发形霞水母触手提取物活性的初步研究

目的 初步分离发形霞水母触手提取物并探讨其生物学活性.方法采用自溶、离心的方法去除发形霞水母触手刺丝囊并获取触手提取物.通过阶段梯度阳离子交换色谱,应用0%.20%,40%,100%B液4种比例洗脱液将触手提取物分成4个组分,分别观察分离各组分的溶血、心血管等活性,并与触手提取物平行对比分析.结果成功分离到具有溶血、心血管活性的发形霞水母触手提取物,4个洗脱组分中,0%B液组分无上述3种活性,20%B液组分具有溶血活性、40%B液组分具有心血管活性,而100%B液组分则含有色素.结论通过阳离子交换色谱.初步将发形霞水母触手提取物中具有溶血活性、心血管活性以及色素等组分分离开来.为后续进一步纯化其单一活性组分打下基础.     

Isolation and characterization of lethal proteins in nematocyst venom of the jellyfish Cyanea nozakii Kishinouye

Cyanea nozakii Kishinouye, a jellyfish widely distributed in coastal areas of China, has garnered attention because of its stinging capacity and the resulting public health hazard. We used a recently developed technique to extract jellyfish venom from nematocysts; the present study investigates the lethality of C. nozakii venom. The nematocyst contents were extremely toxic to the grass carp, Ctenopharyngodon idellus, producing typical neurotoxin toxicity. The ID₅₀ was about 0.6 μg protein/g fish. Toxin samples were stable when kept at −80 ^°C, but after 48 h, an 80% decline in lethality occurred at −20 ^°C. Poor stability of the venom was observed within the range of 65-80 ^°C and at pH 3.5. The venom was hydrolyzed by a proteolytic enzyme, trypsin. Fractionation of the venom yielded two protein bands with molecular weights of 60 kDa and 50 kDa. Our results provide the first evidence that C. nozakii produces lethal toxins. These characteristics highlight the need for the isolation and molecular characterization of new active toxins in C. nozakii.     

发形霞水母触手提取物的蛋白稳定性及其溶血活性

目的探讨不同环境因素和处理方法对发形霞水母（Cyanea capillata）触手提取物（tentacle extract,TE）蛋白稳定性及其溶血活性的影响。方法结合蛋白浓度测定、溶血活性检测和SDS-PAGE分析等方法研究不同环境因素和处理方法对TE蛋白稳定性及其溶血活性的影响。结果 TE溶血活性呈明显的剂量依赖关系,其半数溶血分数HU50=226μg/ml;40℃水浴1 h,能去除TE中大量杂蛋白,但仍保持显著溶血活性;TE在4℃放置28 d,其溶血活性保持稳定,25℃下3 d内其溶血活性变化不明显;p H值对TE溶血活性的影响呈钟形曲线,在p H 6.0-11.0之间活性相对稳定,p H 8.0是TE保持溶血活性的最优p H条件;不同缓冲液对TE稳定性及其溶血活性影响显著,浓度大于26%的硫酸铵溶液对TE溶血蛋白具有良好的盐析作用。结论 40℃水浴1 h预处理可显著减少TE中非活性蛋白组分,并降低样品黏性;4℃和p H 8.0是TE保持蛋白稳定性和溶血活性的最适条件;浓度大于26%的硫酸铵盐析有利于溶血蛋白组分富集。     

不同阳离子对Cyanea capillata水母触手提取物溶血活性的影响

目的 探讨血浆对Cyanea capillata水母触手提取物(TOE)溶血活性的影响和孔道形成在其溶血机制中的作用。方法 在具有相同红细胞浓度的1%稀释的全血和0.5%红细胞悬液中,检测不同浓度(100、200、400 μg/ml)的TOE产生的溶血效应,并在两套体系中分别加入20、50或100 mmol/L的K+、Ca2+、Mg2+、Mn2+、Zn2+、Cu2+、Fe2+、La3+、NH4+等阳离子和离子螯合剂EDTA,观察其对TOE溶血活性的影响。结果 在两套溶血检测体系中,TOE均呈现出剂量依赖性的溶血效应,TOE为200 μg/ml时在红细胞悬液产生的溶血强度高于稀释的全血;Mn2+、Zn2+、La3+、Cu2+、Fe2+和EDTA均明显降低TOE溶血活性(P<0.05),其中以Zn2+的抑制作用最强,而K+、Ca2+、Mg2+、NH4+可不同程度地增强其溶血活性(P<0.05)。结论 血浆对TOE的溶血作用有一定的保护作用,而孔道形成可能在TOE的溶血机制中具有重要作用。     

Preparation and Neutralization Efficacy of Novel Jellyfish Antivenoms against Cyanea nozakii Toxins

Jellyfish stings are a common issue globally, particularly in coastal areas in the summer. Victims can suffer pain, itching, swelling, shock, and even death. Usually, hot water, vinegar, or alumen is used to treat the normal symptoms of a jellyfish sting. However, a specific antivenom may be an effective treatment to deal with severe jellyfish stings. Cyanea nozakii often reach a diameter of 60 cm and are responsible for hundreds of thousands of stings per year in coastal Chinese waters. However, there has been no specific C. nozakii antivenom until now, and so the development of this antivenom is very important. Herein, we collected C. nozakii antisera from tentacle extract venom immunized rabbits and purified the immunoglobulin (IgG) fraction antivenom (AntiCnTXs). Subsequently, two complete procedures to produce a refined F(ab’)₂ type of antivenom (F(ab’)₂-AntiCnTXs) and Fab type of antivenom (Fab-AntiCnTXs) by multiple optimizations and purification were established. The neutralization efficacy of these three types of antivenoms was compared and analyzed in vitro and in vivo, and the results showed that all types of antibodies displayed some neutralization effect on the lethality of C. nozakii venom toxins, with the neutralization efficacy as follows: F(ab’)₂-AntiCnTXs ≥ AntiCnTXs > Fab-AntiCnTXs. This study describes the preparation of novel C. nozakii jellyfish antivenom preparations towards the goal of developing a new, effective treatment for jellyfish stings.     

发形霞水母毒素分离产物溶血活性的比较及其影响因素分析

目的:分离发形霞水母刺丝囊毒素(nematocyst venom,NV)与无刺丝囊触手提取物(tentacle-only extract,TOE),比较二者的溶血活性,分析其影响因素.方法:采用自溶、离心方法分离发形霞水母NV和TOE,比较二者的溶血活性,观察提取物浓度、温度和pH值等因素对二者溶血活性的影响.结果:成功分离到NV与TOE;二者浓度相关溶血活性曲线均呈"S"形,溶血分数50%时所对应的质量浓度(HU50)分别为8μg/ml和67μg/ml,前者溶血活性强度约为后者的8.4倍;温度对二者溶血活性影响较大,最大溶血活性都出现在40℃;pH值对二者溶血活性也有影响,在pH 8处二者都具有最大的溶血活性,但后者较前者更为敏感.结论:发形霞水母毒素分离产物NV和TOE均具有溶血活性,且前者强于后者;二者溶血活性受浓度、温度和pH值的影响.     

The lethality of tentacle-only extract from jellyfish Cyanea capillata is primarily attributed to cardiotoxicity in anaesthetized SD rats

Previous studies in our laboratory have shown that tentacle-only extract (TOE) has similar hypotensive effects with nematocyst venom from jellyfish Cyanea capillata, and the experimental studies on the in vivo cardiovascular effects of TOE were further performed to explore the leading cause of death and analyze the basic physiopathologic change in anaesthztized SD rats. Plots of TOE dose versus time to death showed dose-dependent curvilinear relationship. ECG changed in a dose- and time-dependent manner. Haemodynamic parameters, including the heart rate, mean femoral arterial pressure, left ventricular developed pressure and the first derivative of left ventricular pressures, decreased, but left ventricular end-diastolic pressure did not increase. Arterial partial pressure of oxygen and oxygen saturation did not change. Lactate dehydrogenase, creatine kinase and MB isoenzyme of creatine kinase increased significantly. Histopathological examination showed congestion, haemorrhage, edema and denaturation in the heart; congestion, haemorrhage in the lung and acute congestion in the liver. Transmission electron microscopy examination found that parts of sarcomeric filaments disrupted, dissolved and disappeared, and parts of mitochondria swelled in cardiocytes. Laser scanning confocal microscope examination found that ventricular myocytes from adult rat were deformed and ultimately died within 30 min after TOE treatment. Our results reveal that cardiodepressive effect of C. capillata TOE is the leading cause of death and acute total heart failure is the basic physiopathologic change in anaesthetized SD rats.     

霞水母胶原治疗大鼠佐剂型关节炎的研究

目的 探讨霞水母来源的胶原对大鼠佐剂型关节炎的治疗作用.方法 采用完全弗氏佐剂造成大鼠关节炎模型,致炎后大鼠给予霞水母胶原连续灌胃14 d,观察大鼠双后肢关节肿胀情况,及对血清中一氧化氮(NO),超氧化物歧化酶(SOD)、丙二醛(MDA)的影响.结果 口服霞水母胶原可以降低大鼠的双后肢关节肿胀度,降低大鼠血清中NO和MDA含量,提高大鼠血清中SOD活性.结论 霞水母胶原对佐剂型关节炎有较好的治疗作用,其机理可能与降低体内氧自由基水平及过氧化脂质水平有关.     

Cyanea capillata tentacle-only extract as a potential alternative of nematocyst venom: Its cardiovascular toxicity and tolerance to isolation and purification procedures

As the proteins with cardiovascular toxicity in jellyfish nematocyst venom and tentacle-only extract (TOE) are probably encoded by the same gene, TOE provides a potential alternative of nematocyst venom with much richer source for acquisition of such proteins. In this study, Cyanea capillata nematocyst venom and TOE (5 mg/kg) both exhibited cardiovascular toxicity in rats, and TOE caused blood pressure reduction in slightly greater amplitude than nematocyst venom 3 min after intravenous administration. SDS-PAGE suggested high likeliness that they both contained the same bioactive protein. The activity of TOE was dose-dependent within 1.25-5 mg/kg, but not at higher concentrations. The cardiovascular activity of TOE sustained a major loss after exposure to 60 °C, and was totally abolished after exposure to 80 °C. Within the pH range of 7-11, the activity of TOE was well preserved, and rapidly attenuated in pH below 5. At 4 °C, TOE lost cardiovascular toxicity after preservation for 7 days, which occurred only after an 8-h preservation at 20 °C. Repeated freeze-thawing and freeze-drying did not significantly affect the toxicity of TOE. Buffer solutions obviously affected the toxicity of TOE, and 0.02 mol/L HAc (pH 6.0) was optimal. These results provide experimental data for optimizing the conditions for isolating the proteins with cardiovascular toxicity from jellyfish TOE, which serves as a promising alternative source of nematocyst venom.