柠条单宁通过Toll样受体4/核因子-κB信号通路缓解高精料育肥羔羊肝脏炎性损伤

刘沛楠; 牛晓雨; 母晓佳; 陈治宇; 李慧; 邢媛媛; 李大彪

doi:10.12418/CJAN2025.575

动物营养学报 >

2025 , Vol. 37 >Issue 10: 7062 - 7073

DOI: https://doi.org/10.12418/CJAN2025.575

研究论文

柠条单宁通过Toll样受体4/核因子-κB信号通路缓解高精料育肥羔羊肝脏炎性损伤

刘沛楠 ,
牛晓雨 ,
母晓佳 ,
陈治宇 ,
李慧 ,
邢媛媛 ,
李大彪 ^,^*

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内蒙古农业大学动物科学学院,内蒙古自治区高校动物营养与饲料科学重点实验室,呼和浩特 010018

^*李大彪,教授,博士生导师,E-mail: dkyldb@imau.edu.cn

刘沛楠(2000—),女,内蒙古呼和浩特人,硕士研究生,研究方向为动物营养与饲料科学。E-mail: 1789251937@qq.com

Office editor: 陈鑫

收稿日期: 2025-03-06

网络出版日期: 2025-10-15

基金资助

蒙古自治区科技重大专项项目(2021ZD0024-4)

内蒙古自治区自然科学基金项目(2023MS03023)

收起

Caragana korshinskii Tannin Alleviates Liver Inflammation Injury in High-Concentrate Fattening Lambs through Toll-Like Receptor 4/Nuclear Factor-κB Signaling Pathway

LIU Peinan ,
NIU Xiaoyu ,
MU Xiaojia ,
CHEN Zhiyu ,
LI Hui ,
XING Yuanyuan ,
LI Dabiao ^,^*

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Key Laboratory of Animal Nutrition and Feed Science at Universities of Inner Mongolia Autonomous Region, College of Animal Science, Inner Mongolia Agricultural University, Hohhot 010018, China

^*professor, E-mail: dkyldb@imau.edu.cn

Received date: 2025-03-06

Online published: 2025-10-15

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摘要

本试验旨在研究柠条单宁(CKT)通过Toll样受体4/核因子-κB(TLR4/NF-κB)信号通路缓解高精料育肥羔羊肝脏炎性损伤。试验选取24只3月龄杜蒙杂交公羔,随机分为3组,对照组(CON组)饲喂精粗比30∶70的基础饲粮,高精料组(HC组)饲喂精粗比70∶30的高精料饲粮,CKT组在高精料饲粮的基础上添加2 g/kg CKT,每组8只羊。预试期15 d,正试期60 d。结果表明:1)与CON组相比,HC组和CKT组羔羊末重(FBW)、平均日增重(ADG)显著升高,且料重比(F/G)显著降低(P<0.05)。2)与CON组相比,HC组羔羊血清谷丙转氨酶(ALT)、谷草转氨酶(AST)活性显著升高(P<0.05);与HC组相比,CKT组羔羊血清总蛋白(TP)、白蛋白(ALB)含量显著升高(P<0.05)。3) CON组肝脏细胞细胞质完好,未发现炎症和坏死的现象;HC组肝脏组织出现严重损伤,肝细胞坏死、细胞肿胀以及细胞膜结构被破坏。与HC组相比,CKT组肝脏组织细胞状态得到显著改善。4)与CON组相比,HC组肝脏组织白细胞介素-1β(IL-1β)和脂多糖(LPS)含量显著升高(P<0.05);与HC组相比,CKT组肝脏组织白细胞介素-6(IL-6)、IL-1β和LPS含量显著降低(P<0.05)。5)与CON组相比,HC组肝脏组织IL-1β、肿瘤坏死因子-α(TNF-α)、TLR4、骨髓分化因子88(MyD88)和核转录因子-κB p65(NF-κB p65)的mRNA相对表达量显著升高(P<0.05)。与HC组相比,CKT组肝脏组织TNF-α、IL-1β、MyD88和TLR4的mRNA相对表达量显著降低(P<0.05)。6)与CON组相比,HC组肝脏组织IL-6、TNF-α、IL-1β、MyD88、下游核因子-κB抑制因子α(IκB-α)、NF-κB p65、NF-κB p50和TLR4蛋白表达量显著升高(P<0.05);与HC组相比,CKT组肝脏组织IL-6、IL-1β、MyD88、NF-κB p50蛋白表达量显著降低(P<0.05)。综上所述,在羔羊饲粮中添加2 g/kg CKT可通过下调TLR4/NF-κB信号通路相关基因与蛋白表达,抑制TLR4介导的下游炎症因子转录与蛋白合成,缓解因高精料育肥导致的羔羊肝脏炎性损伤。

关键词： 柠条单宁; 高精料饲粮; 羔羊; 肝脏; 炎性损伤

本文引用格式

刘沛楠 , 牛晓雨 , 母晓佳 , 陈治宇 , 李慧 , 邢媛媛 , 李大彪 . 柠条单宁通过Toll样受体4/核因子-κB信号通路缓解高精料育肥羔羊肝脏炎性损伤[J]. 动物营养学报, 2025 , 37(10) : 7062 -7073 . DOI: 10.12418/CJAN2025.575

Abstract

This experiment aimed to investigate whether Caragana korshinskii tannin (CKT) alleviates liver inflammatory damage in lambs fattened with high-concentrate diets through the Toll-like receptor 4/nuclear factor-κB (TLR4/NF-κB) signaling pathway. A total of 24 three-month-old Duolang crossbred ram lambs were randomly divided into 3 groups: the control group (CON group) fed a basal diet with a concentrate-to-forage ratio of 30∶70; the high-concentrate group (HC group) fed a high-concentrate diet with a concentrate-to-forage ratio of 70∶30; and the CKT group fed the high-concentrate diet supplemented with 2 g/kg CKT, with 8 lambs in each group. The pre-test period lasted 15 days, and the formal test period lasted 60 days. The results showed that: 1) compared with the CON group, the final body weight (FBW) and average daily gain (ADG) of lambs in the HC and CKT groups were significantly increased (P<0.05), while the feed-to-gain ratio (F/G) was significantly decreased (P<0.05). 2) Compared with the CON group, the activities of serum alanine transaminase (ALT) and aspartate transaminase (AST) in the HC group were significantly increased (P<0.05); compared with the HC group, the contents of serum total protein (TP) and albumin (ALB) in the CKT group were significantly increased (P<0.05). 3) The hepatocyte cytoplasm in the CON group was intact, with no signs of inflammation or necrosis; the liver tissue in the HC group showed severe damage, including hepatocyte necrosis, cell swelling, and destruction of cell membrane structure. Compared with the HC group, the state of liver tissue cells in the CKT group was significantly improved. 4) Compared with the CON group, the contents of interleukin-1β (IL-1β) and lipopolysaccharide (LPS) in the liver tissue of the HC group were significantly increased (P<0.05); compared with the HC group, the contents of interleukin-6 (IL-6), IL-1β, and LPS in the liver tissue of the CKT group were significantly decreased (P<0.05). 5) Compared with the CON group, the mRNA relative expression levels of IL-1β, tumor necrosis factor-α (TNF-α), TLR4, myeloid differentiation factor 88 (MyD88), and nuclear factor-κB p65 (NF-κB p65) in the liver tissue of the HC group were significantly increased (P<0.05). Compared with the HC group, the mRNA relative expression levels of TNF-α, IL-1β, MyD88, and TLR4 in the liver tissue of the CKT group were significantly decreased (P<0.05). 6) Compared with the CON group, the protein expression levels of IL-6, TNF-α, IL-1β, MyD88, downstream nuclear factor κB inhibitor-α (IκB-α), NF-κB p65, NF-κB p50, and TLR4 in the liver tissue of the HC group were significantly increased (P<0.05); compared with the HC group, the protein expression levels of IL-6, IL-1β, MyD88, and NF-κB p50 in the liver tissue of the CKT group were significantly decreased (P<0.05). In conclusion, supplementation of 2 g/kg CKT in lamb diets can downregulate the expression of genes and proteins related to the TLR4/NF-κB signaling pathway, inhibit TLR4-mediated downstream inflammatory factor transcription and protein synthesis, and alleviate hepatic inflammatory damage in lambs caused by high-concentrate fattening.

Key words： Caragana korshinskii tannins; high concentrate diet; lambs; liver; inflammatory injury

在畜牧业集约化发展的背景下,为满足羔羊快速生长的能量需求,加之中国优质粗饲料资源短缺等问题,养殖者常采用高精料饲粮的饲养策略。然而,羔羊长期采食高精料饲粮,会致使机体免疫力下降和代谢异常^[1],增加机体对亚急性瘤胃酸中毒和肝脏衰竭等疾病的易感性^[2-3],进而影响生产性能和养殖者效益。此外,过量的过瘤胃淀粉会导致肠道内可挥发性脂肪酸和乳酸含量升高,pH降低,革兰氏阴性菌大量崩解,释放脂多糖(LPS)和肽聚糖等分解产物。LPS能够渗透受损的胃肠道上皮屏障,与组胺等异常代谢产物共同进入血液循环,经由肝门静脉到达肝脏,诱导肝脏枯否细胞释放促炎细胞因子,引发炎症级联反应^[4]。作为反刍动物中最大的代谢器官,肝脏在抗氧化、免疫调节和糖、脂代谢等方面扮演着极为重要的角色^[5]。过量LPS在体内蓄积时,肝脏易遭受到LPS的攻击,从而导致结构受损^[6]。研究表明,Toll样受体4(TLR4)是LPS的特异性识别受体,与LPS结合后活化,将信号传递到细胞内,活化TLR4信号通路关键的下游蛋白——骨髓分化因子88(MyD88),激活核因子-κB (NF-κB)信号通路,启动下游一系列靶基因的转录表达及炎性因子的释放,从而调节免疫应答和炎症反应^[7]。

多酚类物质能够降低机体应激引发的肝脏损伤,调节肝脏免疫功能,使肝脏代谢维持稳定^[8-9]。柠条作为一种非常规饲料资源,广泛分布于中国西北干旱和半干旱地区,富含丰富的营养成分,具有抗氧化、抗菌、抗炎等生物活性^[10]。单宁是锦鸡儿属植物体内重要的次级代谢产物,属多酚类物质,常见于植物的根、茎、皮、叶和果实。柠条单宁(CKT)作为柠条中的主要活性成分,主要成分为儿茶素类缩合单宁^[11]。研究证实,富含儿茶素结构的多酚类物质能通过抑制TLR4信号通路减少炎性细胞因子产生^[12],并且能通过减少活性氧的产生,抑制TLR4蛋白表达发挥抗炎作用^[13]。现有研究表明,CKT能通过调控反刍动物瘤胃菌群,促进瘤胃发酵及营养物质消化吸收,增强机体抗氧化和免疫能力^[14]。且本课题组前期研究发现,给小鼠灌胃100 mg/mL的CKT可以改善LPS诱导的肝脏损伤,增强小鼠血清抗氧化酶活性以及免疫相关因子含量,提高小鼠的免疫与抗氧化能力^[15]。因此,本试验在前期研究的基础上,以高精料育肥羔羊为试验动物,探究CKT对高精料育肥羔羊肝脏炎症损伤的影响及其机制,为羔羊健康养殖及畜牧业绿色可持续发展提供理论依据。

1 材料与方法

1.1 试验设计与动物管理

本试验按照《实验动物福利伦理审查指南》(GB/T 35892—2018)开展动物试验。

选取体重相近的杜蒙杂交公羔24只,随机分为3组,每组8只。对照组(CON组)饲喂精粗比为30∶70的基础饲粮,高精料组(HC组)饲喂精粗比为70∶30的高精料饲粮,CKT组在高精料饲粮的基础上添加2 g/kg CKT。试验羊只单栏饲养,每日早、晚各投料1次,自由采食、饮水。预试期15 d,正试期60 d。CKT含量为27.3%^[15],CKT的单宁单体摩尔比如表1所示。基础饲粮根据《肉羊营养需要量》(NY/T 816—2021)^[16]配制,饲粮为颗粒全混合日粮。基础饲粮组成及营养水平如表2所示。

表1 CKT的单宁单体摩尔比

Table 1 Tannin monomer molar ratio of CKT %

项目 Items	摩尔比 Molar ratio
表没食子儿茶素Epigallocatechin	6.01
儿茶素Catechin	53.37
表没食子儿茶素没食子酸酯 Epigallocatechin gallate	15.94
表儿茶素Epicatechin	9.30
没食子儿茶素Gallocatechin	11.57
表儿茶素-3-O-没食子酸酯 Epicatechin-3-O-gallate	2.97
儿茶素没食子酸酯Catechin gallate	0.84

表2 基础饲粮组成及营养水平(干物质基础)

Table 2 Composition and nutrient levels of basal diets (DM basis) %

项目 Items	对照组 CON group	高精料组 HC group
原料Ingredients
燕麦草Oat grass	31.00
苜蓿Alfalfa	29.00
向日葵壳Sunflower husk	10.00	30.00
玉米Corn	15.00	39.00
玉米皮Corn husk	2.00	8.00
大豆粕Soybean meal	7.50	13.50
菜籽粕Canola meal	3.00	6.00
石粉Limestone	0.00	1.00
磷酸氢钙CaHPO₄	1.00	1.00
食盐NaCl	0.50	0.50
预混料Premix¹⁾	1.00	1.00
合计Total	100.00	100.00
营养水平Nutrient levels²⁾
代谢能ME/(MJ/kg)	7.86	10.04
粗蛋白质CP	15.47	16.57
粗脂肪EE	4.96	6.66
中性洗涤纤维NDF	46.90	30.57
酸性洗涤纤维ADF	28.57	16.75
钙Ca	0.89	1.08
磷P	0.71	0.75

1)预混料为每千克饲粮提供 The premix provided the following per kg of diets: Se 0.30 mg, Cu 25.00 mg, Fe 55.00 mg, I 5.00 mg, Mn 25.00 mg, S 60.00 mg, Zn 16.00 mg, VA 18 00 IU, VD 3 000 IU, VE 576 IU。2)代谢能为计算值^[17],其余为实测值。ME was a calculated value^[17], while the others were measured values.

1.2 样品采集

正试期结束后,使用10 mL无抗凝采血管对试验羔羊进行颈静脉采血,倾斜静置20 min后离心20 min,将血清转移至2 mL离心管中,置于-20 ℃保存,用于血清生化指标的测定。每组选取体重相近的6只羔羊进行屠宰,分离肝脏组织,剪取1 cm×1 cm×1 cm大小的肝脏组织于4%多聚甲醛中固定,用于制作石蜡切片。最后,剪取肝脏组织于2 mL冻存管中,然后放入液氮中,用以炎症相关指标的测定。样品采集结束后转入-80 ℃中保存。

1.3 测定指标与方法

1.3.1 生长性能测定

在正试期第1和60天,对所有羔羊进行称重,记录初重(IBW)、末重(FBW),试验期间记录羔羊每日采食量,正试期结束后计算平均日增重(ADG)、平均日采食量(ADFI)和料重比(F/G)。

1.3.2 饲粮营养水平测定

饲粮中的粗蛋白质(CP)含量参照GB/T 6432—2018的方法测定,干物质(DM)含量参照GB/T 6435—2014的方法测定,粗脂肪(EE)含量参照GB/T 6433—2006的方法测定,中性洗涤纤维(NDF)含量参照GB/T 20806—2022的方法测定,酸性洗涤纤维(ADF)含量参照NY/T 1459—2022的方法测定,钙(Ca)含量参照GB/T 6436—2018的方法测定,磷(P)含量参照GB/T 6437—2018的方法测定。

1.3.3 血清生化指标测定

血清生化指标采用日立全自动生化分析仪(3100,日立公司,日本)检测总蛋白(TP)、白蛋白(ALB)含量及谷丙转氨酶(ALT)、谷草转氨酶(AST)活性。

1.3.4 肝脏组织形态学分析

将组织样本在4%的多聚甲醛溶液中固定,随后进行样本处理、洗涤和脱水,切割成5 μm厚的切片,采用苏木精-伊红(HE)染色技术,利用光学显微镜对肝脏组织细胞进行观察与分析,并在适当的视野下拍摄图像。

1.3.5 肝脏炎症及相关指标测定

肝脏组织中白细胞介素-1β(IL-1β)、白细胞介素-4(IL-4)、白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)和白细胞介素-10(IL-10)含量采用试剂盒测定,所用试剂盒购自南京建成生物工程研究所。肝脏中LPS含量采用显色基质鲎试剂盒测定,所用试剂盒购自厦门鲎试剂生物科技股份有限公司。

1.3.6 肝脏炎症相关基因表达量测定

肝脏炎症相关基因IL-1β、IL-4、IL-6、IL-10、TNF-α、TLR4、MyD88及NF-κB p65表达的检测按照Trizol试剂盒的说明书进行,首先是RNA提取,并通过酶标仪检测总RNA的纯度和浓度,260 nm/280 nm值在1.8~2.0且浓度较高的RNA样品用于后续试验。按照试剂盒说明书进行反转录,合成的cDNA置于-80 ℃保存,进行实时荧光定量PCR分析。该程序为95 ℃预变性循环30 s,然后95 ℃变性5 s,60 ℃延伸30 s,40个循环,最后95 ℃循环5 s,60 ℃退火循环1 min。采用β-肌动蛋白(β-actin)为内参,20 μL反应体系:TB Green 10 μL, ddH₂O 6 μL,上、下游引物各1 μL, cDNA 2 μL。上述免疫相关基因均采用Primer express 5.0引物设计,通过GenBank中Blast检测引物特异性,引物序列见表3。采用2^-△△Ct法计算肝脏组织免疫相关基因的mRNA相对表达量。

表3 引物序列

Table 3 Primer sequences

基因 Genes	引物序列 Primer sequences (5'—3')	GenBank登录号 GenBank accession No.	退火温度 Annealing temperature/℃	长度 Length/bp
白细胞介素-1β IL-1β	F: ACAGATGAAGAGCTGCACCC R: AGACATGTTCGTAGGCACGG	NM_001009465.2	60.0	161
白细胞介素-4 IL-4	F: GTACCAGCCACTTCGTCCAT R: GTGGCTCCTGTAGATACGCC	NM_001009313.3	60.0	200
白细胞介素-6 IL-6	F: CAGTCCTCAAACGAGTGGGT R: TCAGGCTGAACTGCAGGAAAT	NM_001009392.1	60.0	82
白细胞介素-10 IL-10	F: TGCGAAAACAAGAGCAAGGC R: CCCCTCTCTTGGAGCATATTGA	NM_001009327.1	60.0	65
肿瘤坏死因子-α TNF-α	F: CGTTGTAGCCAACATCAGCG R: TGGTAGGAGACTGCAATGCG	NM_001024860.1	60.0	219
Toll样受体4 TLR4	F: GGGTGCGGAATGAACTGGTA R: CTGGGACACCACGACAATCA	NM_001135930.1	60.0	158
髓样分化因子88 MyD88	F: ATTGAGAAGAGGTGCCGTCG R: ACAGACAGTGATGAAGCGCA	NM_001166183.1	60.0	189
核因子-κB p65 NF-κB p65	F: CAGACGAAGGTGGTGAGCTT R: GAGTCTCCATGCCGATCCAG	XM_042238744.1	60.0	71
β-肌动蛋白β-actin	F: TGAGGAGCACCCTGTG R: GTCTCAAACATGATCTGGGT	NM_007393.5	60.0	84

1.3.7 蛋白质免疫印记分析

称取20 mg肝脏组织充分匀浆,12 789×g离心15 min,收集上清液,采用BCA法检测样品蛋白浓度,完成蛋白定量分析。加入等量4×蛋白上样缓冲液后,沸水浴变性10 min。预配制10%分离胶体系(十二烷基硫酸钠-聚丙烯酰胺凝胶),点样后电泳2 h至蛋白分离,转至0.45 μm聚偏氟乙烯(PVDF)膜上30 min,快速封闭液封闭PVDF膜2 h,用Tris缓冲盐溶液(TBST)洗涤5 min,重复3次。对TBST洗涤好的PVDF膜在稀释的IL-6(1∶1 000)、IL-1β(1∶1 000)、TNF-α(1∶1 000)、TLR4(1∶1 000)、NF-κB p65(1∶1 000)、NF-κB p50(1∶1 000)、核因子-κB 抑制蛋白激酶(IKK)(1∶1 000)、核因子-κB 抑制蛋白α(IκBα)(1∶1 000)、MyD88(1∶1 000)及β-actin(1∶10 000)抗体4 ℃孵育过夜。隔天取出,用TBST洗涤5 min,重复3次。室温避光条件下,孵育二抗2 h,再次TBST洗涤5 min,重复3~5次。将经过ECL化学发光液处理的膜在化学发光仪进行曝光,用Image J 1.8.0进行蛋白条带灰度分析。

1.4 数据统计与分析

采用SAS 9.2软件中one-way ANOVA程序进行方差分析,采用Duncan氏法进行多重比较,P<0.05表示差异显著,P>0.05表示差异不显著。

2 结果

2.1 CKT对高精料育肥羔羊生长性能的影响

由表4可知,与CON组相比,HC组羔羊FBW、ADG显著升高(P<0.05),F/G显著降低(P<0.05)。与HC组相比,CKT组羔羊IBW、FBW、ADG、ADFI、F/G均无显著差异(P>0.05)。

表4 CKT对高精料育肥羔羊生长性能的影响

Table 4 Effects of CKT on growth performance of high concentrate fattening lambs

项目 Items	组别Groups			SEM	P值 P-value
项目 Items	CON	HC	CKT	SEM	P值 P-value
始重IBW/kg	31.31	30.23	30.18	0.542	0.279
末重FBW/kg	39.28^b	41.80^a	41.82^a	0.693	0.047
平均日增重ADG/(kg/d)	0.13^b	0.21^a	0.19^a	0.011	0.004
平均日采食量ADFI/(kg/d)	1.40	1.40	1.39	0.013	0.065
料重比F/G	9.34^a	6.78^b	7.24^b	0.459	0.038

	同行数据肩标无字母或相同字母表示差异不显著(P>0.05),不同小写字母表示差异显著(P<0.05)。下表同。
	In the same row, values with no letter or the same letter superscripts mean no significant difference (P>0.05), while with different small letter superscripts mean significant difference (P<0.05). The same as below.

2.2 CKT对高精料育肥羔羊肝脏组织形态结构的影响

由HE染色结果可知,CON组羔羊肝脏组织内肝小叶结构清晰,肝细胞索排列整齐,呈多边形肝细胞。HC组可见大量肝细胞水肿,细胞体积增大、胞质疏松淡染,汇管区周围存在淋巴细胞小灶性浸润。CKT组可见部分肝细胞体积肿大、轻微空泡化,但未见明显的炎性细胞浸润,改善效果显著(图1)。

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图1 CKT对高精料育肥羔羊肝脏组织形态结构的影响

蓝色箭头:肝细胞水肿;橙色箭头:淋巴细胞浸润;棕色箭头:炎性细胞。

Fig.1 Effects of CKT on liver morphological structure of high concentrate fattening lambs (400×)

Blue arrow: hepatocyte edema; orange arrow: lymphocyte infiltration; brown arrows: inflammatory cells.

2.3 CKT对高精料育肥羔羊血清生化指标的影响

由表5可知,与CON组相比,HC组羔羊血清AST、ALT活性显著升高(P<0.05);与HC组相比,CKT组血清TP、ALB含量显著升高(P<0.05)。

表5 CKT对高精料育肥羔羊血清生化指标的影响

Table 5 Effects of CKT on serum biochemical indexes of high concentrate fattening lambs (n=6)

项目 Items	组别Groups			SEM	P值 P-value
项目 Items	CON	HC	CKT	SEM	P值 P-value
总蛋白TP/(g/L)	62.65^c	65.50^b	68.65^a	0.560	<0.001
白蛋白ALB/(g/L)	35.72^b	36.48^b	39.40^a	0.581	0.001
谷草转氨酶AST/(U/L)	112.57^b	119.01^a	92.52^c	1.938	<0.001
谷丙转氨酶ALT/(U/L)	17.00^b	19.56^a	16.76^b	0.502	0.002

2.4 CKT对高精料育肥羔羊肝脏炎症指标的影响

由表6可知,与CON相比,HC组羔羊肝脏组织中IL-1β和LPS含量显著升高(P<0.05),IL-10含量显著降低(P<0.05);与HC组相比,CKT组肝脏组织中IL-6、IL-1β和LPS含量显著降低(P<0.05),且肝脏组织中IL-4和IL-10含量显著升高(P<0.05)。各组间羔羊肝脏组织中TNF-α含量均无显著差异(P>0.05)。

表6 CKT对高精料育肥羔羊肝脏炎症指标的影响

Table 6 Effects of CKT on inflammatory factors in liver of high concentrate fattening lambs (n=6)

项目 Items	组别Groups			SEM	P值 P-value
项目 Items	CON	HC	CKT	SEM	P值 P-value
白细胞介素-4 IL-4/(μg/g prot)	25.53^ab	23.73^b	29.17^a	0.990	0.031
白细胞介素-6 IL-6/(μg/g prot)	24.43^a	24.05^a	20.23^b	0.736	0.042
白细胞介素-10 IL-10/(μg/g prot)	35.84^a	23.65^b	39.37^a	1.906	0.014
白细胞介素-1β IL-1β/(μg/g prot)	8.56^b	10.90^a	6.68^c	0.277	<0.001
肿瘤坏死因子-α TNF-α/(μg/g prot)	54.80	55.53	51.56	1.374	0.369
脂多糖LPS/(ng/mg prot)	3.21^b	4.08^a	3.27^b	0.162	0.031

2.5 CKT对高精料育肥羔羊肝脏TLR4/NF-κB信号通路中相关因子基因表达的影响

由图2可知,HC组羔羊肝脏组织TNF-α、TLR4、MyD88和NF-κB p65的mRNA相对表达量显著高于CON组(P<0.05)。CKT组羔羊肝脏组织TNF-α、IL-1β、TLR4和MyD88的mRNA相对表达量显著低于HC组(P<0.05)。各组间羔羊肝脏组织IL-6的mRNA相对表达量无显著差异(P>0.05)。

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图2 CKT对高精料育肥羔羊肝脏TLR4/NF-κB信号通路中相关因子基因表达的影响

TNF-α:肿瘤坏死因子-α tumor necrosis factor-α;IL-6:白细胞介素-6 interleukin-6;IL-1β:白细胞介素-1β interleukin-1β;TLR4:Toll样受体4 Toll-like receptor 4;MyD88:髓样分化因子88 myeloid differentiation factor 88;NF-κB p65:核因子-κB p65 nuclear factor-κB p65。数据柱标注相同小写字母表示差异不显著(P>0.05),不同小写字母表示差异显著(P<0.05)。下图同。Value columns with the same small letter mean no significant difference(P>0.05), while with different small letters mean significant difference(P<0.05). The same as below.

Fig. 2 Effects of CKT on gene expression of related factors in TLR4/NF-κB signaling pathway in liver of high concentrate fattening lambs

2.6 CKT对高精料育肥羔羊肝脏TLR4/NF-κB信号通路中相关因子蛋白表达的影响

如图3所示,与CON组相比,HC组羔羊肝脏组织IL-6、TNF-α、IL-1β、MyD88、IκB-α、NF-κB p50、 NF-κB p65、IKK、TLR4蛋白表达量显著上调(P<0.05);与HC组相比,CKT组肝脏组织IL-6、IL-1β、MyD88、IκB-α和NF-κB p50蛋白表达量显著下调(P<0.05)。

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图3 CKT对高精料育肥羔羊肝脏TLR4/NF-κB信号通路中相关蛋白表达的影响

IL-6:白细胞介素-6 interleukin-6;TNF-α:肿瘤坏死因子-α tumor necrosis factor-α;IL-1β:白细胞介素-1β interleukin-1β;MyD88:髓样分化因子88 myeloid differentiation factor 88;IκB-α:核因子-κB抑制因子α nuclear factor-κB inhibitor α;NF-κB p50:核因子-κB p50 nuclear factor-κB p50;NF-κB p65:核因子-κB p65 nuclear factor-κB p65;IKK:核因子-κB 抑制蛋白激酶 nuclear factor-κB inhibits protein kinase;TLR4:Toll样受体4 Toll-like receptor 4。A:肝脏TLR4/NF-κB信号通路中相关蛋白的蛋白质免疫印迹图;B:肝脏TLR4/NF-κB信号通路中蛋白相对表达量。A: Western blot of proteins involved in the TLR4/NF-κB signaling pathway in the liver; B: Relative protein expression in the TLR4/NF-κB signaling pathway in liver.

Fig. 3 Effects of CKT on protein expression of related factors in TLR4/NF-κB signaling pathway in liver of high concentrate fattening lambs

3 讨论

近年来,为了快速提升家畜的生产性能,养殖者会在饲粮中大幅提升精料的使用比例以达到生产目的。本试验结果表明,饲喂高精料饲粮可显著提高羔羊ADG和FBW,降低F/G。但反刍动物长期采食高精料饲粮会造成其消化道内源性LPS移位进入肝脏,引发肝脏的氧化应激和炎症反应^[18-19]。肝脏作为反刍动物重要的代谢器官,在免疫应答中也起着重要作用^[20]。据报道,反刍动物长期采食高精料饲粮会使其肝脏出现损伤,肝细胞脓肿并存在大量炎性细胞浸润^[21]。CKT作为一种天然多酚类活性物质,具有抗氧化、抗菌、抗炎特性,有促进育肥羔羊生长和改善羔羊健康状况的作用效果^[22]。本试验结果表明,尽管CKT组肝脏组织仍存在一定病变,但与HC组的肝脏组织相比,病变的程度得到显著缓解。同时,AST和ALT作为评价肝脏功能是否正常的重要指标之一^[23]。

当机体肝脏功能受损时,ALT和AST会从肝细胞释放到血清中,导致血清中ALT和AST活性升高^[24]。血清TP和ALB含量可以直接反映动物肝脏的合成代谢功能是否受到影响。TP含量越高,则代表动物体蛋白质合成代谢的能力越高。牟春堂等^[25]研究证明,在高精料育肥绵羊饲粮中补饲20 mg/(kg BW·d)的葡萄籽原花青素可显著升高血清中TP和ALB含量,降低转氨酶活性,缓解肝脏组织损伤。本研究发现,HC组羔羊血清ALT、AST活性均高于CON组和CKT组,这表明长期饲喂高精料饲粮可能会导致瘤胃代谢异常,造成羔羊肝脏损伤,影响机体健康,与前人研究结果^[26]一致。同时,与HC组相比,CKT组羔羊血清TP和ALB含量升高,这说明添加柠条单宁不仅可以调节高精料饲粮育肥羔羊的肝脏功能,还有望改善羔羊肝脏损伤及提高羔羊生长性能,需进一步研究。

在免疫调控方面,炎性细胞因子作为非特异性免疫应答的重要介质,依据其作用特性可被划分为促炎因子(IL-1β、IL-6、TNF-α)和抗炎因子(IL-4、IL-10)两大类。这些因子通过一系列复杂的信号传导路径和反馈机制相互制约^[27],共同维持免疫系统的动态平衡。李振富^[28]研究表明,饲喂高精料饲粮导致奶牛肝脏组织中IL-1β和TNF-α的含量升高,造成肝脏损伤。周琦璐等^[29]发现,给仔猪饲喂含1 000 mg/kg单宁酸的基础饲粮可以显著降低LPS应激仔猪肝脏组织中IL-6和TNF-α的含量。还有研究报道,单宁可以抑制由LPS诱导的TNF-α、IL-6和IL-1β等促炎细胞因子的释放^[22],从而对LPS刺激所致的炎症反应有缓解作用。本研究表明,与CON组相比,HC组肝脏组织中IL-1β含量显著升高,表明高精料饲喂造成羔羊肝脏发生炎症反应,且CKT组羔羊肝脏中促炎细胞因子IL-1β含量降低,表明这可能与柠条单宁具有良好的抗炎作用有关。

在机体维持免疫稳态的过程中,TLR4/NF-κB信号通路起着关键的作用^[30]。TLR4是能识别外源性或内源性配体,介导免疫细胞活化、炎症细胞因子释放以及组织损伤的受体,被称为与炎症反应关系密切的蛋白质分子^[31]。当机体受到刺激后,TLR4作为天然的跨膜受体,在接收多种炎症信号后,启动炎症反应,促使抑制蛋白IκBα泛素化降解,释放NF-κB p50/p65异源二聚体,易位至细胞核^[32],进而调控一系列炎性细胞因子(如TNF-α、IL-6和IL-1β)含量^[33],从而引发机体炎症反应^[34]。有研究显示,TNF-α与IL-1β激活NF-κB通路,加剧机体免疫应激^[35]。这表明炎性细胞因子含量的增多与NF-κB通路的激活呈正相关关系,而抑制p65的磷酸化可有效阻断NF-κB信号通路的活化^[36],这与本研究结果相似。近年来大量研究结果表明,在动物饲粮中添加适宜剂量的单宁能改善肝脏免疫功能。Yuan等^[37]研究发现,在1日龄爱拔益加肉鸡饲粮中添加300 mg/kg的五倍子单宁能抑制肝脏炎性细胞因子含量增加,并且通过阻断TLR4/NF-κB通路相关基因表达,缓解因LPS诱导的肝脏炎症。因此,推测五倍子单宁对肉鸡肝脏健康的保护作用可能与抑制TLR4/NF-κB信号通路有关。Chen等^[36]研究发现,茶多酚提取物可以通过抑制TLR4蛋白表达,进而减少核苷酸结合寡聚化结构域样受体蛋白3炎症小体(NLRP3)的形成和IL-1β与TNF-α的释放,缓解小鼠在缺氧环境下的神经炎症。研究发现,多酚类化合物能通过抑制血清中IL-1β、IL-6、TNF-α、TLR4和NF-κB的基因表达,阻断TLR4/MyD88信号通路,减轻羔羊免疫应激反应^[38]。本试验结果发现,与HC组相比,CKT组羔羊肝脏TLR4/MyD88/NF-κB信号通路相关基因的mRNA和蛋白表达量显著下调,这表明CKT可有效抑制羔羊肝脏TLR4/MyD88/NF-κB信号通路,缓解肝脏炎性反应,改善机体炎症应答反应。此外,研究表明,表没食子儿茶素-3-没食子酸酯可以通过阻断TLR4/丝裂原活化蛋白激酶(MAPK)信号通路,显著抑制LPS诱导的巨噬细胞中基质金属蛋白酶-9(MMP-9)和单核细胞趋化蛋白-1(MCP-1)的蛋白表达,从而缓解炎症反应^[39]。综上所述,单宁类添加剂通过靶向抑制TLR4及其下游MyD88/MAPK/NF-κB信号通路,下调关键炎症因子(IL-1β、TNF-α、IL-6)及效应分子(MMP-9、MCP-1)的蛋白表达,缓解炎性损伤。

4 结论

综上所述,在高精料育肥羔羊饲粮中添加2 g/kg CKT可降低羔羊肝脏炎症因子含量,抑制免疫相关因子mRNA相对表达量和蛋白表达量,缓解肝脏炎性损伤。

参考文献

原文顺序 | 文献年度倒序 | 文中引用次数倒序

[1]	MAO S Y, HUO W J, ZHU W Y. Microbiome-metabolome analysis reveals unhealthy alterations in the composition and metabolism of ruminal microbiota with increasing dietary grain in a goat model[J]. Environmental Microbiology, 2016, 18(2):525-541.

[2]	SHAH T, MALHI M, KACHIWAL A B, et al. Ameliorative effects of supranutritional selenium on TLR4-NF-κB-TNF-α-mediated hepatic oxidative injury and inflammation in goats fed high concentrate diet[J]. Food Science and Nutrition, 2022, 10(11):3842-3854.

[3]	MA Y, ZHANG Y, ZHANG H, et al. Thiamine alleviates high-concentrate-diet-induced oxidative stress,apoptosis,and protects the rumen epithelial barrier function in goats[J]. Frontiers in Veterinary Science, 2021,8:663698.

[4]	ZHANG H Z, XUE Y, XIE W, et al. Subacute ruminal acidosis downregulates FOXA2,changes oxidative status,and induces autophagy in the livers of dairy cows fed a high-concentrate diet[J]. Journal of Dairy Science, 2023, 106(3):2007-2018.

[5]	TANG J, XU L Q, ZENG Y W, et al. Effect of gut microbiota on LPS-induced acute lung injury by regulating the TLR4/NF-kB signaling pathway[J]. International Immunopharmacology, 2021,91:107272.

[6]	XIE W, XUE Y, ZHANG H Z, et al. A high-concentrate diet provokes inflammatory responses by downregulating forkhead box protein A2 (FOXA2) through epigenetic modifications in the liver of dairy cows[J]. Gene, 2022,837:146703.

[7]	ALLEN M S. Review:control of feed intake by hepatic oxidation in ruminant animals:integration of homeostasis and homeorhesis[J]. Animal, 2020, 14(S1):s55-s64.

[8]	BASIRICÒ L, MORERA P, DIPASQUALE D, et al. (-)-epigallocatechin-3-gallate and hydroxytyrosol improved antioxidative and anti-inflammatory responses in bovine mammary epithelial cells[J]. Animal, 2019, 13(12):2847-2856. DOI PMID

[9]	WU Z H, HUANG S M, LI T T, et al. Gut microbiota from green tea polyphenol-dosed mice improves intestinal epithelial homeostasis and ameliorates experimental colitis[J]. Microbiome, 2021, 9(1):184. DOI PMID

[10]	彭凯, 黄文, 戴文因. 缩合单宁的生物活性及构效关系研究进展[J]. 饲料工业, 2021, 42(24):55-59. PENG K, HUANG W, DAI W Y. Research progress on biological activity and structure-function relationship of condensed tannins[J]. Feed Industry, 2021, 42(24):55-59. (in Chinese)

[11]	解湧芳. 饲粮中不同水平的单宁对绵羊血液生化指标、抗氧化能力和免疫功能的影响[D].硕士学位论文. 呼和浩特: 内蒙古农业大学, 2020. XIE Y F. Effect of different level of tannin in the diet on blood biochemical indexes,antioxidative activity and immune function of sheep[D].Master’s Thesis. Hohhot: Inner Mongolia Agricultural University, 2020. (in Chinese)

[12]	MU C T, HAO X Y, ZHANG X Z, et al. Effects of high-concentrate diet supplemented with grape seed procyanidins on the colonic fermentation,colonic morphology,and inflammatory response in lambs[J]. Animal Feed Science and Technology, 2021,281:115118.

[13]

HOSSEN

, ZHANG

K Q

, WU

, et al. Epigallocatechin gallate (EGCG) inhibits lipopolysaccharide-induced inflammation in RAW 264.7 macrophage cells via modulating nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB) signaling pathway[J]. Food Science & Nutrition, 2023, 11(8):4634-4650.

[14]	王冬梅. 柠条的饲用价值及其对反刍动物生产和经济效益的影响[J]. 饲料研究, 2021, 44(17):154-157. WANG D M. Feeding value of Caragana korshinskii and its effect on ruminants production and economic benefits[J]. Feed Research, 2021, 44(17):154-157. (in Chinese)

[15]	曲魏. 柠条单宁的提取及其对小鼠生长性能,免疫和抗氧化功能的影响[D].硕士学位论文. 呼和浩特: 内蒙古农业大学, 2023. QU W. Extraction of Caragana korshinskii tannin and its effects on growth performance,immunity and antioxidant function in mice[D].Master’s Thesis. Hohhot: Inner Mongolia Agricultural University, 2023. (in Chinese)

[16]	中华人民共和国农业农村部. 肉羊营养需要量:NY/T 816—2021[S]. 北京: 中国农业出版社, 2021. Ministry of Agriculture and Rural Affairs of the People’s Republic of China. Nutrient requirements of meat-type sheep and goat:NY/T 816—2021[S]. Beijing: China Agriculture Press, 2021. (in Chinese)

[17]	刘洁. 肉用绵羊饲料代谢能与代谢蛋白质预测模型的研究[D].博士学位论文. 北京: 中国农业科学院, 2012. LIU J. Prediction of metabolizable energy and metabolizable proteinin feeds for meat sheep[D].Ph.D.Thesis. Beijing: Chinese Academy of Agricultural Sciences, 2012. (in Chinese)

[18]	GAO Z, LI Y J, XU C, et al. Niacin mitigates rumen epithelial damage in vivo by inhibiting rumen epithelial cell apoptosis on a high concentrate diet[J]. Veterinary Research Communications, 2022, 46(3):699-709.

[19]

谢婉. FOXA2对亚急性瘤胃酸中毒引起的肝脏炎症反应、内质网应激和自噬的调控作用及机制研究[D].硕士学位论文. 南京: 南京农业大学, 2023.

XIE

. Research on the regulatory effect and mechanism of FOXA2 on liver inflammatory response,endoplasmic reticulum stress and autophagy caused by subacute rumen acidosis[D].Master’s Thesis. Nanjing: Nanjing Agricultural University, 2023. (in Chinese)

[20]	LEE M M, KIM H G, LEE J S, et al. Synergistic hepatoprotective effects of CGplus on CCl₄-induced acute injury[J]. Journal of Ethnopharmacology, 2020,249:112441.

[21]	GUO J F, CHANG G J, ZHANG K, et al. Rumen-derived lipopolysaccharide provoked inflammatory injury in the liver of dairy cows fed a high-concentrate diet[J]. Oncotarget, 2017, 8(29):46769-46780. DOI PMID

[22]	陈治宇. 柠条单宁对高精料育肥羔羊生长性能,免疫和抗氧化功能的影响[D].硕士学位论文. 呼和浩特: 内蒙古农业大学, 2024. CHEN Z Y. Effects of Caragana korshinskii tannin on growth performance,immune and antioxidant function of lambs fed high concentrate diet[D].Master’s Thesis. Hohhot: Inner Mongolia Agricultural University, 2024. (in Chinese)

[23]	WANG Z, SONG Y, ZHANG F, et al. Early warning for inactive ovaries based on liver function index,serum MDA,IL-6,FGF21 and ANGPTL8 in dairy cows[J]. Italian Journal of Animal Science, 2022, 21(1):113-122.

[24]	ZHOU Y L, TAN F, LI C, et al. White peony (fermented Camellia sinensis) polyphenols help prevent alcoholic liver injury via antioxidation[J]. Antioxidants, 2019, 8(11):524.

[25]

牟春堂, 谢蕴哲, 申玉萍, 等. 葡萄籽原花青素对高精料育肥绵羊肝脏炎症反应、抗氧化功能的影响及代谢组学分析[J]. 动物营养学报, 2024, 36(7):4447-4461.

DOI

C T

, XIE

Y Z

, SHEN

Y P

, et al. Effects of grape seed proanthocyanidins on liver inflammation and antioxidant function of fattening sheep fed a high-concentrate diet and metabolomic analysis[J]. Chinese Journal of Animal Nutrition, 2024, 36(7):4447-4461. (in Chinese)

DOI

[26]	郭峻菲. 亚急性瘤胃酸中毒(SARA)对反刍动物肝脏的炎性损伤与SARA的调控研究[D].博士学位论文. 南京: 南京农业大学, 2017. GUO J F. inflammatory injury in the liver of ruminants by subacute ruminal acidosis (SARA) and manipulation of SARA[D].Ph.D.Thesis. Nanjing: Nanjing Agricultural University, 2017. (in Chinese)

[27]	ZOU G M, LI L, GAO L P, et al. Goniothalamin prevents lipopolysaccharide-induced acute lung injury and inflammation via TLR-4/NF-κB signaling pathway[J]. Journal of Biochemical and Molecular Toxicology, 2023, 37(11):e23461.

[28]	李振富. 高精料日粮条件下反刍动物瘤胃中产生的组胺对肝脏炎性信号通路的影响与调控[D].硕士学位论文. 南京: 南京农业大学, 2016. LI Z F. Effect and regulation of histamineproduced in the rumen of a high concentrate diet on the inflammatory signal pathway of the liver[D].Master’s Thesis. Nanjing: Nanjing Agricultural University, 2016. (in Chinese)

[29]

周琦璐, 刘金松, 吴超, 等. 单宁酸对脂多糖应激仔猪肝组织的功能、抗氧化能力和炎症应答的影响[J]. 畜牧兽医学报, 2024, 55(6):2519-2529.

DOI

ZHOU

Q L

, LIU

J S

, WU

, et al. Effects of tannic acid on liver tissue function,antioxidant ability and inflammatory response in lipopolysaccharide stressed piglets[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(6):2519-2529. (in Chinese)

[30]	SHI Y Y, LI B H, SUN S F, et al. Inhibition of mogroside IIIE on isoproterenol-induced myocardial fibrosis through the TLR4/MYD88/NF-κB signaling pathway[J]. Iranian Journal of Basic Medical Sciences, 2023, 26(1):114-120. DOI PMID

[31]	SHI H, ZHOU P, NI Y Q, et al. In vivo and in vitro studies of Danzhi Jiangtang capsules against diabetic cardiomyopathy via TLR4/MYD88/NF-κB signaling pathway[J]. Saudi Pharmaceutical Journal, 2021, 29(12):1432-1440.

[32]	陶依娆, 朴松伊, 张冬梅. NF-κB在病理性心肌肥厚发生发展中作用的研究进展[J]. 生理科学进展, 2019, 50(5):333-338. TAO Y R, PIAO S Y, ZHANG D M. Roles of NF-κB in the development of pathological cardiac hypertrophy[J]. Progress in Physiological Sciences, 2019, 50(5):333-338. (in Chinese)

[33]	FITZGERALD K A, KAGAN J C. Toll-like receptors and the control of immunity[J]. Cell, 2020, 180(6):1044-1066. DOI PMID

[34]	MOSES T, WAGNER L, FLEMING S D. TLR4-mediated Cox-2 expression increases intestinal ischemia/reperfusion-induced damage[J]. Journal of Leukocyte Biology, 2009, 86(4):971-980.

[35]	贾红豆. 茶多酚通过NF-κB通路缓解LPS诱导的奶牛乳腺上皮细胞炎性反应[J]. 中国兽医学报, 2021, 41(7):1372-1375. JIA H D. Tea polyphenols alleviate LPS-induced inflammation in bovine mammary epithelial cells via the NF-κB pathway[J]. Chinese Journal of Veterinary Science, 2021, 41(7):1372-1375. (in Chinese)

[36]	CHEN G J, CHENG K, NIU Y, et al. (-)-epicatechin gallate prevents inflammatory response in hypoxia-activated microglia and cerebral edema by inhibiting NF-κB signaling[J].Archives of Biochemistry and Biophysics, 2022,729:109393.

[37]	YUAN P, XU H T, MA Y F, et al. Effects of dietary Galla Chinensis tannin supplementation on immune function and liver health in broiler chickens challenged with lipopolysaccharide[J]. Frontiers in Veterinary Science, 2023,10:1126911.

[38]	LIANG Y P, ZHOU J W, JI K X, et al. Protective effect of resveratrol improves systemic inflammation responses in LPS-injected lambs[J]. Animals, 2019, 9(11):872.

[39]

Y F

, WANG

, FAN

, et al. Epigallocatechin-3-gallate inhibits matrix metalloproteinase-9 and monocyte chemotactic protein-1 expression through the 67-κDa laminin receptor and the TLR4/MAPK/NF-κB signalling pathway in lipopolysaccharide-induced macrophages[J]. Cellular Physiology and Biochemistry, 2017, 43(3):926-936.

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