高脂日粮添加胆汁酸对大口黑鲈生长性能、脂代谢和胆汁酸代谢的调节作用

引用本文

杨影, 廖晨蕾, 王力, 康晟超, 秦牧, 李洪运, 张学振. 高脂日粮添加胆汁酸对大口黑鲈生长性能、脂代谢和胆汁酸代谢的调节作用[J]. 中国水产科学, 2023, 30(7): 863-877. DOI: 10.12264/JFSC2023-0065.

YANG Ying, LIAO Chenlei, WANG Li, KANG Shengchao, QIN Mu, LI Hongyun, ZHANG Xuezhen. Effects of dietary bile acid on growth performance, bile acid metabolism and lipid metabolism in largemouth bass (Micropterus salmoides) fed high-fat diets[J]. Journal of Fishery Sciences of China, 2023, 30(7): 863-877. DOI: 10.12264/JFSC2023-0065.

基金项目

国家重点研发计划项目（2019YFD0900303）；中央高校基本科研业务费专项资金项目（2662019FW013）.

作者简介

杨影（1998‒），女，硕士研究生，研究方向为水产健康养殖. E-mail：yingyang@webmail.hzau.edu.cn

通信作者

通信作者：张学振，教授，研究方向为水产健康养殖. E-mail：xuezhen@mail.hzau.edu.cn

文章历史

收稿日期：2023-03-29
修改日期：2023-05-24

Contents Abstract Full text Figures/Tables PDF

高脂日粮添加胆汁酸对大口黑鲈生长性能、脂代谢和胆汁酸代谢的调节作用

杨影^1,2,廖晨蕾^1,2,王力^1,2,康晟超^1,2,秦牧^1,2,李洪运^1,2,张学振^1,2,

1. 华中农业大学水产学院，长江经济带大宗水生生物产业绿色发展教育部工程研究中心，湖北省池塘养殖工程实验室，湖北　武汉　430070
2. 湖北洪山实验室，湖北　武汉　430070

收稿日期：2023-03-29；修改日期：2023-05-24

基金项目：国家重点研发计划项目（2019YFD0900303）；中央高校基本科研业务费专项资金项目（2662019FW013）.

作者简介：杨影（1998‒），女，硕士研究生，研究方向为水产健康养殖. E-mail：yingyang@webmail.hzau.edu.cn.

摘要：为探究胆汁酸对高脂饲料饲喂下大口黑鲈（Micropterus salmoides）生长生理、脂代谢和胆汁酸代谢的影响，配制基础饲料（脂肪含量10.6%，对照组C）、高脂饲料（脂肪含量17.5%，高脂组HF）、高脂饲料+300 mg/kg胆汁酸（HFB1）、高脂饲料+600 mg/kg胆汁酸（HFB2）、高脂饲料+900 mg/kg胆汁酸（HFB3）5种试验饲料。将750尾大口黑鲈随机分为5组，每个组设置3个平行缸，每缸50尾，进行7周养殖实验。结果表明：高脂饲料显著降低大口黑鲈生长性能，而添加胆汁酸后可改善高脂饲料对大口黑鲈生长的不利影响。饲喂高脂饲料组血浆和肝脏中甘油三酯（TG）含量显著升高（P<0.05）。添加胆汁酸后，肝脏中TG含量显著降低（P<0.05），而血浆中TG含量在胆汁酸添加量为900 mg/kg时显著下降（P<0.05）。肝脏和血浆中总胆酸（TBA）含量随着胆汁酸添加量的升高而显著增加（P<0.05）。胆汁酸添加量为600 mg/kg时，肝脏中法尼醇X受体基因（fxr）表达量上调（P<0.05），胆汁酸合成和转运相关基因（cyp7a1、bsep、asbt）均显著上调（P<0.05）。同时对脂代谢相关基因表达进行检测，发现fxr诱导shp的表达，抑制肝脏中srebp1及脂肪合成相关基因（fas、acc2）的表达。此外，胆汁酸增加肝脏中脂肪分解和β氧化相关基因（lpl、hl、hsl、cpt1、acox1）的表达水平。综上所述，高脂饲料中添加胆汁酸可改善大口黑鲈生长性能，通过FXR/SHP/ SREBP1信号通路调节脂质代谢，并促进胆汁酸合成和循环，本研究为进一步研究胆汁酸在鱼类中的营养调控作用奠定了基础。

关键词：大口黑鲈高脂饲料胆汁酸脂代谢胆汁酸合成转运

Effects of dietary bile acid on growth performance, bile acid metabolism and lipid metabolism in largemouth bass (Micropterus salmoides) fed high-fat diets

YANG Ying^1,2,LIAO Chenlei^1,2,WANG Li^1,2,KANG Shengchao^1,2,QIN Mu^1,2,LI Hongyun^1,2,ZHANG Xuezhen^,1,2

1. College of Fisheries, Huazhong Agricultural University; Engineering Research Center of Green Development for Conventional Aquatic Biological Industry in the Yangtze River Economic Belt, Ministry of Education; Hubei Provincial Engineering Laboratory for Pond Aquaculture, Wuhan 430070, China
2. Hubei Hongshan Laboratory, Wuhan 430070, China

Abstract:Bile acids (BAs) are important amphipathic biomolecules that play a key role in the emulsification of lipids and consumption of other fat-soluble nutrients. While fat is an essential energy source for fish growth, excessive dietary fat induces degradation of growth performance, metabolic disorders, and fat accumulation. There is evidence that BAs can promote growth, accelerate lipid breakdown, and maintain bile acid homeostasis in fish fed high-fat diets (HFDs). However, the regulatory mechanisms remain unclear. This study aimed to explore the effects of dietary BA supplementation on BA homeostasis and lipid metabolism in juvenile largemouth bass (Micropterus salmoides) fed HFDs. Five isonitrogenous diets were prepared: a control diet (10.6% lipid, C), high-fat diet (17.53% lipid, HF), and HF diets supplemented with 300, 600, and 900 mg/kg BA (HFB1, HFB2, and HFB3). A 7-week feeding trial was performed, and all the fish were fed to satiety twice daily. Samples were taken at the end of the trial to detect growth and plasma biochemical parameters as well as lipid and BA metabolism-related gene expression levels. The results showed that HF diet significantly decreased final body weight (FBW), specific growth rate (SGR), and weight gain (WG) (P<0.05). In contrast, the addition of BAs to the HF diets improved growth performance, with the growth of largemouth bass significantly increased when supplement with 600 mg/kg BAs (P<0.05). Moreover, the lipid content in muscle was increased by the HF diet (P<0.05) and was decreased by supplementation with BAs (P<0.05). The HF diet increased the triglyceride (TG) contents in the plasma and liver (P<0.05), while BA supplementation decreased both (P<0.05). Furthermore, the addition of BAs significantly decreased the expressions of sterol-regulatory element binding protein 1 (srebp1), fatty acid synthase (fas), and acetyl-CoA carboxylase 2 (acc2) and increased the expressions of lipoprotein lipase (lpl), hormone sensitive lipase (hsl), and hepatic lipase (hl) (P<0.05). Additionally, BA supplementation increased the expressions of carnitine palmitoyltransferase 1 (cpt1) and acyl-CoA oxidase 1 (acox1) (P<0.05), yet had no effect on expression of peroxisome proliferator-activated receptor α (pparα) (P>0.05). These results indicate that BAs increase lipolysis and fatty acid β-oxidation, while decreasing lipogenesis. Moreover, fish fed a BA supplemented-diet showed increased total bile acid (TBA) levels in the liver and plasma (P<0.05). When 600 mg/kg BA was added, the expression of farnesoid X receptor (fxr) was also increased (P<0.05). Therefore, BA supplementation increased the expressions of cholesterol 7 alpha-hydroxylase (cyp7a1), bile salt export protein (bsep), and apical sodium dependent transporter (asbt) via activating fxr (P<0.05). In conclusion, dietary BA supplementation improved growth performance, lipid digestion and absorption, and bile acid metabolism by activating the bile acid nuclear receptor FXR. The effects and possible mechanisms of BAs on lipid and bile acid metabolism were also preliminarily revealed. This study contributes to our understanding of the role of BAs in lipid metabolism in teleosts and enriches our knowledge of bile acid homeostasis under different conditions.

Key words： Micropterus sodmoides high-fat diet bile acids lipid metabolism bile acid synthesis and transport

在适宜范围内，增加饲料脂肪含量可提供能量并且节约蛋白质，因此高脂饲料(high fat diet, HFD)在鱼类养殖中的使用越来越广泛^[1]。然而过量的脂肪会导致鱼类生长性能下降、代谢紊乱和脂肪堆积^[2]。长期饲喂HFD会降低营养物质的消化吸收，引起肝脏损伤和肠道炎症^[3-5]。功能性饲料添加剂可以增强水生动物的体质，促进水生动物的健康生长，以提高水产养殖的经济效益。

胆汁酸(bile acid, BA)是重要的两性分子，主要在肝脏中合成，其合成途径主要包括经典途径和替代途径两种。经典途径由胆固醇7α-羟化酶(cholesterol 7-α-hydroxylase, CYP7A1)启动发生，替代途径由甾醇27-羟化酶(sterol 27A-hydroxlase, CYP27A1)启动发生^[6]。胆汁酸具有促进脂溶性营养素吸收、提高机体抗氧化能力、调节脂质代谢的作用^[7]，因此胆汁酸已经作为一种非营养性饲料添加剂广泛应用于禽畜和水产动物中。饲料中适当添加胆汁酸有利于促进动物生长发育、调控糖脂代谢、维持体内胆汁酸平衡^[8]。在鱼类中，研究表明在罗非鱼(Oreochromis niloticus)和大黄鱼(Larimichthys crocea)饲料中添加胆汁酸可减少肝脏中脂肪含量，改善生长性能^[9-10]。在仔猪饲料中添加胆汁酸，可激活脂肪酶，增强脂肪吸收能力，改善肠道黏膜的保护潜力，促进仔猪生长^[11-12]。研究表明，胆汁酸可以通过激活其受体法尼酯X受体(farnesoid X receptor, FXR)调节机体脂代谢过程。FXR可通过肝脏中的小异源二聚体伴侣(small heterodimer partner, SHP)调控脂代谢过程中的关键转录因子固醇调节因子结合蛋白-1 (sterol-regulatory element binding protein 1, SREBP1)及其下游脂肪合成基因的表达，抑制脂肪合成，减少肝脏脂质含量^[13-15]。FXR不仅可调控脂代谢过程，还可调控胆汁酸的合成与转运，维持体内胆汁酸平衡^[15]。FXR可抑制CYP7A1和甾醇12α-羟化酶(sterol 12-α-hydroxylase, CYP8B1)的转录表达，从而介导BA合成的负反馈^[16]。FXR还通过调节BA转运蛋白的表达来促进BA分泌并抑制肝胆汁摄取^[17]。胆盐输出泵(bile salt export protein, BSEP)和顶端钠依赖性胆汁酸转运蛋白(apical sodium dependent transporter, ASBT)负责胆汁酸在肝脏和肠道之间的循环^[16]。注射牛磺脱氧胆酸可抑制杂交石斑鱼(Epinephelus fuscoguttatus♀×E. lanceolatus♂)肝脏中胆汁酸合成并减少胆汁酸转运^[18]。然而，也有研究表明外源性BA并没有影响乌鳢(Channa argus) BA的合成与转运^[19]。这可能与不同的鱼类和不同的BA种类有关。

BA作为饲料添加剂已广泛应用在水产饲料中，以达到促进生长的作用。然而，胆汁酸对鱼类脂质代谢以及内源性胆汁酸代谢的作用机制尚未完全清楚。本研究以大口黑鲈(Micropterus salmoides)为实验对象，研究高脂饲料中添加胆汁酸对大口黑鲈生长、脂质代谢和胆汁酸合成转运的影响，探讨胆汁酸影响脂代谢和胆汁酸代谢的潜在机制，旨在丰富鱼类不同状态下胆汁酸营养生理学功能的认识，为深入研究胆汁酸对鱼类的代谢调控机制奠定基础，并为大口黑鲈高脂饲料的有效利用提供依据。

1　材料与方法 1.1　实验鱼

本实验所使用的大口黑鲈购自中国水产科学院长江水产研究所，养殖实验在华中农业大学水产学院循环水养殖系统中进行。在养殖实验开始之前，所有大口黑鲈在养殖系统中暂养2周以适应养殖环境。2周后，将750尾大口黑鲈[(6.02± 0.28) g]随机分配到15个(共5组，每组3个平行缸，每缸50尾)养殖缸中。在试验期间，控制水温为(24±0.6) ℃，溶氧7.4~8.6 mg/L; pH6.7~7.5。每天2次饱食投喂(9:00和17:00)。

1.2　实验饲料

本实验共制备5种等氮饲料：基础饲料(Control组，C组，脂肪含量10.6%)、高脂饲料(High fat 组，HF组，脂肪含量17.5%)和胆汁酸含量分别为300 mg/kg、600 mg/kg、900 mg/kg的3种高脂饲料(HFB1、HFB2、HFB3)。基础饲料和高脂饲料配方见表1。胆汁酸购自山东龙昌动物保健品有限公司，其组成及质量比为：猪脱氧胆酸∶鹅脱氧胆酸∶猪胆酸=7∶2∶1。将饲料原料完全研磨并过40目筛，随后将各原料以添加量从低到高的原则逐级添加混匀，最后加入鱼油和豆油混匀。将混合物制成标准直径为2 mm的颗粒，风干后并储存在‒20 ℃。

1.3　样品采集

在7周养殖实验结束时，将大口黑鲈禁食24 h。每个处理组随机选取12尾鱼，用MS-222溶液麻醉后称重；用抗凝剂润湿的一次性注射器从尾静脉获得血液并装入离心管中，在冷冻离心机中快速离心(4 ℃, 4000 r/min, 12 min)以收集血浆，将其储存在‒80 ℃用于生化指数测定；无菌条件下解剖，分离内脏团和肝脏并称重。将肝脏组织在液氮中快速冷冻，并在‒80 ℃下储存，用于酶活性、脂肪酸组成分析和基因表达分析。肠道在液氮中快速冷冻后保存于‒80 ℃用于后续基因表达分析。最后分离背部肌肉组织，取一部分肌肉组织保存于‒80 ℃用于营养成分分析。

1.4　实验方法 1.4.1　生长性能分析

增重率(weight gain, WG, %)=(终末体重–初始体重)/初始体重×100;

表1 　实验饲料配方及化学成分（%干物质） Tab. 1 　Formulation and proximate composition of experimental diets (% dry matter) %

特定生长率(specific growth rate, SGR, %)=(ln 终末体重–ln初始体重)/t×100;

肥满度(condition factor, CF, g/cm³)=终末体重(g)/终末体长³(cm³)×100;

肝体指数(hepatosomatic index, HSI, %)=肝脏重(g)/体重(g)×100;

脏体指数(viscerosomatic index, VSI, %)=内脏重(g)/体重(g)×100。

1.4.2　肌肉基本营养成分测定

饲料和肌肉的基本营养成分测定参考本实验研究方法^[20]：水分采用烘干法测定，粗灰分采用550 ℃灼烧法分析，粗脂肪含量采用索氏抽提法测定，粗蛋白含量采用凯氏定氮法测定。

1.4.3　血浆和肝脏生化指标测定

将血浆逐级解冻直接用于测定。将肝脏样品在生理盐水(8.6 g/L NaCl in dd H₂O)中匀浆3 min，体积为10倍(W/V)，离心15 min (4000 r/min, 4 ℃)，并将上清液保存在‒80 ℃直至分析。使用商业试剂盒检测总甘油三酯(TG)、总胆固醇(TCHO)、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白(LDL-C)和总胆汁酸(TBA)的含量(南京建成生物工程研究所，中国南京)。

1.4.4　肝脏脂肪酸成分分析

肝脏脂肪酸含量的测定采用气相色谱法进行，具体步骤如下：准确称取200 mg肝脏组织放入离心管中，加入1 mL氯仿/甲醇(2∶1, V/V)混合液进行匀浆、离心后，抽吸下层氯仿层以提取脂肪。将吸取的下层液体装入玻璃管中，进行氮吹，吹干后加入1.5 mL三氟化硼甲醇/二氯甲烷(2∶1, V/V)溶液在100 ℃下甲酯化1 h。冷却后，加入0.5 mL 饱和食盐水和1 mL正己烷，涡旋混匀后静置分层，将上层液体吸出后进行氮吹，吹干后按比例加入色谱级正己烷。最后，将处理好的样品在气相色谱仪(岛津，GC-2010 plus，日本东京)进行分析。进样器和检测器(FID)的温度分别设定为250 ℃和260 ℃。温度程序为200 ℃ (40 min)至240 ℃ (15 min)，速度为4 ℃/min。使用高纯度氦气作为载气，流速为1 mL/min。

1.4.5　实时荧光定量PCR

使用TRIZOL试剂(Aidlab，中国北京)提取总RNA。RNA提取方法参考本实验室的方法^[20]。相关基因的转录水平由实时荧光定量PCR (qPCR)检测，使用的试剂盒为Hieff^®qPCR^®Green Master Mix (上海翊圣生物科技有限公司)。所有基因的引物均使用Primer 6.0设计(表2)。以伸长因子1α (ef1α)为内参基因，检测大口黑鲈肝脏和肠道基因的表达。按照Livak等^[21]的方法分析靶基因的相对定量。

1.5　统计学分析

所有数据均以SPSS 19.0 (SPSS Inc., Chicago, IL, USA)进行统计学分析。分别以Shapiro-Willkstest和Levene's test对数据进行正态分布检验和方差齐性检验。对服从正态分布且方差齐性的数据进行单因素方差分析(one-way ANOVA)并以Duncan’s test进行多重比较。对不服从正态分布的数据或方差不齐的数据进行Kruskal-Wallis非参数检验并以Dunn-Bonferroni post-hoc test进行多重比较。本实验中所有数据均表示为均值±标准误($\bar x \pm {\rm{SE}}$)，所有结果统计分析均以P<0.05为显著性水平。

表2 　本研究所用qPCR引物 Tab. 2 　Oligonucleotide primers for qPCR assays in the study

基因gene	引物序列(5ʹ−3ʹ) primer sequence (5ʹ−3ʹ)	长度/bp size	来源source
ef1α	F: GCGCAAGGAGGGTAATGCTA	178	XM_038724777.1
ef1α	R: CATACCGGGCTTCAGGACAC	178	XM_038724777.1
cyp7a1	F: CTGGGCTTCACAGGCTAACAC	153	Chen et al^[22]
cyp7a1	R: TTCAGTGTGGGGTCGTTGGG	153	Chen et al^[22]
cyp27a1	F: ATGCCCGTGTCACTGTTG	176	Chen et al^[22]
cyp27a1	R: GTGCGGCTTGGACTTCTC	176	Chen et al^[22]
cyp8b1	F: TAGACAGCGGCAACCAGGAG	120	Chen et al^[22]
cyp8b1	R: CCGTGCTTTTGTTTCATCCTAC	120	Chen et al^[22]
fxr	F: AGGAAATGCCAGGAGTGTCG	137	XM_038705617.1
fxr	R: GCCTTGGTGTTCTTTCGCAG	137	XM_038705617.1
lxr	F: CAGAGCCAGGACTTGCAGAA	200	XM_038700505.1
lxr	R: TACCGCCGAGATGTTTCGAG	200	XM_038700505.1
shp	F: AGAGCTTGCTTGCATCTGCT	99	XM_038724571.1
shp	R: CAGTCCTTGAACAGCTGGGTT	99	XM_038724571.1
srebp1	F: TGCAGCTCTACGGGTCAAAG	188	XM_038699585.1
srebp1	R: TAGGTGTGCTGCGAATAGCC	188	XM_038699585.1
angptl3	F: CCCTCTGGAATCCTCGGGTA	136	XM_038715308.1
angptl3	R: AAGGAGGCCGTTTGCAAGTA	136	XM_038715308.1
acc2	F: AGTCTCTGGGCACGACCTAT	140	XM_038709726.1
acc2	R: CAGCTCTGTGAAGGTGAGCA	140	XM_038709726.1
apoc2	F: CTGAAAGCTTCCGTGTGCC	115	XM_038710946.1
apoc2	R: ATCACTGGCAGAGTTGACGAA	115	XM_038710946.1
dgact	F: CACGCCTCTTCTTGGAGAAC	126	XM_038724648.1
dgact	R: AATGGTACCCACAGCCAGAC	126	XM_038724648.1
ostβ	F: GACCCGACCACATGGAAGTT	149	XM_038702073.1
ostβ	R: CTCTTCTGAGCCCTAGCAGC	149	XM_038702073.1
asbt	F: AACCCTGGGGCATCATCATC	152	XM_038719691.1
asbt	R: AATGTTGGAGCCGGAACCTC	152	XM_038719691.1
bsep	F: GACTGGAGTGCTGCTGTTGA	118	XM_038720465.1
bsep	R: GCATGTTGACTCATCTGCTCG	118	XM_038720465.1
lpl	F: GTCAAATGTTCCCACGAGCG	148	XM_038715978.1
lpl	R: GCTTGTTGCAGCGGTTCTTT	148	XM_038715978.1
fas	F: GGGCATCGTTAACCTGGACA	164	XM_038735140.1
fas	R: CATCGTAGTGGCGCTTTTCG	164	XM_038735140.1
hsl	F: ATCAGAGCTGGAGCACCCTA	106	XM_038725628.1
hsl	R: GCAGAGGAGAGCAGAAAGGA	106	XM_038725628.1
pparα	F: CCACCGCAATGGTCGATATG	144	XM_038705496.1
pparα	R: TGCTGTTGATGGACTGGGAAA	144	XM_038705496.1
cpt1	F: CCTGGTGGGTTTATGGTAGTG	200	XM_038705334.1
cpt1	R: CGACAACAGGCACTTTAGGA	200	XM_038705334.1
hl	F: GGTGCAGTGACAAAAACGCT	100	XM_038701258.1
hl	R: CCACTGCGAACCCTCTTGAT	100	XM_038701258.1
acox1	F: GCTATCGACCTGGTCAGAGC	116	XM_038713843.1
acox1	R: AGGGTTGACAGCACTGAGTG	116	XM_038713843.1
注：F表示正向引物；R表示反向引物. Note: F indicates forward primer; R indicates reverse primer.

表2 　本研究所用qPCR引物 Tab. 2 　Oligonucleotide primers for qPCR assays in the study

2　结果与分析 2.1　生长性能

如表3所示，与C组相比，HF组中大口黑鲈FBW、WG和SGR显著降低(P<0.05)。补充BA后，FBW、WG和SGR显著增加(P<0.05)。HF饲料显著提高大口黑鲈HSI，补充BA显著降低HIS (P<0.05)。HF组VSI显著增加(P<0.05)，补充胆汁酸后，HFB1和HFB3组大口黑鲈VSI显著降低(P<0.05)。CF没有受到高脂饲料和胆汁酸的影响(P>0.05)。

2.2　肌肉营养成分

如表4所示，与C组相比，HF组中肌肉粗脂肪含量显著增加(P<0.05)。在高脂饲料中添加BA可显著降低肌肉粗脂肪含量(P<0.05)。然而，肌肉中其他营养组成(粗蛋白、水分和粗灰分)在所有处理之间均未受到影响(P>0.05)。

2.3　血浆和肝脏中脂质代谢指标和TBA水平

如表5所示，与C组相比，HF组血浆中TG和LDL-C水平显著提高(P<0.05)。添加BA后，血浆中TG和LDL-C含量显著下降(P<0.05)。HF组HDL-C含量增加(P<0.05)；而饲料BA并没有降低HDL-C的含量(P>0.05)。HF组显著增加肝脏中TG含量(P<0.05)，并且饲料BA降低肝脏中TG含量(P<0.05)。肝脏LDL-C含量在各处理组中无显著变化(P>0.05)。

表3 　胆汁酸对高脂饲料下大口黑鲈生长性能的影响 Tab. 3 　Effects of bile acid on growth performance of Micropterus salmoides fed high-fat diet n=12; $\bar x \pm {\rm{SE}}$

表4 　胆汁酸对高脂饲料下大口黑鲈肌肉营养成分的影响 Tab. 4 　Effects of bile acid on muscle nutritional composition of Micropterus salmoides fed high-fat diet (wet weight %) n=9; $\bar x \pm {\rm{SE}}$; %

表5 　胆汁酸对高脂饲料下大口黑鲈脂质代谢指标的影响 Tab. 5 　Effects of bile acid on biochemical parameters of plasma and liver of Micropterus salmoides fed high-fat diet n=9; $\bar x \pm {\rm{SE}}$

如图1所示，与C组相比，HF组血浆中的TCHO含量显著增加(P<0.05)。随着BA添加量增加，血浆中TCHO含量降低(P<0.05)。然而肝脏中TCHO在各组之间未出现统计学差异(P> 0.05)。HF组血浆和肝脏TBA水平不受脂肪水平影响(P>0.05)。补充胆汁酸后，血浆中TBA水平显著增加，肝脏中TBA水平呈现逐渐上升的趋势(P<0.05)。

图1 　胆汁酸对高脂饲料下大口黑鲈血浆和肝脏总胆固醇和总胆汁酸水平的影响a. 血浆TCHO; b. 肝脏TCHO; c. 血浆TBA; d. 肝脏TBA. 不同小写字母表示处理组间存在显著差异(P<0.05). C：对照组；F：高脂组；HFB1：高脂+300 mg/kg胆汁酸组；HFB2：高脂+600 mg/kg胆汁酸组；HFB3：高脂+900 mg/kg胆汁酸组. 下同. Fig. 1 　Effects of bile acid on the total cholesterol (TCHO) and total bile acid (TBA) level in plasma and liver of Micropterus salmoides fed high-fat dieta. TCHO in plasma; b. TCHO in liver; c. TBA in plasma; d. TBA in liver. Different small letters indicate significant difference among groups (P<0.05). C: control group; HF: High fat diet group; HFB1: high fat diet + 300 mg/kg bile acid; HFB2: high fat diet + 600 mg/kg bile acid; HFB3: high fat diet + 900 mg/kg bile acid. The same below.

2.4　肝脏脂肪酸组成分析

如表6所示，HF组肝脏中C16∶0和C22∶0含量显著增加(P<0.05)，而饲料中添加BA降低C16∶0含量(P<0.05)。HF组中肝脏饱和脂肪酸(SFA)含量显著增加(P<0.05)，而BA的添加降低肝脏中SFA的含量(P<0.05)(图2)。补充BA显著提高单不饱和脂肪酸(MUFA)含量(P<0.05)(图2)。HF组中C14∶1、C16∶1和C18∶1水平显著降低(P<0.05)。BA的添加导致C14∶1和C18∶1浓度呈显著增加趋势(P<0.05)，但C16∶1含量显著降低(P<0.05)。与C组相比，HF组多不饱和脂肪酸(PUFA)含量显著降低，但饲料BA增加HFB1组和HFB2组中肝脏PUFA含量(P<0.05)(图2)。HF组中C18∶2n-6和C20∶2n-6含量下降(P<0.05)，而C22∶6n-3浓度升高(P<0.05)。当BA添加量为900 mg/kg时，C22∶6n-3含量降低(P<0.05)。

表6 　胆汁酸对高脂饲料下大口黑鲈肝脏脂肪酸组成的影响 Tab. 6 　Effect of BA on fatty acid composition (% identified fatty acids) in liver of Micropterus salmoides fed high-fat diet n=9; $\bar x \pm {\rm{SE}}$

脂肪酸fatty acid	组别group
脂肪酸fatty acid	C	HF	HFB1	HFB2	HFB3
C14∶0	0.43±0.03^b	0.37±0.01^c	0.46±0.01^ab	0.47±0.01^a	0.48±0.01^a
C14∶1	1.49±0.08^a	1.16±0.05^b	1.48±0.05^a	1.54±0.11^a	1.47±0.06^a
C16∶0	11.10±0.69^b	16.29±0.67^a	10.24±0.70^b	9.88±0.35^b	9.72±0.38^b
C16∶1	5.24±0.20^a	4.38±0.08^b	3.47±0.13^c	3.24±0.27^c	3.69±0.30^c
C18∶0	4.91±0.17	4.73±0.13	4.92±0.21	5.33±0.34	5.43±0.26
C18∶1	12.74±0.14^b	11.19±0.62^c	13.92±0.12^a	14.05±0.43^a	14.13±0.08^a
C18∶2n-6	16.13±0.28^b	14.48±0.72^b	18.15±0.27^a	18.19±0.84^a	18.44±0.59^a
C18∶3n-6	0.87±0.08^a	0.97±0.17^a	0.73±0.14^ab	0.39±0.08^b	0.94±0.06^a
C18∶3n-3	0.50±0.04^c	0.69±0.10^bc	1.20±0.21^a	0.98±0.13^ab	0.73±0.08^bc
C18∶4n-3	0.70±0.10^a	0.34±0.06^b	0.54±0.03^ab	0.35±0.09^b	0.47±0.03^ab
C20∶0	1.17±0.18^ab	0.75±0.03^c	1.08±0.08^bc	0.90±0.09^bc	1.48±0.11^a
C20∶1n-11	5.25±0.42	5.74±1.05	5.64±0.92	5.40±0.98	6.81±0.81
C20∶2n-6	3.84±0.26^a	2.66±0.12^b	1.47±0.32^c	0.92±0.24^cd	0.61±0.13^d
C20∶3n-3	0.98±0.06	0.58±0.10	0.91±0.24	0.70±0.20	0.94±0.02
C20∶4n-6	1.72±0.11^a	1.35±0.03^b	1.58±0.06^a	1.55±0.06^ab	1.65±0.05^a
C20∶4n-3	0.85±0.12	0.71±0.26	1.08±0.26	1.45±0.38	0.75±0.12
C20∶5n-3	2.42±0.08^a	1.59±0.28^b	1.77±0.41^ab	1.78±0.39^ab	1.31±0.23^b
C22∶0	1.69±0.34^ab	2.25±0.32^ab	1.18±0.13^b	2.60±0.65^a	2.60±0.35^a
C22∶1n-11	1.05±0.08^a	0.84±0.07^b	1.09±0.05^a	1.09±0.02^a	1.17±0.06^a
C22∶5n-3	2.09±0.07^c	2.14±0.20^bc	2.81±0.37^ab	2.94±0.21^a	2.58±0.11^abc
C22∶6n-3	24.71±0.53^b	26.68±0.87^a	26.17±0.53^ab	26.14±0.38^ab	24.51±0.27^b
∑n-3PUFA	32.28±0.47^ab	32.76±1.21^ab	34.52±1.31^a	34.36±0.44^a	31.32±0.17^b
∑n-6PUFA	22.58±0.49^a	19.47±0.86^b	21.96±0.22^a	21.07±0.83^ab	21.65±0.68^a
n-3/n-6PUFA	1.43±0.02^b	1.69±0.26^a	1.57±0.04^ab	1.63±0.07^ab	1.45±0.04^b
注：同一行中不同的上标字母表示处理组间存在显著差异(P<0.05). PUFA为多不饱和脂肪酸. Note: In the same row, different small letter superscripts indicate significant difference among groups (P<0.05). PUFA is polyunsaturated fatty acid.

2.5　胆汁酸代谢相关基因的表达

如图3所示，与C组相比，HF组fxr表达量差异无统计学意义(P>0.05)。当BA水平升高时，fxr的表达水平显著提高(P<0.05)。cyp27a1和cyp7a1的表达水平不受高脂饲料的影响(P>0.05)。cyp7a1的表达水平随BA水平的增加而升高(P<0.05)。然而，cyp27a1的表达随着BA添加水平的增加而降低(P<0.05)。肝脏X受体基因(lxr)和cyp8b1水平在所有处理组中无统计学意义(P>0.05)。

如图4所示，bsep在肝脏中的表达水平随着BA添加水平的增加而上调(P<0.05)。当BA添加水平为600 mg/kg和900 mg/kg时，肠道中asbt表达量增加(P<0.05)。肠道中ostβ的表达量随着BA添加量的增加呈现先降低后升高的趋势(P<0.05)。

2.6　肝脏脂代谢相关基因的表达

如图5所示，饲料BA显著提高shp的表达水平(P<0.05)。同时，添加BA后，甾醇调节元件结合蛋白1 (srebp1)、脂肪酸合成酶(fas)和乙酰辅酶A羧化酶2 (acc2)的表达显著下调(P<0.05)。然而，这种调节作用在二酰基甘油酰基转移酶(dgact)中没有发生。当胆汁酸添加量为900 mg/kg时，dgact的表达量显著上升(P<0.05)。

与脂肪分解相关的基因水平如图6所示。与C组相比，HF饲料对lpl、apoc2和angptl3的表达无影响(P>0.05)。饲料BA显著提高载脂蛋白C2(apoc2)和脂蛋白脂肪酶(lpl)的表达(P<0.05)。HFB1组显著降低血管生成素样蛋白3(angptl3)的转录。此外，饲料BA显著上调肝脏中激素敏感脂肪酶(hsl)和肝脂肪酶(hl)的转录(P<0.05)。hsl和hl mRNA水平随BA水平的增加而上调(P< 0.05)。当高脂饲料中添加胆汁酸，肉碱棕榈酰转移酶1 (cpt1)和酰基辅酶A氧化酶1 (acox1)的表达水平增加(P<0.05)，而过氧化物酶体增殖物激活的受体α (pparα) mRNA水平无显著差异(P>0.05)。

图2 　胆汁酸对高脂饲料下大口黑鲈肝脏中SFA、MUFA和PUFA组成的影响不同小写字母表示处理组间存在显著差异(P<0.05). SFA：饱和脂肪酸；MUFA：单不饱和脂肪酸；PUFA：多不饱和脂肪酸. Fig. 2 　Effects of bile acid on composition of SFA, MUFA and PUFA in liver of Micropterus salmoides fed high-fat dietDifferent small letter indicate significant difference among groups (P<0.05). SFA: saturated fatty acid; MUFA: monounsaturated fatty acid; PUFA: polyunsaturated fatty acid.

图3 　胆汁酸对高脂饲料下大口黑鲈肝脏胆汁酸受体和合成相关基因表达的影响不同小写字母表示处理组间存在显著差异(P<0.05). fxr：法尼酯X受体基因；lxr：肝脏X受体基因；cyp7a1：胆固醇7α-羟化酶基因；cyp8b1：甾醇12α-羟化酶基因；cyp27a1：甾醇27A-羟化酶基因. Fig. 3 　Effects of bile acid on the relative expression of bile acid receptors and biosynthesis markers in the liver of Micropterus salmoides fed high-fat dietDifferent small letters indicate significant difference among groups (P<0.05). fxr: farnesoid X receptor gene; lxr: liver X receptor gene; cyp7a1: cholesterol 7-α-hydroxylase gene; cyp8b1: sterol 12-α-hydroxylase gene; cyp27a1: sterol 27A-hydroxlase gene.

图4 　胆汁酸对高脂饲料下大口黑鲈肝脏和肠道胆汁酸转运受体相关基因表达的影响不同小写字母表示处理组间存在显著差异(P<0.05). bsep：胆盐输出泵基因；asbt：顶端钠依赖性转运蛋白基因；ostβ：有机溶质转运蛋白β基因. Fig. 4 　Effects of bile acid on the relative expression of bile acid transporters in liver and intestine of Micropterus salmoides fed high-fat dietDifferent small letters indicate significant difference among groups (P<0.05). bsep: bile salt export protein gene; asbt: apical sodium dependent transporter gene; ostβ: organic solute transporter β dimer gene.

图5 　胆汁酸对高脂饲料下大口黑鲈肝脏脂肪合成相关基因的影响不同小写字母表示处理组间存在显著差异(P<0.05). shp：小异二聚体伴侣基因；srebp1：固醇调节元件结合蛋白1基因；fas：脂肪酸合成酶基因；acc2：乙酰辅酶A羧化酶基因；dgact：二酰甘油-O-酰基转移酶1基因. Fig. 5 　Effects of bile acid on the relative expression of lipogenesis related mRNAs in liver of Micropterus salmoides fed high-fat dietDifferent small letters indicate significant difference among groups (P<0.05). shp: small heterodimer partner gene; srebp1: sterol-regulatory element binding protein 1 gene; fas: fatty acid synthetase gene; acc2: acetyl-CoA carboxylase 2gene; dgact: diacylglycerol O-acyltransferase 1 gene.

3　讨论 3.1　胆汁酸对高脂饲料下大口黑鲈生长性能的影响

本研究中，HF组的增重率和特定生长率显著低于对照组，这与白姑鱼(Argyrosomus regius)、黄姑鱼(Nibea japonica)和黄河鲤(Cyprinus carpio haematopterus)的研究结果一致，这可能是过量的脂肪水平会降低鱼类摄食欲并降低饲料效率来抑制鱼类生长^[23-25]。本研究中，胆汁酸添加量为600 mg/kg时，大口黑鲈增重率和特定生长率显著提高。研究表明，高脂饲料中添加胆汁酸可以改善罗非鱼(Oreochromis mossambicus)的生长性能^[10]，但不同鱼类的最适添加水平不同，如欧洲鳗鲡(Anguilla anguilla)的最适添加量为500 mg/kg^[26]，齐口裂腹鱼(Schizothorax prenanti)的最适添加量为150 mg/kg^[27]。此外，VSI和HSI的增加也与饲料脂肪水平的增加有关。许多研究表明，高脂质饮食可以增加鱼类HSI，例如梭鱼(Chelon haematocheilus)、杂交鳢(Channa argus×Channa maculata)和星斑川蝶(Platichthys stellatus)^[28-30]，这与本实验结果一致。本研究中，随着胆汁酸的添加水平增加，大口黑鲈HSI先降低后升高。在杂交石斑鱼中，当脱氧胆酸添加量为1200 mg/kg和1500 mg/kg时，肝脏中脂肪含量反而升高^[18]。结果说明合适的胆汁酸水平可以减少肝脏脂肪，而过高的胆汁酸可能会增加肝脏脂肪的沉积。

图6 　胆汁酸对高脂饲料下大口黑鲈肝脏脂肪分解和β氧化相关基因的影响不同小写字母表示处理组间存在显著差异(P<0.05). lpl：脂蛋白脂肪酶基因；apoc2：载脂蛋白C2基因；angptl3：血管生成素样蛋白基因；3; hl：肝脂肪酶基因；hsl：激素敏感脂肪酶基因；pparα：过氧化物酶体增殖物激活受体α基因；cpt1：肉毒碱棕榈酰基转移酶1α基因；acox1：酰基辅酶A氧化酶1基因. Fig. 6 　Effects of bile acid on the relative expression of lipolysis and fatty acids β oxidation related mRNAs in liver of Micropterus salmoides fed high-fat dietDifferent small letter indicate significant difference among groups (P<0.05). lpl: lipoprotein lipase gene; apoc2: apolipoprotein C 2 gene; angptl3: angiopoietin-like protein 3 gene; hl: hepatic lipase gene; hsl: hormone-sensitive lipase gene; pparα: peroxisome proliferator activated receptor α gene; cpt1: carnitine palmitoyltransferase 1α gene; acox1: acyl-coa oxidase 1 gene.

3.2　胆汁酸对高脂饲料下大口黑鲈肝脏脂代谢的影响

当大口黑鲈喂食HF饲料时，血浆和肝脏中TG含量增加，这与在梭鱼和大黄鱼中的研究结果一致^[30-31]。随着饲料BA水平的增加，血浆和肝脏中TG含量降低。补充胆汁酸也缓解了高脂饲料对草鱼的负面影响^[32]。为研究BA如何影响鱼体脂代谢，本研究检测fxr与脂代谢相关基因的表达情况。在本研究中，BA激活fxr并诱导shp表达，从而抑制srebp1的表达。这些结果与之前在哺乳动物中的研究结果一致，激活的FXR通过小鼠FXR-SHP途径抑制SREBP1介导的脂肪生成相关的基因的转录水平^[33-35]。SREBP1是SREBPs家族的一员，主要负责调节细胞脂质代谢和维持体内平衡。SREBP1可激活脂肪生成酶的表达，例如ACC和FAS^[36]。在本研究中，当BA添加量为600 mg/kg时，肝脏中srebp1表达水平降低，并且其下游因子的表达受到抑制，结果说明添加胆汁酸可通过负向调节srebp1基因的表达，减少脂肪生成。SREBP1受到抑制时，会诱导PPARα及其靶基因的表达，促进游离脂肪酸的氧化分解^[37]。在杂交石斑鱼中，高脂饲料中添加胆汁酸诱导ppara和cpt1的表达，促进脂肪分解^[38]。然而，在本研究中，ppara的表达并没有受到HFD和BA的影响。尽管关于鱼类脂代谢表达研究取得一定进展，但在不同鱼类中的研究结果也并不相同。在虹鳟(Oncorhynchus mykiss)中，高脂饲料可引起肝脏中ppara表达增加^[39]；在罗非鱼中，高脂饲料却降低ppara的表达水平^[40]；在尼罗罗非鱼中，高脂饲料也并未引起ppara的表达变化^[41]。关于胆汁酸没有影响ppara的表达水平，这可能与胆汁酸的添加种类和水平、鱼类的大小和种类有关，还需要进一步研究去探讨胆汁酸对pparα表达的影响机制。激活的fxr可通过增加辅激活因子表达水平和降低抑制因子表达水平来增加lpl的表达，促进脂肪酸吸收和甘油三酯的水解^[42-43]。APOC2是LPL的必需辅助因子^[44]。在这项研究中，当BA添加量从600 mg/kg增加到900 mg/kg时，fxr的表达显著上调，通过上调apoc2增加lpl的表达。因此，结果表明饲料BA可以激活FXR，从而增加肝脏LPL活性。HSL是另一种细胞内脂肪酶，可将脂肪组织中的甘油三酯分解成脂肪酸^[45]。当胆汁酸添加量增加至900 mg/kg时，hsl的表达量显著增加。这些结果表明BA通过增加脂肪分解来减少脂质沉积。

3.3　胆汁酸对大口黑鲈胆汁酸代谢的影响

胆汁酸的调控存在负反馈调节机制，当补充外源胆汁酸时，自身的胆汁酸合成受到抑制，避免胆汁酸堆积，造成毒性。然而，在本研究中，血浆和肝脏中总胆汁酸水平随着饲料中胆汁酸添加量的增加而显著提高。鉴于fxr在胆汁酸的负反馈调节中发挥重要作用，因此本实验探究fxr以及其下游靶基因的表达，进一步探索补充胆汁酸对鱼类胆汁酸代谢的作用。CYP7A1和CYP27A1被认为是胆汁酸的重要合成酶^[46]。fxr激活后，会诱导shp的表达，从而抑制cyp7a1和cyp8b1的转录^[47]。值得一提的是，在本研究中，当fxr被胆汁酸激活后，肝脏中shp表达增加，肝脏中cyp7a1的表达也显著上调，表明肝脏中胆汁酸合成酶活力上升。这可能是因为XR/SHP通路降低CYP7A1的能力较差^[48]，并且存在不依赖于SHP的信号通路调节CYP7A1的表达和BA合成^[49]。此外，胆汁酸的负反馈调节机制主要在产生过量的胆汁酸时发挥作用。本实验中900 mg/kg的胆汁酸水平可能仍在大口黑鲈耐受范围内，因此胆汁酸的合成没有受到影响，具体的影响机制还需要进一步探索。肝脏和肠道中的转运受体可以运输胆汁酸，使胆汁酸在肠道和肝脏中循环^[50]。激活的FXR诱导BSEP的表达，BSEP主要负责将胆汁酸从肝脏分泌到肠道中^[51]。OSTα/β将BA从肝脏运输至血液，而ASBT从肝脏吸收胆汁酸^[52]。在本研究中，饲料中添加胆汁酸可激活fxr，并上调bsep和asbt的表达，表明高脂饲料中补充BA可促进肝脏中的胆汁酸外排。多项体外研究表明，鹅去氧胆酸可增加bsep表达水平，促进胆汁酸转运^[53,54]。综上所述，本实验中，饲料中添加胆汁酸可增加肝脏中胆汁酸合成基因cyp7a1和转运相关基因(bsep和asbt)的表达水平，这与总胆汁酸含量的增高一致，说明饲料中添加外源胆汁酸可能会影响胆汁酸代谢，促进胆汁酸的合成和转运。然而，也有研究表明胆汁酸的添加会降低胆汁酸的合成和转运^[18]，因此关于饲料中添加胆汁酸对鱼类内源胆汁酸代谢的影响还需要进一步研究。

4　结论

本研究表明，HF饲料会损害鱼类生长并增加脂质积累。补充BA可改善大口黑鲈生长性能，并降低高脂饲料下大口黑鲈血浆甘油三酯和胆固醇水平。同时，BA可通过FXR/SHP/SREBP1信号通路抑制脂肪合成基因的转录，并增加脂肪分解和β氧化相关基因的表达。此外，饲料中补充BA可促进胆汁酸合成和转运，避免胆汁酸在肝脏中累积。因此，从鱼类健康生长和脂质积累的角度，建议高脂饲料中胆汁酸添加的适宜水平为600 mg/kg。综上所述，本研究初步探讨胆汁酸对高脂饲料下鱼类脂代谢和胆汁酸代谢的影响以及潜在机制，为研究胆汁酸对鱼类胆汁酸代谢的调控作用奠定基础。

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