Analgesic Effects of Tetrodotoxin Combined with Acetylsalicylic Acid on Acetic Acid-induced Abdominal Constriction and Formalin Test in Mice

Aim To study the effects of tetrodotoxin （TTX） combined with acetylsalicylic acid （ASA） on nociceptive stimulus in mice. Methods To assess the antinociceptive effects of TTX, ASA or TTX plus ASA, the acetic acid-induced abdominal constriction test and formalin pain test were used. Results TTX （0.5 - 4.0 μg· kg^-1 ） or ASA （25 - 200 mg· kg^-1 ） im produced a significant inhibition of acetic acid-induced abdominal constriction. The median inhibitory doses （ID508） were 2.1 μg· kg^-1 for TTX（ and 64 mg· kg^-1 for ASA. TTX and ASA also showed a dose-dependent inhibition of the second phase response in the formalin pain model, the ID508, being 2.3μg·kg^-1 and 74.2 mg· kg^-1, respectively. The ihteraction between TTX and ASA was synergistic, as evidenced by the fact that （1） when ASA alone compared with the combination of TTX （0.79 μg · kg^-1 or 0.39μg· kg^-1 ） and ASA, the ID508, of ASA reduced from 64.0 mg· kg^-1 to 5.8 mg· kg^-1 or 12.6 mg· kg^-1, and from 74.2 mg· kg^-1 to 7.4 mg· kg^-1 or 13.0 mg· kg^-1 on tile two models of nociceptive tests, respectively; and that （2） synergism in the analgesic effects was shown by isobiolographic analysis. Conclusion TTX, ASA and the combination of the two drags produce analgesic effects in acetic acid-induced abdominal constriction test and formalin-induced pain test. The interactions between TTX and ASA may be useful in developing novel analgesic agents.

Simultaneous identification and quantification of tetrodotoxin in fresh pufferfish and pufferfish-based products using immunoaffinity columns and liquid chromatography/quadrupole-linear ion trap mass spectrometry

In this study, we established a comprehensive method for simultaneous identification and quantification of tetrodotoxin （TTX） in fresh pufferfish tissues and pufferfish-based products using liquid chromatography/quadrupole-linear ion trap mass spectrometry （LC-QqLIT-MS）. TTX was extracted by 1% acetic acid-methanol, and most of the lipids were then removed by freezing lipid precipitation, followed by purification and concentration using immunoaffinity columns （IACs）. Matrix effects were substantially reduced due to the high specificity of the IACs, and thus, background interference was avoided. Quantitation analysis was therefore performed using an external calibration curve with standards prepared in mobile phase. The method was evaluated by fortifying samples at 1, 10, and 100 ng/g, respectively, and the recoveries ranged from 75.8%--107%, with a relative standard deviation of less than 15%. The TTX calibration curves were linear over the range of 1-1 000 ~tg/L, with a detection limit of 0.3 ng/g and a quantification limit of 1 ng/g. Using this method, samples can be further analyzed using an information- dependent acquisition （IDA） experiment, in the positive mode, from a single liquid chromatography-tandem mass spectrometry injection, which can provide an extra level of confirmation by matching the full product ion spectra acquired for a standard sample with those from an enhanced product ion （EPI） library. The scheduled multiple reaction monitoring method enabled TTX to be screened for, and TTX was positively identified using the IDA and EPI spectra. This method was successfully applied to analyze a total of 206 samples of fresh pufferfish tissues and pufferfish-based products. The results from this study show that the proposed method can be used to quantify and identify TTX in a single run with excellent sensitivity and reproducibility, and is suitable for the analysis of complex matrix pufferfish samples.

Effects of continuous peripheral nerve block by tetrodotoxin on growth associated protein-43 expression during neuropathic pain development

BACKGROUND： Peripheral nerve injury may lead to neuropathic pain and cause a markedly increase expression of growth associated protein-43 （GAP-43） in the spinal cord and dorsal root ganglion, local anesthetics blocking electrical impulse propagation of nerve fibers may also affect the expression of GAP-43 in the spinal cord and dorsal root ganglion. OBJECTIVE： To determine the effects of continuous peripheral nerve block by tetrodotoxin before and after nerve injury on GAP-43 expression in the dorsal root ganglion during the development of neuropathic pain. DESIGN： A randomized controlled animal experiment. SETTINGS： Department of Anesthesiology, the Second Hospital of Xiamen City; Department of Anesthesiology, the Second Affiliated Hospital of Shantou University Medical College. MATERIALS： Thirty-five Spragne Dawley （SD） rats, weighing 200 - 250 g, were randomly divided into four groups： control group （n =5）, simple sciatic nerve transection group （n =10）, peripheral nerve block before and after sciatic nerve transection groups （n =10）. All the sciatic nerve transection groups were divided into two subgroups according to the different postoperative survival periods： 3 and 7 days （n =5） respectively. Mouse anti-GAP-43 monoclonal antibody （Sigma Co., Ltd.）, supervision TM anti-mouse reagent （HRP, Changdao antibody diagnosis reagent Co., Ltd., Shanghai）, and HMIAS-100 image analysis system （Qianping Image Engineering Company, Tongji Medical University） were employed in this study. METHODS： This experiment was carried out in the Department of Surgery and Pathological Laboratory, the Second Affiliated Hospital of Shantou University Medical College from April 2005 to April 2006. ①The animals were anesthetized and the right sciatic nerve was exposed and transected at 1 cm distal to sciatic notch. ② Tetrodotoxin 10 μg/kg was injected percutaneously between the greater trochanter and the posterior superior iliac spine of fight hind limb to block the sciatic nerve proximally at 1 hour before or 4 hours after nerve injury respectively, the injection was repeated in all the rats every 12 hours.③ At 3 or 7 days after nerve injury, immunohistochemistry and image analysis were used to evaluate the expression of GAP-43 in the dorsal root ganglions of L5 to the transected sciatic nerve, and quantitative analysis was also performed. ④ Statistical analysis was performed using one way analysis of variance followed by t test. MAIN OUTCOME MEASURE： Expression of GAP-43 in the fight dorsal root ganglions of L5. RESULTS： All the 35 SD rats were involved in the final analysis of results. In normal rats, there were very low expressions of GAP-43 in the dorsal root ganglions. In simple sciatic nerve transection rats 3 and 7 days after sciatic nerve transection, the average absorbance value of GAP-43 immunopositive neurons were significantly different from that in normal rats （t =8.806, 6.771, P 〈 0.01）. Whereas 3 and 7 days after sciatic nerve transection in rats with peripheral nerve block before and after nerve injury, the average absorbance value of GAP-43 immunopositive neurons were not significantly different from that in normal rats （P 〉 0.05）. CONCLUSION： Local anesthetic continuous peripheral nerve block before or after nerve injury can suppress nerve injury induced high expression of GAP-43 during the development of neuropathic pain.

The development and optimization of ELISA for the determination of tetrodotoxin

Objective： To optimize the ELISA for the determination of tetrodotoxin. Methods： A competitive enzyme-linked immunosorbent assay （ELISA） was used. In the ELISA, 100 μl antigen （1. 0 μg/ml） was coated on the microtiter plate for 60 min at 37 C or over night at 4 C. The plate was then washed 3 times with PBS-T for 3-5 s each time. The optimal incubation time for monoclonal antibody （mAb）, goat anti-mice IgG peroxidase conjugate and OPD were 30 min, 20 min and 10 min at 37 C, re- spectively. Results.. The detection limit is 0. 05 ng in each well. The curve was linear for TTX doses be- tween 5-5 000 ng/ml （0. 25-250 ng for every assay）. The linear regress equation was Y = 0. 30 88X-0.17 41 （R=0.99 01）. The average callback for TTX of muscles and gonads were 99.74% and 100.30%, respectively. The sensitivity of optimization ELISA was 5 times than traditional method and the time of 1.8 h were saved. Conclusion： The optimized ELISA is an ideal method for the determination of tetrodotoxin.

特异性Tetrodotoxin亲和吸附微珠的制备及其提取性能

将横纹东方豚卵巢与醋酸乙醇溶液混合匀浆,经离心,加热,沉淀,脱脂等处理,制得粗提液,活性炭吸附粗提液得到粗毒液;以壳聚糖为载体合成亲和微珠分别来分离纯化粗提液和粗毒液,用传统的小鼠生物法检测TTX毒素量.潮湿活性炭对TTX的吸附量约为2μg/g,潮湿亲和微珠吸附量约为10μg/g;亲和微珠吸附提取粗毒液,回收率达51%,直接吸附粗提液,回收率达到80%,同时蛋白也得到有效的去除.

Effects of tetrodotoxin monoclonal antibody on the blocking action of tetrodotoxin on sodium channels

The effect of tetrodotoxin(TTX) monoclonal antibody (McAb) 8A5 on the blocking action of TTX on sodium channels was studied by using the electrophysiological technique of whole cell recording.We found the specific TTX McAb have the following characterizations: TTX sensitivity to NG108-15 cell was high , with sodium ion current of NG108-15 cell completely blocked by only 10-6 mol/L level of TTX; when the cell was treated with TTX McAb 8A5 for 1 min and 5min, after the sodium current was completely abolished by TTX, the sodium ion current was restored to 79. 44%?. 20% and 73. 89%?. 74% (n=5) of the control values respectively; when the cell was treated for 1 min with 8A5 and TTX which had been mixed for 1 h before added,the sodium ion current was maintained at 89. 21%?. 41% (n=4) of the control. These results indicated that TTX-induced blockage on the sodium ion current could be powerfully antagonized by TTX McAb 8A5 with two distinct administering ways.

Effects of dragon’s blood resin and its component loureirin B on tetrodotoxin-sensitive voltage-gated sodium currents in rat dorsal root ganglion neurons

Using whole-cell patch clamp technique on the membrane of freshly isolated dorsal root ganglion (DRG) neurons, the effects of dragons blood resin and its important component loureirin B on tetrodotoxin-sensitive (TTX-S) voltage-gated sodium currents were observed. The results show that both blood resin and loureirin B could suppress TTX-S voltage-gated sodium currents in a dose-dependent way. The peak current amplitudes and the steady-state activation and inactivation curves are also made to shift by 0.05% blood resin and 0.2 mmol/L loureirin B. These results demonstrate that the effects of blood resin on TTX-S sodium current may contrib-ute to loureirin B in blood resin. Perhaps the analgesic effect of blood resin is caused partly by loureirin B directly interfering with the nociceptive transmission of primary sensory neurons.

Modulation of dragon's blood on tetrodotoxin-resistant sodium currents in dorsal root ganglion neurons and identification of its material basis for efficacy

To clarify the modulation of dragon's blood on the tetrodotoxin-resistant (TTX-R) sodium currents in dorsal root ganglion (DRG) neurons and explore its corresponding material basis for the efficacy, using whole-cell patch clamp technique, the effects of dragon's blood and the combined effects of three components (cochinchinenin A, cochinchinenin B, and loureirin B) extracted from dragon's blood on the TTX-R sodium currents in acute-isolated DRG neurons of rats were observed. According to the operational definition of material basis for the efficacy of TCM established, the material basis of the modulation on the TTX-R sodium currents in DRG neurons of dragon's blood was judged from the experimental results. The drug interaction equation of Greco et al. was used to assess the interaction of the three components extracted from dragon's blood. This investigation demonstrated that dragon's blood suppressed the peak TTX-R sodium currents in a dose-dependent way and affected the activations of TTX-R sodium currents. The effects of the combination of cochinchinenin A, cochinchinenin B, and loureirin B were in good agreement with those of dragon's blood. Although the three components used alone could modulate TTX-R sodium currents, the concentrations of the three components used alone were respectively higher than those used in combination when the inhibition rates on the TTX-R sodium currents of them used alone and in combination were the same. The combined effects of the three components were synergistic. These results suggested that the interference with pain messages caused by the modulation of dragon's blood on TTX-R sodium currents in DRG neurons may explain some of the analgesic effect of dragon's blood and the corresponding material basis for the efficacy is the combination of cochinchinenin A, cochinchinenin B, and loureirin B.

产河豚毒素微生物的研究进展

【背景】河豚毒素(TTX)是已知毒性较强的海洋毒素之一,广泛分布于河鲀等海洋生物体内。作为典型的钠离子通道阻断剂,TTX在镇痛、戒毒和抗心律失常等医疗领域展现出巨大的应用价值。然而,因获取困难和产量低下,TTX在医药领域的应用受到制约。因此,利用微生物发酵生产TTX成为了一个备受关注的研究方向。【目的】了解TTX的概况、产TTX微生物的分布和分类学多样性、TTX的发酵生产研究及TTX在微生物和环境中的迁移机制。【进展】目前,学界对TTX的理化性质已有较为清晰的认识,但其来源仍无明确定论。自1986年以来,科研人员陆续从河鲀、蓝环章鱼(Octopus maculosus)等生物及其栖息环境中分离出约150株产TTX菌株,其中以弧菌属、芽胞杆菌属为主,这些发现不仅为TTX的微生物起源说提供了强有力的理论支撑,也为后续利用微生物发酵生产TTX的研究奠定了坚实的基础。许多学者对TTX菌株发酵产毒中关键影响因素以及TTX迁移机制进行了探究,并取得了一定的成果。【展望】未来研究可重点考虑定向改造产TTX菌株、完善菌株大规模培养条件、探索影响菌株产TTX能力的关键因素以及分析微生物群落的相互作用对其合成TTX的影响,从而为TTX生物合成机制的阐明提供借鉴。【意义】通过对产TTX微生物的综述,阐明TTX的生物合成途径,为实现TTX规模化生产,解决TTX获取困难、成本高的难题,推动其在医药领域的应用提供理论参考。

HPLC-柱后衍生荧光法测定水产品中河豚毒素含量结果的不确定度评定

通过建立HPLC-柱后衍生荧光法测定水产品中河豚毒素含量的不确定度评定方法,量化分析测定结果的不确定度分量,并对各分量相对贡献进行比较。结果表明:测定结果的不确定度主要来源于标准品的校准过程、样品称量、实验过程移取及定容、样品重复实验误差、前处理过程中的回收率不同等。其中校准不确定度中标准溶液配制的不确定度贡献最多,称样量不确定度最小;当水产品中河豚毒素的含量为90.6μg/kg时,其扩展不确定度为0.69μg/kg(包含因子k=2)。

Inhibitive effect of loureirin B plus capsaicin on tetrodotoxin-resistant sodium channel

OBJECTIVE: To investigate whether the effect of loureirin B plus capsaicin on tetrodotoxin-resistant(TTX-R) sodium channel.METHODS: By using whole-cell patch-clamp recordings, in acutely isolated dorsal root ganglion(DRG) neurons, the combined effects of loureirin B and capsaicin on TTX-R sodium channel were observed. Based on the data, the interaction between loureirin B and capsaicin in their modulation on TTX-R sodium channel was assessed.RESULTS: Loureirin B could not induce transient inward TRPV1 current. Capsazepine, a transient receptor potential vanilloid l(TRPV1) antagonist, could not attenuate the block of 0.64 mmol/L loureirin B on TTX-R sodium channel. There was no significant difference(P > 0.05) between IC_(50) of loureirin B(0.37 mmol/L) on TTX-R sodium channel in capsaicin-sensitive DRG neurons and that(0.38 mmol/L)in capsaicin-insensitive DRG neurons. However,there was a significant difference(P < 0.05) between the IC_(50) of capsaicin(0.28 μmol/L) on TTX-R sodium channel in capsaicin-sensitive DRG neurons and that(52.24 μmol/L) in capsaicin-insensitive DRG neurons. Four combinations composed of various concentrations of loureirin B and capsaicin could all inhibit TTX-R sodium currents but have different interactions between loureirin B and capsaicin.CONCLUSION: Loureirin B plus capsaicin could produce double blockage on TRPV1 and modulation on TTX-R sodium channel. The action of loureirin B onTTX-R sodium channel was independent ofTRPV1 but similar with that of capsaicin on TTX-R sodium channel in capsaicin-insensitive DRG neurons.

Adsorption of tetrodotoxin by flexible shape-memory polymers synthesized from silica-stabilized Pickering high internal phase emulsion

Flexible shape-memory polymers were synthesized by Pickering high internal phase emulsion(HIPE)polymerization and used to adsorb and separate tetrodotoxin(TTX)from an aqueous solution.SiO_(2)nanoparticles were used to stabilize the Pickering oil-in-water(O/W)HIPEs.We introduced imidazolium-modified bromobutyl rubber(IBR)with excellent mechanical properties and high viscosity into the emulsion system as the shape-memory monomer.The properties,such as shape memory and morphology,were characterized by various methods,and batches of static adsorption experiments were conducted to analyze the adsorption performance of SiO_(2)@IBR on TTX.The characterization revealed that the SiO_(2)@IBR had a porous structure and good shape memory.Thus,the combination of SiO_(2)particles and IBR prevented shedding of SiO_(2)and enhanced the mechanical and adsorption properties of SiO_(2)@IBR.The results of the adsorption experiments indicated that the SiO_(2)@IBR had good adsorption of TTX.Both the Langmuir and Freundlich models fitted the isothermal adsorption experiment process.The TTX adsorption capacity of SiO_(2)@IBR was about 290.44 mg/g at 308 K.The fitting results of the pseudo-first-order and pseudosecond-order kinetic models showed that the adsorption process involved both chemical bonding and physical adsorption.After 10 adsorption and desorption experiments,the adsorption capacity of SiO_(2)@IBR decreased less than 0.03%,indicating that it had good adsorption and regeneration performance.

Rapid Determination of Tetrodotoxin in Human Plasma by Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry

A sensitive analytical method was developed to determine tetrodotoxin（TTX） in human plasma samples using protein precipitation, followed by ultra performance liquid chromatography（UPLC） analysis coupled with tandem mass spectrometry（MS/MS） using 11-deoxytetrodotoxin（11-deoxyTTX） as an internal standard. The plasma samples were prepared using protein precipitation prior to being analyzed by UPLC-MSfMS to identify TTX over a zwitterionic-hydrophilic interaction liquid chromatography column. The retention time values of TTX and 11-deoxyTTX were 4.12 and 3.67 min, respectively. TTX and 11-deoxyTTX were monitored and quantitated on the basis of their ion transitions for their respective precursor ions to their product ions（i.e., m/z 320.0→162. l for TTX and m/z 304.0→176.0 for 11-deoxyTTX） in the multiple reaction-monitoring mode. The lower limit of quantification of this method was determined to be 0.0199 ug/mL. This method showed good linearity for plasma samples that contained TTX concentrations in the range of 0.0199--1.99 ng/mL. The specificity, precision, accuracy, matrix effect, and stability characteristics of this method were also examined. The intra-assay precision and accuracy ranged from 1.89% to 6.00% and from 92.21% to 100.00%, whereas the inter-assay precision and accuracy ranged from 0.64% to 7.75% and from 99.38% to 101.26%, respectively. This new method therefore represents a rapid, accurate, reliable, and highly sensitive method for the qualitative and quantitative analyses of a trace amount of TTX in human plasma samples.

HILIC-UPLC-MS/MS在两起河豚中毒事件病因确证和病情预测中的应用研究

目的 建立河豚毒素(TTX)亲水液相色谱串联质谱检测方法,查明两起食物中毒事件原因,根据TTX摄入量和体内残留量对患者病情进行预测,并实施抢救,为此类中毒事件处置提供参考。方法 运用亲水液相色谱串联质谱技术对食物和人体样本中的TTX进行检测,评估患者毒素摄入量和体内残留量,预测患者病情和预后情况。结果 方法准确性、重现性和灵敏度良好。TTX在10~200 ng/mL水平,线性相关系数r>0.999。血液中TTX回收率为80%~103%,鱼肉中TTX回收率为75%~87%,相对标准偏差为5.3%~9.2%。血液和鱼肉中TTX检出限分别为0.2 ng/mL和3.7μg/kg。案例1中患者A的TTX摄入量约为0.28 mg,患者B摄入量约为0.04 mg。洗胃后,二者血液中TTX降为摄入量的10%,远远低于致死剂量(0.5 mg),患者预后良好。相反,案例2中,洗胃后患者C血液中TTX残留量为1.9 mg,远远高于致死剂量(0.5 mg),患者死亡。结论 该研究建立的亲水液相色谱串联质谱技术不仅可以为河豚中毒的诊断提供定性确证依据,还可以测定食品及人体中TTX水平,为TTX摄入量、体内残留量判断以及病情和预后评估提供依据。

河鲀毒素及其在河鲀体内积累研究进展

河鲀毒素(tetrodotoxin,TTX)神经毒性极高,广泛存在于多种水生生物中。河鲀为含TTX的代表性生物,食用不当存在一定的安全隐患,极大地限制了河鲀产业的发展。随着近年河鲀产业的有条件放开,组学和分析化学等技术的不断进步,关于河鲀TTX来源和认识逐渐深入,为系统研究河鲀积累代谢TTX的调控机制提供了理论基础。本文综述了TTX的特性分布、毒性机制及在河鲀体内的积累等最新资料,总结了TTX在河鲀体内积累代谢分子机制的研究进展,为全面了解河鲀体内TTX积累途径和机制,及防控养殖河鲀食品安全风险提供参考。

胶体金免疫层析法快速检测水产品中河豚毒素的研究

该文将特异性识别河豚毒素的单克隆抗体加以胶体金标记用作示踪物,建立了豚毒素的胶体金免疫层析技术快速检测方法。优化了胶体金体系的pH值、抗原抗体浓度、离子浓度、表面活性剂种类以及样品前处理方法。结果显示:在最优条件下,建立的胶体金免疫层析法对河豚毒素的定量检出限为0.5 ng/mL,线性范围为0.8~10.6 ng/mL,定性检出限(裸眼判别)为12.0 ng/mL。河豚、织纹螺等样品的加标回收率为70.5%~110%,相对标准偏差为3.7%~7.1%,检测结果与LC-MS/MS法一致。所建立的胶体金免疫层析技术在现场快速检测方面具有良好的可行性和实用性,可用于大量样品的快速筛查和河豚毒素中毒后的快速诊断。

河鲀毒素——一种具有缓解癌性疼痛潜力的药物

河鲀毒素(tetrodotoxin,TTX)是一种剧毒的生物碱类天然神经毒素,具有选择性阻断钠离子通道作用。人体摄入TTX后会产生致命影响,对人类的致死剂量范围1.5~2.0 mg(血药浓度9μg/L)。但以远低于其半数致死量(LD50)给患者施用TTX时,却可以治疗多种疾病,包括海洛因和可卡因戒断症状、脊髓损伤、脑外伤、肿瘤、神经性疼痛和内脏疼痛等。研究人员经过几十年不懈地探索,在TTX应用于生物医学领域,尤其是治疗癌症相关疼痛方面取得可喜进展,本文着重介绍TTX作为缓解癌性疼痛药物的临床开发、作用的有效性和安全性等最新结果,展示TTX缓解癌性疼痛的前景。

河鲀毒素的检测技术与应用

河鲀毒素(tetrodotoxin,TTX)是毒性极强的小分子生物碱类毒素,包括中国在内的亚洲沿海国家因误食TTX污染食品而中毒的事件时有发生,其发病迅速且无特效解毒剂,对环境安全、食品安全与社会安全造成极大的威胁。通过检测食品与环境中的TTX含量可以实现TTX的风险预警,可有效防范TTX中毒事件的发生。本文梳理了4类TTX的检测技术,分析比较了传统的生物检测法、化学检测法、免疫检测法之间的优势、不足与实际应用进展,介绍了基于适配体技术的新型检测技术的兴起、发展与广阔的应用前景,对生物安全领域中TTX风险的管理与控制有现实意义。

产河豚毒素菌株的筛选、鉴定及透析培养

河豚毒素(Tetrodotoxin, TTX)在镇静、镇痛、麻醉、戒毒等神经性疾病治疗方面效果显著且无致瘾性,因此具有极好的应用前景,但由于其从河豚体内提取的成本过高,所以难以大量推广应用。越来越多的证据表明TTX来源于河豚体内共生的微生物。我们从野生横纹东方鲀(Takifugu oblongus)肝脏中分离得到了一株产TTX较高的芽孢杆菌3G2(Bacillus sp. 3G2),通过ELISA和胶体金的方法检测到其发酵液TTX含量为35 ng/mL。将该菌株接种于专用透析培养器中,不断流加新鲜培养基实现连续培养,同时实现简化分离工艺,对发酵液进行小鼠生物活性检测、TTX试纸条检测,并通过河豚毒素抗体免疫共沉淀纯化得到TTX粗品后进行高效液相色谱鉴定均表明发酵液中含有TTX且含量较高。本研究通过筛选产TTX的优质菌株进一步了证实河豚毒素外源性假说,并初步验证了透析法生产河豚毒素的优越性,为后续商业应用提供实践性基础。