海藻硫酸多糖对铜绿假单胞菌感染的凡纳滨对虾抗氧化防御系统的影响

Effects of seaweed sulfate polysaccharides on the antioxidant defense system of Pseudomonas aeruginosa-infected Litopenarus vannamei

为了研究海藻硫酸多糖对铜绿假单胞菌(Pseudomonas aeruginosa,PA)感染的凡纳滨对虾(Litopenaeus vannamei)抗氧化防御系统的影响,试验先采用磷钼络合物法测定5种海藻硫酸多糖(PE-Ⅰ、PE-Ⅱ、LJP-Ⅰ、LJP-Ⅱ、LJP-Ⅲ)总抗氧化活性[以二丁基羟基甲苯(BHT)为阳性对照],采用滤纸片法测定5种海藻硫酸多糖的抑菌圈直径,同时测定PA对凡纳滨对虾的半数致死浓度(LC_(50))。将健康的对虾随机分为3组,分别为模型组、试验组及对照组,每组20尾,对照组与模型组饲喂基础日粮,试验组饲喂添加抑菌效果最好的海藻硫酸多糖的基础日粮,饲喂14 d后模型组和试验组腹腔注射PA菌悬液(浓度为攻毒48 h后的LC_(50))30μL,对照组不做任何处理。攻毒后第24小时、48小时、7天、14天统计死亡率,收集攻毒48 h后各组对虾肠道内容物,计数PA的菌落总数。利用试剂盒测定血淋巴中血浆总蛋白(TP)质量浓度、酚氧化酶(PO)活性,肝胰腺组织中总超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和谷胱甘肽过氧化物酶(GSH-Px)等抗氧化防御酶活性及丙二醛(MDA)含量;采集对虾肝胰腺组织用苏木精-伊红(H.E.)染色试剂盒染色进行组织病理学观察。结果表明:6种物质的总抗氧化活性大小为LJP-Ⅱ>LJP-Ⅰ>LJP-Ⅲ>BHT>PE-Ⅰ>PE-Ⅱ,其中LJP-Ⅱ、LJP-Ⅰ、LJP-Ⅲ总抗氧化活性显著高于BHT(P<0.05);海藻硫酸多糖LJP-Ⅱ在浓度150 mg/mL时对PA的抑菌圈直径最大,达到(15.05±0.04)mm,因此选取LJP-Ⅱ作为日粮添加物,研究其对PA感染的凡纳滨对虾抗氧化防御系统的影响。当攻毒剂量为30μL时,PA对凡纳滨对虾48 h的LC_(50)为7.96×10~(6)cfu/mL。与模型组相比,攻毒后第14天试验组的死亡率显著下降(P<0.05);血淋巴中TP质量浓度、PO活性,肝胰腺组织中SOD、CAT、GSH-Px活性均显著升高(P<0.05),MDA含量显著下降(P<0.05)。饲喂添加海藻硫酸多糖LJP-Ⅱ日粮可明显改善PA感染对虾肝胰腺组织、管腔和小管损伤程度,形态结构趋于正常。说明海藻硫酸多糖LJP-Ⅱ可显著提升抗氧化防御系统生化指标水平,减轻PA对肝胰腺组织的损伤。

In order to study the effects of seaweed sulfate polysaccharides on the antioxidant defense system of Pseudomonas aeruginosa(PA)-infected Litopenarus vannamei,in the experiment,the total antioxidant activity of five seaweed sulfate polysaccharides(PE-Ⅰ,PE-Ⅱ,LJP-Ⅰ,LJP-Ⅱ,LJP-Ⅲ)was determined by phosphomolybdenum complex method;BHT was used as the positive control;the diameter of the circle of inhibition of the five seaweed sulfate polysaccharides was determined by the filter paper sheet method;and the half lethal concentration(LC_(50))of PA for Litopenarus vannamei was also determined.Healthy shrimp were randomly divided into three groups,namely the model group,the experimental group and the control group;with 20 shrimps in each group consisted.The control and model groups were fed with basal diets,while the experimental group was fed with basal diets supplemented with seaweed sulfate polysaccharides,which had the best bacteriostatic effect.After 14 days of feeding,the model and control groups were injected intraperitoneally with 30μL of PA bacterial suspension(LC_(50)after 48 h of challenge);the control group was left untreated.Mortality was counted at the 24th,48th hours,7th,and 14th day after challenge;the intestinal contents of shrimp in each group were collected 48 h after challenge;and the total number of colonies of PA was counted.The mass concentration of total plasma protein(TP)and phenoloxidase(PO)activity in hemolymph,antioxidant defense enzyme activities such as total superoxide dismutase(SOD),catalase(CAT),and glutathione peroxidase(GSH-Px),and malondialdehyde(MDA)content in hepatopancreatic tissue were determined by using the kits.Shrimp hepatopancreatic tissues were collected and stained with hematoxylin-eosin(H.E.)staining kit for histopathological observation.The results showed that the magnitude of the total antioxidant activity of the six substances was LJP-Ⅱ>LJP-Ⅰ>LJP-Ⅲ>BHT>PE-Ⅰ>PE-Ⅱ,in which the total antioxidant activity of LJP-Ⅰ,LJP-Ⅱ,and LJP-Ⅲwas significantly higher than that of BHT(P<0.05).The highest diameter of the circle of inhibition of PA was achieved by LJP-Ⅱ,a brown alga kelp polysaccharide with sulfate,at a concentration of 150 mg/mL,which reached(15.05±0.04)mm;therefore,LJP-Ⅱwas selected as a dietary additive to study its effect on the antioxidant defense system of PA-infected Litopenarus vannamei.At an attack dose of 30μL,the LC_(50)of PA on Litopenarus vannamei for 48 h was 7.96×10~6 cfu/mL.Compared with the model group,the mortality rate of the experimental group for 14th day after challenge was significantly decreased(P<0.05);TP mass concentration and PO activity in hemolymph,and SOD,CAT,and GSH-Px activities in hepatopancreatic tissues were significantly increased(P<0.05);and MDA content was significantly decreased(P<0.05).PA-infected shrimp fed diets supplemented with fucoidan kelp sulfate polysaccharide LJP-Ⅱsignificantly improved the degree of hepatopancreatic tissue,lumen,and tubule damage,and normalized the morphologic structure of the shrimp.The results indicated that the fucoidan kelp polysaccharide LJP-Ⅱcould significantly improve the level of biochemical indexes of antioxidant defense system and reduce the damage of hepatopancreatic tissues caused by PA.