EFFECTS OF BACILLUS SUBTILIS ON THE HEPATIC LIPID METABOLISM OF CTENOPHARYNGODN IDELLUS
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摘要: 为研究益生枯草芽孢杆菌(Bacillus subtilis)对草鱼(Ctenopharyngodon idella)肝脏脂质代谢及抗氧化功能的影响, 实验设置对照组(Con组)、Aeromonas hydrophila组(Ah组)、Aeromonas hydrophila+Bacillus subtilis组(Ah+Bs组)、Bacillus subtilis+Aeromonas hydrophila组(Bs+Ah组), 三个实验组均腹腔注射1×105 CFU/fish嗜水气单胞菌(Aeromonas hydrophila), 枯草芽孢杆菌饲料含菌量为1×107 CFU/g, 实验周期为56d, 并于第28和第56天取样。结果表明, 与Ah组相比, 投喂枯草芽孢杆菌饲料后, (1)体增重率、特定生长率显著增加(P<0.05); (2) 28d时肝脏油红O染色脂滴面积及脂肪含量显著下降(P<0.05); (3)调节血脂代谢及缓解肝脏损伤: 血清胆固醇、甘油三酯、高密度脂蛋白胆固醇、低密度脂蛋白胆固醇含量升高, 谷草转氨酶和谷丙转氨酶活性显著降低(P<0.05); (4)调节脂质代谢: 28d时乙酰辅酶A羧化酶的表达水平下调, 脂蛋白脂酶及脂肪甘油三酯脂肪酶的表达水平上调; (5)增强肝脏抗氧化能力、减少脂质过氧化的发生: 肝脏超氧化物歧化酶、过氧化氢酶、谷胱甘肽、总抗氧化能力提高; 丙二醛及过氧化氢含量降低。综上, 在饲料中添加益生枯草芽孢杆菌可以增强草鱼的抗氧化能力, 缓解机体因嗜水气单胞菌感染造成的肝脏损伤, 调节肝脏脂质代谢功能, 减少脂质在肝脏中的积累, 并促进草鱼的生长。Abstract: To investigate the effects of Bacillus subtilis on the hepatic lipid metabolism and antioxidant function, the grass carp were randomly divided into 4 groups: control group (Con), Aeromonas hydrophila group (Ah), Aeromonas hydrophila+Bacillus subtilis (Ah+Bs) group and Bacillus subtilis+Aeromonas hydrophila group (Bs+Ah), and the three experimental groups were intraperitoneally injected with 1×105 CFU/fish A. hydrophila, and B. subtilis diet contained 1×107 CFU/g for a trial of 56 days. The results showed that two groups fed with B. subtilis had higher weight gain rate and special growth rate compared with the Ah group (P<0.05). B. subtilis significantly decreased liver lipid droplet size and the hepatic lipid content at day 28 (P<0.05), increased the content of serum cholesterol, triglyceride, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol and reduced activities of aspartate aminotransferaseand alanine aminotransferase. B. subtilis downregulated the expression of acetyl-CoA carboxylase alpha and upregulated the expression of lipoprotein lipase and adipose triglyceride lipase on day 28. B. subtilis enhanced superoxide dismutase, catalase, glutathione and total antioxidant capacity, and diminished malondialdehyde and hydrogen peroxide. In conclusion, probiotic B. subtilis supplementation can promote growth, enhance the antioxidant function, reduce the liver damage caused by infection of A. hydrophila, regulate the hepatic lipid metabolism, and reduce the hepatic lipid accumulation in grass carp.
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Keywords:
- Grass carp /
- Aeromonas hydrophila /
- Bacillus subtilis /
- Lipid metabolism /
- Antioxidant function
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草鱼(Ctenopharyngodon idella)是我国重要的淡水养殖鱼类之一[1, 2], 随着集约化养殖的发展, 脂肪肝在养殖草鱼中发病率不断上升, 已成为水产养殖业关注的严重问题[3]。“二次打击”学说在脂肪肝病的发病机制中受到认可并得到深入研究, 学说表明氧化应激在脂肪肝病发生发展进程中起重要作用[4]。嗜水气单胞菌(Aeromonas hydrophila)广泛存在于养殖水体中, 是淡水鱼类养殖过程中的主要致病菌。本实验室研究表明, 感染嗜水气单胞菌可降低草鱼抗氧化能力, 诱发肝脏脂质过氧化[5]和肝脏脂质沉积[6]。研究表明, 益生菌可显著减轻哺乳动物肝脏脂肪肝等症状, 改善肝功能[7, 8]。益生枯草芽孢杆菌(Bacillus subtilis)广泛应用于水产养殖, 可调节水质[9]、增强水产动物的免疫力和抗氧化能力[5, 10]、调节肠道菌群及增强消化酶活性[11, 12]等, 但对其在鱼类肝脏脂质代谢方面研究甚少。
因此, 本研究分别在感染嗜水气单胞菌前后投喂含益生枯草芽孢杆菌的饲料, 探究枯草芽孢杆菌对肝脏脂质代谢的作用及可能机制, 为益生菌在草鱼“保肝护肝”方面的作用提供依据。
1. 材料与方法
1.1 实验菌株
实验用嗜水气单胞菌Ah1菌株分离自细菌败血症银鲫; 枯草芽孢杆菌Ch9菌株分离自草鱼肠道。以上菌株均有由华中农业大学水产学院水产动物医学实验室提供和保存。
1.2 实验饲料
本实验两种饲料配方及营养成分实测值见表 1, 饲料原料购自湖北海大饲料有限公司。饲料原料经粉碎后过60目筛, 制作含枯草芽孢杆菌的饲料时将枯草芽孢杆菌菌液代替无菌水, 所有原料混合均匀后经小型制粒机加工成直径为2.0 mm的颗粒饲料, 风干后置于–20℃冰箱中保存。采用平板计数法测定饲料中枯草芽孢杆菌的含量约为1×107 CFU/g。
表 1 实验饲料配方及基本成分Table 1. Compositions of experimental diets原料Ingredient 含量Content (g/kg) 营养成分Composition 含量Content
(% DM)鱼粉Fish meal 80 基础饲料Basal diet 豆粕Soybean meal 240 粗蛋白Crude protein 29.83 菜粕Rapeseed meal 340 粗脂肪Crude lipid 4.39 小麦粉Wheat Flour 250 水分Moisture 10.16 豆油Soybean oil 6 灰分Ash 8.32 磷酸二氢钙Ca(H2PO4)2 20 维生素预混料Vitamin premix 1 1 枯草芽孢杆菌饲料B. subtilis diet 矿物质预混料Mineral premix 2 3 粗蛋白Crude protein 29.74 氯化钠NaCl 2 粗脂肪Crude lipid 4.37 氯化胆碱Choline chloride 2 水分Moisture 10.13 纤维素Cellulose 56 灰分Ash 8.39 总量Total 1000 注: 1维生素预混料(mg/kg): 维生素A, 6500 IU; 维生素D3, 4500 IU; 维生素C, 120 mg; 维生素E, 25 mg; 维生素K3, 5 mg; 维生素B1, 12.5 mg; 维生素 B2, 12.5 mg; 维生素B6, 15.0 mg; 维生素B12, 0.025 mg; 烟酰胺, 50 mg; 泛酸, 40 mg; 肌醇, 75 mg; 叶酸, 2.5 mg; 生物素, 0.08 mg; 2矿物质预混料(mg/kg): 氯化钠, 1.0; 硫酸镁, 15.0; 磷酸二氢钠, 25.0; 六水合氯化铝, 0.06; 磷酸二氢钾, 32.0; 磷酸二氢钙, 20.0; 柠檬酸铁, 2.5; 乳酸钙, 3.5; 七水硫酸锌, 0.353; 四水硫酸锰, 0.162; 五水硫酸铜, 0.031; 六水合氯化钴, 0.001; 碘酸钾, 0.003; 纤维素, 0.39Note: 1 Vitamin premix (mg/kg): vit. A 6500 IU, vit. D3 4500 IU, vit. C 120 mg, vit. E 25 mg, vit. K3 5 mg, vit. B1 12.5 mg, vit. B2 12.5 mg, vit. B6 15.0 mg, vit. B12 0.025 mg, niacinamide 50 mg, pantothenate 40 mg, inositol 75 mg, folic acid 2.5 mg, biotin 0.08 mg; 2 Mineral premix (mg/kg): NaCl 1.0; MgSO4 15.0; NAh2PO4·2H2O 25.0; AlCl3·6H2O 0.06; KH2PO4 32.0; Ca(H2PO4)2·H2O 20.0; C6H5FeO7·10H2O 2.5; CaC6H10CaO6·5H2O 3.5; ZnSO4·7H2O 0.353; MnSO4·4H2O 0.162; CuSO4·5H2O 0.031; CoCl2·6H2O 0.001; KIO3·6H2O, 0.003; cellulose, 0.39 1.3 实验设计
实验用草鱼购自湖北省黄冈市团风县百容水产良种有限公司。暂养4周后选取外观健康、规格一致的草鱼(50.53±0.70) g随机分配到12个容量为300 L的养殖缸(一共4个组, 每组3个重复), 每个缸投放密度为25尾。
实验分为4个组, 具体分组情况如表 2所示, 实验开始时对照组每尾鱼腹腔注射0.1 mL 0.01mol/L灭菌PBS, 其余各组每尾鱼腹腔注射0.1 mL 1.0×106 CFU/mL 嗜水气单胞菌菌液。嗜水气单胞菌的浓度为LC50的10%(半致死浓度为1×106 CFU/fish)。实验用鱼均在华中农业大学水产学院养殖实验基地进行循环水养殖, 整个实验期间水质条件: 水温(25±1)℃、pH 7.5±0.3、溶解氧(7±0.45) mg/L、氨氮含量(0.015±0.002) mg/L以及亚硝酸盐氮(0.05±0.008) mg/L, 光周期为自然光周期。养殖实验持续56d, 每天饱食投喂饲料2次(8:30和16:30), 未吃完的剩余饲料在投喂之后的2h使用虹吸管收集, 并将其在烘箱中以60℃干燥12h至恒重, 以确定各组实验鱼的实际摄食量。
表 2 实验分组设计Table 2. The experimental design分组Group 暂养期间饲料Diet during the temporary rearing period 腹腔注射Intraperitoneal injection 注射后投喂饲料Diet after intraperitoneal injection 对照Control 基础饲料 PBS 基础饲料 Ah 基础饲料 1×105 CFU/fish A. hydrophila 基础饲料 Ah+Bs 基础饲料 1×105 CFU/fish A. hydrophila B. subtilis饲料 Bs+Ah B. subtilis饲料 1×105 CFU/fish A. hydrophila 基础饲料 1.4 样品采集
分别在感染嗜水气单胞菌后的第28和第56天, 将草鱼饥饿24h。用MS-222(10 mg/L)麻醉后, 将每缸鱼计数并称重。每组随机选择24尾鱼, 其中6尾鱼测量个体的体长、体重、内脏重量和肝脏重量, 及从尾静脉取血以获得血清; 6尾鱼用于总RNA提取及抗氧化酶的测定; 6尾鱼用于肝脏脂质含量的测定; 6尾鱼取得的肝脏放入多聚甲醛固定液固定以备后续冰冻切片。
1.5 样品分析
脂肪含量的测定及油红染色分析 脂肪含量的测定: 将肝脏样品在–50℃冷冻干燥24h, 然后用索氏抽提法测定肝脏的粗脂肪含量。
油红O染色分析: 用于油红O染色的肝脏样本用多聚甲醛固定48h后, 进行脱水、包埋、切片(厚度8 μm)、染色和拍照。从每个样本中随机选取10个视野, 用Image-Pro Plus 6.0软件计算肝组织中染成红色的脂滴的相对面积, 采用双盲法统计各图, 汇总得到结果。
血清生化指标测定 血清胆固醇(CHO)、甘油三酯(TG)、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)含量以及血清谷草转氨酶(AST)、谷丙转氨酶(ALT)活性采用荷兰威图全自动生化分析仪测定, 试剂盒均购自中生北控生物科技股份有限公司。
脂质代谢相关基因表达分析 从草鱼肝脏中提取RNA, 然后反转录为cDNA, 以β-肌动蛋白(β-actin)为内参, 使用Roche Light Cycler 480 real-time PCR仪进行实时荧光定量PCR, 引物见表 3(由武汉天一辉远生物科技有限公司合成)。反应总体系为20 μL, 包括cDNA 2 μL、上下游引物各0.5 μL、焦碳酸二乙酯(Diethylpyrocarbonate, DEPC)水7 μL、2×SYBR Green qPCR Mix 10 μL(Aidlab, PC5902)。
表 3 RT-PCR引物序列Table 3. Primers used for real-time PCR基因Gene 登录号GenBank accession No. 引物序列Primer sequence(5′—3′) 退火温度Annealing temperature (℃) 脂肪酸合酶FAS HM802556.1 GTCCACAGGGTGTCGTTC 58 GAGGTCTTGGGCTCTTTATT 乙酰辅酶A羧化酶α ACCα GU908475 AGTATCGCAGTGGCATCA 58 TGTCCCCTTTGTTTTCCT 肉碱棕榈酰基转移酶Iα1a CPTIα1a KJ816747 TTTACGACGGACGGTTGC 58 GCTTGTTCTTCCCACGACT 脂蛋白脂酶LPL FJ436077 AGCCCTGTATGAACGAGA 58 CACATCCTTGCCCACTAG 脂肪甘油三酯脂肪酶ATGL HQ845211 TATTGTGGTTTAATCCCTCC 58 CAGTGCCTTGCTCAGTCT 激素敏感脂酶HSL FJ843081 CCGACAAGGACAGGACAGT 58 ATGACCAGGCAGGGAGAA 肝型脂肪酸结合蛋白L-FABP EU220990.1 GGGAAAACCATCACTAACTC 58 TCAGGGTCTCAACCATCTC 脂肪酸移位酶FAT/CD36 KU821103.1 CTTCCCCACTTCCTCTATG 58 TAATCGGTTCCACATCCA 固醇调控元件结合蛋白-1c SREBP-1c GU339498 GGATTGAGGTGAGCCGACAT 58 TGAGGAAAGCCATTGACTACATT 过氧化物酶体增殖物激活受体γ PPARγ GQ220296 AATGCACCTTTCGTTATCC 58 GAGCGTCACTTGGTCGTTC 过氧化物酶体增殖物激活受体α PPARα FJ595500 TGTCAATACTGCCGTTTCC 58 GACTGGTGCTCCTCTTTCC 肌动蛋白β-actin M25013 CCTTCTTGGGTATGGAGTCTTG T AGAGTATTTACGCTCAGGTGGG 抗氧化指标测定 取得的肝脏样品在冰上解冻后, 制成匀浆液, 用于测定总抗氧化能力(T-AOC)、超氧化物歧化酶(SOD)、氧化氢酶(CAT)、还原型谷胱甘肽(GSH)、丙二醛(MDA)、过氧化氢(H2O2), 以上指标均按照试剂盒说明书进行检测, 试剂盒均购自南京建成生物工程研究所。
生长性能及形体指标数据计算 成活率(Survival rate, SR, %)=100×(终尾数/初尾数)
体增重率(Weight gain rate, WGR, %)=100×(末体重–初体重)/初体重
特定生长率(Special growth rate, SGR, %/d)=100×(Ln末体重–Ln初体重)/饲养天数
摄食量(Feed intake, FI, g/fish)=饲料消耗量(g, 干重)/鱼数量
饲料系数(Feed conversion rate, FCR)=每缸投喂饲料总量/每缸鱼体总增重量
肥满度(Condition factor, CF, g/cm3)=100×体重(g)/[体长(cm)]3
肝胰脏指数(Hepatosomatic index, HSI, %)=100×肝重(g)/体重(g)
内脏指数(Viscera index, VSI, %)=100×内脏重(g)/体重(g)
1.6 统计分析
实验数据用平均值±标准误(Mean±SEM)表示, 采用SPSS 22.0软件进行处理, 相同取样天数的不同分组采用单因素方差分析, 差异显著时通过Duncan’s检验方法进行多重比较, 以P<0.05为差异显著性标准。比较同一分组的两个取样时间点之间的显著性采用独立样本T检验, 以P<0.05为差异显著性标准。
2. 结果
2.1 枯草芽孢杆菌对草鱼生长性能、摄食量及形态学参数的影响
如表 4所示, Ah+Bs组及Bs+Ah组WGR、SGR于第28d和第56天时, 均显著高于Ah组(P<0.05), 并于第56天时显著高于对照组(P<0.05), 且Ah+Bs组FCR显著低于Ah组(P<0.05)。在两个取样时间点, CF、HSI和VSI在各组之间无显著差异(P>0.05, 表 5)。
表 4 益生枯草芽孢杆菌对草鱼生长性能及摄食量的影响Table 4. Effects of B. subtilis on growth performance and food intake of grass carps指标Index 对照Control Ah Ah + Bs Bs + Ah IW 50.77±0.83 50.61±0.60 50.34±0.38 50.42±1.26 28d SR 98.67±2.31 96.00±4.00 98.67±2.31 97.33±4.62 FW 68.76±1.62 67.53±2.66 70.97±1.06 70.69±2.73 WGR 35.46±3.49ab 33.42±4.16a 40.98±1.28b 40.17±1.89b SGR 1.08±0.09ab 1.03±0.11a 1.23±0.03b 1.21±0.05b FI 31.45±0.26 30.62±1.57 31.74±1.19 32.31±1.04 FCR 1.76±0.14ab 1.83±0.20b 1.54±0.01a 1.60±0.07ab 56d SR 97.31±2.57 95.98±1.09 97.37±0.54 96.71±0.61 FW 103.77±2.17ab 102.71±3.86a 115.36±6.31c 110.87±2.98bc WGR 104.37±0.98a 102.96±7.21a 129.12±11.00b 119.91±1.71b SGR 1.28±0.01a 1.26±0.06a 1.48±0.09b 1.41±0.01b FI 104.70±0.83a 105.14±7.41a 123.34±15.76b 121.30±4.92b FCR 1.98±0.05ab 2.02±0.01b 1.89±0.07a 2.01±0.03b 注: 表中数值(平均值±标准误)为样本的平均值(n=3重复缸)。每行数值后上标的不同字母表示差异显著(P<0.05); 下同Note: Values are the mean±SEM (n=3). Values within the same row with different letters are significantly different (P<0.05); The same applies below 表 5 益生枯草芽孢杆菌对草鱼形态学参数的影响Table 5. Effects of B. subtilis on morphological parameters of grass carps指标Index 对照Control Ah Ah + Bs Bs + Ah 28d CF 1.91±0.05 1.84±0.07 1.90±0.17 1.86±0.03 HSI 2.13±0.13 2.03±0.09 2.06±0.09 2.15±0.06 VSI 10.30±1.06 10.50±1.64 11.84±1.31 10.23±1.15 56d CF 2.00±0.09 1.90±0.16 1.94±0.06 1.96±0.18 HSI 2.49±0.23 2.39±0.11 2.53±0.17 2.43±0.10 VSI 11.83±1.05 11.15±1.48 10.93±1.19 11.10±1.43 注: 表中数值(平均值±标准误)为样本的平均值(n=6)Note: Values are the mean ± SEM (n=6) 2.2 枯草芽孢杆菌对草鱼肝脏脂肪含量的影响
经染色后, 脂质呈红色, 细胞核呈蓝色。图 1中油红O染色结果(A-H)及对油红O染色的脂滴所占相对面积统计结果(图 1-I)显示, 28d时油红O染色的脂滴面积在Ah组显著增加(P<0.05), 而在Bs+Ah组及Ah+Bs组变化无统计学差异(P>0.05); 第56天时各组脂滴面积无显著差异(P>0.05)。图 1-J为肝脏脂肪含量测定结果: 第28天时, Ah组的脂肪含量显著高于对照组(P<0.05), Ah+Bs和Bs+Ah组脂肪含量与Ah组相比显著降低(P<0.05)。第56天时, 各组肝脏脂肪含量均无统计学差异(P>0.05)。对照组、Bs+Ah组及Ah+Bs组在第56天时的肝脏脂肪含量显著高于第28天时的脂肪含量(P<0.05)。
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图 1 益生枯草芽孢杆菌对草鱼肝脏脂质沉积的影响A—H(400×). 分别为第28天(A—D图)和第56天(E—H)的对照组、Ah组、Ah+Bs组和Bs+Ah组油红染色结果; I图. 用Image-Pro Plus 6.0软件统计的油红染色区域面积结果; J图. 肝脏的脂肪含量；图中数值(平均值±标准误)为样本的平均值(n=6)；不同的上标字母表示不同处理组间差异显著(P<0.05)；*表示28d和56d之间有显著差异(P<0.05), 下同Figure 1. The effect of B. subtilis on hepatic lipid content in grass carpA—H. The oil red O staining of the control group, Ah group, Ah+Bs group and Bs+Ah group on the 28d (A—D) and 56d (E—H); I. The relative areas of the lipid droplets after oil-red O staining analysed by Image-Pro Plus 6.0; J. The effect of the B. subtilis diet on the hepatic lipid content in grass carp. Values are the mean±SEM (n=6). Different letters indicate significant differences among groups (P<0.05). Asterisks indicate significant differences between 28d and 56d (P<0.05). The same applies below2.3 枯草芽孢杆菌对草鱼血清生化指标的影响
如表 6所示, 第28天时, TG和HDL-C含量在Ah+Bs组较Ah组有升高的趋势, 在Bs+Ah组显著高于Ah组(P<0.05); AST活性在Ah+Bs组较Ah组显著降低(P<0.05); 此外, TG、HDL-C含量及AST活性在Ah+Bs及Bs+Ah组与对照组均无显著差异(P>0.05)。第56天时, CHO、HDL-C和LDL-C含量在Bs+Ah组较Ah组显著升高(P<0.05), 在Ah+Bs组有升高的趋势, 且均与对照组无显著差异(P>0.05)。AST和ALT活性在Ah组显著高于对照组(P<0.05), 而在Ah+Bs和Bs+Ah组则与对照组无显著差异(P>0.05)。
表 6 益生枯草芽孢杆菌对草鱼血液生化参数的影响Table 6. Effects of B. subtilis on blood biochemical parameters of grass carps指标Index 对照Control Ah Ah+Bs Bs+Ah 28d CHO
(mmol/L)6.52±0.17ab 6.05±0.26a 6.29±0.43a 6.79±0.16b TG (mmol/L) 5.76±0.38b 5.14±0.31a 5.59±0.47ab 5.73±0.42b HDL-C (mmol/L) 2.25±0.25ab 1.89±0.16a 2.22±0.21ab 2.47±0.20b LDL-C (mmol/L) 2.39±0.40 2.27±0.13 2.43±0.28 2.29±0.25 AST (U/L) 52.83±12.69ab 67.04±9.31b 49.50±7.32a 55.93±14.13ab ALT (U/L) 6.59±0.25 6.85±0.33 6.69±0.92 6.96±0.15 56d CHO (mmol/L) 6.07±0.49b 5.39±0.49a 5.86±0.54ab 6.02±0.34b TG (mmol/L) 5.56±0.42 6.02±0.70 5.79±0.42 5.56±0.49 HDL-C (mmol/L) 2.19±0.39ab 1.94±0.16a 2.06±0.14ab 2.30±0.20b LDL-C (mmol/L) 2.54±0.25b 2.10±0.20a 2.33±0.24ab 2.46±0.21b AST (U/L) 52.52±6.32a 81.48±18.54b 50.73±9.13a 58.72±6.08a ALT (U/L) 6.78±0.39a 7.41±0.45b 6.71±0.29a 6.58±0.61a 2.4 枯草芽孢杆菌对草鱼肝脏脂代谢相关基因表达的影响
如图 2所示, 第28天时, Ah组ACCα表达水平较对照组显著上调(P<0.05), LPL、ATGL、SREBP-1c、PPARγ表达水平显著下调(P<0.05), 但这些基因表达水平在Ah+Bs及Bs+Ah组较对照组无显著差异(P>0.05)。PPARα表达水平在Ah+Bs和Bs+Ah组均较Ah组显著上调(P<0.05)且与对照组比无显著差异(P>0.05)。第56天时, FAS、ACCα、CPTIα1a、HSL、SPEBP-1c、PPARγ、PPARα的表达水平在Ah组显著下调(P<0.05), 但在Ah+Bs和Bs+Ah组较Ah组显著上调或有上调的趋势。与对照组比, ATGL表达水平在Ah组有下调的趋势; 但在Ah+Bs组和Bs+Ah组较Ah组显著上调(P<0.05)。
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图 2 益生枯草芽孢杆菌对草鱼肝脏脂代谢基因表达的影响Figure 2. Effects of B. subtilis on mRNA levels of lipid metabolism genes of grass carp2.5 枯草芽孢杆菌对草鱼肝脏抗氧化能力的影响
如图 3所示, 第28天时CAT、SOD活性及第56天时T-AOC, 在Ah组较对照组显著降低(P<0.05), 在Ah+Bs组和Bs+Ah组较Ah组显著升高(P<0.05)。第28天时, GSH含量在Bs+Ah组较Ah组显著升高(P<0.05); H2O2含量在Ah组显著升高(P<0.05), 在Bs+Ah组显著低于Ah组(P<0.05)。Ah组MDA含量在两个取样时间点均显著高于对照组(P<0.05), 在Ah+Bs组和Bs+Ah组与对照组无显著差异(P>0.05)。
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图 3 益生枯草芽孢杆菌对草鱼肝脏抗氧化酶活性及MDA和H2O2含量的影响Figure 3. Effects of B. subtilis on activities of antioxidant enzymes and content of MDA and H2O2 in grass carp3. 讨论
3.1 枯草芽孢杆菌对草鱼生长性能及形态学参数的影响
本研究表明, 感染嗜水气单胞菌28d后, 草鱼WGR、SGR有下降的趋势, FCR有增加的趋势。非洲鲶(Clarias gariepinus)在感染嗜水气单胞菌后, 其SGR显著下降, FCR显著增加[13], 与本研究结果一致。本研究投喂益生菌饲料的草鱼表现出更好的生长性能, 且在第56天时Ah+Bs组的FCR最小, 表明在饲料中添加枯草芽孢杆菌Ch9能促进草鱼生长, 提高饲料的效率。在点带石斑鱼(Epinephelus coioides)[14]、里海鳟(Salmo caspius)[15]等鱼类也有类似的报道。这可能是与芽孢杆菌能够提高消化酶活性以及能产生酶, 减少抗营养因子, 提高动物对营养的利用等有关[11, 16, 17]。
3.2 枯草芽孢杆菌对草鱼肝脏脂肪含量的影响
肝脏的脂肪含量及油红染色结果表明, 嗜水气单胞菌能够造成草鱼肝脏的脂质积累, 而通过饲料中添加枯草芽孢杆菌可以改善这种状况, 减少脂质在草鱼肝脏中的积累。具体体现为第28天时, Ah+Bs和Bs+Ah组草鱼肝脏脂肪含量显著低于Ah组, 且与对照组肝脏脂肪含量处于同一水平。在哺乳动物中, 已经有许多文献表明一些益生菌能减少肝脏中脂质的积累, 治疗脂肪肝病[18—20]。在鱼类中, Shewanella属的两个益生菌菌株(Pdp11及Pdp13)及鼠李糖乳杆菌(Lactobacillus rhamnosus)在降低肝脏脂质方面也有很大潜力[21—24]。
3.3 枯草芽孢杆菌对草鱼血清生化指标的影响
肝脏在脂质代谢过程中具有许多作用(如摄取、转运、合成及分解等), 进入肝脏的脂肪酸, 能酯化成为TGs, 并能以脂滴的形式储存在肝细胞中, 或者以富含TGs的脂蛋白的形式分泌到血液中[25]; 当肝细胞受到一定的损害时, 脂类代谢的平衡会被打破, 并影响血清中的血脂水平[26]。因此, 血脂水平等血清生化指标可以在一定程度上反映出肝脏的健康状态和机体脂质代谢等状况。本研究血清生化指标结果显示, 在注射嗜水气单胞菌后第28天, TG显著降低, 至第56天时CHO和LDL-C显著降低, HDL-C也有降低的趋势; 而投喂枯草芽孢杆菌饲料, 均能使血脂恢复到与对照组相同的水平。另外, 在添加枯草芽孢杆菌后, 血清中AST和ALT活性较Ah组显著下降, 表明枯草芽孢杆菌Ch9可能减少了嗜水气单胞菌对肝脏的损害。已有研究表明, 鼠李糖乳杆菌、粪肠球菌(Enterococcus faecalis)能降低罗非鱼血清中AST和ALT的活性[27]。对尼罗罗非鱼(Oreochromis niloticus)饲喂含有枯草芽孢杆菌的饲料, 血清中AST和ALT活性显著下降[28]。因此, 枯草芽孢杆菌在减少因嗜水气单胞菌的感染对肝脏的损伤及其对脂质代谢的影响, 改善因嗜水气单胞菌感染造成的血脂下降方面具有一定潜力。
3.4 枯草芽孢杆菌对草鱼肝脏脂代谢相关基因表达的影响
为分析感染嗜水气单胞菌及投喂枯草芽孢杆菌饲料对草鱼肝脏脂肪合成、分解与调控相关基因mRNA表达水平的影响, 采用qPCR技术检测草鱼肝脏脂代谢相关基因mRNA表达水平。重要的脂肪生成相关酶, 如FAS、ACC, 以及重要的转录因子SREBP-1c和PPARγ可以促进肝脏脂质的合成; PPARα可诱导CPTI表达, 促进脂肪酸分解; HSL、ATGL、LPL均参与脂肪的水解[29—36], FAT/CD36除了与脂肪酸转运相关, 还在脂肪酸氧化代谢过程中发挥了重要作用[37—39]。本研究表明, 投喂枯草芽孢杆菌在维持肝脏正常脂质代谢方面具有一定作用。第28天时Ah组ACCα较对照组显著地上调, LPL、ATGL、FAT/CD36显著地下调, 血清TG在第28天显著降低, 表明感染嗜水气单胞菌诱导草鱼脂质合成增加、脂质分解及输出减少, 导致肝脏脂质积累; 在投喂枯草芽孢杆菌后, ACCα的mRNA表达水平较Ah组下调, 甘油三酯水解相关酶LPL及ATGL mRNA表达水平均上调, 并与对照无显著差异。因此脂肪酸合成的减少及脂质分解的增加造成了Bs+Ah组及Ah+Bs组脂肪含量低于Ah组且与对照无显著差异。相关研究表明, 高脂饲料诱导的脂肪肝罗非鱼LPL表达水平显著下调, 在高脂饲料中添加垂盆草提取物后则显著上调LPL表达水平[3]; 用咖啡因治疗斑马鱼(Danio rerio)的脂肪肝, 其肝脏ACC1表达水平显著地下调[40], 这些均与本研究相似。56d时Ah组FAS、ACCα、CPTIα1a、HSL、SPEBP-1c、PPARγ和PPARα表达水平较对照均显著地下调, 表明Ah组肝脏脂质合成和分解均减少, 脂质代谢异常。在投喂Cu过量饲料后, 黄颡鱼ACCα和ATGL表达水平均显著下调, 而肝脏脂肪含量减少[41], 与本研究结果相似。在投喂枯草芽孢杆菌饲料后, 这些基因表达与对照无显著差异, 表明在感染嗜水气单胞菌前后投喂枯草芽孢杆菌饲料均能够减少因感染嗜水气单胞菌对草鱼脂质代谢的影响。
3.5 枯草芽孢杆菌对草鱼肝脏抗氧化能力的影响
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4. 结论
以上结果表明, 在饲料中添加枯草芽孢杆菌可减少嗜水气单胞菌感染对草鱼肝脏功能的损害, 有利于调节肝脏正常的脂质合成、分解、转运等功能, 减少脂质在肝脏中的积累, 并促进草鱼的生长及降低饲料系数。
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图 1 益生枯草芽孢杆菌对草鱼肝脏脂质沉积的影响
A—H(400×). 分别为第28天(A—D图)和第56天(E—H)的对照组、Ah组、Ah+Bs组和Bs+Ah组油红染色结果; I图. 用Image-Pro Plus 6.0软件统计的油红染色区域面积结果; J图. 肝脏的脂肪含量；图中数值(平均值±标准误)为样本的平均值(n=6)；不同的上标字母表示不同处理组间差异显著(P<0.05)；*表示28d和56d之间有显著差异(P<0.05), 下同
Figure 1. The effect of B. subtilis on hepatic lipid content in grass carp
A—H. The oil red O staining of the control group, Ah group, Ah+Bs group and Bs+Ah group on the 28d (A—D) and 56d (E—H); I. The relative areas of the lipid droplets after oil-red O staining analysed by Image-Pro Plus 6.0; J. The effect of the B. subtilis diet on the hepatic lipid content in grass carp. Values are the mean±SEM (n=6). Different letters indicate significant differences among groups (P<0.05). Asterisks indicate significant differences between 28d and 56d (P<0.05). The same applies below
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图 2 益生枯草芽孢杆菌对草鱼肝脏脂代谢基因表达的影响
Figure 2. Effects of B. subtilis on mRNA levels of lipid metabolism genes of grass carp
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图 3 益生枯草芽孢杆菌对草鱼肝脏抗氧化酶活性及MDA和H2O2含量的影响
Figure 3. Effects of B. subtilis on activities of antioxidant enzymes and content of MDA and H2O2 in grass carp
表 1 实验饲料配方及基本成分
Table 1 Compositions of experimental diets
原料Ingredient 含量Content (g/kg) 营养成分Composition 含量Content
(% DM)鱼粉Fish meal 80 基础饲料Basal diet 豆粕Soybean meal 240 粗蛋白Crude protein 29.83 菜粕Rapeseed meal 340 粗脂肪Crude lipid 4.39 小麦粉Wheat Flour 250 水分Moisture 10.16 豆油Soybean oil 6 灰分Ash 8.32 磷酸二氢钙Ca(H2PO4)2 20 维生素预混料Vitamin premix 1 1 枯草芽孢杆菌饲料B. subtilis diet 矿物质预混料Mineral premix 2 3 粗蛋白Crude protein 29.74 氯化钠NaCl 2 粗脂肪Crude lipid 4.37 氯化胆碱Choline chloride 2 水分Moisture 10.13 纤维素Cellulose 56 灰分Ash 8.39 总量Total 1000 注: 1维生素预混料(mg/kg): 维生素A, 6500 IU; 维生素D3, 4500 IU; 维生素C, 120 mg; 维生素E, 25 mg; 维生素K3, 5 mg; 维生素B1, 12.5 mg; 维生素 B2, 12.5 mg; 维生素B6, 15.0 mg; 维生素B12, 0.025 mg; 烟酰胺, 50 mg; 泛酸, 40 mg; 肌醇, 75 mg; 叶酸, 2.5 mg; 生物素, 0.08 mg; 2矿物质预混料(mg/kg): 氯化钠, 1.0; 硫酸镁, 15.0; 磷酸二氢钠, 25.0; 六水合氯化铝, 0.06; 磷酸二氢钾, 32.0; 磷酸二氢钙, 20.0; 柠檬酸铁, 2.5; 乳酸钙, 3.5; 七水硫酸锌, 0.353; 四水硫酸锰, 0.162; 五水硫酸铜, 0.031; 六水合氯化钴, 0.001; 碘酸钾, 0.003; 纤维素, 0.39Note: 1 Vitamin premix (mg/kg): vit. A 6500 IU, vit. D3 4500 IU, vit. C 120 mg, vit. E 25 mg, vit. K3 5 mg, vit. B1 12.5 mg, vit. B2 12.5 mg, vit. B6 15.0 mg, vit. B12 0.025 mg, niacinamide 50 mg, pantothenate 40 mg, inositol 75 mg, folic acid 2.5 mg, biotin 0.08 mg; 2 Mineral premix (mg/kg): NaCl 1.0; MgSO4 15.0; NAh2PO4·2H2O 25.0; AlCl3·6H2O 0.06; KH2PO4 32.0; Ca(H2PO4)2·H2O 20.0; C6H5FeO7·10H2O 2.5; CaC6H10CaO6·5H2O 3.5; ZnSO4·7H2O 0.353; MnSO4·4H2O 0.162; CuSO4·5H2O 0.031; CoCl2·6H2O 0.001; KIO3·6H2O, 0.003; cellulose, 0.39 
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表 2 实验分组设计
Table 2 The experimental design
分组Group 暂养期间饲料Diet during the temporary rearing period 腹腔注射Intraperitoneal injection 注射后投喂饲料Diet after intraperitoneal injection 对照Control 基础饲料 PBS 基础饲料 Ah 基础饲料 1×105 CFU/fish A. hydrophila 基础饲料 Ah+Bs 基础饲料 1×105 CFU/fish A. hydrophila B. subtilis饲料 Bs+Ah B. subtilis饲料 1×105 CFU/fish A. hydrophila 基础饲料
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表 3 RT-PCR引物序列
Table 3 Primers used for real-time PCR
基因Gene 登录号GenBank accession No. 引物序列Primer sequence(5′—3′) 退火温度Annealing temperature (℃) 脂肪酸合酶FAS HM802556.1 GTCCACAGGGTGTCGTTC 58 GAGGTCTTGGGCTCTTTATT 乙酰辅酶A羧化酶α ACCα GU908475 AGTATCGCAGTGGCATCA 58 TGTCCCCTTTGTTTTCCT 肉碱棕榈酰基转移酶Iα1a CPTIα1a KJ816747 TTTACGACGGACGGTTGC 58 GCTTGTTCTTCCCACGACT 脂蛋白脂酶LPL FJ436077 AGCCCTGTATGAACGAGA 58 CACATCCTTGCCCACTAG 脂肪甘油三酯脂肪酶ATGL HQ845211 TATTGTGGTTTAATCCCTCC 58 CAGTGCCTTGCTCAGTCT 激素敏感脂酶HSL FJ843081 CCGACAAGGACAGGACAGT 58 ATGACCAGGCAGGGAGAA 肝型脂肪酸结合蛋白L-FABP EU220990.1 GGGAAAACCATCACTAACTC 58 TCAGGGTCTCAACCATCTC 脂肪酸移位酶FAT/CD36 KU821103.1 CTTCCCCACTTCCTCTATG 58 TAATCGGTTCCACATCCA 固醇调控元件结合蛋白-1c SREBP-1c GU339498 GGATTGAGGTGAGCCGACAT 58 TGAGGAAAGCCATTGACTACATT 过氧化物酶体增殖物激活受体γ PPARγ GQ220296 AATGCACCTTTCGTTATCC 58 GAGCGTCACTTGGTCGTTC 过氧化物酶体增殖物激活受体α PPARα FJ595500 TGTCAATACTGCCGTTTCC 58 GACTGGTGCTCCTCTTTCC 肌动蛋白β-actin M25013 CCTTCTTGGGTATGGAGTCTTG T AGAGTATTTACGCTCAGGTGGG
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表 4 益生枯草芽孢杆菌对草鱼生长性能及摄食量的影响
Table 4 Effects of B. subtilis on growth performance and food intake of grass carps
指标Index 对照Control Ah Ah + Bs Bs + Ah IW 50.77±0.83 50.61±0.60 50.34±0.38 50.42±1.26 28d SR 98.67±2.31 96.00±4.00 98.67±2.31 97.33±4.62 FW 68.76±1.62 67.53±2.66 70.97±1.06 70.69±2.73 WGR 35.46±3.49ab 33.42±4.16a 40.98±1.28b 40.17±1.89b SGR 1.08±0.09ab 1.03±0.11a 1.23±0.03b 1.21±0.05b FI 31.45±0.26 30.62±1.57 31.74±1.19 32.31±1.04 FCR 1.76±0.14ab 1.83±0.20b 1.54±0.01a 1.60±0.07ab 56d SR 97.31±2.57 95.98±1.09 97.37±0.54 96.71±0.61 FW 103.77±2.17ab 102.71±3.86a 115.36±6.31c 110.87±2.98bc WGR 104.37±0.98a 102.96±7.21a 129.12±11.00b 119.91±1.71b SGR 1.28±0.01a 1.26±0.06a 1.48±0.09b 1.41±0.01b FI 104.70±0.83a 105.14±7.41a 123.34±15.76b 121.30±4.92b FCR 1.98±0.05ab 2.02±0.01b 1.89±0.07a 2.01±0.03b 注: 表中数值(平均值±标准误)为样本的平均值(n=3重复缸)。每行数值后上标的不同字母表示差异显著(P<0.05); 下同Note: Values are the mean±SEM (n=3). Values within the same row with different letters are significantly different (P<0.05); The same applies below
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表 5 益生枯草芽孢杆菌对草鱼形态学参数的影响
Table 5 Effects of B. subtilis on morphological parameters of grass carps
指标Index 对照Control Ah Ah + Bs Bs + Ah 28d CF 1.91±0.05 1.84±0.07 1.90±0.17 1.86±0.03 HSI 2.13±0.13 2.03±0.09 2.06±0.09 2.15±0.06 VSI 10.30±1.06 10.50±1.64 11.84±1.31 10.23±1.15 56d CF 2.00±0.09 1.90±0.16 1.94±0.06 1.96±0.18 HSI 2.49±0.23 2.39±0.11 2.53±0.17 2.43±0.10 VSI 11.83±1.05 11.15±1.48 10.93±1.19 11.10±1.43 注: 表中数值(平均值±标准误)为样本的平均值(n=6)Note: Values are the mean ± SEM (n=6)
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表 6 益生枯草芽孢杆菌对草鱼血液生化参数的影响
Table 6 Effects of B. subtilis on blood biochemical parameters of grass carps
指标Index 对照Control Ah Ah+Bs Bs+Ah 28d CHO
(mmol/L)6.52±0.17ab 6.05±0.26a 6.29±0.43a 6.79±0.16b TG (mmol/L) 5.76±0.38b 5.14±0.31a 5.59±0.47ab 5.73±0.42b HDL-C (mmol/L) 2.25±0.25ab 1.89±0.16a 2.22±0.21ab 2.47±0.20b LDL-C (mmol/L) 2.39±0.40 2.27±0.13 2.43±0.28 2.29±0.25 AST (U/L) 52.83±12.69ab 67.04±9.31b 49.50±7.32a 55.93±14.13ab ALT (U/L) 6.59±0.25 6.85±0.33 6.69±0.92 6.96±0.15 56d CHO (mmol/L) 6.07±0.49b 5.39±0.49a 5.86±0.54ab 6.02±0.34b TG (mmol/L) 5.56±0.42 6.02±0.70 5.79±0.42 5.56±0.49 HDL-C (mmol/L) 2.19±0.39ab 1.94±0.16a 2.06±0.14ab 2.30±0.20b LDL-C (mmol/L) 2.54±0.25b 2.10±0.20a 2.33±0.24ab 2.46±0.21b AST (U/L) 52.52±6.32a 81.48±18.54b 50.73±9.13a 58.72±6.08a ALT (U/L) 6.78±0.39a 7.41±0.45b 6.71±0.29a 6.58±0.61a
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