Isolation, identification and electrophysiological activity of BmK M2 from Buthus martensii Karsch venom / 中国药科大学学报

@#This study aimed to isolate and identify novel toxin peptides targeting voltage-gated sodium channels (VGSGs) from the venom of the Buthus martensii Karsch (BmK) scorpion. Using G50-gel filtration, HPLC, peptide fingerprinting and amino acid sequencing, a novel sodium channel modulator, BmK M2, was identified from BMK scorpion. BmK M2 is a relatively abundant long chain polypeptide toxin in BmK scorpion venom with a molecular weight of 7 235.59, consisting of 64 amino acids and 4 pairs of disulfide bonds.Sequence alignment showed that the amino acid sequence of BmK M2 had high sequence and structural similarity to that of the discovered sodium channel toxins of BmK M1, BmK M3 and BmK M9, etc.BmK M2 is a potential new sodium channel modulator.Electrophysiological results revealed that BmK M2 can significantly enhance the activation, delay the steady-state inactivation and closed-state inactivation of Nav1.7, but has no activity on Nav1.8.BmK M2 can be used as a novel peptide probe for the study of the structure and function of Nav1.7 and the development of drugs targeting Nav1.7.

Sodium Channel NaV1. 7 and Neuropathic Pain / 中国生物化学与分子生物学报

Sodium channel Nav1. 7 is one of the subtypes of voltage-gated sodium channel. Most of it is expressed on the nociceptors of small C fibers in dorsal root ganglion (DRG). It has the characteristics of slow opening and slow closing of inactivation. It can produce a large amount of ramp current, reduce the threshold of action potential in sensory neurons, and amplify the external small and slow depolarization ramp current. Thus, it can increase the excitability of neurons and play a key role in the generation, transmission and regulation of pain. With the deepening of genetic research, Nav1. 7 channel has become a particularly attractive drug target in new analgesic therapy due to its function acquired mutation and function deletion mutation. However, the study found that Nav1. 7 channel improves neuronal excitability and participates in neuropathic pain through different ways in neuropathic pain caused by different factors, which has brought great obstacles to the research and development of Nav1. 7 selective inhibitors. Although the existing Nav1. 7 selective inhibitors have effective analgesic effects without obvious side effects or addiction problems, it is extremely difficult to find Nav1. 7 selective ligands. In addition, the existing Nav1. 7 selective inhibitors also differ in inhibitory efficacy, targeting, safety and feasibility due to different types of neuropathic pain. It is suggested that finding the general mechanism of Nav1. 7 channel acting on different neuropathic pain or the specific receptor binding site of Nav1. 7 channel may be the main direction of the research and development of Nav1. 7 selective inhibitors in the future. This paper briefly reviews the main role of Nav1. 7 channel in neuropathic pain caused by different factors.

Design, synthesis and biological evaluation of novel α-aminoamide derivatives / 国际药学研究杂志

Objective: To design and synthesize novel α-aminoamide derivatives for the treatment of neuropathic pain. Methods: Ralfinamide was used as a lead compound, and it's ring A was replaced by a benzoheterocyclic moiety(2, 3-dihydrobenzofuran or 2, 3-dihydrobenzo[b][1, 4]dioxin)to obtain the new compounds 5a-5f. The in vivo and in vitro analgesic activities of the new compounds were evaluated by the formalin test and the whole-cell patch clamp assay, respectively. Results: In the formalin test in vivo, compound 5e exhibited higher analgesic activities(60% higher)than ralfinamide(P<0.05). The whole-cell patch clamp assay showed that the inhibitory effect of all tested compounds, except for 5d, on sodium channel Nav1.7 was much weaker than ralfinamide (P<0.05 or P<0.01). Conclusion: A set of new compounds with better analgesic activities than ralfinamide were obtained via optimizing the structure of ring A of ralfinamide. The reults of this study can provide useful references for relative research.

Structure-based assessment of disease-related mutations in human voltage-gated sodium channels

Voltage-gated sodium (Na) channels are essential for the rapid upstroke of action potentials and the propagation of electrical signals in nerves and muscles. Defects of Na channels are associated with a variety of channelopathies. More than 1000 disease-related mutations have been identified in Na channels, with Na1.1 and Na1.5 each harboring more than 400 mutations. Na channels represent major targets for a wide array of neurotoxins and drugs. Atomic structures of Na channels are required to understand their function and disease mechanisms. The recently determined atomic structure of the rabbit voltage-gated calcium (Ca) channel Ca1.1 provides a template for homology-based structural modeling of the evolutionarily related Na channels. In this Resource article, we summarized all the reported disease-related mutations in human Na channels, generated a homologous model of human Na1.7, and structurally mapped disease-associated mutations. Before the determination of structures of human Na channels, the analysis presented here serves as the base framework for mechanistic investigation of Na channelopathies and for potential structure-based drug discovery.

Efficacy of Anti-NaV1.7 Antibody on the Sensory Nervous System in a Rat Model of Lumbar Intervertebral Disc Injury

PURPOSE: The pathophysiology of discogenic low back pain is not fully understood. Tetrodotoxin-sensitive voltage-gated sodium (NaV) channels are associated with primary sensory nerve transmission, and the NaV1.7 channel has emerged as an analgesic target. Previously, we found increased NaV1.7 expression in dorsal root ganglion (DRG) neurons innervating injured discs. This study aimed to examine the effect of blocking NaV1.7 on sensory nerves after disc injury. MATERIALS AND METHODS: Rat DRG neurons innervating the L5/6 disc were labeled with Fluoro-Gold (FG) neurotracer. Twenty-four rats underwent intervertebral disc puncture (puncture group) and 12 rats underwent sham surgery (non-puncture group). The injury group was divided into a saline infusion group (puncture+saline group) and a NaV1.7 inhibition group, injected with anti-NaV1.7 antibody (puncture+anti-NaV1.7 group); n=12 per group. Seven and 14 days post-surgery, L1 to L6 DRGs were harvested and immunostained for calcitonin gene-related peptide (CGRP) (an inflammatory pain marker), and the proportion of CGRP-immunoreactive (IR) DRG neurons of all FG-positive neurons was evaluated. RESULTS: The ratio of CGRP-IR DRG neurons to total FG-labeled neurons in the puncture+saline group significantly increased at 7 and 14 days, compared with the non-puncture group, respectively (p<0.05). Application of anti-NaV1.7 into the disc significantly decreased the ratio of CGRP-IR DRG neurons to total FG-labeled neurons after disc puncture at 7 and 14 days (40% and 37%, respectively; p<0.05). CONCLUSION: NaV1.7 antibody suppressed CGRP expression in disc DRG neurons. Anti-NaV1.7 antibody is a potential therapeutic target for pain control in patients with lumbar disc degeneration.

ReIationships between sodium channeI NaV 1.7 and pain / 中国药理学与毒理学杂志

Voltage-gated sodium channels (NaV1.1-NaV1.9) play important roles in the generation and maintenance of electrical excitability. NaV1.7 is preferentially expressed in peripheral somatic sensory neurons and sympathetic ganglion neurons. ln humans, gain-of-function mutations of SCN9A gene, which encodes NaV1.7, cause inherited neuropathic pain, whereas loss-of-function mutations result in a congenital indifference to pain without motor, cognitive and cardiac deficits. The effects of some analge-sics are associated, at least in part, with the NaV1.7 and selective NaV1.7 inhibitors have also been demonstrated to be analgesic in animal models. NaV1.7 has emerged as a potential target for the treat-ment of pain.

Effect of down-regulation of voltage-gated sodium channel Nav1.7 on activation of astrocytes and microglia in DRG in rats with cancer pain

Objective:To evaluate the effect of down-regulation of Nav1.7 on the activation of astrocytes and microglia in DRG of rats with cancer pain, and explore the transmission of the nociceptive information.Methods:Lentiviral vector harboring RNAi sequence targeting theNav1.7gene was constructed, and Walker 256 breast cancer cell and morphine was injected to build the bone cancer pain model and morphine tolerance model in rats. Lentiviral vector was injected. Rats in each model were divided into 4 groups: model group, PBS group, vehicle group and LV-Nav1.7 group. The expression levels of GFAP and OX42 in dorsal root ganglia (DRG) were measured.Results: After the animal model was built,the level of Nav1.7, GFAP and OX42 was improved obviously with the time prolonged, which was statistically significant (P<0.05). The expression level of GFAP and OX42 in the DRG in the LV-Nav1.7 group declined obviously compared to the model group, PBS group and vehicle group (P<0.05).Conclusions:Intrathecal injection of Navl.7 shRNA lentiviral vector can reduce the expression of Nav1.7 and inhibit the activation of astrocytes and microglia in DRG. The effort is also effective in morphine tolerance bone cancer pain model rats.

Effect of down-regulation of voltage-gated sodium channel Nav1.7 on activation of astrocytes and microglia in DRG in rats with cancer pain

OBJECTIVE@#To evaluate the effect of down-regulation of Nav1.7 on the activation of astrocytes and microglia in DRG of rats with cancer pain, and explore the transmission of the nociceptive information.@*METHODS@#Lentiviral vector harboring RNAi sequence targeting the Nav1.7 gene was constructed, and Walker 256 breast cancer cell and morphine was injected to build the bone cancer pain model and morphine tolerance model in rats. Lentiviral vector was injected. Rats in each model were divided into 4 groups: model group, PBS group, vehicle group and LV-Nav1.7 group. The expression levels of GFAP and OX42 in dorsal root ganglia (DRG) were measured.@*RESULTS@#After the animal model was built, the level of Nav1.7, GFAP and OX42 was improved obviously with the time prolonged, which was statistically significant (P<0.05). The expression level of GFAP and OX42 in the DRG in the LV-Nav1.7 group declined obviously compared to the model group, PBS group and vehicle group (P<0.05).@*CONCLUSIONS@#Intrathecal injection of Navl.7 shRNA lentiviral vector can reduce the expression of Nav1.7 and inhibit the activation of astrocytes and microglia in DRG. The effort is also effective in morphine tolerance bone cancer pain model rats.

Effect of down-regulation of voltage-gated sodium channel Nav1.7 on activation of astrocytes and microglia in DRG in rats with cancer pain

Objective: To evaluate the effect of down-regulation of Nav1.7 on the activation of astrocytes and microglia in DRG of rats with cancer pain, and explore the transmission of the nociceptive information. Methods: Lentiviral vector harboring RNAi sequence targeting the Nav1.7 gene was constructed, and Walker 256 breast cancer cell and morphine was injected to build the bone cancer pain model and morphine tolerance model in rats. Lentiviral vector was injected. Rats in each model were divided into 4 groups: model group, PBS group, vehicle group and LV-Nav1.7 group. The expression levels of GFAP and OX42 in dorsal root ganglia (DRG) were measured. Results: After the animal model was built, the level of Nav1.7, GFAP and OX42 was improved obviously with the time prolonged, which was statistically significant (P<0.05). The expression level of GFAP and OX42 in the DRG in the LV-Nav1.7 group declined obviously compared to the model group, PBS group and vehicle group (P<0.05). Conclusions: Intrathecal injection of Navl.7 shRNA lentiviral vector can reduce the expression of Nav1.7 and inhibit the activation of astrocytes and microglia in DRG. The effort is also effective in morphine tolerance bone cancer pain model rats.

Effect of Curing-injury Cataplasma on Analgesia and Expression of Nav1 . 7 in Model Rats with Formaldehyde-induced Inflammatory Pain / 世界科学技术-中医药现代化

This study was aimed to observe the analgesia of curing-injury Cataplasma and discuss the Nav1 . 7 expression in dorsal root ganglion ( DRG ) in model rats with formaldehyde-induced inflammatory pain . A total of 36 Sprague-Dawley rats were divided into three groups, which were the blank control group (n = 12), model group ( n = 12 ) , and treatment group ( n = 12 ) . The blank control group was without any treatment . The model group was injected with 0 . 1 mL 5% saline formalin on the left rear foot . The treatment group was applied with curing-injury Cataplasma on the left rear foot 24 h before the injection of 0 . 1 mL 5% saline formalin in the establishment of animal model . The behavior reactions to pain of model rats were observed . Dubuisson score was recorded and compared . Meanwhile , L3-6 DRG was collected from rats in each group . The expres-sion of Nav1 . 7 was detected by real-time quantitative PCR and western blot . The results showed that the pain reaction integral in the treatment group was lower than the model group ( P < 0 . 05 ) . Results from the real-time quantitative PCR showed that the relative expression of Nav1 . 7 mRNA in the model group was more than the treatment group . And the relative expression of Nav1 . 7 mRNA in the treatment group was more than the blank control group . There was significant difference among three groups ( P < 0 . 05 ) . There was no statistical difference at the three time points within three groups. Results from the western blot showed that the relative expression of Nav1 . 7 in the model group was more than the treatment group . And the expression of Nav1 . 7 in the treatment group was more than the blank control group . There was significant difference among three groups (P < 0.05). There was no statistical difference at the three time points within three groups. It was concluded that the curing-injury Cataplasma can alleviate inflammatory pain response in rats, and have certain analgesia effect . Meanwhile , it can influence Nav1 . 7 expression in DRG in model rats with formaldehyde-induced inflam-matory pain .