谷胱甘肽对凡纳滨对虾抗氧化防御的调控机理
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摘要
凡纳滨对虾(Litopenaeus Vannamei)是世界上最优良的虾类养殖品种,在我国沿海地区已进行了大规模的人工养殖,目前是我国对虾的主导养殖品种和重要的出口水产品之一。但在集约化养殖过程中,对虾面临着大量的应激因素(如：拥挤、营养、环境、代谢等)。激烈的应激往往会引起对虾抗病力减弱,疾病爆发和流行,导致养殖者过度使用兽药和抗生素饲料添加剂。长期、大量使用抗生素会造成对虾肠道内菌群失调,破坏微生态环境,产生药物残留。药物残留会导致人类过敏反应、免疫抑制、致畸、致癌、致突变等。近几年我国发生了多起食品安全的重大事件,严重地挫伤了消费者的信心。随着国际贸易中绿色壁垒的逐渐加强,我国农产品特别是水产品出口受到的影响越来越大,所有这些严重威胁着我国对虾养殖业的可持续发展。因此,抗生素替代品的研究与开发已成为人们关注的热点。研制高效安全的抗氧化饲料添加剂,通过营养调控提高或激活对虾自身的抗氧化防御能力,是解决上述难题的有效的新途径之—
本论文以凡纳滨对虾为研究对象,通过“体内”(in vivo)和“体外”(in vitro)实验,系统研究了还原型谷胱甘肽(GSH)对凡纳滨对虾生长性能、抗氧化系统以及非特异免疫因子的影响；对凡纳滨对虾原代培养肝胰腺细胞的增殖、生理生化功能以及相关抗氧化酶的影响；并从分子水平上揭示了GSH在凡纳滨对虾上的抗氧化机理。本论文主要包括如下四个部分：
1.凡纳滨对虾组织抗氧化酶活性和脂质过氧化产物含量对氨氮胁迫的反应特点
本文选择南方有代表性的凡纳滨对虾(Litopenaeus vannamei)(初始体重为6.322±0.221g),分别测定了凡纳滨对虾在氨氮胁迫前后,腮丝、肌肉、肝胰腺和血清中超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GSH-Px)活性和GSH、丙二醛(MDA)含量以及机体总抗氧化能力(T-AOC)的变化情况。
结果显示,经离子铵氮胁迫后,凡纳滨对虾鳃丝中除GSH-Px (p<0.05)外,肌肉中除SOD(p<0.05)外,其它各抗氧化酶活性、T-AOC、GSH和MDA含量,在胁迫前后变化不大；在肝胰腺中,除T-AOC外,各项指标和胁迫前相比,有显著的变化(p<0.05)；在血清中,SOD、GSH-Px和MDA含量在氨氮胁迫前后有显著差异(p<0.05)。其中肝胰腺中CAT、GSH,血清中GSH-Px活性与胁迫前相比,差异极显著(p<0.01)。结果表明,血清和肝胰腺是凡纳滨对虾对铵氮造成胁迫的敏感组织,抗氧化酶SOD、CAT和GSH-Px、总抗氧化能力和MDA含量可作为衡量凡纳滨对虾氧化应激状态的敏感指标。
2.谷胱甘肽对凡纳滨对虾生长性能、抗氧化和非特异免疫功能的影响
在基础日粮中分别添加0、60、120、180、240和300mg/kg还原型谷胱甘肽(GSH),饲喂初始平均体重(initial body weight, IBW)为1.123±0.007g凡纳滨对虾(litopenaeus vannamei)(分别记为G0、G60、G120、G180、G240和G300组),研究饲料中添加GSH对凡纳滨对虾生长性能、机体抗氧化水平、脂质过氧化物含量和非特异免疫功能的影响。养殖试验持续8周。停止喂料24 h后,全部称重,统计耗料、死亡。每个重复随机取15只虾,取样。剩余部分放回原循环系统,加入20mg/L浓度的氯化铵处理1周,停止循环水,试验各组继续投喂试验饲料,第9周末,停喂24h后,统计成活率,取样。
试验结果显示：
(1)饲料中添加GSH能显著提高凡纳滨对虾的增重率、成活率和饲料转化效率。试验各组凡纳滨对虾成活率较对照组提高8.53%-31.69%(p<0.05)；增重率随GSH添加量的增加而增加,当添加量为180mg/kg时达到高峰；随着GSH添加量的进一步增加,增重率呈下降趋势(p<0.05)。饲料中GSH添加量不低于120 mg/kg时,显著地降低饵料系数(p<0.05)；饲料中添加GSH提高了凡纳滨对虾血清、肝胰腺和肌肉中的蛋白浓度。当GSH添加量不低于180 mg/kg时,血清及肝胰腺蛋白浓度显著高于对照组(p<0.05)；肌肉蛋白浓度在GSH添加量180 mg/kg时达到最高(p<0.05)；鳃丝蛋白浓度试验各组差异不显著(p>0.05)；试验各组凡纳滨对虾的血淋巴细胞计数均显著高于对照组(p<0.05),且与GSH添加量呈剂量-效应关系；当饲料中GSH的添加量分别60、120和180mg/kg时,凡纳滨对虾肝胰腺中GSH浓度显著升高(p<0.05)。以增重率为评价指标,GSH在凡纳滨对虾饲料中的适宜添加量为174.13 mg/kg。
(2)饲料中添加一定量的GSH能提高凡纳滨对虾肝胰腺抗氧化能力和降低脂质过氧化物含量。饲料GSH能显著提高凡纳滨对虾肝胰腺中抗氧化酶活性(p<0.05)：其中120 mg/kg、180 mg/kg和300 mg/kg组的SOD活性,120 mg/kg、180 mg/kg和240 mg/kg组的GSH-Px活性,60 mg/kg、120 mg/kg组的GR活性,显著高于对照组(p<0.05)；各试验组肝胰腺中GSH含量和总抗氧化能力T-AOC比对照组分别提高了8.93%-52.57%和3.02%-37.03%,且呈剂量-效应关系(p<0.05)。随着饲料GSH含量的升高,肝胰腺氧自由基(ROS)和脂质过氧化物MDA的含量呈下降趋势,分别在添加量为240 mg/kg和300 mg/kg时达到最低,且显著低于对照组(p<0.05)。对虾成活率和肝胰腺抗O2能力,均呈现先升后降的趋势,各项指标分别在120 mg/kg、180 mg/kg组达到最高值,并显著高于对照组(p<0.05)。
(3)在凡纳滨对虾的肝胰腺中,髓过氧化物酶(MPO)、溶菌酶(LSZ)、碱性磷酸酶(AKP)、酸性磷酸酶(ACP)活性随着饲料中GSH添加量的增加,均呈先升后降的趋势,在120-240 mg/kg之间达到最高值,且显著高于对照组(p<0.05),但随着GSH量继续增加(高于240mg/kg时),各种酶的活性急剧下降。而在血清中,LSZ、AKP和ACP活性先随饲料GSH添加量的增加而增加,分别在180 mg/kg、240 mg/kg和240mg/kg达到最高值,然后急剧下降。血清和肝胰脏中的谷草转氨酶(AST/GOT)和谷丙转氨酶(ALT/GPT)呈下降趋势,在240 mg/kg组达到最低,且显著低于对照组(p<0.05),但在300mg/kg组又有所回升。凡纳滨对虾经过离子铵胁迫1周后,发现一定添加量的GSH能提高氨氮胁迫凡纳滨对虾成活率,并对免疫因子有积极的影响。但不同的免疫因子,在不同组织敏感程度不一样,作用剂量不同。从本试验结果看,血清和肝胰腺中的LSZ、AST/GOT、ALT/GPT\ACP都是能反应凡纳滨对虾非特异免疫因子的敏感指标,与成活率结果比较一致；而MPO和肝胰腺中的AKP相对而言,规律性不强。
由上述可见,饲料中添加一定量的GSH能显著提高凡纳滨对虾的生长性能,提高凡纳滨对虾肝胰腺抗氧化能力和降低脂质过氧化物含量,有效提高凡纳滨对虾血清和肝胰腺中髓过氧化物酶、溶菌酶和磷酸酶活性,影响转氨酶的活性,并影响凡纳滨对虾机体的免疫因子水平,从而激发凡纳滨对虾的非特异免疫功能,提高凡纳滨对虾成活率,改善离子铵对凡纳滨对虾造成的氧化胁迫。
3.凡纳滨对虾肝胰腺线粒体脂质过氧化模型建立与GSH的抗氧化损伤作用
首先,建立凡纳滨对虾肝胰腺线粒体的制备方法。采用三种不同的差速离心法,提取凡纳滨对虾肝胰腺线粒体,用中性红—詹纳斯绿B (Jana's green B)染色鉴定,以OD260/OD280比值检查线粒体纯度。第二,建立凡纳滨对虾肝胰腺线粒体氧化损伤模型。分别用VC/FeSO4、NADH来诱导线粒体氧化,并确定各种催化剂的反应浓度,筛选反应时间和反应条件,建立两种诱导凡纳滨对虾肝胰腺线粒体的氧化模型。第三,研究GSH对凡纳滨对虾线粒体氧化损伤的抑制作用。在所建立的线粒体氧化损伤模型中加入不同浓度(0、0.1、0.2、0.4、0.8、1.2mmol/L)的GSH,以反应体系中MDA含量、线粒体膨胀度和DNA双链百分比为指标来观察GSH对线粒体脂质过氧化的抑制效果。研究结果显示：
(1)凡纳滨对虾肝胰腺线粒体制备方法为：按肝胰腺(鲜重)：缓冲液=1：15加入缓冲液STE (mmol/L:Sucrose 250, Tris-HCl 10, EDTA 1, pH8.0)进行匀浆,4℃1000g离心15min；4℃10000g离心20min,用缓冲液STM (mmol/L:Sucrose 250, Tris-HCl 50, MgCl2 5, pH7.4)重悬浮纯化,中性红—詹纳斯绿B染色,高倍镜观察为亮绿色,OD260/OD280=1.73。
(2)建立的VC/FeSO4氧化模型为：在线粒体蛋白浓度1mg/ml, VC浓度0.2mmol/L, FeSO4浓度4μmol/L, pH7.4HEPES缓冲体系中,30℃,经30min反应。当外源GSH添加浓度为0.4mmol/L时,对线粒体脂质过氧化的抑制作用最显著。建立的NADH模型为：线粒体蛋白浓度1.0mg/ml, FeCl30.04mmol/L, ADP 4.0mmol/L,在25mmol/L HEPES/NaOH缓冲液pH7.4(含0.15mol/LKCl)条件下,加入NADH120μmol/L启动反应,30℃水浴,振荡30min后加入20%三氯乙酸终止反应。当外源GSH添加浓度为0.4mmol/L时,对线粒体的保护效果最好。
(3)通过对VC/FeSO4和NADH模型的比较发现,两种模型都能显著(p<0.05)激发凡纳滨对虾肝胰腺线粒体的脂质过氧化,二者之间差异不显著(p>0.05)。与对照组相比,两个模型MDA含量分别为2.10倍和1.43倍,线粒体膨胀度分别为1.71倍和1.38倍,DNA双链百分比分别是60.07%和55.13%。从MDA和线粒体膨胀度来看,NADH模型的效果更好；以DNA双链百分比作为指标,VC/FeSO4模型效果更佳。
4.凡纳滨对虾肝胰腺细胞原代培养、谷胱甘肽对细胞生长、生理生化和抗氧化功能以及抗氧化酶mRNA表达量的影响
首先建立凡纳滨对虾肝胰腺细胞原代培养方法。第二,研究GSH对细胞增殖、生理生化和抗氧化功能的影响。用含有不同浓度(0、0.1、0.2、0.4、0.8、1.2mmol/L)GSH的培养基对凡纳滨对虾肝胰腺细胞进行原代培养,分别在第24h、48h和72h,收集细胞培养上清液,测定离体肝胰腺细胞的活力、RNA/DNA比、白蛋白含量、一氧化氮合酶(NOS)、胰岛素样生长因子-I(IGF-Ⅰ)、ATPase、谷丙转氨酶(ALT/GPT)和谷草转氨酶(AST/GOT)活性。分别在第24h、48h和72h收集细胞,匀浆后,测定细胞匀浆上清液中SOD、GSH-Px、CAT活性、MDA和H202含量。第三,研究GSH对离体肝胰腺细胞SOD和CAT mRNA表达量的影响。收集原代培养72h的肝胰腺细胞,提取细胞RNA,测定SODmRNA和CATmRNA表达量。结果如下：
(1)建立的凡纳滨对虾肝胰腺细胞原代培养方法为：采用高锰酸钾浸泡、冰冷灭菌PBS溶液冲洗、75%乙醇体表消毒后取出肝胰腺,用0.05%Ⅱ型胶原酶27℃消化10min,低温离心(1200r/min, 10min; 1000r/min,5min),27℃、5%CO2和饱和湿度下进行培养(接种密度：5×105个细胞/m1)。72 h后,细胞状态良好,外表光滑,折光性好,经测定细胞活性高。
(2)GSH能促进凡纳滨对虾离体肝胰腺细胞增殖、提高RNA/DNA比,促进IGF-Ⅰ分泌,表明GSH能通过对生长有关激素的调控来促进细胞的生长,GSH除了本身的抗氧化功能外,还具有营养作用。GSH能促进离体肝胰腺细胞白蛋白和NOS酶分泌,提高体外培养肝细胞的生物活性。能通过提高ATPase活性,保护细胞膜通透性,维持膜的正常生理功能。
(3)培养72h后,添加GSH对抗氧化酶和MDA含量等的影响分别为：明显提高肝胰腺细胞匀浆中SOD活性,呈现先升后降,最后上升趋于平稳,除1.2 mmol/L组外,试验各组分别比对照组提高了17.28%、9.05%、37.04%和45.27%,其中0.8mmol/L组达到显著水平(p<0.05)；各试验组GSH-Px活性均高于对照组,但试验各组组间差异不显著(p>0.05)；MDA含量除1.2 mmol/L组外,试验各组均显著低于对照组(p<0.05),在0.2 mmol/L组达到最低水平；H202含量在各试验组显著降低,各组依次降低了4.42%、17.85%、10.22%、25.58%和16.48%,其中试验0.8 mmol/L组能达到显著水平(p<0.05)。
(4)添加GSH明显提高凡纳滨对虾离体肝胰腺细胞SODmRNA表达量,除0.1 mmol/L组外,各试验组的SODmRNA表达水平均显著高于对照组(p<0.05)。与对照组相比,除1.2 mmol/L组外,添加GSH各组肝胰腺细胞CATmRNA的表达量均有所升高(p>0.05)。结果表明,GSH能提高离体肝胰腺细胞中SOD酶活力,降低脂质过氧化产物含量,影响SOD和CATmRNA的表达。
5.结论
1) GSH在体内能改善凡纳滨对虾的生长性能,提高饲料转化效率和存活率,提高对虾机体的抗氧化水平和抗氧化能力,缓解离子铵引起的氧化应激。以增重率为评价指标,GSH在凡纳滨对虾饲料中的适宜添加量为174.13 mg/kg。
2)GSH在体外能抑制凡纳滨对虾肝胰腺线粒体的脂质过氧化,当GSH添加浓度为0.4mmol/L时,对线粒体的保护效果最好。外源GSH促进了离体肝胰腺原代培养细胞的生长、增殖,提高原代培养肝胰腺细胞的生物活性。能通过调节与生长代谢相关的酶和抗氧化酶的活性,降低MDA含量来保护细胞膜通透性,维持膜的正常生理功能。并提高了凡纳滨对虾离体肝胰腺细胞SODmRNA表达量,GSH的添加量为0.8 mmol/L时效果达显著水平。
Litopenaeus vannamei is one of the excellent aquaculture varieties in the world, and aquaculture of this white shrimp has become an important export industry in developing countries in Asia and America. In China's around-sea area, Litopenaeus vannamei has been developed to be the dominant cultured products which is one of the most important exported commodities. However, the white shrimp Litopenaeus vannamei are challenged with crowded, nutritional, environmental, and metabolic stress during the intensive aquaculture processes, resulting in significantly reduced disease resistance, thus disease broken epidemicaly and new plague emerged uninterruptedly. It's too difficult for veterinarian to diagnose and therapy these maladies so that the farmers keen on abusing the antibiotics in feedstuff. Long-time and large-dose antibiotics abusing in shrimp would lead to many side-effects, such as dysbacteriosis in intestinal tract, disruption microenvironment, drug residues ect. Eventually due to humankind's allergic reaction, immunosuppression, distortion, canceration, mutation, thus the sustainable development of Litopenaeus vannamei aquaculture industry is threaten by its susceptibility to disease outbreak.
Now, it is considered that one of the effective ways to solve the problem is to increase or activate the antioxidative capacities of Litopenaeus vannamei by nutritional regulation, such as using highly efficient and safe antioxidative feed additives.
In this research, we emphasized on the roles of reduced glutathione (GSH) playing in: (1) improving the growth performance of Litopenaeus vannamei; protecting the shrimp from oxidative stress; increasing non-specific immunologic factors level in vivo; (2) being an accelerator proliferation of primary cultured hepatopancreas cells; mediating the diverse biological effects of primary cultured hepatopancreas cells of Litopenaeus vannamei; (3) improving the expression of mRNA in SOD and CAT in vitro. This research comprises of four parts.
1. Distribution of peroxidative enzymes and lipid peroxidation product in the tissues of litopenaeus vannamei before and after ammonia stress
In order to reveal the distributed characteristics of the antioxidant enzymes ----superoxide dismutase (SOD), catalas (CAT), glutathione peroxidase (GSH-Px); total antioxidant capacity (T-AOC), glutathione (GSH) and lipid peroxidation product-malonydialdehyde (MDA) in various tissues of Litopenaeus vannamei, such as gills, muscles, hepatopancreas and serum, an experiment was designed to assess the changes of these indexes before and after ammonia stress.72 shrimps, with initial weight of 6.322±0.221g were randomly selected from the representative South China Litopenaeus vannamei to sample their gills, muscles, hepatopancreas and serum to analyze the activities of SOD, GSH-Px, CAT, T-AOC, the content of GSH and MDA before and after ammonia stress.
The results indicated that hepatopancreas and serum were the sensitive tissues while gills and muscles were not as so sensitive as others. Ammonium-N stress experiments showed that CAT, GSH-PX activities, T-AOC, GSH and MDA content were not sensitive in gills and muscles to NH4Cl treatment whereas all these indexes (except T-AOC) were significantly (p<0.05) changed in hepatopancreas and SOD, GSH-Px and MDA were significantly (p<0.05) changed in serum of Litopenaeus vannamei. Furthermore, CAT activities and GSH content in hepatopancreas and GSH-Px activities in serum were significantly (p<0.01) changed after stress. These results suggest that hepatopancreas and serum are the tissues sensitive to ammonium-N stress, therefore, CAT、GSH-PX、T-AOC、GSH and MDA content could be regarded as antioxidative stress indexes of Litopenaeus vannamei and these antioxidant enzymes and lipid peroxidation product in hepatopancreas and serum could be regarded as the effective indexes of Litopenaeus vannamei when the shrimp suffer from a stress.
2. Effects of dietary GSH on the growth performance, antioxidant indexes and lipid peroxidation content, nonspecific immune factors in Litopenaeus vannamei
6 levels of glutathione (GSH) (0,60,120,180,240 and 300mg/kg), being named G0, G60, G120, G180, G240 and G300 respectively, were supplemented to a basal diet to test the effects of different dietary GSH levels on the growth performance, hepatopancreas antioxidant indexes and lipid peroxidation content, non-specific immune factors and activities of AKP, ACP, AST/GOT and ALT/GPT in litopenaeus vannamei (IBW of 1.12±0.01g).
The feeding experiment ran for 8 weeks. After 24hs the shrimp were weighed, taken into account total dead rate, total feed consumption. Then,15 shrimps were sampled per replication. The rest shrimps were sent back to the circulation system. The Litopenaeus vannamei were treated with 20 mg/L of NH4Cl without water circulating for one week. At the end of 9th week, the feeding was stopped for 24 hours, the dead rate was calculated and samples were taken. The Litopenaeus vannamei tissues were collected for assessing viability and immune factor levels.
The results showed that:
(1) weight gain rate (WGR), survival rate and feed conversion efficiency (FCE) of the shrimp fed dietary GSH were significantly increased than those of control (p<0.05). Survival rate was increased for 8.53%-31.69%(p<0.05);WGR increased with dietary GSH increasing and reached the highest when dietary GSH was 180mg/kg, but tent to decrease when dietary GSH increasing above 180mg/kg (p<0.05); When dietary GSH was above 120mg/kg, FCE was significantly increased (p<0.05). Protein content in serum, hepatopancreas, muscle of the shrimp were increased when dietary GSH was supplemented, being significantly higher in serum and hepatopancreas than that of control when dietary GSH was above 180mg/kg (p<0.05); Protein content in gills had no significant difference among the 6 groups (p>0.05). The numbers of haemolymphs of the shrimp fed dietary GSH were significantly higher than those of the control (p<0.05), showing a dose-dependent relationship (p<0.05). GSH content in hepatopancreas was increased when dietary GSH was above 60mg/kg (p<0.05). In summation, the optimal level of dietary GSH for litopenaeus vannamei was 174.13 mg/kg.
(2) Dietary GSH improved the activities of antioxidant enzymes and total antioxidation capacities, significantly decreased the content of MDA (p<0.05). The increasing dietary GSH had a significant effect on the activities of SOD and GSH-Px when the supplementary GSH was above 120 mg/kg (p<0.05), while had a significant effect on GR activities in 60mg/kg and 120mg/kg (p<0.05). Dietary GSH raised the content of GSH and the total antioxidant ability (T-AOC) in hepatopancreas and showed a dose-dependent relationship, the increase rate was 8.93%-52.57% and 3.02%-37.03%, respectively. With increasing dietary GSH, ROS and MDA level tent to decrease, reached the lowest at 240mg/kg and 300mg/kg (p<0.05) respectively. The ROS in group 180mg/kg,240mg/kg and the MDA content in each experiment group was significantly lower than those of the control (p<0.05). Dietary GSH significantly improved the survival rate and the anti-O2-ability in group 120mg/kg and 180mg/kg (p<0.05).
(3) In hepatopancreas, with the increasing of dietary GSH level, activities of MPO, LSZ, AKP and ACP of the shrimp increased, reaching the highest at 120 mg/kg,240 mg/kg,240 mg/kg,120 mg/kg respectively, then significantly decreased at a higher level (when more than 240 mg/kg). While in serum, activities of LSZ, AKP and ACP increased, reaching the highest at 180 mg/kg,240 mg/kg and 240 mg/kg respectively, then significantly decreased. AST/GOT and ALT/GPT activities, both in serum and hepatopancreas of shrimp fed dietary GSH, decreased significantly and achieved the bottom at 240 mg/kg (p<0.05), then increased at 300 mg/kg.
After a week's stress of NH4+, the survival of the shrimp fed with high level of dietary GSH was significantly increased than that of the control and the low dose group (p<0.05). Furthermore, the appropriate dietary GSH had a positive effect on nonspecific immunity factors. But for the different immunity factors, there had the different effects and had different sensitive tissues in Litopenaeus vannamei. LSZ, AST/GOT, ALT/GPT and ACP activities both in hepatopancreas and serum could be the sensitive indexes to reflect the state of non-specific immunity factors in Litopenaeus vannamei, nevertheless, the MPO and the AKP in hepatopancrea was lacking regularity.
In conclusion, dietary GSH could increase body weight gain and feed conversion efficiency of Litopenaeus vannamei. Optimal supplementation of GSH in shrimp diet could improve antioxidant capacities and decrease lipid peroxidation products, enhance the immune factors of Litopenaeus vannamei, and improve the antioxidant stress caused by ionized ammon and activate the non-specific immune functions of Litopenaeus vannamei.
3. Extraction method of hepatopancreas mitochondria, establishment of lipid peroxidation model and the effect of GSH on the oxidative damage in mitochondria of Litopenaeus vannamei
In this study, the extraction method of hepatopancreas mitochondria of Litopenaeus vannamei was studied. By using different oxidants (VC/FeSO4、NADH) at different conditions (concentration, time, and pH), two lipid peroxidation models to induce hepatopancreas mitochondrial oxidation of the Litopenaeus vannamei were established. The optimized conditions and the effective elicitor to induce hepatopancreas mitochondrial oxidation of the Litopenaeus vannamei were elucidated by analyzing the lipid peroxidation products, swelling rate of mitochondria and DNA double chain ratio. These mitochondrial oxidation models could be suitable for studying the hepatopancreas mitochondrial damage caused by lipid peroxidation, and the roles in protecting oxidation damage conferred by GSH in vitro at the subcellular level.
This research includes two parts. Firstly, the hepatopancreas mitochondria of Litopenaeus vannamei were extracted with three steps of ultracentrifugation. The mitochondria were authenticated by neutral red-Jana's green B, inspected the purity with the index of OD260/OD280 to choose the suitable extraction method of hepatopancreas mitochondria of Litopenaeus vannamei. Secondly, VC/FeSO4 and NADH were used to induce the peroxidation of hepatopancreas mitochondria of Litopenaeus vannamei, the concentration of different catalysts, the optimal time and conditions for the reaction were established to build two models. In these two models, various concentration of GSH (0, 0.1,0.2,0.4,0.8 and 1.2 mmol/L) were added to the reaction system to observe its inhibition effects of GSH. Finally, these two models were compared objectively, the MDA contents in the reaction system, the swelling rate and double chain of DNA in hepatopancreas mitochondria of Litopenaeus vannamei were the indexes to evaluate these two models.
The results showed that:(1) After homogenated, the hepatopancreas with a wet weight:buffer rate is 1:15, in a buffer of STE (mmol/L:Sucrose 250, Tris-HCl 10, EDTA 1, pH8.0), then, centrifuged at 4℃1000gx5 min,10000gx20 min and purified with buffer STM (mmol/L:Sucrose 250, Tris-HCl 50, MgCl25, pH7.4). The hepatopancreas mitochondria were in light green color in the electron microscopy at high magnification after being dyed by neutral red-Jana's green B, and the OD260/OD280 was 1.73, which is proximity to 1.8.
(2) In model VC/FeSO4, when the protein content of hepatopancreas mitochondria was lmg/ml, the VC 0.2mmol/L, the FeSO4 41/4mol/L, the reaction temperature 30℃, reacting for 30min in the buffer of pH 7.4 HEPES, the VC/FeSO4 system induced the peroxidation of hepatopancreas mitochondria effectively. While in the model of NADH, the best reaction conditions were:the protein content of hepatopancreas mitochondria was 1.0mg/ml, FeCl3 0.04mmol/L, ADP 4.0mmol/L, in 25mmol/L HEPES/NaOH buffer system with pH7.4, the start factor was NADH 120μmol/L, the reaction condition was 30℃,30min, then ended the reaction with 20%trichloroacetic acid. The exogenous GSH at 0.4mmol/L inhibited the peroxidation in both of these models obviously.
(3) Comparing the NADH with the VC/FeSO4 model of hepatopancreas mitochondrial oxidation of the Litopenaeus vannamei revealed that both the NADH and the VC/FeSO4 could significantly activate mitochondrial lipid peroxidation of Litopenaeus vannamei (p<0.05), whereas there were no significant differences between the two models (p>0.05). In the VC/FeSO4 model, the MDA content, swelling rate of mitochondria and DNA double strands percentage were respectively 2.10 times,1.71 times and 60.07% of the control; in the NADH model, the three parameters were 1.43 times,1.38 times and 55.13%, respectively. From MDA and mitochondrial swelling rate, it was suggested that the NADH model functioned better; however, the percentage of double strand DNA indicated that the VC/FeSO4 model confered better inhibitory effect.
4. Study of method for primary culture of hepatopancreatic cells of Litopenaeus vannamei, effects of GSH on the proliferation, physiological and biochemical characteristics and functions, the anti-oxidative effects, and expression of mRNA of SOD and CAT in primary culture cells in Litopenaeus vannamei
In order to reveal the effects of GSH on the proliferation, physiological and biochemical characteristics and functions, on the antioxidant affects and on the expression of anti-oxidase in vitro, thus to have the light on the mechanism insight of micronutrient in regulating aquatic organism's antioxidant stress at molecular level, we designed the experiment as follows.
Firstly, the method for improving the protocol of primary cultured hepatopancreatic cells of Litopenaeus vannamei were studied, including:(1) optimizing the methods of separating, digesting and purifying the hepatopancreatic cells of Litopenaeus vannamei;
(2) choosing the culture medium for primary cultured hepatopancreatic cells; (3) selecting the most suitable salt rate and the pH of culture and the seeding density of hepatopancreatic cells of Litopenaeus vannamei.
Secondly, the primary cultured hepatopancreatic cells were cultured in the medium with the final concentration of GSH by 0,0.1,0.2,0.4,0.8 and 1.2mmol/L, separately. We collected the supernatant of hepatopancrea cells of all the groups at 24h,48h and 72h separately, to measure the activities of cells, the RNA/DNA, the content of albumin, the nitricoxide synthase (NOS), Insulin-like growth factor-Ⅰ(IGF-Ⅰ), ATPase, alanine aminotransferase (ALT/GPT) and aspartate aminotransferase (AST/GOT).
Thirdly, the cells were collected and crushed in the culture media by microwave and then centrifuged (4℃,3000 r/min, 10min) and the supernatants were taken to analysis the antioxidant index, include:SOD, GSH-Px, CAT, MDA and H2O2 quantitatively at 24h,48h and 72h, separately.
Finally, after the hepatopancreatic cells were primary cultured in medium with different concentration of GSH for 72 hours, the cells were collected, the RNA of hepatopancreatic cells was extracted and the mRNA was detected by RT-PCR to measure the expression levels of SOD mRNA and CAT mRNA.
The results showed that:
(1) The method for primary culture of hepatopancreatic cells of Litopenaeus vannamei was established. After being soaked in potassium permanganate, washed by cold PBS and sterilized totally in 75% ethanol, the hepatopancreas were removed away from the Litopenaeus vannamei. The hepatopancreatic cells were isolated in 0.05% typeⅡcollagenase at 27℃for 10 min, then purified with a centrifugation method at 1200 r/min,10 min+1000 r/min,5min, and cultured in the box of 5% CO2, saturated degree of humidity,27℃at a seeding density of 5×105 cells/ml. The cells were at an excellent status characterized by smooth cell surface, better refractive, and higher activities of hepatocytes.
(2) GSH boosted the proliferation of Litopenaeus vanhnamei's hepatocyte in vitro, increased RNA/DNA ratio, accelerated the hepatocytes to excrete IGF-1, indicating that GSH could facilitate the up-growth of cells through regulating the relevant hormone. GSH could take on nutrition action and antioxidant function. GSH could improve the content of albumin and the secretion of nitricoxide synthase (NOS) in vitro, thus enhance the bioactivity of hepatopancreatic cells in vitro. Boost the ATPase activity, and protect the penetrating power of cell membrane while keep the well-balanced physiological function. GSH could reduce the damnification hurt by environment factor, and then accelerate the organizational recovery when the hepatopancrea was damnified.
(3) After being cultured for 72h, GSH addition increased the SOD activities in all the experiment groups except 1.2 mmol/L group. The the increase rate was 17.28%、9.05%、37.04% and 45.27% respectively and the group 0.8 mmol/L affected the SOD activities significantly compared with the control (p<0.05). In conclusion, the proper GSH addition affected the SOD activities in the hepatopancreatic cells of Litopenaeus vannamei in vitro, showing a trend of increasing first then declining and increasing slowly until reaching to a platform, finally. After being cultured for 72h, GSH addition improved the GSH-Px activities in all experiment groups, but had no difference between all the groups (p>0.05). Whlie, the CAT hadn't been detected in some groups because of the insensitivity of the methods for testing in the supernatant preparations from the hepatopancreatic cells in vitro.
MDA contents had a obvious decline trend in all groups at 72h obviously (p<0.05) and reach the lowest content at 0.2 mmol/L group. After being cultured for 72h, the GSH addition can decrease the H2O2 contents in all the experiment groups significantly (p<0.05). The the decrease rate was 4.42%,17.85%,10.22%,25.58% and 45.27% respectively and had a difference at distinguish level of group 0.8 mmol/L (p<0.05).
(4) Adding GSH evidently boosted the SOD genie expression of litopenaeus vannamei's hepatopancreatic cells in vitro. Except group 0.1mmol/L, the SOD mRNA expression level were all observably higher than the experiment group (p<0.05). Except group 1.2 mmol/L, adding GSH in hepatopancreatic cells elevated CATmRNA expression level (p>0.05). GSH hoisted SOD enzymatic activities in hepatic separate cells, depressed the lipid peroxide content, effected the expression of SOD and CAT mRNA.
The results described above suggests that the supplementation of GSH in culture media be capable of reducing lipid peroxide and modulating the activities of SOD and CAT in hepatopancreatic cells of Litopenaeus vannamei in vitro. Through increasing the effect of the expression capacity of antioxidant enzyme CAT and SOD, especially SODase, regulating antioxidant level, that purged the free radicals, efficiently regulated the free radicals metabolism, prevented lipid peroxidation.
In Conclusion, GSH can ameliorate the growth performance of Litopenaeus vannamei in vivi, boost the efficiency of feedstuff transforming, enhance the anti-oxidative level and anti-oxidative capability of Litopenaeus vannamei's, organic tissue, ease up the oxidation allergic response caused by ammonium stress. The optimal level of dietary GSH for litopenaeus vannamei was 174.13 mg/kg. GSH can also restrain the lipid peroxide of Litopenaeus vannamei hepatopancreatic mitochondrion in vitro, when the culture medium GSH concentration is 0.4mmol/L, there is best protection effect for mitochondrion. Exogenous GSH can improve the growth and proliferation of the hepatopancreatic cell in vitro, enhance it biologic activity. It can protect the cell membrane penetration by regulating the correlative metabolic enzyme and anti-oxidative enzyme, depressing the content of MDA, maintaining the well-balanced physiology function. It can boost the hepatopancreatic cell SODmRNA expression level in Litopenaeus vannamei. The effect reach the prominent level when the culture medium GSH is 0.8 mmol/L.
本论文以凡纳滨对虾为研究对象,通过“体内”(in vivo)和“体外”(in vitro)实验,系统研究了还原型谷胱甘肽(GSH)对凡纳滨对虾生长性能、抗氧化系统以及非特异免疫因子的影响；对凡纳滨对虾原代培养肝胰腺细胞的增殖、生理生化功能以及相关抗氧化酶的影响；并从分子水平上揭示了GSH在凡纳滨对虾上的抗氧化机理。本论文主要包括如下四个部分：
1.凡纳滨对虾组织抗氧化酶活性和脂质过氧化产物含量对氨氮胁迫的反应特点
本文选择南方有代表性的凡纳滨对虾(Litopenaeus vannamei)(初始体重为6.322±0.221g),分别测定了凡纳滨对虾在氨氮胁迫前后,腮丝、肌肉、肝胰腺和血清中超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GSH-Px)活性和GSH、丙二醛(MDA)含量以及机体总抗氧化能力(T-AOC)的变化情况。
结果显示,经离子铵氮胁迫后,凡纳滨对虾鳃丝中除GSH-Px (p<0.05)外,肌肉中除SOD(p<0.05)外,其它各抗氧化酶活性、T-AOC、GSH和MDA含量,在胁迫前后变化不大；在肝胰腺中,除T-AOC外,各项指标和胁迫前相比,有显著的变化(p<0.05)；在血清中,SOD、GSH-Px和MDA含量在氨氮胁迫前后有显著差异(p<0.05)。其中肝胰腺中CAT、GSH,血清中GSH-Px活性与胁迫前相比,差异极显著(p<0.01)。结果表明,血清和肝胰腺是凡纳滨对虾对铵氮造成胁迫的敏感组织,抗氧化酶SOD、CAT和GSH-Px、总抗氧化能力和MDA含量可作为衡量凡纳滨对虾氧化应激状态的敏感指标。
2.谷胱甘肽对凡纳滨对虾生长性能、抗氧化和非特异免疫功能的影响
在基础日粮中分别添加0、60、120、180、240和300mg/kg还原型谷胱甘肽(GSH),饲喂初始平均体重(initial body weight, IBW)为1.123±0.007g凡纳滨对虾(litopenaeus vannamei)(分别记为G0、G60、G120、G180、G240和G300组),研究饲料中添加GSH对凡纳滨对虾生长性能、机体抗氧化水平、脂质过氧化物含量和非特异免疫功能的影响。养殖试验持续8周。停止喂料24 h后,全部称重,统计耗料、死亡。每个重复随机取15只虾,取样。剩余部分放回原循环系统,加入20mg/L浓度的氯化铵处理1周,停止循环水,试验各组继续投喂试验饲料,第9周末,停喂24h后,统计成活率,取样。
试验结果显示：
(1)饲料中添加GSH能显著提高凡纳滨对虾的增重率、成活率和饲料转化效率。试验各组凡纳滨对虾成活率较对照组提高8.53%-31.69%(p<0.05)；增重率随GSH添加量的增加而增加,当添加量为180mg/kg时达到高峰；随着GSH添加量的进一步增加,增重率呈下降趋势(p<0.05)。饲料中GSH添加量不低于120 mg/kg时,显著地降低饵料系数(p<0.05)；饲料中添加GSH提高了凡纳滨对虾血清、肝胰腺和肌肉中的蛋白浓度。当GSH添加量不低于180 mg/kg时,血清及肝胰腺蛋白浓度显著高于对照组(p<0.05)；肌肉蛋白浓度在GSH添加量180 mg/kg时达到最高(p<0.05)；鳃丝蛋白浓度试验各组差异不显著(p>0.05)；试验各组凡纳滨对虾的血淋巴细胞计数均显著高于对照组(p<0.05),且与GSH添加量呈剂量-效应关系；当饲料中GSH的添加量分别60、120和180mg/kg时,凡纳滨对虾肝胰腺中GSH浓度显著升高(p<0.05)。以增重率为评价指标,GSH在凡纳滨对虾饲料中的适宜添加量为174.13 mg/kg。
(2)饲料中添加一定量的GSH能提高凡纳滨对虾肝胰腺抗氧化能力和降低脂质过氧化物含量。饲料GSH能显著提高凡纳滨对虾肝胰腺中抗氧化酶活性(p<0.05)：其中120 mg/kg、180 mg/kg和300 mg/kg组的SOD活性,120 mg/kg、180 mg/kg和240 mg/kg组的GSH-Px活性,60 mg/kg、120 mg/kg组的GR活性,显著高于对照组(p<0.05)；各试验组肝胰腺中GSH含量和总抗氧化能力T-AOC比对照组分别提高了8.93%-52.57%和3.02%-37.03%,且呈剂量-效应关系(p<0.05)。随着饲料GSH含量的升高,肝胰腺氧自由基(ROS)和脂质过氧化物MDA的含量呈下降趋势,分别在添加量为240 mg/kg和300 mg/kg时达到最低,且显著低于对照组(p<0.05)。对虾成活率和肝胰腺抗O2能力,均呈现先升后降的趋势,各项指标分别在120 mg/kg、180 mg/kg组达到最高值,并显著高于对照组(p<0.05)。
(3)在凡纳滨对虾的肝胰腺中,髓过氧化物酶(MPO)、溶菌酶(LSZ)、碱性磷酸酶(AKP)、酸性磷酸酶(ACP)活性随着饲料中GSH添加量的增加,均呈先升后降的趋势,在120-240 mg/kg之间达到最高值,且显著高于对照组(p<0.05),但随着GSH量继续增加(高于240mg/kg时),各种酶的活性急剧下降。而在血清中,LSZ、AKP和ACP活性先随饲料GSH添加量的增加而增加,分别在180 mg/kg、240 mg/kg和240mg/kg达到最高值,然后急剧下降。血清和肝胰脏中的谷草转氨酶(AST/GOT)和谷丙转氨酶(ALT/GPT)呈下降趋势,在240 mg/kg组达到最低,且显著低于对照组(p<0.05),但在300mg/kg组又有所回升。凡纳滨对虾经过离子铵胁迫1周后,发现一定添加量的GSH能提高氨氮胁迫凡纳滨对虾成活率,并对免疫因子有积极的影响。但不同的免疫因子,在不同组织敏感程度不一样,作用剂量不同。从本试验结果看,血清和肝胰腺中的LSZ、AST/GOT、ALT/GPT\ACP都是能反应凡纳滨对虾非特异免疫因子的敏感指标,与成活率结果比较一致；而MPO和肝胰腺中的AKP相对而言,规律性不强。
由上述可见,饲料中添加一定量的GSH能显著提高凡纳滨对虾的生长性能,提高凡纳滨对虾肝胰腺抗氧化能力和降低脂质过氧化物含量,有效提高凡纳滨对虾血清和肝胰腺中髓过氧化物酶、溶菌酶和磷酸酶活性,影响转氨酶的活性,并影响凡纳滨对虾机体的免疫因子水平,从而激发凡纳滨对虾的非特异免疫功能,提高凡纳滨对虾成活率,改善离子铵对凡纳滨对虾造成的氧化胁迫。
3.凡纳滨对虾肝胰腺线粒体脂质过氧化模型建立与GSH的抗氧化损伤作用
首先,建立凡纳滨对虾肝胰腺线粒体的制备方法。采用三种不同的差速离心法,提取凡纳滨对虾肝胰腺线粒体,用中性红—詹纳斯绿B (Jana's green B)染色鉴定,以OD260/OD280比值检查线粒体纯度。第二,建立凡纳滨对虾肝胰腺线粒体氧化损伤模型。分别用VC/FeSO4、NADH来诱导线粒体氧化,并确定各种催化剂的反应浓度,筛选反应时间和反应条件,建立两种诱导凡纳滨对虾肝胰腺线粒体的氧化模型。第三,研究GSH对凡纳滨对虾线粒体氧化损伤的抑制作用。在所建立的线粒体氧化损伤模型中加入不同浓度(0、0.1、0.2、0.4、0.8、1.2mmol/L)的GSH,以反应体系中MDA含量、线粒体膨胀度和DNA双链百分比为指标来观察GSH对线粒体脂质过氧化的抑制效果。研究结果显示：
(1)凡纳滨对虾肝胰腺线粒体制备方法为：按肝胰腺(鲜重)：缓冲液=1：15加入缓冲液STE (mmol/L:Sucrose 250, Tris-HCl 10, EDTA 1, pH8.0)进行匀浆,4℃1000g离心15min；4℃10000g离心20min,用缓冲液STM (mmol/L:Sucrose 250, Tris-HCl 50, MgCl2 5, pH7.4)重悬浮纯化,中性红—詹纳斯绿B染色,高倍镜观察为亮绿色,OD260/OD280=1.73。
(2)建立的VC/FeSO4氧化模型为：在线粒体蛋白浓度1mg/ml, VC浓度0.2mmol/L, FeSO4浓度4μmol/L, pH7.4HEPES缓冲体系中,30℃,经30min反应。当外源GSH添加浓度为0.4mmol/L时,对线粒体脂质过氧化的抑制作用最显著。建立的NADH模型为：线粒体蛋白浓度1.0mg/ml, FeCl30.04mmol/L, ADP 4.0mmol/L,在25mmol/L HEPES/NaOH缓冲液pH7.4(含0.15mol/LKCl)条件下,加入NADH120μmol/L启动反应,30℃水浴,振荡30min后加入20%三氯乙酸终止反应。当外源GSH添加浓度为0.4mmol/L时,对线粒体的保护效果最好。
(3)通过对VC/FeSO4和NADH模型的比较发现,两种模型都能显著(p<0.05)激发凡纳滨对虾肝胰腺线粒体的脂质过氧化,二者之间差异不显著(p>0.05)。与对照组相比,两个模型MDA含量分别为2.10倍和1.43倍,线粒体膨胀度分别为1.71倍和1.38倍,DNA双链百分比分别是60.07%和55.13%。从MDA和线粒体膨胀度来看,NADH模型的效果更好；以DNA双链百分比作为指标,VC/FeSO4模型效果更佳。
4.凡纳滨对虾肝胰腺细胞原代培养、谷胱甘肽对细胞生长、生理生化和抗氧化功能以及抗氧化酶mRNA表达量的影响
首先建立凡纳滨对虾肝胰腺细胞原代培养方法。第二,研究GSH对细胞增殖、生理生化和抗氧化功能的影响。用含有不同浓度(0、0.1、0.2、0.4、0.8、1.2mmol/L)GSH的培养基对凡纳滨对虾肝胰腺细胞进行原代培养,分别在第24h、48h和72h,收集细胞培养上清液,测定离体肝胰腺细胞的活力、RNA/DNA比、白蛋白含量、一氧化氮合酶(NOS)、胰岛素样生长因子-I(IGF-Ⅰ)、ATPase、谷丙转氨酶(ALT/GPT)和谷草转氨酶(AST/GOT)活性。分别在第24h、48h和72h收集细胞,匀浆后,测定细胞匀浆上清液中SOD、GSH-Px、CAT活性、MDA和H202含量。第三,研究GSH对离体肝胰腺细胞SOD和CAT mRNA表达量的影响。收集原代培养72h的肝胰腺细胞,提取细胞RNA,测定SODmRNA和CATmRNA表达量。结果如下：
(1)建立的凡纳滨对虾肝胰腺细胞原代培养方法为：采用高锰酸钾浸泡、冰冷灭菌PBS溶液冲洗、75%乙醇体表消毒后取出肝胰腺,用0.05%Ⅱ型胶原酶27℃消化10min,低温离心(1200r/min, 10min; 1000r/min,5min),27℃、5%CO2和饱和湿度下进行培养(接种密度：5×105个细胞/m1)。72 h后,细胞状态良好,外表光滑,折光性好,经测定细胞活性高。
(2)GSH能促进凡纳滨对虾离体肝胰腺细胞增殖、提高RNA/DNA比,促进IGF-Ⅰ分泌,表明GSH能通过对生长有关激素的调控来促进细胞的生长,GSH除了本身的抗氧化功能外,还具有营养作用。GSH能促进离体肝胰腺细胞白蛋白和NOS酶分泌,提高体外培养肝细胞的生物活性。能通过提高ATPase活性,保护细胞膜通透性,维持膜的正常生理功能。
(3)培养72h后,添加GSH对抗氧化酶和MDA含量等的影响分别为：明显提高肝胰腺细胞匀浆中SOD活性,呈现先升后降,最后上升趋于平稳,除1.2 mmol/L组外,试验各组分别比对照组提高了17.28%、9.05%、37.04%和45.27%,其中0.8mmol/L组达到显著水平(p<0.05)；各试验组GSH-Px活性均高于对照组,但试验各组组间差异不显著(p>0.05)；MDA含量除1.2 mmol/L组外,试验各组均显著低于对照组(p<0.05),在0.2 mmol/L组达到最低水平；H202含量在各试验组显著降低,各组依次降低了4.42%、17.85%、10.22%、25.58%和16.48%,其中试验0.8 mmol/L组能达到显著水平(p<0.05)。
(4)添加GSH明显提高凡纳滨对虾离体肝胰腺细胞SODmRNA表达量,除0.1 mmol/L组外,各试验组的SODmRNA表达水平均显著高于对照组(p<0.05)。与对照组相比,除1.2 mmol/L组外,添加GSH各组肝胰腺细胞CATmRNA的表达量均有所升高(p>0.05)。结果表明,GSH能提高离体肝胰腺细胞中SOD酶活力,降低脂质过氧化产物含量,影响SOD和CATmRNA的表达。
5.结论
1) GSH在体内能改善凡纳滨对虾的生长性能,提高饲料转化效率和存活率,提高对虾机体的抗氧化水平和抗氧化能力,缓解离子铵引起的氧化应激。以增重率为评价指标,GSH在凡纳滨对虾饲料中的适宜添加量为174.13 mg/kg。
2)GSH在体外能抑制凡纳滨对虾肝胰腺线粒体的脂质过氧化,当GSH添加浓度为0.4mmol/L时,对线粒体的保护效果最好。外源GSH促进了离体肝胰腺原代培养细胞的生长、增殖,提高原代培养肝胰腺细胞的生物活性。能通过调节与生长代谢相关的酶和抗氧化酶的活性,降低MDA含量来保护细胞膜通透性,维持膜的正常生理功能。并提高了凡纳滨对虾离体肝胰腺细胞SODmRNA表达量,GSH的添加量为0.8 mmol/L时效果达显著水平。
Litopenaeus vannamei is one of the excellent aquaculture varieties in the world, and aquaculture of this white shrimp has become an important export industry in developing countries in Asia and America. In China's around-sea area, Litopenaeus vannamei has been developed to be the dominant cultured products which is one of the most important exported commodities. However, the white shrimp Litopenaeus vannamei are challenged with crowded, nutritional, environmental, and metabolic stress during the intensive aquaculture processes, resulting in significantly reduced disease resistance, thus disease broken epidemicaly and new plague emerged uninterruptedly. It's too difficult for veterinarian to diagnose and therapy these maladies so that the farmers keen on abusing the antibiotics in feedstuff. Long-time and large-dose antibiotics abusing in shrimp would lead to many side-effects, such as dysbacteriosis in intestinal tract, disruption microenvironment, drug residues ect. Eventually due to humankind's allergic reaction, immunosuppression, distortion, canceration, mutation, thus the sustainable development of Litopenaeus vannamei aquaculture industry is threaten by its susceptibility to disease outbreak.
Now, it is considered that one of the effective ways to solve the problem is to increase or activate the antioxidative capacities of Litopenaeus vannamei by nutritional regulation, such as using highly efficient and safe antioxidative feed additives.
In this research, we emphasized on the roles of reduced glutathione (GSH) playing in: (1) improving the growth performance of Litopenaeus vannamei; protecting the shrimp from oxidative stress; increasing non-specific immunologic factors level in vivo; (2) being an accelerator proliferation of primary cultured hepatopancreas cells; mediating the diverse biological effects of primary cultured hepatopancreas cells of Litopenaeus vannamei; (3) improving the expression of mRNA in SOD and CAT in vitro. This research comprises of four parts.
1. Distribution of peroxidative enzymes and lipid peroxidation product in the tissues of litopenaeus vannamei before and after ammonia stress
In order to reveal the distributed characteristics of the antioxidant enzymes ----superoxide dismutase (SOD), catalas (CAT), glutathione peroxidase (GSH-Px); total antioxidant capacity (T-AOC), glutathione (GSH) and lipid peroxidation product-malonydialdehyde (MDA) in various tissues of Litopenaeus vannamei, such as gills, muscles, hepatopancreas and serum, an experiment was designed to assess the changes of these indexes before and after ammonia stress.72 shrimps, with initial weight of 6.322±0.221g were randomly selected from the representative South China Litopenaeus vannamei to sample their gills, muscles, hepatopancreas and serum to analyze the activities of SOD, GSH-Px, CAT, T-AOC, the content of GSH and MDA before and after ammonia stress.
The results indicated that hepatopancreas and serum were the sensitive tissues while gills and muscles were not as so sensitive as others. Ammonium-N stress experiments showed that CAT, GSH-PX activities, T-AOC, GSH and MDA content were not sensitive in gills and muscles to NH4Cl treatment whereas all these indexes (except T-AOC) were significantly (p<0.05) changed in hepatopancreas and SOD, GSH-Px and MDA were significantly (p<0.05) changed in serum of Litopenaeus vannamei. Furthermore, CAT activities and GSH content in hepatopancreas and GSH-Px activities in serum were significantly (p<0.01) changed after stress. These results suggest that hepatopancreas and serum are the tissues sensitive to ammonium-N stress, therefore, CAT、GSH-PX、T-AOC、GSH and MDA content could be regarded as antioxidative stress indexes of Litopenaeus vannamei and these antioxidant enzymes and lipid peroxidation product in hepatopancreas and serum could be regarded as the effective indexes of Litopenaeus vannamei when the shrimp suffer from a stress.
2. Effects of dietary GSH on the growth performance, antioxidant indexes and lipid peroxidation content, nonspecific immune factors in Litopenaeus vannamei
6 levels of glutathione (GSH) (0,60,120,180,240 and 300mg/kg), being named G0, G60, G120, G180, G240 and G300 respectively, were supplemented to a basal diet to test the effects of different dietary GSH levels on the growth performance, hepatopancreas antioxidant indexes and lipid peroxidation content, non-specific immune factors and activities of AKP, ACP, AST/GOT and ALT/GPT in litopenaeus vannamei (IBW of 1.12±0.01g).
The feeding experiment ran for 8 weeks. After 24hs the shrimp were weighed, taken into account total dead rate, total feed consumption. Then,15 shrimps were sampled per replication. The rest shrimps were sent back to the circulation system. The Litopenaeus vannamei were treated with 20 mg/L of NH4Cl without water circulating for one week. At the end of 9th week, the feeding was stopped for 24 hours, the dead rate was calculated and samples were taken. The Litopenaeus vannamei tissues were collected for assessing viability and immune factor levels.
The results showed that:
(1) weight gain rate (WGR), survival rate and feed conversion efficiency (FCE) of the shrimp fed dietary GSH were significantly increased than those of control (p<0.05). Survival rate was increased for 8.53%-31.69%(p<0.05);WGR increased with dietary GSH increasing and reached the highest when dietary GSH was 180mg/kg, but tent to decrease when dietary GSH increasing above 180mg/kg (p<0.05); When dietary GSH was above 120mg/kg, FCE was significantly increased (p<0.05). Protein content in serum, hepatopancreas, muscle of the shrimp were increased when dietary GSH was supplemented, being significantly higher in serum and hepatopancreas than that of control when dietary GSH was above 180mg/kg (p<0.05); Protein content in gills had no significant difference among the 6 groups (p>0.05). The numbers of haemolymphs of the shrimp fed dietary GSH were significantly higher than those of the control (p<0.05), showing a dose-dependent relationship (p<0.05). GSH content in hepatopancreas was increased when dietary GSH was above 60mg/kg (p<0.05). In summation, the optimal level of dietary GSH for litopenaeus vannamei was 174.13 mg/kg.
(2) Dietary GSH improved the activities of antioxidant enzymes and total antioxidation capacities, significantly decreased the content of MDA (p<0.05). The increasing dietary GSH had a significant effect on the activities of SOD and GSH-Px when the supplementary GSH was above 120 mg/kg (p<0.05), while had a significant effect on GR activities in 60mg/kg and 120mg/kg (p<0.05). Dietary GSH raised the content of GSH and the total antioxidant ability (T-AOC) in hepatopancreas and showed a dose-dependent relationship, the increase rate was 8.93%-52.57% and 3.02%-37.03%, respectively. With increasing dietary GSH, ROS and MDA level tent to decrease, reached the lowest at 240mg/kg and 300mg/kg (p<0.05) respectively. The ROS in group 180mg/kg,240mg/kg and the MDA content in each experiment group was significantly lower than those of the control (p<0.05). Dietary GSH significantly improved the survival rate and the anti-O2-ability in group 120mg/kg and 180mg/kg (p<0.05).
(3) In hepatopancreas, with the increasing of dietary GSH level, activities of MPO, LSZ, AKP and ACP of the shrimp increased, reaching the highest at 120 mg/kg,240 mg/kg,240 mg/kg,120 mg/kg respectively, then significantly decreased at a higher level (when more than 240 mg/kg). While in serum, activities of LSZ, AKP and ACP increased, reaching the highest at 180 mg/kg,240 mg/kg and 240 mg/kg respectively, then significantly decreased. AST/GOT and ALT/GPT activities, both in serum and hepatopancreas of shrimp fed dietary GSH, decreased significantly and achieved the bottom at 240 mg/kg (p<0.05), then increased at 300 mg/kg.
After a week's stress of NH4+, the survival of the shrimp fed with high level of dietary GSH was significantly increased than that of the control and the low dose group (p<0.05). Furthermore, the appropriate dietary GSH had a positive effect on nonspecific immunity factors. But for the different immunity factors, there had the different effects and had different sensitive tissues in Litopenaeus vannamei. LSZ, AST/GOT, ALT/GPT and ACP activities both in hepatopancreas and serum could be the sensitive indexes to reflect the state of non-specific immunity factors in Litopenaeus vannamei, nevertheless, the MPO and the AKP in hepatopancrea was lacking regularity.
In conclusion, dietary GSH could increase body weight gain and feed conversion efficiency of Litopenaeus vannamei. Optimal supplementation of GSH in shrimp diet could improve antioxidant capacities and decrease lipid peroxidation products, enhance the immune factors of Litopenaeus vannamei, and improve the antioxidant stress caused by ionized ammon and activate the non-specific immune functions of Litopenaeus vannamei.
3. Extraction method of hepatopancreas mitochondria, establishment of lipid peroxidation model and the effect of GSH on the oxidative damage in mitochondria of Litopenaeus vannamei
In this study, the extraction method of hepatopancreas mitochondria of Litopenaeus vannamei was studied. By using different oxidants (VC/FeSO4、NADH) at different conditions (concentration, time, and pH), two lipid peroxidation models to induce hepatopancreas mitochondrial oxidation of the Litopenaeus vannamei were established. The optimized conditions and the effective elicitor to induce hepatopancreas mitochondrial oxidation of the Litopenaeus vannamei were elucidated by analyzing the lipid peroxidation products, swelling rate of mitochondria and DNA double chain ratio. These mitochondrial oxidation models could be suitable for studying the hepatopancreas mitochondrial damage caused by lipid peroxidation, and the roles in protecting oxidation damage conferred by GSH in vitro at the subcellular level.
This research includes two parts. Firstly, the hepatopancreas mitochondria of Litopenaeus vannamei were extracted with three steps of ultracentrifugation. The mitochondria were authenticated by neutral red-Jana's green B, inspected the purity with the index of OD260/OD280 to choose the suitable extraction method of hepatopancreas mitochondria of Litopenaeus vannamei. Secondly, VC/FeSO4 and NADH were used to induce the peroxidation of hepatopancreas mitochondria of Litopenaeus vannamei, the concentration of different catalysts, the optimal time and conditions for the reaction were established to build two models. In these two models, various concentration of GSH (0, 0.1,0.2,0.4,0.8 and 1.2 mmol/L) were added to the reaction system to observe its inhibition effects of GSH. Finally, these two models were compared objectively, the MDA contents in the reaction system, the swelling rate and double chain of DNA in hepatopancreas mitochondria of Litopenaeus vannamei were the indexes to evaluate these two models.
The results showed that:(1) After homogenated, the hepatopancreas with a wet weight:buffer rate is 1:15, in a buffer of STE (mmol/L:Sucrose 250, Tris-HCl 10, EDTA 1, pH8.0), then, centrifuged at 4℃1000gx5 min,10000gx20 min and purified with buffer STM (mmol/L:Sucrose 250, Tris-HCl 50, MgCl25, pH7.4). The hepatopancreas mitochondria were in light green color in the electron microscopy at high magnification after being dyed by neutral red-Jana's green B, and the OD260/OD280 was 1.73, which is proximity to 1.8.
(2) In model VC/FeSO4, when the protein content of hepatopancreas mitochondria was lmg/ml, the VC 0.2mmol/L, the FeSO4 41/4mol/L, the reaction temperature 30℃, reacting for 30min in the buffer of pH 7.4 HEPES, the VC/FeSO4 system induced the peroxidation of hepatopancreas mitochondria effectively. While in the model of NADH, the best reaction conditions were:the protein content of hepatopancreas mitochondria was 1.0mg/ml, FeCl3 0.04mmol/L, ADP 4.0mmol/L, in 25mmol/L HEPES/NaOH buffer system with pH7.4, the start factor was NADH 120μmol/L, the reaction condition was 30℃,30min, then ended the reaction with 20%trichloroacetic acid. The exogenous GSH at 0.4mmol/L inhibited the peroxidation in both of these models obviously.
(3) Comparing the NADH with the VC/FeSO4 model of hepatopancreas mitochondrial oxidation of the Litopenaeus vannamei revealed that both the NADH and the VC/FeSO4 could significantly activate mitochondrial lipid peroxidation of Litopenaeus vannamei (p<0.05), whereas there were no significant differences between the two models (p>0.05). In the VC/FeSO4 model, the MDA content, swelling rate of mitochondria and DNA double strands percentage were respectively 2.10 times,1.71 times and 60.07% of the control; in the NADH model, the three parameters were 1.43 times,1.38 times and 55.13%, respectively. From MDA and mitochondrial swelling rate, it was suggested that the NADH model functioned better; however, the percentage of double strand DNA indicated that the VC/FeSO4 model confered better inhibitory effect.
4. Study of method for primary culture of hepatopancreatic cells of Litopenaeus vannamei, effects of GSH on the proliferation, physiological and biochemical characteristics and functions, the anti-oxidative effects, and expression of mRNA of SOD and CAT in primary culture cells in Litopenaeus vannamei
In order to reveal the effects of GSH on the proliferation, physiological and biochemical characteristics and functions, on the antioxidant affects and on the expression of anti-oxidase in vitro, thus to have the light on the mechanism insight of micronutrient in regulating aquatic organism's antioxidant stress at molecular level, we designed the experiment as follows.
Firstly, the method for improving the protocol of primary cultured hepatopancreatic cells of Litopenaeus vannamei were studied, including:(1) optimizing the methods of separating, digesting and purifying the hepatopancreatic cells of Litopenaeus vannamei;
(2) choosing the culture medium for primary cultured hepatopancreatic cells; (3) selecting the most suitable salt rate and the pH of culture and the seeding density of hepatopancreatic cells of Litopenaeus vannamei.
Secondly, the primary cultured hepatopancreatic cells were cultured in the medium with the final concentration of GSH by 0,0.1,0.2,0.4,0.8 and 1.2mmol/L, separately. We collected the supernatant of hepatopancrea cells of all the groups at 24h,48h and 72h separately, to measure the activities of cells, the RNA/DNA, the content of albumin, the nitricoxide synthase (NOS), Insulin-like growth factor-Ⅰ(IGF-Ⅰ), ATPase, alanine aminotransferase (ALT/GPT) and aspartate aminotransferase (AST/GOT).
Thirdly, the cells were collected and crushed in the culture media by microwave and then centrifuged (4℃,3000 r/min, 10min) and the supernatants were taken to analysis the antioxidant index, include:SOD, GSH-Px, CAT, MDA and H2O2 quantitatively at 24h,48h and 72h, separately.
Finally, after the hepatopancreatic cells were primary cultured in medium with different concentration of GSH for 72 hours, the cells were collected, the RNA of hepatopancreatic cells was extracted and the mRNA was detected by RT-PCR to measure the expression levels of SOD mRNA and CAT mRNA.
The results showed that:
(1) The method for primary culture of hepatopancreatic cells of Litopenaeus vannamei was established. After being soaked in potassium permanganate, washed by cold PBS and sterilized totally in 75% ethanol, the hepatopancreas were removed away from the Litopenaeus vannamei. The hepatopancreatic cells were isolated in 0.05% typeⅡcollagenase at 27℃for 10 min, then purified with a centrifugation method at 1200 r/min,10 min+1000 r/min,5min, and cultured in the box of 5% CO2, saturated degree of humidity,27℃at a seeding density of 5×105 cells/ml. The cells were at an excellent status characterized by smooth cell surface, better refractive, and higher activities of hepatocytes.
(2) GSH boosted the proliferation of Litopenaeus vanhnamei's hepatocyte in vitro, increased RNA/DNA ratio, accelerated the hepatocytes to excrete IGF-1, indicating that GSH could facilitate the up-growth of cells through regulating the relevant hormone. GSH could take on nutrition action and antioxidant function. GSH could improve the content of albumin and the secretion of nitricoxide synthase (NOS) in vitro, thus enhance the bioactivity of hepatopancreatic cells in vitro. Boost the ATPase activity, and protect the penetrating power of cell membrane while keep the well-balanced physiological function. GSH could reduce the damnification hurt by environment factor, and then accelerate the organizational recovery when the hepatopancrea was damnified.
(3) After being cultured for 72h, GSH addition increased the SOD activities in all the experiment groups except 1.2 mmol/L group. The the increase rate was 17.28%、9.05%、37.04% and 45.27% respectively and the group 0.8 mmol/L affected the SOD activities significantly compared with the control (p<0.05). In conclusion, the proper GSH addition affected the SOD activities in the hepatopancreatic cells of Litopenaeus vannamei in vitro, showing a trend of increasing first then declining and increasing slowly until reaching to a platform, finally. After being cultured for 72h, GSH addition improved the GSH-Px activities in all experiment groups, but had no difference between all the groups (p>0.05). Whlie, the CAT hadn't been detected in some groups because of the insensitivity of the methods for testing in the supernatant preparations from the hepatopancreatic cells in vitro.
MDA contents had a obvious decline trend in all groups at 72h obviously (p<0.05) and reach the lowest content at 0.2 mmol/L group. After being cultured for 72h, the GSH addition can decrease the H2O2 contents in all the experiment groups significantly (p<0.05). The the decrease rate was 4.42%,17.85%,10.22%,25.58% and 45.27% respectively and had a difference at distinguish level of group 0.8 mmol/L (p<0.05).
(4) Adding GSH evidently boosted the SOD genie expression of litopenaeus vannamei's hepatopancreatic cells in vitro. Except group 0.1mmol/L, the SOD mRNA expression level were all observably higher than the experiment group (p<0.05). Except group 1.2 mmol/L, adding GSH in hepatopancreatic cells elevated CATmRNA expression level (p>0.05). GSH hoisted SOD enzymatic activities in hepatic separate cells, depressed the lipid peroxide content, effected the expression of SOD and CAT mRNA.
The results described above suggests that the supplementation of GSH in culture media be capable of reducing lipid peroxide and modulating the activities of SOD and CAT in hepatopancreatic cells of Litopenaeus vannamei in vitro. Through increasing the effect of the expression capacity of antioxidant enzyme CAT and SOD, especially SODase, regulating antioxidant level, that purged the free radicals, efficiently regulated the free radicals metabolism, prevented lipid peroxidation.
In Conclusion, GSH can ameliorate the growth performance of Litopenaeus vannamei in vivi, boost the efficiency of feedstuff transforming, enhance the anti-oxidative level and anti-oxidative capability of Litopenaeus vannamei's, organic tissue, ease up the oxidation allergic response caused by ammonium stress. The optimal level of dietary GSH for litopenaeus vannamei was 174.13 mg/kg. GSH can also restrain the lipid peroxide of Litopenaeus vannamei hepatopancreatic mitochondrion in vitro, when the culture medium GSH concentration is 0.4mmol/L, there is best protection effect for mitochondrion. Exogenous GSH can improve the growth and proliferation of the hepatopancreatic cell in vitro, enhance it biologic activity. It can protect the cell membrane penetration by regulating the correlative metabolic enzyme and anti-oxidative enzyme, depressing the content of MDA, maintaining the well-balanced physiology function. It can boost the hepatopancreatic cell SODmRNA expression level in Litopenaeus vannamei. The effect reach the prominent level when the culture medium GSH is 0.8 mmol/L.
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