Effects of Chinese Herbal Extracts Added to Low-Fish Meal Diet on Growth Performance, Mammalian Target of Rapamycin Signaling Pathway and Antioxidant Capacity of Litopenaeus vannamei

HU Junru; ZHONG Yongfeng; DENG Jingming; LIU Xiaolei; LI Zhiwen; WEI Zhenhong; LU Huijie; PENG Kai; ZHANG Yufan; LI Baosheng; HUANG Wen

doi:10.12418/CJAN2025.271

2025 , Vol. 37 >Issue 5: 3293 - 3303

DOI: https://doi.org/10.12418/CJAN2025.271

RESEARCH PAPER

Effects of Chinese Herbal Extracts Added to Low-Fish Meal Diet on Growth Performance, Mammalian Target of Rapamycin Signaling Pathway and Antioxidant Capacity of Litopenaeus vannamei

HU Junru ^,¹ ,
ZHONG Yongfeng ¹ ,
DENG Jingming ² ,
LIU Xiaolei ¹ ,
LI Zhiwen ³ ,
WEI Zhenhong ³ ,
LU Huijie ¹ ,
PENG Kai ¹ ,
ZHANG Yufan ⁴ ,
LI Baosheng ⁵ ,
HUANG Wen ^,¹^,^*

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¹ Guangdong Key Laboratory of Animal Breeding and Nutrition, Key Laboratory of Animal Nutrition and Feed Science in South China of Ministry of Agriculture and Rural Affairs, Institute of Animal Science, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China
² Guangzhou Danong Biotechnology Co., Ltd., Guangzhou 510640, China
³ Guangdong Wangyu Biotechnology Co., Ltd., Yangjiang 529599, China
⁴ Beijing Alltech Biological Products Co., Ltd., Beijing 101499, China
⁵ Tongwei Agricultural Development Co., Ltd., Chengdu 610000, China

^* professor, E-mail: huangwen549@126.com

Received date: 2024-10-12

Online published: 2025-05-14

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Abstract

The objective of this study was to investigate the effects of Chinese herbal extracts added to low-fish meal diet with fish meal content of 8% on growth performance, mammalian target of rapamycin (mTOR) signaling pathway and antioxidant capacity of Litopenaeus vannamei. The five groups were supplemented with 0, 0.2, 0.4, 0.8, and 1.6 g/kg Chinese herbal extract in the basal diets, respectively, which were designated as G0, G1, G2, G3 and G4. A total of 600 Litopenaeus vannamei with an average initial body weight of approximately 0.80 g were randomly assigned into 5 groups with 3 replicates in each group and 40 shrimp in each replicate. The shrimp were fed the corresponding experimental diets respectively and feeding for 42 days. The results showed as follows: 1) the survival rate of G1 to G4 groups was significantly higher than that of G0 group (P<0.05). The final average weight, weight gain rate and specific growth rate of G3 group were the highest, and the feed coefficient was the lowest. 2) The urea content of G2 and G4 groups was significantly higher than that of G0 group (P<0.05). 3) The hepatopancreas malondialdehyde content of G1 and G4 groups was significantly lower than that of G0 group (P<0.05). 4) The gene relative expression levels of superoxide dismutase (SOD) and heme oxygenase-1 in hepatopancreas and intestine of G1 to G4 groups were significantly higher than those of G0 group (P<0.05). 5) The gene relative expression levels of phosphatidylinositol 3-kinase (PI3K), mTOR, p38 mitogen-activated protein kinases (p38 MAPK), phosphatase and tensin homology deleted on chromosometen (PTEN), ribosomal protein S6 kinase 1 (S6K1) and eukaryotic translation initiation factor 4E binding protein 1 (4EBP1) of mTOR signaling pathway in hepatopancreas and intestine of G2 and G3 groups were significantly higher than those of G0 group (P<0.05). In conclusion, adding 0.8 g/kg Chinese herbal extract in the low-fish meal diet can improve the growth rate and survival rate, activate the hepatopancreas and intestinal mTOR signaling pathways, and improve the body antioxidant capacity of Litopenaeus vannamei.

Key words： Chinese herbal extract; Litopenaeus vannamei; growth; mTOR signaling pathway; antioxidant capacity

Cite this article

HU Junru , ZHONG Yongfeng , DENG Jingming , LIU Xiaolei , LI Zhiwen , WEI Zhenhong , LU Huijie , PENG Kai , ZHANG Yufan , LI Baosheng , HUANG Wen . Effects of Chinese Herbal Extracts Added to Low-Fish Meal Diet on Growth Performance, Mammalian Target of Rapamycin Signaling Pathway and Antioxidant Capacity of Litopenaeus vannamei[J]. Chinese Journal of Animal Nutrition, 2025 , 37(5) : 3293 -3303 . DOI: 10.12418/CJAN2025.271

2022年全球虾类养殖产量近1 120万t,凡纳滨对虾是最具代表性的品种,占总产量的52%^[1]。鱼粉因具有营养价值高、适口性好的特点,是凡纳滨对虾配合饲料的主要原料来源^[2]。然而,由于鱼粉的可持续供应以及成本过高,减少对虾饲料中鱼粉的使用一直是对虾养殖业可持续发展的要求^[3]。在过去的几十年里,已经开展了大量的研究用以提高包括对虾在内的许多水产养殖物种饲料中鱼粉替代的问题^[4-5],但研究显示在许多情况下,饲料中低于10%的鱼粉对对虾的养殖仍然是一项挑战,主要表现为生长性能下降、饲料消化率降低以及机体健康受损等^[6-8];在凡纳滨对虾中具体体现为显著降低肠道褶皱高度、褶皱宽度、环形肌层厚度,破坏肠道结构以及降低肠道菌群的多样性和丰富度,引起菌群失调;下调免疫和蜕皮相关基因的表达,从而抑制对虾的生长^[7-8]。

中草药含有酚类、多酚类、生物碱、醌类、萜类、卵磷脂和多肽化合物,具有促进生长、抗菌、刺激食欲和抗应激的特性^[9],因其价格低、毒性低、副作用少、不易产生耐药性等优势,中草药已成为埃及、日本、印度、印度尼西亚、韩国、墨西哥、尼日利亚和泰国等许多国家研究和开发的热点^[10]。在水产养殖中,中草药也被用作预防药物和饲料添加剂使用^[11]。研究发现,中草药可以有效改善由饲料原料改变对水产动物机体造成的负面影响,提高非鱼粉原料在饲料中的使用比例,实现促进水产动物生长、提高饲料利用率和机体免疫力的作用。Mo等^[12]研究发现,黄芪和枸杞提取物添加在使用食物废弃物(即厨房废物,如蔬菜、肉制品、食品加工废物)部分取代鱼粉、豆粕等的基础饲料中,可提高草鱼(Ctenopharyngodon idella)和尼罗罗非鱼(Oreochromis niloticus)的生长性能和非特异性免疫力。饲料中添加0.06%中草药缓解了由低鱼粉高豆粕饲料引起的黄颡鱼(Pelteobagrus fulvidraco)氧化应激,改善了肠道结构,提高了黄颡鱼生长速度和存活率^[13]。大黄提取物联合凝结芽孢杆菌对缓解低鱼粉导致的罗氏沼虾(Macrobrachium rosenbergii)生长性能下降、提高抗氧化能力、改善肠道菌群结构具有积极的作用^[14]。此外,研究还发现饲料中添加4%~6%复方中草药改善了小球藻替代鱼粉时大口黑鲈(Micropterus salmoides)的体色与肉色,提高了鱼体肌肉营养成分含量及质构^[15]。上述研究结果为中草药在低鱼粉饲料中的应用提供了有益的价值参考。

黄芩、大黄、黄柏、黄芪、黄连是我国传统的中药材,这些中药材主要通过黄酮类、生物碱、鞣质类等有效成分发挥促进生长、调节肠道微生物群落、抗炎和抗氧化的作用^[16-18]。在凡纳滨对虾中,黄芩提取物通过诱导血细胞中活性氧的产生以及激活酚氧化酶(proPO)系统和超氧化物歧化酶(SOD)、α-2-巨球蛋白(A2M)、脂多糖结合蛋白(LGBP)等免疫基因表达,发挥免疫刺激作用和杀菌活性^[19]。黄芩苷亦可通过与N-乙酰氨基葡萄糖苷酶(NAGase)相互作用调节凡纳滨对虾的周期性蜕壳及疾病防御^[20]。而大黄则通过提高凡纳滨对虾白细胞和血细胞的吞噬活性、溶菌酶活性提高抗菌能力^[21]。然而中草药的作用效果具有物种特异性^[22],此外,具有不同药理作用的中草药组成复方制剂较单一制剂可以获得药效互补、疗效增强的作用^[23]。因此,本试验在上述研究的基础上进一步探讨饲料中添加由黄芩、黄芪、大黄、黄柏、黄连组成的复方中草药提取物对低鱼粉养殖凡纳滨对虾生长性能、哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)信号通路和抗氧化能力的影响,以期为凡纳滨对虾低鱼粉饲料配制提供科学依据,同时为不断增长的对虾养殖业低成本饲料的经济可持续性提供借鉴。

1 材料与方法

1.1 试验饲料

中草药组分为黄芩、大黄、黄柏、黄芪、黄连,比例依次为30%、30%、30%、5%、5%。采用水提法提取有效成分,具体步骤为:先用水浸泡1 h,然后加入10倍水保持沸腾2 h,反复水提2次,水提液经过浓缩后喷成干粉制得中草药提取物。中草药提取物的主要活性成分为黄芩苷和总蒽醌(芦荟大黄素、大黄酸、大黄素、大黄酚),检测含量分别为2.2%和0.3%(以干燥品计)。

由进口鱼粉、豆粕、花生粕、虾粉、鸡肉粉、膨化大豆粕、面粉、鱼油等作为主要原料,在基础饲料中分别添加0、0.2、0.4、0.8、1.6 g/kg中草药提取物,制作5种试验饲料,依次记作G0、G1、G2、G3、G4。试验饲料组成及营养水平见表1。饲料原料粉碎后过80目筛网,维生素和微量元素等微量成分与其余粉状原料逐级混合直至均匀,然后加入鱼油和水进行捏合,捏合后的物料加入SLX-80型双螺杆挤压机,挤压制成直径为0.5 mm的颗粒料。湿颗粒料平铺于托盘中,置于烘箱中55 ℃烘干,自然冷却后收集过筛,筛除粉尘,将颗粒料用密封袋封装,置于-20 ℃冰箱中保存备用。

表1 试验饲料组成及营养水平(干物质基础)

Table 1 Composition and nutrient levels of experimental diets (DM basis) %

项目 Items	饲料Diets
项目 Items	G0	G1	G2	G3	G4
原料Ingredients
进口鱼粉Imported fish meal	8.00	8.00	8.00	8.00	8.00
豆粕Soybean meal	24.50	24.50	24.50	24.50	24.50
花生粕Peanut meal	20.00	20.00	20.00	20.00	20.00
虾粉Shrimp shell powder	5.00	5.00	5.00	5.00	5.00
鸡肉粉Chicken meal	5.00	5.00	5.00	5.00	5.00
膨化大豆粕Puffed soybean meal	4.00	4.00	4.00	4.00	4.00
血球蛋白粉Blood globulin powder	2.00	2.00	2.00	2.00	2.00
面粉Flour	24.82	24.80	24.78	24.74	24.66
鱼油Fish oil	3.00	3.00	3.00	3.00	3.00
磷酸二氢钙Ca(H₂PO₄)₂	1.50	1.50	1.50	1.50	1.50
复合预混合饲料Composite premixed feed¹⁾	1.00	1.00	1.00	1.00	1.00
氯化胆碱Choline chloride	0.15	0.15	0.15	0.15	0.15
L-抗坏血酸-2-磷酸脂L-ascorbate-2-phosphate	0.03	0.03	0.03	0.03	0.03
腐植酸钠Sodium humate	1.00	1.00	1.00	1.00	1.00
中草药提取物Chinese herbal extract		0.02	0.04	0.08	0.16
合计Total	100.00	100.00	100.00	100.00	100.00
营养水平Nutrient levels²⁾
粗蛋白质Crude protein	39.85	39.91	39.77	39.94	39.82
粗脂肪Crude lipid	5.97	5.90	6.11	6.07	6.14
粗灰分Ash	9.96	9.88	9.73	9.81	9.92

1)每千克复合预混合饲料包含 One kg of composite premixed feed contained the following: VA 4 000 000 IU,VD₃ 2 000 000 IU,VE 30 g,VK₃ 10 g,VB₁ 5 g,VB₂ 15 g,VB₆ 8 g,D-泛酸 D-pantothenic acid 25 g,叶酸 folic acid 2.5 g,生物素 biotin 0.08 g,烟酸 nicotinic acid 40 g,VB₁₂ 0.02 g,肌醇 inositol 150 g,MgSO₄·H₂O 12 g,MgCl₂ 90 g,Met-Cu 3 g,FeSO₄·H₂O 1 g,ZnSO₄·H₂O 10 g,Ca(IO₃)₂ 0.06 g,Met-Co 0.16 g,Na₂SeO₃ 0.003 6 g。

2)实测值 Measured values。

1.2 试验设计与养殖管理

动物试验伦理审批机构:广东省农业科学院动物科学研究所(水产研究所)实验动物伦理委员会;批准编号:2022002。

虾苗购自番禺区某养殖场,试验开始前将虾苗在水泥池中暂养3~5 d。养殖试验在广东省农业科学院动物科学研究所内养殖系统循环中进行挑选规格均匀、体格健壮、初始均重约为0.80 g左右的凡纳滨对虾600尾,随机分成5组(G0~G4组),每组3个重复,每个重复40尾。每缸放养40尾虾作为1个重复,投喂对应的试验饲料。分别于每日08:30、14:30和20:30饱食投喂1次,投喂量视虾的摄食情况而定,每次根据摄食积极性采用多轮投喂,尽量保证不剩料,若有剩料及时捞出,烘干称重。养殖前期每3 d换水1次,养殖后期每1~2 d换水1次,每天记录水质指标情况。养殖期间采用自然光照,水温27~32 ℃,盐度4.5‰~5.5‰,氨氮含量<0.20 mg/L,亚硝酸盐含量<0.01 mg/L,溶氧量>5.0 mg/L,pH 7.8~8.2。养殖时长为42 d。

1.3 样品采集

养殖42 d后,采样前对虾饥饿12 h,然后记录每缸的尾数和总重。随后,从每缸中随机选捞取15尾虾,用1 mL无菌注射器于围心腔采集血淋巴,血淋巴于4 ℃冰箱静置4 h后,置于冷冻离心机于2 680×g离心10 min,对分离出的血清样品进行分装,超低温保存备用。每缸抽取6尾虾,于无菌条件下采集肝胰腺和肠道组织,其中每3尾混合置于1支无酶管中,-80 ℃保存备用,用于抗氧化和免疫指标及基因相对表达量测定。

1.4 样品检测

生长性能指标计算公式如下:

$末均重 (\mathrm{g})= 终末体重 / 尾数；特定生长率（ \% / \mathrm{d} ） =100 \times （ \ln 终末体重 -\ln 初始体重）／养殖天数；增重率（％） ~ 100 \times （终末体重—初始体重）／初始体重；饲料系数 = 总耗料量／（末总重 + 死亡总重 - 初总重）；存活率 (\%)=100 \times （终末尾数／初始尾数）。$

分别采用凯氏定氮法(GB 5009.5—2016)、索氏抽提法(GB 5009.6—2016)、550 ℃马弗炉灼烧法(GB 5009.4—2016)、恒温干燥法(GB 5009.3—2016)测定饲料中粗蛋白质、粗脂肪、粗灰分和水分含量。

采用罗氏全自动生化分析仪(Cobas-8000 c702,美国)测定血清总蛋白(total protein,TP)、白蛋白(albumin,ALB)、球蛋白(globulin,GLOB)、胆固醇(cholesterol,CHOL)、甘油三酯(triglyceride,TG)、葡萄糖(glucose,GLU)、尿素(urea,UA)含量^[24],所用试剂购自威特曼(南京)生物科技有限公司。

肝胰腺过氧化氢酶(catalase,CAT)、SOD活性以及过氧化氢(hydrogen peroxide,H₂O₂)和丙二醛(malondialdehyde,MDA)含量所用试剂购自南京建成生物工程研究所,按照说明书进行操作。

使用TaKaRa MiniBEST通用RNA提取试剂盒(TaKaRa Biomedical Technology,美国)提取总RNA。使用TransScript^®One-Step gDNA Removal and cDNA Synthesis Kit(TransGen Biotech,美国)合成第一链cDNA。实时荧光定量PCR(RT-qPCR)采用LineGene K实时荧光定量PCR系统(Bioer Technology)测定。热循环进行如下:94 ℃,30 s,然后40个温度循环(94 ℃,5 s;60 ℃,30 s)。以β-肌动蛋白(β-actin)为内参基因,使用2^-△△Ct法计算目的基因的相对表达量。目的基因包括mTOR、磷脂酰肌醇-3-激酶(phosphatidylinositol 3-kinase,PI3K)、p38丝裂原活化蛋白激酶(p38 mitogen-activated protein kinases,p38 MAPK)、磷酸酶与张力蛋白同源物(phosphatase and tensin homology deleted on chromosometen,PTEN)、核糖体蛋白S6激酶1(ribosomal protein S6 kinase 1,S6K1)、真核起始因子4结合蛋白1(eukaryotic translation initiation factor 4E binding protein 1,4EBP1)、SOD、血红素氧合酶-1(heme oxygenase-1,HO-1)。引物序列如表2所示。

表2 引物序列

Table 2 Primer sequences

基因Genes	序列Sequence(5'—3')
β-肌动蛋白β-actin	F: GCCCATCTACGAGGGATA R: GGTGGTCGTGAAGGTGTAA
哺乳动物雷帕霉素靶蛋白mTOR	F: TGCCAACGGGTGGTAGA R: GGGTGTTTGTGGACGGA
磷脂酰肌醇-3-激酶PI3K	F: CGTGTGCCAAGACCAGTTTT R: GCTGAGTGTCATGCCAGAGAA
p38丝裂原活化蛋白激酶p38 MAPK	F: GTCGGCTCGCAACTACATAC R: CCGTTACACGCCTTTCACT
磷酸酶与张力蛋白同源物PTEN	F: CTCAGAAGTTGGAGGGTGTGTAC R: CAATCAGAGGCGGTGGGTTA
核糖体蛋白S6激酶1 S6K1	F: GCAAGAGGAAGACGCCATA R: CCGCCCTTGCCCAAAACCT
真核起始因子4结合蛋1 4EBP1	F: ATGTCTGCTTCGCTCGCTCGCC R: GGTTCTTGGGTGGGCTCTT
超氧化物歧化酶SOD	F: CTGGTTCCGTTGCTTGGC R: CGCTCATTCACGTTCTCCC
血红素氧合酶-1 HO-1	F: GCATGGCAGTGACCGAGATTGA R: GTCGCTGCTTCGTCTCCTCATC

1.5 数据统计分析

试验数据采用SPSS 19.0软件进行统计分析。首先对数据进行方差齐性检验,若满足则进行单因素方差分析(one-way ANOVA),并采用Duncan氏检验方法进行多重比较;若不满足则采用Dunnett's T3检验法进行多重比较。试验数据用平均值±标准误表示,P<0.05为差异显著。

2 结果

2.1 中草药提取物对凡纳滨对虾生长性能的影响

由表3可知,G1~G3组的末均重、增重率和特定生长率均高于G0组,但差异不显著(P>0.05),G3组上述指标均为最高;G4组的末均重、增重率和特定生长率均显著低于G1和G3组(P<0.05)。G1~G4组的存活率显著高于G0组(P<0.05)。各组之间饲料系数差异不显著(P>0.05),以G3组最低。

表3 中草药提取物对凡纳滨对虾生长性能的影响

Table 3 Effects of Chinese herbal extracts on growth performance of Litopenaeus vannamei (n=3)

组别 Groups	末均重 FBW/g	增重率 WGR/%	特定生长率 SGR/(%/d)	存活率 SR/%	饲料系数 FC
G0	5.37±0.09^ab	571.10±10.79^ab	4.53±0.04^ab	73.33±3.33^a	2.46±0.37
G1	5.67±0.33^b	608.98±40.93^b	4.66±0.14^b	89.17±4.41^b	2.12±0.16
G2	5.44±0.76^ab	578.86±94.30^ab	4.54±0.34^ab	90.00±2.50^b	2.16±0.45
G3	5.93±0.24^b	641.23±29.97^b	4.77±0.10^b	90.00±0.00^b	1.92±0.22
G4	4.32±0.21^a	440.05±25.94^a	4.01±0.12^a	87.50±0.00^b	2.28±0.13

	同列数据肩标不同小写字母表示差异显著(P<0.05),相同或无字母表示差异不显著(P>0.05)。下表同。
	In the same column, values with different small letter superscripts mean significant difference (P<0.05), while with the same or no letter superscripts mean no significant difference (P>0.05). The same as below.

2.2 中草药提取物对凡纳滨对虾血清生化指标的影响

由表4可知,G2和G4组的血清UA含量显著高于G0组(P<0.05)。各组之间其他血清生化指标差异不显著(P>0.05)。

表4 中草药提取物对凡纳滨对虾血清生化指标的影响

Table 4 Effects of Chinese herbal extracts on serum biochemical indices of Litopenaeus vannamei (n=3)

组别 Groups	总蛋白 TP/(g/L)	白蛋白 ALB/(g/L)	球蛋白 GLOB/(g/L)	葡萄糖 GLU/ (mmol/L)	胆固醇 CHOL/ (mmol/L)	甘油三酯 TG/ (mmol/L)	尿素 UA/ (mmol/L)
G0	82.87±9.73	9.60±1.86	73.27±7.94	1.20±0.15	0.47±0.10	0.59±0.14	0.76±0.34^a
G1	85.03±7.57	9.27±0.88	75.77±6.75	1.53±0.18	0.55±0.10	0.69±0.06	1.71±0.34^ab
G2	60.10±21.33	6.60±2.01	53.50±19.66	1.40±0.44	0.41±0.14	0.57±0.26	2.39±0.10^b
G3	80.87±6.92	7.67±0.76	73.20±6.16	1.70±0.21	0.44±0.07	0.84±0.08	1.84±0.50^ab
G4	76.20±4.31	10.33±3.10	65.87±2.80	1.33±0.33	0.42±0.17	0.65±0.18	2.32±0.43^b

2.3 中草药提取物对凡纳滨对虾肝胰腺抗氧化指标及肝胰腺和肠道抗氧化基因相对表达量的影响

由表5可知,G1和G4组的肝胰腺MDA含量显著低于G0组(P<0.05)。各组之间肝胰腺CAT、SOD活性和H₂O₂含量差异不显著(P>0.05)。

表5 中草药提取物对凡纳滨对虾肝胰腺抗氧化指标的影响

Table 5 Effects of Chinese herbal extracts on hepatopancreas antioxidant indices of Litopenaeus vannamei (n=3)

组别 Groups	过氧化氢酶 CAT/(U/mg prot)	超氧化物歧化酶 SOD/(U/g prot)	过氧化氢 H₂O₂/(mmol/g prot)	丙二醛 MDA/(nmol/mg prot)
G0	0.65±0.24	21.62±4.62	158.27±5.03	4.17±0.59^b
G1	0.59±0.09	37.76±0.35	115.88±27.65	0.75±0.15^a
G2	1.38±0.53	27.46±2.84	102.65±13.31	2.29±0.12^ab
G3	1.73±0.36	27.12±3.96	90.80±28.25	2.08±1.48^ab
G4	0.87±0.26	26.27±5.10	113.10±15.56	0.48±0.13^a

由表6可知,G1~G4组的肝胰腺和肠道SOD和HO-1基因相对表达量均显著高于G0组(P<0.05)。

表6 中草药提取物对凡纳滨对虾肝胰腺和肠道抗氧化基因相对表达量的影响

Table 6 Effects of Chinese herbal extracts on relative expression levels of antioxidant genes in hepatopancreas and intestine of Litopenaeus vannamei (n=3)

组别 Groups	肝胰腺Hepatopancreas		肠道Intestine
组别 Groups	超氧化物歧化酶SOD	血红素氧合酶-1 HO-1	超氧化物歧化酶SOD	血红素氧合酶-1 HO-1
G0	1.00±0.06^a	1.00±0.05^a	1.02±0.13^a	1.02±0.05^a
G1	37.58±1.45^b	24.19±0.35^d	45.43±5.10^b	40.76±2.19^c
G2	33.37±4.36^b	14.35±2.46^bc	42.96±6.95^b	16.95±3.23^b
G3	30.25±4.10^b	10.54±0.98^b	53.71±7.71^b	12.63±1.08^b
G4	43.39±7.92^b	16.49±2.08^c	89.84±15.28^c	18.41±1.80^b

2.4 中草药提取物对凡纳滨对虾肝胰腺和肠道mTOR信号通路基因相对表达量的影响

由表7可知,G1~G4组的肝胰腺mTOR信号通路中PI3K、mTOR、p38 MAPK、PTEN、S6K1和4EBP1基因相对表达量均显著高于G0组(P<0.05)。G2和G3组的肠道mTOR信号通路中PI3K、mTOR、p38 MAPK、PTEN、S6K1和4EBP1基因相对表达量均显著高于G0组(P<0.05)。

表7 中草药提取物对凡纳滨对虾肝胰腺和肠道mTOR信号通路基因相对表达量的影响

Table 7 Effects of Chinese herbal extracts on relative expression levels of mTOR signaling pathway genes in hepatopancreas and intestine of Litopenaeus vannamei (n=3)

组别 Groups	磷脂酰肌醇- 3-激酶 PI3K	哺乳动物雷帕霉素靶蛋白 mTOR	p38丝裂原活化蛋白激酶 p38 MAPK	磷酸酶与张力蛋白同源物 PTEN	核糖体蛋白 S6激酶1 S6K1	真核起始因子4结合蛋1 4EBP1
肝胰腺Hepatopancreas
G0	1.00±0.06^a	1.00±0.05^a	1.02±0.13^a	1.02±0.05^a	1.02±0.14^a	1.02±0.13^a
G1	37.58±1.45^b	24.19±0.35^d	45.43±5.10^b	40.76±2.19^c	25.47±2.14^c	29.22±1.64^d
G2	33.37±4.36^b	14.35±2.46^bc	42.96±6.95^b	16.95±3.23^b	19.26±0.68^bc	18.41±1.53^c
G3	30.25±4.10^b	10.54±0.98^b	53.71±7.71^b	12.63±1.08^b	16.67±0.94^b	8.24±1.51^b
G4	43.39±7.92^b	16.49±2.08^c	89.84±15.28^c	18.41±1.80^b	23.42±2.51^bc	16.74±0.25^c
肠道Intestine
G0	1.00±0.00^a	1.00±0.09^a	1.01±0.11^a	1.01±0.07^a	1.00±0.03^a	1.00±0.07^a
G1	0.50±0.10^a	0.71±0.13^a	0.53±0.07^a	1.28±0.15^a	1.12±0.18^a	2.47±1.96^ab
G2	3.76±0.00^c	4.09±0.12^b	6.45±0.01^c	4.46±0.00^b	6.53±0.48^b	7.38±0.24^c
G3	2.84±0.32^b	6.64±0.06^c	3.58±0.61^b	5.88±0.78^c	9.14±1.23^c	8.67±0.58^c
G4	0.79±0.02^a	0.67±0.04^a	1.26±0.09^a	1.19±0.18^a	2.05±0.06^a	4.54±0.14^b

3 讨论

饲料中鱼粉过低往往会降低凡纳滨对虾生长性能,增加机体炎症反应和病害风险,导致死亡率升高^[25]。为了弥补低鱼粉产生的负面影响,研究者建议在饲料中添加植物活性物质,因其可以提高水产动物的营养保留,改善生长性能和抗病性^[26-27],并改善陆生动物的饲料适口性、肠道功能和抗氧化能力^[28-29]。本研究显示,在低鱼粉饲料中添加0.2~0.8 g/kg中草药提取物提高了凡纳滨对虾的增重率和存活率。类似的发现,在含有植物性蛋白质来源(豆粕和全麦)的低鱼粉(5%)饲料中添加植物精油混合物使凡纳滨对虾的存活率、饲料转化率、血细胞总数和呼吸爆发均提高到饲喂高鱼粉饲料组的水平,弥补了低鱼粉饲料对对虾造成的负面影响和健康后果^[30]。

黄芩作为传统中草药,已证实其提取物对凡纳滨对虾^[19]、珍珠比目鱼(Paralichthys olivaceus)^[31-32]和珍珠龙胆石斑鱼(Epinephelus fuscoguttatus♀×Epinephelus lanceolatus

)^[33]具有较好的促生长作用。关于中草药如何发挥促生长的作用,一部分研究认为中草药中含有独特气味的活性物质可以作用于鱼类的味觉和嗅觉,从而通过刺激食欲增加采食量来提高生长性能^[34-36];也有研究认为中草药改善了鱼类的肠道结构,提高消化吸收能力,从而促进了营养物质的利用效率,实现体重的增加^[37-38]。黄芩的促生长作用主要来源于黄芩苷等有效成分^[39],在珍珠比目鱼中研究表明黄芩主要通过促进骨骼、肝脏和肌肉中碱性磷酸酶和生长相关因子的表达,刺激消化酶活性,促进生长^[32]。

中草药添加量与生长之间存在剂量效应关系。本研究发现,当低鱼粉饲料中添加0.2、0.4、0.8 g/kg复方中草药时,促进了凡纳滨对虾生长;而当添加量提高到1.6 g/kg时,生长性能降低。同样地,Maurus等^[19]也发现当黄芩提取物添加量在0.2%~1.0%时,凡纳滨对虾具有较好的生长效果;而当添加量达到5%时,增重率下降。Cho等^[31]也发现,珍珠比目鱼的增重率随着黄芩提取物添加量的增加出现先升高后降低的趋势。

血清GLU、蛋白质、脂质和酶作为广泛的临床指标被广泛用于鱼类健康评估^[40-41]。UA是蛋白质分解代谢的主要含氮代谢产物,无脊椎动物中UA来源于精氨酸的水解及尿酸的氧化2个代谢途径^[42],研究证实甲壳类体内的UA参与渗透压调节^[43]。研究发现,当凡纳滨对虾在遭受白斑综合症病毒感染或盐度、氨氮胁迫时,对虾血淋巴UA含量出现异常升高或降低^[44-45],说明对虾血淋巴渗透压调节能力出现异常。也有研究指出,甲壳动物UA排放量可能与其代谢的蛋白质的质和量有关^[46]。Malmolf^[47]认为饲料氨基酸平衡且蛋白质代谢良好时,血清UA含量降低,而当肝脏、肾脏发生炎症、功能异常时,血清UA含量往往升高。本研究显示,低鱼粉饲料中添加中草药提取物对凡纳滨对虾血清TP含量影响不显著,但是提高了血清UA含量,尤其是在添加量为0.4和1.6 g/kg时,说明高剂量的中草药提取物可能影响了凡纳滨对虾的渗透压调节。

mTOR信号通路在细胞代谢和生长调控领域受到了相当广泛的关注。mTOR信号通路通过刺激生物合成途径如蛋白质、脂质和核苷酸的产生以及抑制自噬途径抑制细胞的分解代谢促进细胞生长和增殖^[48]。Shao等^[49]研究发现,低鱼粉饲料导致mTOR信号通路中mTOR和S6K1基因被显著抑制,凡纳滨对虾增重率和特定生长率显著降低,说明低鱼粉对凡纳滨对虾生长造成负反馈作用。本研究发现,中草药提取物显著提高了凡纳滨对虾mTOR信号通路中PI3K、mTOR、p38 MAPK、PTEN、S6K1和4EBP1基因相对表达量,此外也发现这些基因相对表达量在G2和G3组肠道中同样也显著提高,说明中草药提取物通过激活mTOR信号通路改善低鱼粉对凡纳滨对虾生长造成的负作用。目前鲜有关于黄芩通过mTOR信号通路调控动物生长性能的报导,较多的研究主要集中在黄芩通过mTOR信号通路调控疾病的发生,如黄芩苷能够通过激活mTOR信号通路,促进小鼠肝细胞增殖,并抑制乙酰氨基酚诱导的自噬,从而促进肝修复等^[50]。

中草药含有多种有效成分,如多糖、生物碱或类黄酮^[51]。复合中草药提取物可作为免疫刺激剂增强鱼虾的免疫反应^[10]。研究报道,黄芩能提高凡纳滨对虾体内溶菌酶和CAT活性^[21]。黄芩苷通过降低罗非鱼血清和肝脏中MDA含量以及提高SOD活性和T-AOC缓解由H₂O₂诱导的肝脏损伤和氧化应激^[52]。在分子层面,采用固液萃取法获得的黄芩提取物显著提高了凡纳滨对虾SOD基因相对表达量^[19]。本研究中,添加中草药提取物后,凡纳滨对虾肝胰腺MDA含量降低,肝胰腺和肠道SOD基因相对表达量显著升高,与上述研究结果一致。HO-1是核转录因子E2相关因子2(Nrf2)下游的主要抗氧化基因,可有效介导抗氧化应激^[53]。本研究中,中草药提取物能够显著提高肝胰腺和肠道HO-1基因相对表达量,说明中草药提取物可激活HO-1基因,从而提高低鱼粉养殖凡纳滨对虾机体的抗氧化能力。

4 结论

在鱼粉用量为8%、蛋白质水平为39%的低鱼粉饲料中添加0.8 g/kg的中草药提取物可提高凡纳滨对虾的生长速度和存活率,激活肝胰腺和肠道mTOR信号通路,提高机体的抗氧化能力。

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