一种复方中草药免疫增强剂对牙鲆免疫力及抗病力的影响

Effects of a Compound Chinese Herbal Immune Enhancer on the Immunity and Disease Resistance in Japanese Flounders Paralichthys olivaceus

关键词:  中草药  免疫增强剂  酶活性  免疫相关基因  牙鲆

Key words:  Chinese herbal medicine  immuno-enhancer  enzyme activity  immuno-related gene  Japanese flounder

摘要:

为研究中草药复方制剂对牙鲆Paralichthys olivaceus免疫力及抗病力的影响，实验基于中草药药物功效与配伍原则，研制出一种复方中草药免疫增强剂（YLK），配方包含黄芪、茯苓、党参、蒲公英、鱼腥草、白花蛇舌草、金银花、玉竹、甘草、五味子等；将复方中草药分别以1%（W：W）和3%（W：W）的添加量拌入基础配合饲料中，连续投喂牙鲆30 d，然后用迟钝爱德华氏菌Edwardsiella tarda攻毒，分别于攻毒后24 h和72 h取鱼血清进行免疫相关酶活性测定，同时在攻毒后6，12，24，48，72 h取鱼脾脏进行免疫相关基因表达水平的检测，并观察记录鱼体的累计死亡情况。结果表明，在迟钝爱德华氏菌感染后24 h和72 h，3%中草药添加组的牙鲆血清中碱性磷酸酶（Alkaline Phosphatase，ALP）、酸性磷酸酶（Acid Phosphatase，ACP）、超氧化物歧化酶（Superoxide Dismutase，SOD）以及溶菌酶（Lysozyme，LSZ）活性水平均显著高于仅投喂基础饲料的对照组；1%中草药添加组除ALP活性以外，其他3种酶活水平也显著高于对照组（P<0.05），2组不同中草药添加组鱼体血清酶活整体上差异不显著性；荧光定量PCR检测结果显示，各投喂组牙鲆脾脏组织中白介素1β（IL-1β）、白介素6（IL-6）、TCRα受体及C型溶菌酶基因表达量均出现不同程度的上调，而2个中草药投喂组牙鲆在感染后多个时间点的基因表达水平均高于单独投喂饲料组的牙鲆（P<0.05）；添加3%中草药和1%中草药对牙鲆抗迟钝爱德华氏菌感染的相对保护率分别为34.2%和26.3%。研制的复方中草药制剂具有明显的免疫增强效果，能显著提高牙鲆抗迟钝爱德华氏菌感染能力，研究结果将为水产养殖中草药防病技术研究提供重要参考。

Abstract:

In order to study the effect of Chinese herbal medicine compound preparation on Paralichthys olivaceus immunity and disease resistance,based on the efficacy and compatibility of Chinese herbal medicine,the experiment developed a compound Chinese herbal medicine immune enhancer.The formula contained Astragalus membranaceus,Poria cocos,Codonopsis pilosula,Taraxacum mongolicum,Houttuynia cordata,Herba Hedyotidis diffusae,Lonicera japonica,Polygonatum odoratum,Glycyrrhiza uralensis and Schisandra chinensis etc. The compound Chinese herbal medicines were mixed into the basic compound feed at the dosages of 1% (W:W) and 3% (W:W) respectively,and were continuously fed to flounder for 30 d.Then the fish was challenged by intramuscular injection with Edwardsiella tarda.The fish serum was taken at 24 h and 72 h after challenge to measure the activity of immune related enzymes. At the same time,at 6,12,24,48,72 h after challenge,the spleen of the fish was taken to detect the expression level of immune-related genes,and the cumulative death of the fish was observed and recorded. The results showed that 24 h and 72 h after E.tarda infection,in the serum of P.olivaceus in the 3% Chinese herbal supplement group,the activity levels of alkaline phosphatase (ALP),acid phosphatase (ACP),superoxide dismutase (SOD) and lysozyme(LSZ) were significantly higher than that in control group fed only by basic feed. In addition to the ALP activity in the 1% Chinese herbal medicine addition group,the other three enzyme activities were also significantly higher than those in the control group (P<0.05). There was no significant difference in serum enzyme activity between the two groups with different Chinese herbal medicines. The results of fluorescent quantitative PCR showed that the expression levels of interleukin 1β (IL-1β),interleukin 6 (IL-6),TCRα receptor and C-type lysozyme genes in the spleen tissue of P.olivaceus in each feeding group were up-regulated to varying degrees. However,the gene expression level of P.olivaceus in the two Chinese herbal medicine fed groups at multiple time points after infection was higher than that of the P.olivaceus group fed alone (P<0.05). The relative protection rates of adding 3% Chinese herbal medicine and 1% Chinese herbal medicine to P.olivaceus against E.tarda infection were 34.2% and 26.3%,respectively.These results indicated that the Chinese herbal formula could enhance the immunity of flounders and significantly improve the resistance of flounder to the infection of E.tarda,which could provide an important reference for the research on the disease prevention technology of Chinese herbal medicine in aquaculture.