甘草总黄酮对高脂条件下罗非鱼肝损伤的保护作用

doi:10.3969/j.issn.1004-1524.2021.10.05

浙江农业学报 ›› 2021, Vol. 33 ›› Issue (10): 1826-1835.DOI: 10.3969/j.issn.1004-1524.2021.10.05

甘草总黄酮对高脂条件下罗非鱼肝损伤的保护作用

杜金梁¹^,²^,³(), 曹丽萍¹^,², 贾睿¹^,², 顾郑琰⁴, 何勤⁴, 徐跑¹^,²^,⁴, JENEYGalina²^,⁵, 马玉忠³^,^*(), 殷国俊¹^,²^,⁴^,^*()

1.中国水产科学研究院淡水渔业研究中心/农业农村部淡水渔业和种质资源利用重点实验室,江苏无锡 214081
2.中国水产科学研究院淡水渔业研究中心/鱼类免疫药理学国际联合实验室,江苏无锡 214081
3.河北农业大学动物医学院,河北保定 071001
4.南京农业大学无锡渔业学院,江苏无锡 214081
5.匈牙利水产与渔业研究所国家农业研究中心,匈牙利索尔沃什 5440

收稿日期:2020-11-18 出版日期:2021-10-25 发布日期:2021-11-02
通讯作者: 马玉忠,殷国俊
作者简介:*马玉忠,E-mail: dkma@hebau.edu.cn;
杜金梁(1982—),男,河北沧州人,博士研究生,研究方向为鱼类免疫学与药理学。E-mail: dujl@ffrc.cn
基金资助:
中央级公益性科研院所基本科研业务费专项(2019JBFM10);江苏省自然科学基金(BK20201143)

Protective effects of Glycyrrhiza total flavones on liver injury of tilapia (Oreochromis niloticus) under high fat condition

DU Jinliang¹^,²^,³(), CAO Liping¹^,², JIA Rui¹^,², GU Zhengyan⁴, HE Qin⁴, XU Pao¹^,²^,⁴, JENEY Galina²^,⁵, MA Yuzhong³^,^*(), YIN Guojun¹^,²^,⁴^,^*()

1. Key Laboratory of Freshwater Fisheries and Germplasm Resources Utilization, Ministry of Agriculture and Rural Affairs/Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi 214081, China
2. International Joint Research Laboratory for Fish Immunopharmacology/Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi 214081, China
3. College of Veterinary Medicine, Hebei Agricultural University, Baoding 071001, China
4. Wuxi Fisheries College, Nanjing Agricultural University, Wuxi 214081, China
5. National Agricultural Research Center, Research Institute for Fisheries and Aquaculture, Szarvas 5440, Hungary

Received:2020-11-18 Online:2021-10-25 Published:2021-11-02
Contact: MA Yuzhong,YIN Guojun

摘要/Abstract

摘要：

为了研究甘草总黄酮对高脂饲料造成的罗非鱼(Oreochromis niloticus)脂肪肝损伤是否具有保护作用,随机将180尾健康无伤罗非鱼分成6组,即对照组、高脂饲料模型组和4个不同浓度甘草总黄酮组(0.05、0.10、0.50、1.00 g·kg^-1甘草总黄酮),每组30尾。饲喂90 d后,采集罗非鱼各实验组肝,测定肝匀浆中超氧化物歧化酶(SOD)活性、还原型谷胱甘肽(GSH)活性、丙二醛(MDA)含量、甘油三酯(TG)含量、总胆固醇(TC)含量、总抗氧化能力(T-AOC)水平,Western blotting法检测肝中核转录因子-κB P65(NF-κB P65)和核转录因子-κB C-Rel (NF-κB C-Rel)蛋白表达水平,RT-PCR法测定肝中微粒体甘油三酸酯转移蛋白(MTTP)和载脂蛋白B100(ApoB100)基因表达情况。结果表明:与对照组相比,高脂饲料模型组罗非鱼肝匀浆中TG、TC、MDA含量极显著(P<0.01)升高,GSH活性、SOD活性、T-AOC水平均极显著(P<0.01)降低;MTTP和ApoB100 mRNA表达水平降低(P<0.01或P<0.05);Western blotting结果显示,NF-κB P 65和C-Rel蛋白表达水平升高。饲料中加入甘草总黄酮后,与高脂饲料模型组相比,罗非鱼肝中GSH活性、SOD活性、T-AOC水平不同程度提高,TC、TG、MDA含量和NF-κB P65、C-Rel蛋白表达水平不同程度降低,MTTP和ApoB100的mRNA表达水平升高,以0.50 g·kg^-1甘草总黄酮组效果较好。说明甘草总黄酮对于罗非鱼脂肪肝损伤具有一定的保护作用,可以缓解高脂饲料造成的脂肪肝损伤。

关键词: 甘草, 总黄酮, 罗非鱼, 脂肪肝, 基因表达, Western blotting

Abstract:

In order to evaluate protective effects of Glycyrrhiza total flavones (GTF) on liver of tilapia (Oreochromis niloticus) after fatty liver injury, 180 healthy and uninjured tilapia were randomly divided into six experimental groups. Namely, control group (CK), high-fat diet (HFD) model group and four different concentrations (0.05, 0.10, 0.50, 1.00 g·kg ^-1) of Glycyrrhiza total flavones treatment groups, each group 30 tails. After 90 days of feeding, liver were collected for determination activities of superoxide dismutase (SOD), reduced glutathione (GSH), contents of malondialdehyde (MDA), triglyceride (TG) and total cholesterol (TC), and level of total antioxidant capacity (T-AOC) in liver homogenate. Western blotting method was used to measure the protein levels of nuclear transcription factor-κB C-rel (NF-κB C-Rel) and nuclear transcription factor-κB P 65 (NF-κB P65) in liver. Quantitative real-time PCR method was used to measure mRNA levels of microsomal triglyceride transfer protein (MTTP) and apolipoprotein B100 (ApoB100). The results showed that: compared with the control group, contents of TG, TC and MDA in tilapia liver homogenate in high-fat diet group significantly increased (P< 0.01), activities of GSH, SOD and T-AOC level in liver homogenate significantly decreased (P<0.01), and mRNA levels of MTTP, ApoB100 down-regulated (P<0.01 or P<0.05), NF-κB P65 and C-rel protein levels increased. After adding Glycyrrhiza total flavones to the diet, activities of GSH, SOD and T-AOC level in liver were increased in different degrees, contents of TC, TG, MDA, and NF-κB C-Rel, NF-κB P65 protein levels were decreased in different degrees, mRNA levels of MTTP and ApoB100 showed an up-regulated trend, and concentration of Glycyrrhiza total flavones at 0.50 g·kg ^-1 showed the best effects. Overall, these results indicated that Glycyrrhiza total flavones had certain effects on tilapia fatty liver and could alleviate the fatty liver injury caused by high-fat diet.

Key words: Glycyrrhiza uralensis Fisch, total flavonoids, tilapia, fatty liver, gene expression, western blotting

中图分类号:

S965.125

杜金梁, 曹丽萍, 贾睿, 顾郑琰, 何勤, 徐跑, JENEYGalina, 马玉忠, 殷国俊. 甘草总黄酮对高脂条件下罗非鱼肝损伤的保护作用[J]. 浙江农业学报, 2021, 33(10): 1826-1835.

DU Jinliang, CAO Liping, JIA Rui, GU Zhengyan, HE Qin, XU Pao, JENEY Galina, MA Yuzhong, YIN Guojun. Protective effects of Glycyrrhiza total flavones on liver injury of tilapia (Oreochromis niloticus) under high fat condition[J]. Acta Agriculturae Zhejiangensis, 2021, 33(10): 1826-1835.

图/表 9

表1 实验组日粮组成

Table 1 Formation of experimental diets

成分 Ingredients/ (g·kg^-1)		鱼粉 Fish meal (CP 53.5%)/ (g·kg^-1)	酪蛋白 Casein/ (g·kg^-1)	明胶 Gelatin/ (g·kg^-1)	玉米淀粉 Corn starch/ (g·kg^-1)	维生素预混料¹⁾ Vitamin premix/ (g·kg^-1)	矿物质预混料²⁾ Mineral premix/ (g·kg^-1)	纤维素 Cellulose/ (g·kg^-1)	羧甲基纤维素钠 Carboxymethylc- ellulose sodium/ (g·kg^-1)
对照组Control group		90	200	70	440.00	10	20	80	30
高脂组High-fat-fed group		90	200	70	282.00	10	20	80	30
不同浓度(g·kg^-1)	0.05	90	200	70	281.95	10	20	80	30
甘草总黄酮组	0.10	90	200	70	281.90	10	20	80	30
Different concentrations	0.50	90	200	70	281.50	10	20	80	30
(g·kg^-1) of Glycyrrhiza	1.00	90	200	70	281.00	10	20	80	30
total flavones groups
成分 Ingredients/ (g·kg^-1)		氯化胆碱 Choline chloride/ (g·kg^-1)	猪油 Lard/ (g·kg^-1)	胆固醇 Choleste- rol/(g· kg^-1)	大豆油 Soybean oil/(g· kg^-1)	甘草总黄酮 GTF/ (g· kg^-1)	粗蛋白质 Crude protein/ %	粗脂肪 Crude lipid/%	灰分 Ash/%
对照组Control group		5	0	0	55	0	28	6.0	2.7
高脂组High-fat-fed group		5	135	5	73	0	28	21.1	2.8
不同浓度(g·kg^-1)	0.05	5	135	5	73	0.05	28	21.1	2.8
甘草总黄酮组	0.10	5	135	5	73	0.10	28	21.1	2.8
Different concentrations	0.50	5	135	5	73	0.50	28	21.1	2.8
(g·kg^-1) of Glycyrrhiza	1.00	5	135	5	73	1.00	28	21.1	2.8
total flavones groups

表2 qRT-PCR引物信息

Table 2 Information of primers used for qRT-PCR

基因 Genes	引物序列 Primer sequence (5'-3')	Genbank登录号 GenBank accession No.
MTTP	F: TGCATGTGGCTTTGCTTCAC R: CTTGACCTGCTCCACGGTAG	XM_005458713.4
ApoB100	F: ACTAGAAGGCAGACCAACCA R: CCTGCCATTTCAACACCCATT	XM_003446376.4
β-actin	F: CCTGAGCGTAAATACTCCGTCTG R: AAGCACTTGCGGTGGACGAT	KJ126772.1

图1 不同处理组罗非鱼肝中总胆固醇、甘油三酯含量 CK,对照组;HFD,高脂组;GTF1、GTF2、GTF3、GTF4分别为0.05、0.10、0.50、1.00 g·kg-1甘草总黄酮处理组。*、**分别表示与高脂组相比在P<0.05和P<0.01水平差异显著。下同。

Fig.1 Contents of total cholesterol and triglyceride in liver of tilapia in different treatments CK, Control group; HFD, high-fat-fed group; GTF1、GTF2、GTF3、GTF4 were 0.05, 0.10, 0.50, 1.00 g·kg-1 Glycyrrhiza total flavones groups, respectively. * and ** meant significant differences at the levels of P<0.05 and P<0.01 compared with the HFD group, respectively. The same as below.

图2 不同处理组罗非鱼肝中还原型谷胱甘肽活性

Fig.2 Activity of reduced glutathione in liver of tilapia in different treatments

图3 不同处理组罗非鱼肝中的超氧化物歧化酶活性

Fig.3 Activity of superoxide dismutase in liver of tilapia in different treatments

图4 不同处理组罗非鱼肝中总抗氧化能力水平

Fig.4 Level of total antioxidant capacity in liver of tilapia in different treatments

图5 不同处理组罗非鱼肝中的丙二醛含量

Fig.5 Contents of malondialdehyde in liver of tilapia in different treatments

图6 不同处理组罗非鱼肝中核转录因子-κB C-rel和P65蛋白的相对表达水平

Fig.6 Relative expression levels of NF-κB C-Rel and NF-κB P65 protein in liver of tilapia in different treatments

图7 不同处理组罗非鱼肝中微粒体甘油三酸酯转移蛋白、载脂蛋白B100的mRNA表达水平

Fig. 7 mRNA levels of microsomal triglyceride transfer protein and apolipoprotein B100 in liver of tilapia in different treatments

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甘草总黄酮对高脂条件下罗非鱼肝损伤的保护作用

Protective effects of Glycyrrhiza total flavones on liver injury of tilapia (Oreochromis niloticus) under high fat condition

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