Update on Pathophysiology, Diagnosis, and Therapeutic Advances

Sarcopenia is an age-related disorder characterized by progressive and generalized loss of skeletal muscle tissue. This condition affects approximately 9.9-40.4% of older adults, 2-34% of outpatients, and about 56% of hospitalized patients. Sarcopenia is classified into primary and secondary types, with primary sarcopenia resulting from the natural aging process and secondary sarcopenia caused by various factors such as sedentary lifestyle, disease, and nutrition. The pathophysiology of sarcopenia involves cellular mechanisms, including genetic alterations, telomere erosion, and proteostasis disruption. Mitochondrial dysfunction, epigenetic modifications, and metabolic factors also contribute to the condition. Neurological aspects, such as motor neuron loss and sodium channel dysfunction, play a crucial role in the development of sarcopenia. Prevention and treatment strategies include exercise interventions, nutritional strategies, and pharmacological interventions. Vitamin D supplementation, testosterone replacement therapy, and selective androgen receptor modulators (SARMs) are among the treatments explored. However, these treatments come with potential

Urothelium-derived prostanoids enhance contractility of urinary bladder smooth muscle and stimulate bladder afferent nerve activity in the mouse.

The transitional epithelial cells (urothelium) that line the lumen of the urinary bladder form a barrier between potentially harmful pathogens, toxins, and other bladder contents and the inner layers of the bladder wall. The urothelium, however, is not simply a passive barrier, as it can produce signaling factors, such as ATP, nitric oxide, prostaglandins, and other prostanoids, that can modulate bladder function. We investigated whether substances produced by the urothelium could directly modulate the contractility of the underlying urinary bladder smooth muscle. Force was measured in isolated strips of mouse urinary bladder with the urothelium intact or denuded. Bladder strips developed spontaneous tone and phasic contractions. In urothelium-intact strips, basal tone, as well as the frequency and amplitude of phasic contractions, were 25%, 32%, and 338% higher than in urothelium-denuded strips, respectively. Basal tone and phasic contractility in urothelium-intact bladder strips were abolished by the cyclooxygenase (COX) inhibitor indomethacin (10 µM) or the voltage-dependent Ca2+ channel blocker diltiazem (50 µM), whereas blocking neuronal sodium channels with tetrodotoxin (1 µM) had no effect. These results suggest that prostanoids produced in the urothelium enhance smooth muscle tone and phasic contractions by activating voltage-dependent Ca2+ channels in the underlying bladder smooth muscle. We went on to demonstrate that blocking COX inhibits the generation of transient pressure events in isolated pressurized bladders and greatly attenuates the afferent nerve activity during bladder filling, suggesting that urothelial prostanoids may also play a role in sensory nerve signaling.NEW & NOTEWORTHY This paper provides evidence for the role of urothelial-derived prostanoids in maintaining tone in the urinary bladder during bladder filling, not only underscoring the role of the urothelium as more than a barrier but also contributing to active regulation of the urinary bladder. Furthermore, cyclooxygenase products greatly augment sensory nerve activity generated by bladder afferents during bladder filling and thus may play a role in perception of bladder fullness.

Quercetin, Main Active Ingredient of Moutan Cortex, Alleviates Chronic Orofacial Pain via Block of Voltage-Gated Sodium Channel.

Chronic orofacial pain (COP) therapy is challenging, as current medical treatments are extremely lacking. Moutan Cortex (MC) is a traditional Chinese medicine herb widely used for chronic inflammatory diseases. However, the mechanism behind MC in COP therapy has not been well-established. The purpose of this study was to identify the active ingredients of MC and their specific underlying mechanisms in COP treatment. In this study, the main active ingredients and compound-target network of MC in COP therapy were identified through network pharmacology and bioinformatics analysis. Adult male Sprague-Dawley rats received oral mucosa lipopolysaccharide (LPS) injection to induce COP. Pain behaviors were evaluated by orofacial mechanical nociceptive assessment after intraganglionar injection. In vitro inflammatory cytokines in LPS-pretreated human periodontal ligament stem cells (hPDLSCs) and rat primary cultural trigeminal ganglion (TG) neurons were quantified by real-time quantitative polymerase chain reaction (RT-qPCR). Schrödinger software was used to verify the molecular docking of quercetin and critical targets. Whole-cell recording electrophysiology was used to evaluate the effect of quercetin on voltage-gated sodium (Na v ) channel in rat TG neurons. The assembled compound-target network consisted of 4 compounds and 46 targets. As 1 of the active components of MC correlated with most related targets, quercetin alleviated mechanical allodynia in LPS-induced rat model of COP (mechanical allodynia threshold median [interquartile range (IQR) 0.5 hours after drug administration: vehicle 1.3 [0.6-2.0] g vs quercetin 7.0 [6.0-8.5] g, P = .002). Gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that immune response and membrane functions play essential roles in MC-COP therapy. Five of the related targets were identified as core targets by protein-protein interaction analysis. Quercetin exerted an analgesic effect, possibly through blocking Na v channel in TG sensory neurons (peak current density median [IQR]: LPS -850.2 [-983.6 to -660.7] mV vs LPS + quercetin -589.6 [-711.0 to -147.8] mV, P = .006) while downregulating the expression level of proinflammatory cytokines-FOS (normalized messenger RNA [mRNA] level mean ± standard error of mean [SEM]: LPS [2. 22 ± 0.33] vs LPS + quercetin [1. 33 ± 0.14], P = .034) and TNF-α (normalized mRNA level mean ± SEM: LPS [8. 93 ± 0.78] vs LPS + quercetin [3. 77 ± 0.49], P < .0001). Identifying Na v as the molecular target of quercetin clarifies the analgesic mechanism of MC, and provides ideas for the development of novel selective and efficient chronic pain relievers.

Non-dravet syndrome epilepsy in 4 polish patients with SCN1A de novo mutations

Background: The spectrum of neurological disorders associated with mutations in SCN1A, which encodes the type 1 subunit of the neuronal voltage-gated sodium channel, is broad and can manifest as both epileptic (a heterogeneous group of epileptic syndromes) and non-epileptic conditions. Dravet syndrome (DRVT) and Genetic Epilepsy with Febrile Seizures plus (GEFS+) are the most frequent. However, there exist less common phenotypes with varying severity of clinical course that authors would like to highlight.

ATP-Induced Contractile Response of Esophageal Smooth Muscle in Mice.

The tunica muscularis of mammalian esophagi is composed of striated muscle and smooth muscle. Contraction of the esophageal striated muscle portion is mainly controlled by cholinergic neurons. On the other hand, smooth muscle contraction and relaxation are controlled not only by cholinergic components but also by non-cholinergic components in the esophagus. Adenosine triphosphate (ATP) is known to regulate smooth muscle contraction and relaxation in the gastrointestinal tract via purinergic receptors. However, the precise mechanism of purinergic regulation in the esophagus is still unclear. Therefore, the aim of the present study was to clarify the effects of ATP on the mechanical responses of the esophageal muscle in mice. An isolated segment of the mouse esophagus was placed in a Magnus's tube and longitudinal mechanical responses were recorded. Exogenous application of ATP induced contractile responses in the esophageal preparations. Tetrodotoxin, a blocker of voltage-dependent sodium channels in neurons and striated muscle, did not affect the ATP-induced contraction. The ATP-evoked contraction was blocked by pretreatment with suramin, a purinergic receptor antagonist. RT-PCR revealed the expression of mRNA of purinergic receptor genes in the mouse esophageal tissue. The findings suggest that purinergic signaling might regulate the motor activity of mouse esophageal smooth muscle.

Modeling toxin effects on sodium channels in neurons using real-time dynamic clamp

Modeling toxin effects on sodium channels in neurons using real-time dynamic clamp

Investigating the Mechanisms Underlying U46619-Induced Contraction on Porcine Lower Esophageal Sphincter.

Gastroesophageal reflux disease (GERD) is associated with an incompetent lower esophageal sphincter (LES), resulting in the reflux of gastric contents into the esophagus. U46619, a thromboxane A2 (TXA2) receptor agonist, induces contractions in various smooth muscles. Therefore, this study aimed to investigate the effects and mechanisms of action of U46619 on the porcine LES. To achieve this, contractions of the clasp and sling strips of the porcine LES, induced by U46619, were measured using isometric transducers. Furthermore, the contractile mechanism of U46619 in the porcine LES was investigated by pretreating the strips with atropine (a muscarinic receptor antagonist), tetrodotoxin (a neuronal sodium channel blocker), nifedipine (a calcium channel blocker), and Ca2+-free Krebs-Henseleit solution. Additionally, reverse transcription polymerase chain reaction (PCR) and immunohistochemistry (IHC) were performed to determine the presence of the TXA2 receptor in porcine LES. The results of this study demonstrated that U46619 caused marked concentration-dependent contractions in both porcine sling and clasp strips. The mechanism of U46619-induced contraction of the porcine LES was found to be related to calcium channels. Furthermore, the reverse transcription PCR analysis and IHC revealed that the TXA2 receptor was expressed in the clasp and sling fibers of porcine LES. Consequently, this study suggests that U46619 mediates the contraction of porcine LES through calcium channels and has potential as a therapeutic approach for treating GERD. SIGNIFICANCE STATEMENT: This study establishes that U46619 induces concentration-dependent contractions in porcine LES, primarily mediated by calcium channels. The presence of the TXA2 receptor in LES clasp and sling fibers is confirmed. These findings highlight U46619's potential as a GERD therapeutic by targeting calcium channels for LES contraction modulation.

REPRINTED WITH PERMISSION OF IASP – PAIN 164 (2023) 3–13: Ambroxol for neuropathic pain: hiding in plain sight?

Ambroxol is a multifaceted drug with primarily mucoactive and secretolytic actions, along with anti-inflammatory, antioxidant, and local anaesthetic properties. It has a long history of use in the treatment of respiratory tract diseases and has shown to be efficacious in relieving sore throat. In more recent years, ambroxol has gained interest for its potential usefulness in treating neuropathic pain. Research into this area has been slow, despite clear preclinical evidence to support its primary analgesic mechanism of action—blockade of voltage-gated sodium (Nav) channels in sensory neurons. Ambroxol is a commercially available inhibitor of Nav1.8, a crucial player in the pathophysiology of neuropathic pain, and Nav1.7, a particularly exciting target for the treatment of chronic pain. In this review, we discuss the analgesic mechanisms of action of ambroxol, as well as proposed synergistic properties, followed by the preclinical and clinical results of its use in the treatment of persistent pain and neuropathic pain symptoms, including trigeminal neuralgia, fibromyalgia, and complex regional pain syndrome. With its well-established safety profile, extensive preclinical and clinical drug data, and early evidence of clinical effectiveness, ambroxol is an old drug worthy of further investigation for repurposing. As a patent-expired drug, a push is needed to progress the drug to clinical trials for neuropathic pain. We encourage the pharmaceutical industry to look at patented drug formulations and take an active role in bringing an optimized version for neuropathic pain to market.

Differential Pharmacokinetic Interplay of Atorvastatin on Lacosamide and Levetiracetam on Experimental Convulsions in Mice.

The beneficial effects of statins, other than their hypocholesterolemia role, have been well documented, however, their use as an adjuvant drug with other antiseizure drugs, in the treatment of epilepsy is poorly understood. This study aimed to investigate the symbiotic effect of ATOR along with either lacosamide (LACO) or levetiracetam (LEVE) on experimentally induced epilepsy (Maximal electro-shock-MES or pentylenetetrazol- PTZ) in mice models. Conventional elevated-maze (EPM) and rotarod methods were performed to observe the behavioral effects. In both the animal models, we found that co-administration of ATOR along with LACO showed a significant reduction in hind-limb extension (HLE) and clonic convulsion (CC) responses, respectively, but not in the ATOR+LEVE treated group. Intriguingly, comparable Straub tail response and myoclonic convulsion as the diazepam (DIA) group were observed only in the ATOR+LACO treated group. Moreover, a significant muscle-grip strength was observed in both groups. Also, pharmacokinetic analysis has indicated that the mean plasma concentration of ATOR peaked at 2ndhr in the presence of LACO but marginally peaked in the presence of LEVE. An Insilico study has revealed that ATOR has a higher binding affinity toward neuronal sodium channels. This study has demonstrated that the plasma concentration of ATOR was potentiated in the presence of LACO, but not in the presence of LEVE and it has provided significant protection against both the electro and chemo-convulsive models in mice. This could be due to the symbiotic pharmacokinetic interplay of ATOR with LACO, and possibly, this interplay may interfere with sodium channel conductance.

In-silico Studies on Phthalimide GABA Analogs for Anticonvulsant Activity Against Sodium Channel and GABA-AT

Introduction: GABA is a type of inhibitory neurotransmitter; it blocks or inhibits certain nerve transmission. Our nervous system is calmed by GABA, thus preventing excessive fear or anxiety is a simple and rapid approach to raise and maintain healthy mental health. Some medical conditions associated with a change in the level of GABA are anxiety and mood disorders, schizophrenia, autism spectrum disorder, depression, epilepsy, and seizures. The aim of this study to increase the activity of GABA which can be enhanced by the incorporation of anthracene, phthalimide, benzene, and thalidomide. Materials and methods: Insilico Docking study was carried out by using Auto Dock 4.0 against GABA-AT protein and the binding energy was found out to be -7.61kcal/mol. GABA-Phthalimide substitution, derivatives were designed as they possess a similar degree of anticonvulsant potency due to their phenytoin-like profile. The phthalimide pharmacophores interaction with voltage dependent sodium channels in neurons was investigated. Results and conclusion: This suggests the substitution of GABA as a possible lead compound. Therefore, in the present study, N, N-phthaloyl GABA combine with aniline a Noval compound 4-(1,3-dioxoisoindolin-2-yl)-N-phenylbutanamide is designed with improved anticonvulsant properties.

Unusual stroke-like symptoms with oxaliplatin use.

Oxaliplatin has become the mainstay of treatment for many cancers, but its use can be accompanied by unusual side effects. We describe herein a 74-year-old patient with pancreatic cancer who developed severe motor weakness affecting lower extremities after starting treatment with oxaliplatin on three separate occasions. Our patient also experienced slurred speech, with decreased ability to phonate and word-finding difficulty. Brain imaging studies did not suggest recent brain ischemia, and the symptoms resolved within 15-20 h. Oxaliplatin had to be discontinued due to suboptimal tolerance and a short-lived clinical response. After discontinuation of oxaliplatin, she did not experience any more similar symptoms. A score of 9 on the Naranjo nomogram supported a definite causality relationship between oxaliplatin and the observed neurologic toxicity. Rare reports of stroke-like events have previously been described with oxaliplatin. While the exact mechanism of these phenomena is not known, alterations in neuronal sodium channels might be involved. Clinicians, pharmacists, and patients need to be aware of these rare but important side effects of oxaliplatin. Nonetheless, work-up for a cerebrovascular accident is still warranted as hypercoagulability related to malignancy can also predispose the patients to strokes.

Direct fluorescent labeling of NF186 and NaV1.6 in living primary neurons using bioorthogonal click chemistry.

The axon initial segment (AIS) is a highly specialized neuronal compartment that regulates the generation of action potentials and maintenance of neuronal polarity. Live imaging of the AIS is challenging due to the limited number of suitable labeling methods. To overcome this limitation, we established a novel approach for live labeling of the AIS using unnatural amino acids (UAAs) and click chemistry. The small size of UAAs and the possibility of introducing them virtually anywhere into target proteins make this method particularly suitable for labeling of complex and spatially restricted proteins. Using this approach, we labeled two large AIS components, the 186 kDa isoform of neurofascin (NF186; encoded by Nfasc) and the 260 kDa voltage-gated Na+ channel (NaV1.6, encoded by Scn8a) in primary neurons and performed conventional and super-resolution microscopy. We also studied the localization of epilepsy-causing NaV1.6 variants with a loss-of-function effect. Finally, to improve the efficiency of UAA incorporation, we developed adeno-associated viral (AAV) vectors for click labeling in neurons, an achievement that could be transferred to more complex systems such as organotypic slice cultures, organoids, and animal models.

Generation of a human iPSC line (ZJSHDPi001-A) from peripheral blood mononuclear cells of a patient with Developmental epileptic encephalopathy-47 carrying FGF12 gene mutation (c.334G > A)

Developmental epileptic encephalopathy-47 (DEE47) is a nervous system disease characterized by the onset of intractable seizures that appear the first days or weeks after birth. FGF12 is the disease-causing gene of DEE47 that encodes a small cytoplasm protein, which is a member of the fibroblast growth factor homologous factor (FGF) family. The FGF12-encoded protein interacts with the cytoplasmic tail of voltage-gated sodium channels to enhance the voltage dependence of rapid inactivation of sodium channels in neurons. This study used non-insertion Sendai virus transfection to establish the induced pluripotent stem cells(iPSCs)line with FGF12 mutation. The cell line was obtained from a 3-year-old boy carrying the c.334G > A heterozygous mutation in the FGF12 gene. This iPSC line could facilitate the investigations of pathogeneses of complex nervous system diseases such as developmental epileptic encephalopathy.

Argentatin C Analogues with Potential Antinociceptive Activity and Other Triterpenoid Constituents from the Aerial Parts of Parthenium incanum.

Four new triterpenes, 25-dehydroxy-25-methoxyargentatin C (1), 20S-hydroxyargentatin C (2), 20S-hydroxyisoargentatin C (3), and 24-epi-argentatin C (4), together with 10 known triterpenes (5-14) were isolated from the aerial parts of Parthenium incanum. The structures of 1-4 were elucidated by detailed analysis of their spectroscopic data, and the known compounds 5-14 were identified by comparison of their spectroscopic data with those reported. Since argentatin C (11) was found to exhibit antinociceptive activity by decreasing the excitability of rat and macaque dorsal root ganglia (DRG) neurons, 11 and its new analogues 1-4 were evaluated for their ability to decrease the excitability of rat DRG neurons. Of the argentatin C analogues tested, 25-dehydroxy-25-methoxyargentatin C (1) and 24-epi-argentatin C (4) decreased neuronal excitability in a manner comparable to 11. Preliminary structure-activity relationships for the action potential-reducing effects of argentatin C (11) and its analogues 1-4, and their predicted binding sites in pain-relevant voltage-gated sodium and calcium channels (VGSCs and VGCCs) in DRG neurons are presented.

Sympathetic Sudomotor Ganglionopathy Following Envenomation from a Northern Australia Jellyfish Irukandji Syndrome

The oceans of northern Australia coastal beaches harbor numerous venomous jellyfish. Several species cause Irukandji Syndrome, a severe disorder (developing within an hour after a sting) consisting painful muscle cramps, vomiting, sweating, agitation, vasoconstriction, prostration, hypertension, cardiac irregularities. The jellyfish toxin may modulate neuronal sodium channels leading to a massive release of endogenous catecholamines. Segmental sympathetic cardiovascular hyperactivity has been emphasized but this case report portrays a dramatic effect on segmental sympathetic sudomotor function.

Melatonin attenuated chronic visceral pain by reducing Nav1.8 expression and nociceptive neuronal sensitization.

Irritable bowel syndrome (IBS) is a common functional gastrointestinal disorder, and its specific pathogenesis is still unclear. We have previously reported that TTX-resistant (TTX-R) sodium channels in colon-specific dorsal root ganglion (DRG) neurons were sensitized in a rat model of visceral hypersensitivity induced by neonatal colonic inflammation (NCI). However, the detailed molecular mechanism for activation of sodium channels remains unknown. This study was designed to examine roles for melatonin (MT) in sensitization of sodium channels in NCI rats. Colorectal distention (CRD) in adult male rats as a measure of visceral hypersensitivity. Colon-specific dorsal root ganglion (DRG) neurons were labeled with DiI and acutely dissociated for measuring excitability and sodium channel current under whole-cell patch clamp configurations. Western blot and Immunofluorescence were employed to detect changes in expression of Nav1.8 and MT2. The results showed that rats exhibited visceral hypersensitivity after NCI treatment. Intrathecal application of melatonin significantly increased the threshold of CRD in NCI rats with a dose-dependent manner, but has no role in the control group. Whole-cell patch clamp recording showed that melatonin remarkably decreased the excitability and the density of TTX-R sodium channel in DRG neurons from NCI rats. The expression of MT2 receptor at the protein level was markedly lower in NCI rats. 8MP, an agonist of MT2 receptor, enhanced the distention threshold in NCI rats. Application of 8MP reversed the enhanced hypersensitivity of DRG neurons from NCI rats. 8MP also reduced TTX-R sodium current density and modulated dynamics of TTX-R sodium current activation. These data suggest that sensitization of sodium channels of colon DRG neurons in NCI rats is most likely mediated by MT2 receptor, thus identifying a potential target for treatment for chronic visceral pain in patients with IBS.

Pathogenic SCN2A variants cause early-stage dysfunction in patient-derived neurons.

Pathogenic heterozygous variants in SCN2A, which encodes the neuronal sodium channel NaV1.2, cause different types of epilepsy or intellectual disability (ID)/autism without seizures. Previous studies using mouse models or heterologous systems suggest that NaV1.2 channel gain-of-function typically causes epilepsy, whereas loss-of-function leads to ID/autism. How altered channel biophysics translate into patient neurons remains unknown. Here, we investigated iPSC-derived early-stage cortical neurons from ID patients harboring diverse pathogenic SCN2A variants [p.(Leu611Valfs*35); p.(Arg937Cys); p.(Trp1716*)] and compared them with neurons from an epileptic encephalopathy (EE) patient [p.(Glu1803Gly)] and controls. ID neurons consistently expressed lower NaV1.2 protein levels. In neurons with the frameshift variant, NaV1.2 mRNA and protein levels were reduced by ~ 50%, suggesting nonsense-mediated decay and haploinsufficiency. In other ID neurons, only protein levels were reduced implying NaV1.2 instability. Electrophysiological analysis revealed decreased sodium current density and impaired action potential (AP) firing in ID neurons, consistent with reduced NaV1.2 levels. In contrast, epilepsy neurons displayed no change in NaV1.2 levels or sodium current density, but impaired sodium channel inactivation. Single-cell transcriptomics identified dysregulation of distinct molecular pathways including inhibition of oxidative phosphorylation in neurons with SCN2A haploinsufficiency and activation of calcium signaling and neurotransmission in epilepsy neurons. Together, our patient iPSC-derived neurons reveal characteristic sodium channel dysfunction consistent with biophysical changes previously observed in heterologous systems. Additionally, our model links the channel dysfunction in ID to reduced NaV1.2 levels and uncovers impaired AP firing in early-stage neurons. The altered molecular pathways may reflect a homeostatic response to NaV1.2 dysfunction and can guide further investigations.

Phytol from Faeces Bombycis alleviated migraine pain by inhibiting Nav1.7 sodium channels.

Faeces Bombycis (silkworm excrement, called Cansha in Chinese), is the dried faeces of the larvae of silkworm. According to the theories of traditional Chinese medicine recorded in "Compendium of Materia Medica", Faeces Bombycis has often been prescribed in traditional Chinese medicine for the treatment of recurrent headache, rheumatalgia, rubella and itching et al. However, the bioactive components and their exact mechanisms underlying the pain-relieving effects remain to be revealed. The present study aimed to evaluate the analgesic effect of Faeces Bombycis extract (FBE) on migraine, explore the main active constituents and investigate the pharmacological mechanisms for its pain relief. The bioactivity of different extracts from Faeces Bombycis was tracked by the nitroglycerin (NTG)-induced migraine model on rats and identified by NMR spectroscopic data. Whole-cell patch clamp technique, an electrophysiological method, was used to screen the potential targets and study the mechanism of action for the bioactive compound. The following targets have been screened and studied, including Nav1.7 sodium channels, Nav1.8 sodium channels, TRPV1 channels and TRPA1 channels. The trigeminal ganglion neurons were further used to study the effects of the identified compound on neuronal excitability. By testing the bioactivity of the different extracts proceedingly, fraction petroleum ether showed higher anti-migraine activity. Through further step-by-step isolations, 7 compounds were isolated. Among them, phytol was identified with the highest yield and displayed a potent anti-migraine effect. By screening the potential ion channel targets for migraine, phytol was found to preferentially block the inactivated state of Nav1.7 sodium channels with half-inhibition concentration 0.32±0.05μM. Thus, the effects of phytol on the biophysical properties of Nav1.7 sodium channels were further characterized. Phytol induced a hyperpolarizing shift of voltage-dependent inactivation and slowed the recovery from inactivation. The affinity of phytol became weaker in the inactivation-deficient Nav1.7 channels (Nav1.7-WCW). And such an effect was independent on the local anesthetic site (Nav1.7 F1737A). Consistent with the data from recombinant channels, the compound also displayed state-dependent inhibition on neuronal sodium channels and further decreased the neuronal excitability in trigeminal ganglion neurons. Moreover, besides Nav1.7 channel, phytol also antagonized the activation of TRPV1 and TRPA1 channels at micromolar concentrations with a weaker affinity. Our results demonstrated that phytol is the major anti-migraine ingredient of Faeces Bombycis and alleviates migraine behaviors by acting on Nav1.7 sodium channels in the trigeminal ganglion neurons. This study provided evidences for the therapeutic application of Faeces Bombycis and phytol on migraine disease.