基于代谢组学的黄芩水提物防治罗非鱼无乳链球菌病作用机制研究

Study on the prevention and treatment mechanism of Streptococcus agalactiae disease of Tilapia by water extract of Scutellaria baicalensis based on metabolomics

为了探讨黄芩水提物防治罗非鱼无乳链球菌病的作用机制,试验将104尾体重60 g左右的吉富罗非鱼随机分为4组,分别为空白对照组、阴性对照组、防治组和模型组,每组26尾。阴性对照组和防治组均饲喂含5%黄芩水提物的日粮,空白对照组和模型组饲喂基础日粮。于饲喂后第8天进行攻毒试验,空白对照组和阴性对照组腹腔注射灭菌生理盐水,防治组和模型组腹腔注射浓度为1.5×10~9 cfu/mL的无乳链球菌菌液,0.3 mL/尾,观察10 d,试验过程中记录各组临床症状及死亡情况,对濒死的试验鱼进行尾静脉采血,并对死亡试验鱼进行剖检,攻毒试验结束后计算各组的死亡率及5%黄芩水提物对防治组的保护率,并对其他试验鱼进行尾静脉采血。对各组血液进行液相色谱质谱(LC-MS)分析,通过构建主成分分析(PCA)模型和偏最小二乘判别分析(PLS-DA)模型筛选差异代谢物,导入京都基因和基因组百科全书(KEGG)数据库后进行通路富集分析。结果表明:攻毒后,空白对照组和阴性对照组食欲佳,未出现异常症状,死亡率均为0,剖检未见明显异常。模型组行动迟缓,食欲减退,体表破溃出血,眼球突出,机体失去平衡,出现侧游或转圈游动等行为;剖检可见肝脏、脾脏、肠组织出现不同程度的水肿、出血,死亡率为95.45%。防治组先是出现与模型组相同的症状,部分鱼死亡,部分鱼症状有所缓解(食欲慢慢恢复正常,精神状态良好),死亡率为46.15%;经计算5%黄芩水提物对罗非鱼的保护率为51.65%。在正负离子模式下5组共检测到7652个离子峰,鉴定到998种代谢物。其中模型组与空白对照组之间的差异代谢物有348种,阴性对照组与空白对照组之间的差异代谢物有257种,治疗组与空白对照组之间的差异代谢物有241种,治疗组与模型组之间的差异代谢物有275种,治疗组与阴性对照组之间的差异代谢物有190种,阴性对照组与模型组之间的差异代谢物有377种。与空白对照组比较,模型组血清中N′-胆酰甘氨酸、喹啉-4,8-二醇、色氨酸、吲哚-3-乙酰胺、5-羟吲哚乙酸、雌二醇-17β-3-硫酸酯、可的松、N′-甲酰基犬尿氨酸、牛磺胆酸相对丰度不同程度降低,19-羟基烯二酮、脱氧皮质酮、11b-羟孕酮相对丰度不同程度升高。给予5%黄芩水提物干预后,防治组这12种代谢物相对丰度均发生了显著或极显著回调(P<0.05或P<0.01、P<0.001)。这个过程涉及色氨酸代谢、类固醇激素的生物合成、胆固醇代谢及胆汁分泌等代谢通路。说明5%黄芩水提物能够有效减轻感染无乳链球菌的罗非鱼的临床症状并降低死亡率,且黄芩主要通过调节色氨酸代谢、类固醇激素的生物合成、胆固醇代谢、胆汁分泌等代谢通路发挥防治罗非鱼无乳链球菌病的作用。

In order to explore the mechanism of prevention and treatment of Streptococcus agalactiae disease of Tilapia by water extract of Scutellaria baicalensis,A total of 104 GIFT Tilapia with body weight of about 60 g were randomly divided into 4 groups:blank control group,negative control group,control group and model group,with 26 fish in each group.The negative control group and control group were fed a diet containing 5%Scutellaria baicalensis water extract,and the blank control group and model group were fed a basic diet.The challenge test was conducted on the 8th day after feeding;the blank control group and the negative control group were injected with sterile normal saline peritonelly,and the control group and the model group were injected with 1.5x10°efu/mL of Streptococcus agalactiae bacteriae solution,O.3 mL/tail,and observed for 10 days.During the experiment,clinical symptoms and death of each group were recorded,and blood was collected from the tail vein of dying test fish.The dead test fish were dissected.After the experiment,the mortality rate of each group and the protection rate of 5%Scutellaria baicalensis water extract to the control group were calculated,and the tail vein blood was collected for other test fish.The blood of each group was analyzed by liquid chromatography mass spectrometry(LC-MS),and the differential metabolites were screened by constructing principal component analysis(PCA)model and partial least squares discriminant analysis(PLS-DA)model,and the pathway enrichment analysis was performed after the KEGG database was imported.The results showed that the blank control group and the negative control group had good appetite and no abnormal symptoms,and the mortality rate was O.In the model group,the movement was slow;the appetite was decreased;the body surface was broken and bleeding;the eyeball was protruding;the body was out of balance,and the behavior of side swimming or circle swimming appeared.Necropsy showed edema and bleeding in liver,spleen and intestinal tissues of different degrees,and the mortality rate was 95.45%.The symptoms of the control group were the same as those of the model group.Some fish die;some fish were relieved(the appetite slowly returned to normal,and the mental state was good).The mortality rate was 46.15%.The protection rate of 5%scutellaria scutellaria water extract to Tilapia was 51.65%.In the positive and negative ion mode,7652 ion peaks were detected and 998 metabolites were identified.Among them,there were 348 different metabolites between the model group and the blank control group,257 different metabolites between the negative control group and the blank control group,241 different metabolites between the treatment group and the blank control group,275 different metabolites between the treatment group and the model group,and 190 different metabolites between the treatment group and the negative control group.There were 377 different metabolites between negative control group and model group.Compared with the blank control group,the relative abundations of N'-cholinylglycine,quinoline-4,8-diol,tryptophan,indole3 acetamide,5-hydroxyindoleacetic acid,estradiol 17β-3-sulfate,cortisone,N'-formylkynurenine and taurocholic acid in serum in the model group decreased to varions degrees.The relative abundance of 19-hydroxyenedione,deoxycorticosterone and llb-hydroxyprogesterone increased to different degrees.After the intervention of 5%Scutellaria baicalensis water extract,the relative abundance of these 12 metabolites in the control group was significantly or extremely significantly reversed(P<0.05 or P<0.01,P<0.001).This process involved tryptophan metabolism,steroid hormone biosynthesis,cholesterol metabolism,bile secretion and other metabolic pathways.These results indicated that 5%water extract of scutellaria baicalensis could effectively reduce the clinical symptoms and mortality of Tilapia infected with Streptococcus agalactis.Scutellaria baicalensis played a role in the prevention and treatment of Streptococcus agalactiae disease of Tilapia by regulating metabolism of tryptophan,biosynthesis of steroid hormones,cholesterol metabolism,bile secretion and other metabolic pathways.