地塞米松诱导肉鸡脂肪肝模型的构建及效果分析

doi:10.3864/j.issn.0578-1752.2023.20.015

摘要/Abstract

摘要：

【背景】腹脂沉积过多和脂肪肝综合征是家禽养殖中面临的重要脂代谢紊乱产业问题，肝脏是家禽体内从头合成脂肪酸的主要部位，也在物质代谢、胆汁生成、解毒等的调节中发挥重要作用。因此解析脂代谢紊乱机理将为家禽产业的健康发展提供参考依据，但畜禽动物发病机理的探究往往需要模型为前提。【目的】通过试验评价地塞米松诱导肉鸡脂肪肝的效果，为家禽脂代谢紊乱相关模型的构建提供参考。【方法】选用体重相近的35日龄AA肉鸡16只，随机分为对照组和地塞米松组，每组8个重复，地塞米松组连续7 d颈部皮下注射地塞米松磷酸钠溶液（4.5 mg·kg^-1体重），对照组注射同体积的生理盐水，7 d后观测肉鸡肝脏病理性变化并对其进行油红O染色，同时检测血清生化与机体抗氧化指标，采用实时荧光定量PCR方法检测肝脏中与脂质代谢和炎症反应相关的基因表达。【结果】与对照组相比，地塞米松组肉鸡肝脏指数显著提高（P<0.05），油红O切片显示肉鸡肝组织有大量红色脂滴出现，且肝脏中TG和TC含量均高于对照组（P<0.05）。血清生化结果显示地塞米松组谷草转氨酶（AST）、总胆红素（TBIL）、尿酸（UA）、总蛋白（TP）、高密度脂蛋白胆固醇（HDL-c）、低密度脂蛋白胆固醇（LDL-c）、总胆固醇（TC）和甘油三酯（TG）水平显著高于对照组（P<0.05），但谷丙转氨酶（ALT）、葡萄糖（GLU）含量无显著变化（P>0.05）。地塞米松注射显著提高了肉鸡肝脏ACC、FAS、SCD1、PPARγ、ChREBP、SREBP-1c、IGF2、GR等脂质合成相关基因的表达（P<0.05），但ELOVL6基因的表达量无显著变化（P>0.05）;降低了脂质分解代谢相关基因CPT1、LPL、PPARα的mRNA丰度（P<0.05）;但地塞米松组与脂质水解相关的ATGL基因以及与脂质转运相关CETP、MTTP基因表达量显著高于对照组（P<0.05）;除此之外，地塞米松注射显著提高了肉鸡肝脏MyD88、NFκB、IL-6、TNF-α等与炎症发生相关的基因表达（P<0.05）。另一方面，抗氧化的检测结果显示肉鸡肝脏和血清的总抗氧化能力无显著变化（P>0.05），但地塞米松组血清中的丙二醛含量显著高于对照组（P<0.05）。【结论】颈部皮下注射地塞米松可以诱导肉鸡产生脂代谢紊乱，并引发机体氧化应激和肝脏炎症反应，与脂肪肝疾病特征相似，可用来较快速地建立肉鸡脂肪肝模型。

关键词: 地塞米松, 肉鸡, 脂肪肝综合征

Abstract:

【Background】Excessive abdominal fat deposition and fatty liver syndrome are important industrial problems of lipid metabolism disorder in poultry breeding. The liver is the main site of de novo fatty acid synthesis in poultry, which plays a vital function in nutrients metabolism, formation of bile acids and detoxication. Therefore, the mechanism analysis of lipid metabolism disorder will provide reference for the healthy development of poultry industry, but the disease model is necessary for pathogenesis research. 【Objective】This experiment was conducted to evaluate the effect of dexamethasone-induced fatty liver in broilers, aiming to provide the reference for the construction of lipid metabolism disorder model in poultry. 【Method】Sixteen 35-day-old AA broilers with similar body weight were randomly divided into control group and dexamethasone group (DXM), with 8 replicates in each group. The birds in the DXM were injected subcutaneously with dexamethasone sodium phosphate (4.5 mg·kg^-1) for 7 days, and the birds in the control group were given the administration of normal saline injection. After 7 days, oil red O staining was used to analyze the histopathological changes of the liver, and serum biochemical and antioxidant indexes were detected. RT-PCR was applied to detect genes expression related to lipid metabolism and inflammatory response in the liver. 【Result】Compared with the control group, the liver index of broilers in the DXM was significantly increased (P<0.05). Oil red O sections showed a large number of red lipid droplets, and the contents of TG and TC in the liver were higher than those in the control group (P<0.05). Serum biochemical results showed that aspartate amino transferase (AST), total bilirubin (TBIL), uric acid (UA), total protein (TP), high density lipoprotein cholesterol (HDL-c), low density lipoprotein cholesterol (LDL-c), total cholesterol (TC) and triglyceride (TG) levels in the DXM were significantly higher than those in the control group (P<0.05), but there was no significant difference about alanine aminotransferase (ALT) and glucose (GLU) (P>0.05). Dexamethasone injection significantly increased genes expression about lipid synthesis, such as ACC, FAS, SCD1, PPARγ, ChREBP, SREBP-1c, IGF2 and GR in the liver (P<0.05), but ELOVL6 expression was not affected (P>0.05). In addition, genes expression about lipid catabolism such as CPT1, LPL and PPARα were deceased in DXM group (P<0.05). However, the expression of ATGL gene related to lipid hydrolysis, CETP and MTTP gene related to lipid transport were significantly higher in DXM group (P<0.05). On the other hand, DXM injection significantly increased the genes expression of MyD88, NFκB, IL-6, TNF-α in the liver of broilers (P<0.05). Antioxidant analysis showed that there was no significant change in the total antioxidant capacity of liver and serum (P>0.05), but the content of malondialdehyde in serum was significantly higher in DXM group (P<0.05). 【Conclusion】Neck subcutaneous injection of DXM could cause lipid metabolism disorders in broilers, and lead to oxidative stress and liver inflammation, which was similar to the characteristics of fatty liver disease, indicating that this method could be used to establish the fatty liver model in broilers quickly.

Key words: dexamethasone, broiler, fatty liver syndrome

王超慧, 孙喜, 王强刚, 刘芮兵, 李妮, 杨小军, 刘艳利. 地塞米松诱导肉鸡脂肪肝模型的构建及效果分析[J]. 中国农业科学, 2023, 56(20): 4115-4124.

WANG ChaoHui, SUN Xi, WANG QiangGang, LIU RuiBing, LI Ni, YANG XiaoJun, LIU YanLi. Fatty Liver Model Construction and Its Effectiveness Evaluation Induced by Dexamethasone in Broilers[J]. Scientia Agricultura Sinica, 2023, 56(20): 4115-4124.

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参考文献 33

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引物 Primer	登录号 Accession number	引物序列 Primer sequences (5′ - 3′)	产物大小 Product size(bp)
β-actin	L_08165	F:ATTGTCCACCGCAAATGCTTC R:AAATAAAGCCATGCCAATCTCGTC	113
ACC	XM_046929960	F:GCTTCCCATTTGCCGTCCTA R:GCCATTCTCACCACCTGATTACTG	185
FAS	NM_205155	F:TTTGGTGGTTCGAGGTGGTA R:CAAAGGTTGTATTTCGGGAGC	212
SCD1	NM_204890	F:GTTTCCACAACTACCACCATACATT R:CCATCTCCAGTCCGCATTTT	175
PPARγ	NM_001001460	F:CCAAGGCAGCGGCAAAATAA R:GTGCCCATAAATGATGGCCTAA	188
ELOVL6	XM_046916529	F:GGTGGTCGGCACCTAATGAA R:TCTGGTCACACACTGACTGC	169
ChREBP	EU_152408	F: ATTGACCCGACCCTGACG R: CATACTGGATGTACCACGCTCT	160
SREBP1c	XM_046927256	F: GCCCTCTGTGCCTTTGTCTTC R: ACTCAGCCATGATGCTTCTTC	130
IGF2	NM_001030342	F: ATTGTCCACCGCAAATGCTTC R: AAATAAAGCCATGCCAATCTCGTC	131
GR	NM_001037826	F: TCCTGTGCTTTGCACCAGAT R: GATGGCCTTTCCCAGCTCTT	245
ATGL	NM_001113291	F: TCCTAGGGGCCTACCACATC R: CCAGGAACCTCTTTCGTGCT	195
CETP	NM_001034814	F: AGTCTCGCCCTTCCTGAGAT R: GCAGCTTGGATAGTGACCGT	149
CPT1	XM_046918285	F: TAGAGGGCGTGGACCAATAA R: TGGGATGCGGGAGGTATT	229
LPL	NM_205282	F: CCGATCCCGAAGCTGAGATG R: ACATTCCTGTCACCGTCCAC	186
MTTP	NM_001109784	F: GCAGATGGACAGAGTTGGCT R: ACACCAAAAGTGCAAGGTGC	224
PPARα	XM_046906400	F: TTTAACGGAGTTCCAATCGC R: AACCCTTACAACCTTCACAAGC	224
IL-6	NM_204628	F: CTCGTCCGGAACAACCTCAA R: GGAGAGCTTCGTCAGGCATT	96
MyD88	NM_001030962	F: CGTGCCAAAGACTTCAGAGC R: CTGGCAAGACATCCCGATCA	191
NFκB	NM_001396038	F: CAGCCCATCTATGACAACCG R: CAGCCCAGAAACGAACCTC	151
TNFα	XM_046927265	F: TGTGTATGTGCAGCAACCCGTAGT R: GGCATTGCAATTTGGACAGAAGT	229

项目 Item	对照组 Control	地塞米松组Dexamethasone	P值 P-value
谷丙转氨酶ALT（U·L^-1）	0.86±0.34	2.00±0.53	0.096
谷草转氨酶AST（U·L^-1）	317.33±14.72	466.43±52.36*	0.029
总胆红素TBIL（µmol·L^-1）	20.13±1.06	34.14±2.45**	<0.001
尿酸UA（µmol·L^-1）	318.43±37.52	532.86±48.32**	0.004
血清总蛋白TP（g·L^-1）	30.44±1.42	45.00±1.30**	<0.001
血清葡萄糖GLU（mmol·L^-1）	12.11±0.25	15.04±1.29	0.063
高密度脂蛋白胆固醇HDL-c（mmol·L^-1）	1.71±0.09	4.42±0.21**	<0.001
低密度脂蛋白胆固醇LDL-c（mmol·L^-1）	0.48±0.26	1.53±0.14**	<0.001
总胆固醇TC（mmol·L^-1）	2.75±0.14	7.93±0.34**	<0.001
甘油三酯TG（mmol·L^-1）	0.65±0.05	1.54±0.28*	0.018