Purification And Analgesic Activity Of Ctxn From Naja Naja Atra Venom

Background: Snake venoms are largely a complex mixture of proteins,polypeptides,enzymes and other small molecular substances. Cytotoxins(CTXs),neurotoxins(NTs) and PLA2 are the main toxins. Cytotoxins ( CTXs ) are highly basic polypeptides consisting of 60-63 amino acid residues, These peptides show a three-finger motif in the three-dimensional structure. There are marked variations within intraspeces of snake venoms for theirs complicated components.Venom constitutions occur great variability even within the same snake species owing to their geographical differences , season of venom collection, diet ,sexes and age of snakes. Mukherjee et al. found that there existed high variations in two closely related snakes, Naja kaouthia and Naja naja collected in the same place and in the venom composition of Naja naja from three neighboring districts of India excluding age, sex and seasonal variation. Naja naja atra from the Elapidae family is broadly distributed in Southern China, Taiwan Northern Laos and Vietnam. Detection of the geographical variation of venom constitutions show great significance for the separation and purification of venom and emergency treatment of snakebite.Chronic neuropathic pain results from the disorders or injuries to the peripheral or central nervous system. The major clinical symptoms of neuropathic pain are hyperalgesia, allodynia and spontaneous ongoing pain. Currently, there are not completely effective treatment to relieve neuropathic pain of this condition, including the use of analgesic drugs such as non-steroidal anti-inflammatory drugs (NSAIDs) and opioids,both of which have limitations or side effects. Thereby, novel modalities of treatment and drugs are necessary to be investigated. Animal models of peripheral nerve injury, such as Chronic constriction injury model (CCI), Spared nerve injury model (SNI), Spinal nerve ligation model (SNL), which reproduce some of the symptoms of human neuropathic pain, are useful to elucidate the mechanisms underlying neuropathic painand to make improvements to current clinical treatments .Snake venoms have been demonstrated antinociceptive activity for a long time.and cobrotoxin,isolated neurotoxins from Naja naja atra have demonstrated significant analgesia in animal models. Crotalphine, from the venom of the South American rattlesnake crotalus durissus terrificus, mediated by activation of peripheralκopioid receptors,induces a potent antinociceptive effect in chronic constriction of sciatic nerve. Crotoxin, another major neurotoxin of Crotalus durissus terrificus venom, induces analgesia in a model of neuropathic pain induced by rat sciatic nerve transection mediated by activation of central muscarinic receptors and by activation of 5-HT receptors. Further studies showed that cardiotoxin has various pharmacological functions such as hemolysis, cardiac toxicity,cytotoxicity, cell apoptosis and anti-tumor cells. Recently, Dexian Donga and Jiang WJ reported the analgesia activity of snake cytotoxins,respectively.Compared with neurotoxins, Cytotoxins show abound application future.The analgesia activity and mechanism of snake cytotoxins had become the new research targets of the antinociception of snake toxin. The present study was undertaken to evaluate the analgesia effect of snake cytotoxins .The possible mechanisms involved in the antinociceptive effect of the venom were also evaluated.Objectives: To analyze the components variation of Naja naja atra venoms from five regions in central south of china with RP-HPLC ; Then separate and purify Naja naja atra venom from Yichun region,Jiangxi Province,where the contents of CTXs is abound in naja naja atra venom and select the contents with antinociceptive activity primarily. After the physical and chemical identification, The analgesia induced by CTXs was investigated using the acute pain models and the neuropathic pain models.Meanwhile, The possible mechanisms involved in the antinociceptive effect of the venom were also evaluated by microdialysis and RP-HPLC.Methods:1. Purification and identification of CTXs from Naja naja atra venom1.1 To analyze the venom samples from six regions in central south of china with RP-HPLC The snake venom samples from six regions in central south of china were collected and gathered. Then the geographical variation of snake venom constitutions were detected with RP-HPLC.1.2 Purification and identification of CTXs from Naja naja Atra venom Naja naja Atra venoms were separated and purified by Sephadex G-75 chromatography and ion-exchange chromatography.Then the isolated fractions with antinociceptive activity primarily were selected. The purity analysis,MALDI-TOF mass spectrometry and protein N-terminal sequences were determined about the effective components.2. Evaluation of the toxicity and analgesia activity of CTXn 2.1 Toxicological experiments To detect the LD₅₀ of CTXn according to the Bliss methods, the drug tolerance and dependence of CTXn were assessed by the drug tolerance model,indirect ELISA assay,natural drug withdrawal test,precipitated withdrawal test and conditioned place preference(CPP)assay.2.2 Acute pain experiments The antinociceptive action was determined using the hot plate test and the acetic acid writhing test in mice and rats.2.3 Neuropathic pain experiments The antinociceptive effect of CTXn in neuropathic pain model induced by L5 spinal nerve ligation (SNL) was evaluated. The involvement of cholinergic and the opioid peptidergic systems in CTXn-induced analgesia were examined by pretreatment of animals with atropine or naloxone.2.4 Spontaneous activity test Possible changes in locomotor activity induced by CTXn were vestigated in the open field test.3. Detection the effect of CTXn on the release of monosmines in spinal dorsal horn in rats3.1 Detection the response of CTXn on the release of NA and 5-HT in spinal dorsal horn in SNL rats by microdialysis and RP-HPLC in real time.3.1 Evaluation the reaction of pretreatment with naloxone and atropine on the release of NA and 5-HT in spinal dorsal horn in CTXn-induced analgesia rats.4. Statistical analysisData were analyzed using one-way analysis of variance followed by the Dunnett's t-test and SPSS software.Results:1. The naja naja atra venom samples from six regions in central south of china were analyzed with RP-HPLC. The results showed that the main components in different regions were similar,however, there were geographical variation on the quantities of the components in different regions. The content level of CTXn was the highest in Jiangxi area, which couldn't be found in the area of Guangxi.2. The naja naja Atra venoms were separated and purified by Sephadex G-75 chromatography and ion-exchange chromatography.The better effect of separation was acquired.And the purity of isolated fractions was above 95%.3. The primarily antinociceptive test and the physicochemical properties showed that CTXn had a good analgesia activity and a high therapy index.4. The toxicological experiments detected that the LD₅₀ of CTXn was 19.61±2.05 mg/kg. Prolong treatment with CTXn could induce the drug tolerance, ELISA assay could detect the antibody in rats after CTXn intraperitoneal administration two weeks.While intrathecal administration with CTXn couldn't induce the drug tolerance. The natural drug withdrawal test,precipitated withdrawal test and conditioned place preference(CPP)assay showed that Prolong treatment with CTXn couldn't result in the drug dependence.5. The acute pain experiments proved that CTXn(0.50mg/kg,1.0mg/kg and 2.0mg/kg.ip.) had antinociceptive action in hot plate test and the acetic acid writhing test with dose dependence.6. The neuropathic pain experiments results indicated that CTX induced a long lasting antinociceptive effect in neuropathic pain, The effect of CTX was blocked by naloxone (3 mg/kg, i.p.) ,but not by atropine (10 mg/kg, i.p.).7. The open field test indicated that CTXn with treatment dose had no effect on the spontaneous activity of rats.8. The microdialysis and RP-HPLC results showed that CTXn could increase the release of NA and 5-HT in spinal dorsal horn in rats. The effect of CTX was blocked by naloxone (3 mg/kg, i.p.).Conclusions:1. There were geographical variations on the quantities of the naja naja atra venom components from six regions in central south of china.The content level of CTXs were similar in adjacent areas.2. The better effect of separation of naja naja Atra venoms and high qualities was acquired by Sephadex G-75 chromatography and ion-exchange chromatography.3. Prolong treatment with CTXn could induce the drug tolerance, maybe associated with the production and neutralization of the antibody. While prolong intrathecal administration with CTXn couldn't induce the drug tolerance.4. CTXn had antinociceptive action in the acute pain experiments. CTX induced a long lasting antinociceptive effect in neuropathic pain, which is mediated by activation of central opioid receptors and by increase the release of NA and 5-HT derived from the Brainstem descending inhibitory system pathway modulate the action of CTXn. The central muscarinic system may not involve in the antinociception activity of CTXn.