#These authors contributed equally to this work.
本文旨在研究地衣芽孢杆菌(
选择24头28日龄健康状况良好的三元杂交断奶仔猪(杜×长×大),随机分成4个试验组:对照组(control, CON),500 mg/kg地衣芽孢杆菌HJ0135组(BL)、LPS应激组(LPS)以及LPS+500 mg/kg地衣芽孢杆菌HJ0135组(BL-LPS),在试验第28天,给LPS组和BL-LPS组仔猪腹腔注射1.5 mL浓度为1 mg/mL的LPS,给CON组和BL组仔猪注射等量生理盐水,1.5 h后屠宰并采样。
与CON组相比,BL组血清谷草转氨酶(aspartate aminotransferase, AST)和谷丙转氨酶(alanine aminotransferase, ALT)的含量降低,肝脏的总抗氧化能力(total antioxidant capacity, T-AOC)提升且过氧化氢酶(catalase, CAT)和总超氧化物歧化酶(total superoxide dismutase, T-SOD)活性均增强。肝脏中促炎因子白细胞介素-6 (interleukin-6, IL-6)含量减少,同时肿瘤坏死因子-α (tumor necrosis factor-α, TNF-α)含量下降,肝脏
断奶仔猪日粮中添加地衣芽孢杆菌HJ0135,能够激活Nrf2和TLR4相关基因表达,降低脂多糖刺激引起的肝脏损伤和炎症因子的分泌,提高断奶仔猪抗氧化能力。
To investigate the protective effects of
A total of 24 healthy 28-day-old weaned piglets (Duroc×Landrace×Yorkshire) were randomly assigned into 4 groups: control (CON) group, 500 mg/kg
Compared with the CON group, BL treatment decreased the content of aspartate aminotransferase (AST) and alanine aminotransferase (ALT), increased the total antioxidant capacity (T-AOC), the activities of catalase (CAT) and total superoxide dismutase (T-SOD), reduced the levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in the liver, and up-regulated the expression of
Dietary
早期断奶可诱导仔猪氧化应激[
目前,益生菌作为一种绿色健康、提高生长性能并能够有效改善动物肠道健康的饲料添加剂已经得到了市场的广泛认可。Lan等[
本实验室前期筛选了地衣芽孢杆菌(
试验选择24头体重相近、健康状况良好的28日龄三元杂交断奶仔猪(杜×长×大),将其随机分为4个试验组,每组6头仔猪,试验期为28 d。对照组(CON),饲喂基础饲粮;地衣芽孢杆菌组(BL),饲喂基础饲粮+500 mg/kg地衣芽孢杆菌HJ0135;LPS应激组(LPS),饲喂基础饲粮;LPS+500 mg/kg地衣芽孢杆菌HJ0135组(BL-LPS),饲喂基础饲粮+500 mg/kg地衣芽孢杆菌HJ0135,其中添加的地衣芽孢杆菌中含1×109 CFU/g活菌数。在试验第28天,给LPS组和BL-LPS组仔猪腹腔注射1.5 mL浓度为1 mg/mL的LPS,给CON组和BL组仔猪注射等量生理盐水,1.5 h后屠宰并采样。试验在杭州正兴牧业有限公司进行,试验期间,不限制试验仔猪采食与饮水,采食与饮水均可自由进行,并且按照养殖场的常规流程和制度对场所及物品进行消毒。断奶仔猪饲喂的基础日粮组成及营养水平如
基础饲粮组成及营养水平(风干基础)
Composition and nutrition levels of basal diets (air-dry basis)
Ingredients | Content (%) | Nutrient level | Content |
1: Supplied the following per kg of diet. Vitamin A, 10 000 IU; Vitamin D3, 400 IU; Vitamin E, 10 mg; Pantothenic acid, 15 mg; Vitamin B6, 2 mg; Biotin, 0.3 mg; Folic acid, 3 mg; Vitamin B12, 0.009 mg; Ascorbic acid, 40 mg; Fe, 150 mg; Cu, 130 mg; Mn, 60 mg; Zn, 120 mg; I, 0.3 mg; and Se, 0.25 mg. | |||
Corn | 55.00 | DE, MJ/kg | 14.17 |
Wheat midding | 3.50 | CP, % | 20.35 |
Phospholipid | 2.00 | Lys, % | 1.34 |
Whey powder | 5.00 | Met+Cys, % | 0.77 |
Extruded soybean | 7.30 | Thr, % | 0.80 |
Soybean meal | 18.50 | Ca, % | 0.95 |
Fish meal | 5.00 | TP, % | 0.65 |
Dicalcium phosphate | 1.00 | AP, % | 0.48 |
Limestone | 1.10 | ||
NaCl | 0.10 | ||
l-lysine HCl | 0.35 | ||
dl-methionine | 0.15 | ||
Vitamin-Mineral premix1 | 1.00 | ||
Total | 100.00 |
在第28天对断奶仔猪前腔静脉注射LPS或生理盐水,1.5 h后对断奶仔猪进行前腔静脉采血,之后屠宰采集肝脏并进行称重,剪取部分仔猪肝脏置于冻存管中,将血清和肝脏保存于−80 ℃冰箱,用于后续各项指标的测定。
血清肝脏功能相关指标血清谷草转氨酶(aspartate aminotransferase, AST)和谷丙转氨酶(alanine aminotransferase, ALT)采用从南京建成生物工程研究所购买的试剂盒检测。
取0.1 g肝脏组织样品,加入1 mL生理盐水,使用匀浆机进行充分匀浆,12 000×
ELISA试剂盒检测肝脏组织样炎症因子,包括白细胞介素-1β (interleukin-1β, IL-1β)、白细胞介素-6 (interleukin-6, IL-6)、肿瘤坏死因子-α (tumor necrosis factor-α, TNF-α)和白细胞介素-10 (interleukin-10, IL-10)。上述指标交由南京奥青生物科技有限公司检测。
使用RNAiso Plus试剂(TaKaRa)提取肝脏总RNA。使用超微量分光光度计对提取的RNA浓度进行检测,再使用PrimeScriptTM RT reagent Kit with gDNA Eraser试剂盒将1 μL浓度为500 ng/μL的RNA反转录成cDNA。cDNA的合成按照试剂盒的操作说明书进行,最后将cDNA储存在−80 ℃冰箱备用。以cDNA为模板,分别选取与肝脏抗氧化功能、肝脏炎症因子和TLR4/MyD88信号通路相关的基因进行荧光定量PCR,引物见
荧光定量PCR引物序列
Sequence of primers of real-time PCR
Gene names | Forward primer (5 |
Reverse primer (5 |
Accession number |
AGCTTTGCCCTTGCACAAAC | TCCATATCCGTTCATGTGCCTGTG | NM_214301.2 | |
AAGGCCGTGTGTGTGCTGAA | GATCACCTTCAGCCAGTCCTTT | NM_001190422.1 | |
GGCCTACGTGAACAACCTGA | TGATTGATGTGGCCTCCACC | NM_214127.2 | |
CCTCAAGTACGTCCGACCAG | GTGAGCATTTGCGCCATTCA | NM_214201.1 | |
ACCTGGACCTTGGTTCTC | GGATTCTTCATCGGCTTC | NM_214055.1 | |
TTCAGTCCAGTCGCCTTCT | GTGGCATCACCTTTGGCATCTTCTT | NM_214399.1 | |
ACGCTCTTCTGCCTACTGC | TCCCTCGGCTTTGACATT | NM_214022.1 | |
CACTGCTCTATTGCCTGATCTTCC | AAACTCTTCACTGGGCCGAAG | NM_214041.1 | |
CATGGCGGTCAGAAAAGCAC | ATGGCATACAGGTCCGACAC | NM_001159613.1 | |
GCCCCTGGAAGCGTTAAAC | GGACTGTATCCCCAGAAGGTTGT | XM_003133500.5 | |
ACGACGTGGAGACAGAAACGT | GCTTCGCCGATGCTTCA | NM_001114671.1 | |
TGGATTTATCCAGCCAGGACG | GCTGGGACACCACGACAATA | NM_001293317.1 | |
CACCATTCGAGATGACCCCC | CTAGCAATGGACCAGACGCA | NM_001099923.1 | |
TTCTGGACCGCTTGGGTAAC | CACCGTTGGGGTGGTTGATA | NM_001048232.1 | |
CTACACCGCTACCAGTTCGC | TAGGAGTCCTTCTGGCCCAT | DQ452569.1 |
采用SPSS 25.0软件对数据进行单因素方差分析(analysis of variance, ANOVA),用最小显著差异法(least significant difference, LSD)多重比较法分析数据间的显著性差异。采用GraphPad Prism 8进行作图,试验结果用平均值±标准误表示,
由
添加地衣芽孢杆菌HJ0135对LPS应激断奶仔猪肝脏指数的影响
Effects of
由
添加地衣芽孢杆菌HJ0135对LPS应激断奶仔猪血清AST (A)和ALT (B)的影响
Effects of
由
添加地衣芽孢杆菌HJ0135对LPS应激断奶仔猪肝脏抗氧化功能的影响
Effects of
由
添加地衣芽孢杆菌HJ0135对LPS应激断奶仔猪肝脏炎症因子的影响
Effects of
由
添加地衣芽孢杆菌HJ0135对LPS应激断奶仔猪肝脏基因表达的影响
Effects of
肝脏是机体营养代谢和解毒的主要中枢器官,更易受到体内毒素和代谢物产生的氧化应激和炎症的影响[
氧化应激主要是由氧自由基的积累引起的,细胞活性氧(reactive oxygen species, ROS)的产生和消除之间的动态平衡在维持身体健康中起着至关重要的作用[
诸多研究表明,细胞因子在仔猪的炎症反应中发挥重要作用。地衣芽孢杆菌可诱导调节性T淋巴细胞向浆细胞分化,从而增强免疫球蛋白分泌,促进抗炎作用[
本研究显示地衣芽孢杆菌通过抑制炎症和氧化应激的作用减轻了LPS导致的肝脏损伤。为了充分阐明其中的保护机制,我们研究了两个参与抗炎和抗氧化的关键信号通路,即NF-κB和Nrf2信号通路。NF-κB和Nrf2通路形成复杂的信号网络,调节肠道炎症和氧化还原状态。LPS刺激介导的TLR4信号通路,可以激活NF-κB,从而调节炎症细胞因子分泌,在免疫系统应对革兰氏阴性菌入侵中发挥重要作用[
饲粮中添加地衣芽孢杆菌HJ0135可以激活Nrf2和TLR4信号通路,缓解LPS刺激引起的肝脏损伤和炎症因子的分泌,提高断奶仔猪抗氧化能力。
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