Chinese Journal of Animal Nutrition >

2025 , Vol. 37 >Issue 10: 6651 - 6661

DOI: https://doi.org/10.12418/CJAN2025.541

RESEARCH PAPER

Mitigating Effect of Fagopyrum dibotrys Extract Against Lipopolysaccharide/D-Galactosamine-Induced Liver Injury in Broilers

  • CHEN Jiang , 1 ,
  • SONG Qiongli , 1, * ,
  • AI Gaoxiang 1 ,
  • XIONG Pingwen 1 ,
  • SONG Wenjing 1 ,
  • XU Chuanhui 1 ,
  • SU Weide 1 ,
  • LIU Guohua 2 ,
  • ZHENG Aijuan 2 ,
  • CHEN Xiaolian 1 ,
  • ZOU Zhiheng 1 ,
  • WEI Qipeng 1
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  • 1 Jiangxi Province Key Laboratory of Animal Green and Healthy Breeding, Institute of Animal Husbandry and Veterinary Science, Jiangxi Academy of Agricultural Sciences, Nanchang 330200, China
  • 2 Key Laboratory of Feed Biotechnology of the Ministry of Agriculture, Institute of Feed Research, Chinese Academy of Agricultural Sciences, Beijing 100081, China
*associate professor, E-mail:

Received date: 2025-03-03

  Online published: 2025-10-15

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Abstract

This experiment aimed to study the mitigating effect of Fagopyrum dibotrys extract on liver injury in broilers, using lipopolysaccharide (LPS)/D-galactosamine (D-GalN) induced liver injury model in broilers. One hundred and eighty 7-day-old Shengze 901 female broilers were randomly divided into three groups (6 replicates per group, and 10 chickens per replicate): blank control group (CON group), LPS/D-GalN model group (LPSD group), LPS/D-GalN+Fagopyrum dibotrys treatment group (JQM group). CON group and LPSD group were fed with a basal diet, and the JQM group was added with 500 mg/kg of Fagopyrum dibotrys extract to the basal diet. The broilers were fed the corresponding experimental diet starting from 7 days of age. At 16, 18 and 20 days of age, the LPSD group and JQM group were injected intraperitoneally with 0.5 mg/kg BW of LPS and 150 mg/kg BW of D-GalN (LPS and D-GalN dissolved in saline to a final concentration of 0.5 and 150 mg/mL, respectively), while the CON group was injected with saline (1 mL/kg BW). The samples were collected at 21 days of age. The results showed as follows: 1) compared with the CON group, the average daily feed intake (ADFI) and average daily gain (ADG) were significantly decreased in the LPSD group (P<0.05), and the ADG was significantly decreased in the JQM group (P<0.05). The ADG was significantly increased in the JQM group compared with the LPSD group (P<0.05). 2) Compared with the CON group, the liver index of broilers in the LPSD group and JQM group was significantly increased (P<0.05); compared with the LPSD group, the liver index of broilers in the JQM group was reduced to a certain extent, although there was no significant difference (P>0.05). 3) Compared with the CON group, the activities of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) of broilers in the LPSD group were significantly increased (P<0.05); the ALT activity was significantly reduced in the JQM group compared with the LPSD group (P<0.05). 4) Serum interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) contents were significantly increased of broilers in the LPSD group compared with the CON group (P<0.05), while the serum IL-6, TNF-α and IL-1β contents in the JQM group showed no significant differences compared with either the CON group or the LPSD group (P>0.05). 5) Pathological observation results showed that the liver lobules in the LPSD group were accompanied by focal infiltration of lymphocytes and focal hepatic sinusoidal stasis, and more connective tissue hyperplasia was seen, while occasional connective tissue hyperplasia and infiltration of lymphocytes were seen in the JQM group. Transmission electron microscopy results showed that the LPSD group hepatocytes moderate damage, mainly manifested in mitochondria, endoplasmic reticulum and other organelles swelling or damage; JQM group organelle swelling and damage were alleviated. 6) Compared with the LPSD group, the relative abundances of Bacteroidota and Bacteroides were significantly decreased (P<0.05), while the relative abundance of Firmicutes was significantly increased in the JQM group (P>0.05). In conclusion, Fagopyrum dibotrys extract has a mitigating effect on LPS/D-GalN-induced liver injury in broilers, indicating that Fagopyrum dibotrys extract has a hepatoprotective effect.

Key words： Fagopyrum dibotrys extract; lipopolysaccharide; D-galactosamine; broilers; liver injury

Cite this article

CHEN Jiang , SONG Qiongli , AI Gaoxiang , XIONG Pingwen , SONG Wenjing , XU Chuanhui , SU Weide , LIU Guohua , ZHENG Aijuan , CHEN Xiaolian , ZOU Zhiheng , WEI Qipeng . Mitigating Effect of Fagopyrum dibotrys Extract Against Lipopolysaccharide/D-Galactosamine-Induced Liver Injury in Broilers[J]. Chinese Journal of Animal Nutrition, 2025 , 37(10) : 6651 -6661 . DOI: 10.12418/CJAN2025.541

随着家禽养殖业规模化、集约化程度不断提升,家禽因饲料问题、环境压力、疾病感染和药物滥用导致的肝脏损伤问题日益凸显,给养禽业造成巨大的经济损失,因此迫切需要寻找绿色、无公害的饲料添加剂来预防和保护家禽肝脏、提高家禽生产性能及改善家禽健康。脂多糖(LPS)和D-氨基半乳糖(D-GalN)联用是构建动物肝脏损伤模型的常用药物,被广泛应用于探索急性肝脏损伤机制和保肝药物开发[1]。LPS作为革兰氏阴性菌的外膜成分,可引起肝脏损伤,D-GalN对肝脏具有特异性毒性并导致肝脏损伤[2],D-GalN与LPS联用可增强肝脏对LPS的敏感性,且与家禽受细菌侵害诱发肝脏损伤发病机制相似,因此可用LPS/D-GalN诱导的肝脏损伤模型研究家禽肝脏损伤的潜在机制及研发新型保肝添加剂[3]。研究发现,中草药植物提取物含有丰富的具有抗病特性的生物碱、抗氧化特性的黄酮类和酚类等物质[4],这些活性成分可通过调节免疫机能、增强抗氧化性能及减轻炎症反应等途径维护肝细胞正常功能,具有良好的保肝护肝作用[5-8]。随着对无抗添加剂的深入研究,金荞麦(Fagopyrum dibotrys)因其抗炎抑菌、抗氧化和抗癌等多种生理功能受到越来越多的研究人员关注[9]。金荞麦作为蓼科多年生药饲兼用草本植物,其制剂和提取物中含有抗炎杀菌等活性成分,具有促生长、防疾病、提高肉品质及免疫功能等作用[10]。同时,金荞麦提取物中的黄酮类物质可通过调节Toll样受体4(TLR4)/核因子-κB(NF-κB)/信号传导及转录激活因子(STAT)信号通路对LPS诱导的人外周单核血细胞发挥抗炎作用[11]。然而,有关应用金荞麦提取物对雏鸡肝脏损伤保护效果的研究尚未见报道。因此,本试验以金荞麦提取物作为研究材料,通过LPS/D-GalN诱导雏鸡肝脏损伤模型,探究金荞麦提取物对LPS/D-GalN诱导的肉鸡肝脏损伤的缓解作用,为合理评价金荞麦提取物的饲用价值以及开发新型保肝饲料添加剂提供理论依据。

1 材料与方法

1.1 试验材料

试验所用金荞麦提取物中黄酮含量为9.34 mg/g,总酚含量为4.21 mg/g。

1.2 试验设计与试验饲粮

选取180只7日龄圣泽901雌性肉仔鸡,随机分成3组(每组6个重复,每个重复10只鸡),分别为空白对照组(CON组)、LPS/D-GalN模型组(LPSD组)、LPS/D-GalN+金荞麦提取物处理组(JQM组)。CON组与LPSD组饲喂基础饲粮,JQM组在基础饲粮中添加500 mg/kg金荞麦提取物。试验鸡从7日龄开始饲喂对应试验饲粮,在16、18、20日龄时,LPSD组与JQM组按照鸡只体重腹腔注射0.5 mg/kg LPS与150 mg/kg D-GalN(LPS与D-GalN混合溶于生理盐水,使LPS终浓度为0.5 mg/mL,D-GalN终浓度为150 mg/mL),CON组按照鸡只体重腹腔注射生理盐水(1 mL/kg),于21日龄时进行采样。基础饲粮营养标准参考NY/T 33—2004设计,其组成及营养水平见表1。分别采用GB/T 6432—2018、GB/T 6437—2018和GB/T 6436—2018中方法检测饲粮中粗蛋白质、总磷和钙含量,饲粮中代谢能、有效磷、赖氨酸和蛋氨酸含量参照《中国饲料成分及营养价值表(2024年第35版)》进行计算。本试验由江西省农业科学院实验动物伦理委员会批准,试验伦理批准编号为2024-JXAAS-XM-08。
表1 基础饲粮组成及营养水平(饲喂基础)

Table 1 Composition and nutrient levels of the basal diet (as-fed basis) %

项目Items 含量Content
原料Ingredients
玉米Corn 50.31
豆粕Soybean meal 34.92
大豆油Soybean oil 5.35
菜籽粕Rapeseed meal 5.00
L-赖氨酸盐酸盐L-Lys·HCl(79%) 0.15
DL-蛋氨酸DL-Met(99%) 0.18
苏氨酸Threonine 0.07
石粉Limestone 1.87
磷酸氢钙CaHPO4 0.85
食盐NaCl 0.30
预混料Premix1) 1.00
合计Total 100.00
营养水平Nutrient levels2)
代谢能ME/(MJ/kg) 12.55
粗蛋白质CP 21.08
钙Ca 0.97
总磷TP 0.54
有效磷AP 0.27
赖氨酸Lys 1.15
蛋氨酸Met 0.50

1)预混料为每千克饲粮提供 The premix provided the following per kg of the diet:Cu 20 mg,Fe 320 mg,Mn 100 mg,Zn 200 mg,I 0.60 mg,Se 0.30 mg,VA 20 000 IU,VD 2 500 IU,VE 25 mg,VK3 3 mg,VB1 5 mg,VB2 10 mg,VB6 12 mg,VB12 0.03 mg,D-泛酸 D-pantothenic acid 20 mg,烟酸 niacin 30 mg,生物素 biotin 0.10 mg,叶酸 folic acid 3 mg。2)饲粮中粗蛋白质、钙、总磷为实测值,代谢能、有效磷、赖氨酸和蛋氨酸为计算值。CP, Ca and TP were measured values, while ME, AP, Lys and Met were calculated values.

1.3 饲养管理

饲养试验在江西省农业科学院畜牧兽医研究所试验鸡场进行,采用3层立体笼养,每笼5只(笼尺寸:70 cm深×100 cm宽×50 cm高)。在入舍前1周对整个鸡舍包括试验用具进行彻底清扫消毒。在整个试验期间,鸡只可以自由进食和饮水,鸡舍自然通风,温度前3 d保持在33 ℃,此后每周降2 ℃,光照强度为5 lx,24 h不间断照明。饲养过程中做好各种记录,发现异常情况及时处理。

1.4 检测指标与方法

1.4.1 生长性能

肉鸡在断料供水12 h后,试验开始(7日龄)和结束当天(21日龄)对每个重复肉鸡进行称重,计算平均日增重(ADG),同时统计每个重复的总采食量,计算平均日采食量(ADFI)和料重比(F/G)。

1.4.2 血清指标

在21日龄对肉鸡进行翅静脉采血,将血清分装在1.5 mL的EP管中,以离心力为1 005×g离心10 min,吸取血清储存于-80 ℃低温冰箱,用于血清指标检测。用全自动生化分析仪检测血清谷草转氨酶(AST)、谷丙转氨酶(ALT)活性与总胆红素(TBIL)、直接胆红素(DBIL)含量;利用上海酶联生物科技有限公司的鸡特异性酶联免疫吸附试验(ELISA)试剂盒测定血清白细胞介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)含量,各指标测定方法均根据试剂盒说明书要求进行。

1.4.3 肝脏指数及组织病理学观察

肉鸡采完翅静脉血后,通过水浴电击致昏屠宰,检查肝脏变化,称重,计算肝脏指数(肝脏重量与采样体重之比)。严格按照病理实验检测程序获得镜检合格的肝脏组织样片,每张病理图片中用不同颜色箭头标注其典型病变特征。

1.4.4 肝脏组织透射电镜观察

取新鲜的肝脏组织切成1 mm3大小,用1 mol/L磷酸缓冲液(pH 7.4)漂洗3次,然后用1%锇酸避光室温固定,室温条件下经过脱水、渗透包埋、聚合、超薄切片、染色等过程,用HT7800透射电子显微镜(日立,日本)采集图像进行分析。

1.4.5 肠道菌群组成分析

无菌操作收集肉鸡盲肠内容物,置于液氮保存,由上海美吉生物医药科技有限公司完成16S rDNA高通量测序分析,质控优化原始数据后得到有效数据,在美吉生物云平台上进行物种注释分析、物种差异比较分析。

1.5 统计分析

试验数据采用SPSS 24.0统计软件进行单因素方差分析(one-way ANOVA),在差异显著的基础上采用Duncan氏法进行组间多重比较,P<0.05表示差异显著。

2 结果与分析

2.1 金荞麦提取物对LPS/D-GalN诱导的肝脏损伤肉鸡生长性能的影响

由表2可知,与CON组相比,LPSD组F/G没有显著差异(P>0.05),但ADFI和ADG显著降低(P<0.05);JQM组F/G和ADFI没有显著差异(P>0.05),但ADG显著降低(P<0.05)。与LPSD组相比,JQM组ADG显著提高(P<0.05),ADFI在一定程度上有所提升。上述结果说明,饲粮中添加金荞麦提取物对LPS/D-GalN诱导的肝脏损伤肉鸡的生长性能有一定改善作用。
表2 金荞麦提取物对LPS/D-GalN诱导的肝脏损伤肉鸡生长性能的影响

Table 2 Effects of Fagopyrum dibotrys extract on growth performance of broilers with LPS/D-GalN induced liver injury

项目
Items		 组别Groups SEM P值
P-value
CON LPSD JQM
7日龄体重BW at 7 days of age/g 140.22 140.78 140.35 3.324 0.961
21日龄体重BW at 21 days of age/g 848.82a 789.10b 823.83ab 9.132 0.011
平均日增重ADG/(g/d) 51.34a 46.08c 48.78b 0.697 0.001
平均日采食量ADFI/(g/d) 72.69a 68.00b 70.82ab 0.686 0.003
料重比F/G 1.42 1.48 1.46 0.012 0.064

同行数据肩标不同小写字母表示差异显著(P<0.05),无字母或相同小写字母表示差异不显著(P>0.05)。下表同。

In the same row, values with different small letter superscripts mean significant difference (P<0.05), while with no letter or the same letter superscripts mean no significant difference (P>0.05). The same as below.

2.2 金荞麦提取物对LPS/D-GalN诱导的肝脏损伤肉鸡肝脏指数的影响

由表3可知,与CON组相比,LPSD组和JQM组肝脏指数显著升高(P<0.05);与LPSD组相比,JQM组肝脏指数虽无显著差异(P>0.05),但在一定程度上有所降低。上述结果说明,饲粮中添加金荞麦提取物对LPS/D-GalN诱导的肝脏损伤肉鸡的肝脏指数有一定的改善作用。
表3 金荞麦提取物对LPS/D-GalN诱导的肝脏损伤肉鸡肝脏指数的影响

Table 3 Effects of Fagopyrum dibotrys extract on liver index of broilers with LPS/D-GalN induced liver injury

项目
Items		 组别Groups SEM P值
P-value
CON LPSD JQM
采样体重Sample body weight/g 867.82a 806.67b 829.20ab 8.504 0.007
肝脏重量Liver weight/g 23.95b 29.07a 27.07ab 0.691 0.005
肝脏指数Liver index/% 2.76b 3.60a 3.26a 0.086 0.001

2.3 金荞麦提取物对LPS/D-GalN诱导的肝脏损伤肉鸡血清肝脏功能指标的影响

由表4可知,与CON组相比,LPSD组血清ALT和AST活性显著升高(P<0.05),JQM组血清ALT活性显著升高(P<0.05),LPSD组和JQM组血清TBIL和DBIL含量均无显著变化(P>0.05);与LPSD组相比,JQM组血清ALT活性显著降低(P<0.05)。
表4 金荞麦提取物对LPS/D-GalN诱导的肝脏损伤肉鸡血清肝脏功能指标的影响

Table 4 Effects of Fagopyrum dibotrys extract on serum liver function indexes of broilers with LPS/D-GalN induced liver injury

项目
Items		 组别Groups SEM P值
P-value
CON LPSD JQM
谷草转氨酶ALT/(U/L) 3.81c 5.31a 4.63b 0.161 0.001
谷丙转氨酶AST/(U/L) 332.14b 414.12a 393.49ab 14.656 0.046
总胆红素TBIL/(μmol/L) 10.22 11.02 9.79 0.232 0.118
直接胆红素DBIL/(μmol/L) 7.80 8.16 8.12 0.212 0.751

2.4 金荞麦提取物对LPS/D-GalN诱导的肝脏损伤肉鸡血清炎症因子含量的影响

由表5可知,与CON组相比,LPSD组血清TNF-α、IL-6和IL-1β含量显著升高(P<0.05);JQM组血清TNF-α、IL-6和IL-1β含量与LPSD组相比虽无显著差异(P>0.05),但均有不同程度的降低,且JQM组与CON组的差异亦不显著(P>0.05)。上述结果说明,饲粮中添加金荞麦提取物对LPS/D-GalN所致的肉鸡血清TNF-α、IL-6和IL-1β含量升高表现出降低作用。
表5 金荞麦提取物对LPS/D-GalN诱导的肝脏损伤肉鸡血清炎症因子含量的影响

Table 5 Effects of Fagopyrum dibotrys extract on serum inflammatory factor contents of broilers with LPS/D-GalN induced liver injury pg/mL

项目
Items		 组别Groups SEM P值
P-value
CON LPSD JQM
白细胞介素-1β IL-1β 28.23b 49.68a 36.86ab 3.162 0.015
肿瘤坏死因子-α TNF-α 5.54b 18.44a 10.76ab 1.947 0.017
白细胞介素-6 IL-6 3.02b 4.28a 3.75ab 0.196 0.022

2.5 金荞麦提取物对LPS/D-GalN诱导的肝脏损伤肉鸡肝脏组织病理形态的影响

肝脏剖检及苏木精-伊红(HE)染色病理图见图1。从肝脏剖检结果看,与CON组相比,LPSD组肝脏明显肿大、呈土黄色、脂肪蓄积、易碎,可初步判断造模成功;与LPSD组相比,JQM组相应病理变化明显减轻。对肝脏组织病理切片进行观察,CON组无炎症细胞浸润和坏死灶;LPSD组肝小叶分界不明显,伴随淋巴细胞灶性浸润,偶见坏死细胞碎片,并可见较多结缔组织增生,局灶性肝窦淤血;JQM组偶见结缔组织增生和淋巴细胞浸润,胞浆内空泡减少,但仍有少量肝细胞细胞核碎裂或溶解,但肝脏组织基本结构清晰,未见明显病理变化。
图1 肝脏剖检及HE染色病理图

橙色箭头所示为淋巴细胞灶性浸润,紫色箭头所示为坏死细胞碎片,绿色箭头所示为结缔组织增生,红色箭头所示为局灶性肝窦淤血。

Fig.1 Liver dissection and HE-stained pathological diagrams

Orange arrows indicated focal infiltration of lymphocytes, purple arrow indicated necrotic cell debris, green arrows indicated connective tissue hyperplasia, and red arrows indicated hepatic sinusoidal stasis.

2.6 金荞麦提取物对LPS/D-GalN诱导的肝脏损伤肉鸡肝脏超微结构的影响

由图2可知,CON组肝细胞较正常,主要表现为胞内基质均匀,胞内细胞器分布均匀,细胞核呈椭圆形,核膜完整;线粒体形态正常,数量较多,呈椭圆形或圆柱形,膜完整,嵴平行排列;粗面内质网丰富,未见明显损伤。LPSD组肝细胞中度损伤,主要表现为细胞器肿胀,细胞膜模糊,细胞核呈不规则形,核膜模糊,异染色质增多;线粒体数量较多可见明显肿胀,膜模糊,基质溶解不均匀,嵴断裂;粗面内质网数量减少,囊膜扩张,有较多断裂;胞内有少量脂滴。JQM组肝细胞轻微损伤,主要表现为部分细胞器肿胀,细胞核呈皱缩,核膜完整,异染色质增多,核仁可见;线粒体数量较多,呈椭圆形或圆柱形,膜完整,个别线粒体轻微肿胀,粗面内质网丰富,未见明显损伤。
图2 肝脏超微结构透射电镜图

N:细胞核 nucleus;Nu:核仁 nucleolus;M:线粒体 mitochondria;RER:内质网 endoplasmic reticulum;ASS: 溶酶体 autolysosome;D:脂滴 lipid droplet。

Fig.2 Transmission electron micrograph of liver ultrastructure (10 000×)

2.7 金荞麦提取物对LPS/D-GalN诱导的肝脏损伤肉鸡肠道菌群组成的影响

由图3可知,在门水平上,各组盲肠菌群中均以厚壁菌门(Firmicutes)和拟杆菌门(Bacteroidota)为主要的菌门;Firmicutes相对丰度在46.57%~61.97%,其中以JQM组Firmicutes的相对丰度最高,显著高于LPSD组(P<0.05);Bacteroidota的相对丰度在34.83%~51.83%,其中以LPSD组Bacteroidota的相对丰度最高,显著高于JQM组(P<0.05);其他菌门的相对丰度各组间无显著差异(P>0.05),其中弯曲杆菌门(Campilobacterota)和变形菌门(Proteobacteria)的相对丰度均以JQM组最低,其他组变化不大,均在1.00%以下。
图3 金荞麦提取物对LPS/D-GalN诱导的肝脏损伤肉鸡肠道菌群门水平上组成的影响

Firmicutes:厚壁菌门;Bacteroidota:拟杆菌门;Campilobacterota:弯曲杆菌门;Proteobacteria:变形菌门;Actinobacteriota:放线菌门;Desulfobacterota:脱硫杆菌门;Cyanobacteria:蓝藻菌门;Synergistota:互养菌门;Deferribacterota:脱铁杆菌门;Others:其他。“*”表示差异显著(P<0.05)。下图同。“*” represented significant difference (P<0.05). The same as below.

Fig.3 Effects of Fagopyrum dibotrys extract on gut microbiota composition on phylum level of broilers with LPS/D-GalN induced liver injury

由图4可知,在属水平上,各组盲肠菌群中均以拟杆菌属(Bacteroides)为主要的菌属,其相对丰度在29.76%~49.22%;JQM组Bacteroides的相对丰度显著低于LPSD组(P<0.05),其他菌属的相对丰度各组间差异不显著(P>0.05)。
图4 金荞麦提取物对LPS/D-GalN诱导的肝脏损伤肉鸡肠道菌群属水平上组成的影响

Bacteroides:拟杆菌属;Faecalibacterium:粪杆菌属:Ruminococcus_torques_group;瘤胃球菌属torques群:Clostridia_UCG-014:梭菌纲UCG-014;Alistipes:另枝菌属;Phascolarctobacterium:考拉杆菌属;Norank_f_Ruminococcaceae:未分类瘤胃球菌科;Megamonas:巨单胞菌属;Lactobacillus:乳杆菌属;Eubacterium:真杆菌属;Others:其他菌属。

Fig.4 Effects of Fagopyrum dibotrys extract on gut microbiota composition on genus level of broilers with LPS/D-GalN induced liver injury

3 讨论

3.1 金荞麦提取物对LPS/D-GalN诱导的肝脏损伤肉鸡生长性能的影响

金荞麦是一种药饲两用草本植物,具有丰富的营养成分和药理活性成分,能很好地促进动物的生长发育,增强机体抗病能力[10]。张冰坤等[12]研究发现,将金荞麦有效成分富集制成的金荞麦粗提物能降低雌性三黄仔鸡的F/G,增加ADG,从而提高生长性能。本研究中,腹腔注射LPS/D-GalN后,LSPD组肉鸡表现为食欲不振,采食及行为活动能力降低,ADFI和ADG明显降低;在饲粮中添加金荞麦提取物基础上腹腔注射LPS/D-GalN后,肉鸡21日龄体重和ADFI在一定程度上有所提高,且ADG显著提高,表明添加金荞麦提取物对LPS/D-GalN诱导的肝脏损伤肉鸡的生长性能有一定的改善作用。其原因可能是金荞麦提取物中含有丰富的黄酮类与多酚类等抗炎活性物质,对肉鸡肝脏及肠道具有调节作用,从而改善了肉鸡的生长性能。

3.2 金荞麦提取物对LPS/D-GalN诱导的肝脏损伤肉鸡肝脏组织病理形态与结构的影响

脏器指数作为评价动物脏器功能状态的重要指标,其变化能够反映脏器的生理病理状况,可以作为组织病理学改变的重要佐证[13]。具体而言,脏器指数的减小可能暗示脏器萎缩或退行性改变等现象的存在,而脏器指数的增大则可能意味着水肿、充血、增生等病理现象的出现[14]。本研究发现,LSPD组肉鸡的肝脏指数显著升高,肉眼观察肝脏呈肿胀易碎状态,这一现象表明LPS/D-GalN刺激导致肉鸡肝脏受损。本试验中,饲粮中添加金荞麦提取物的LPS/D-GalN诱导的肝脏损伤肉鸡肝脏指数呈现出降低的趋势;从肝脏剖检变化图可以观察到,肝脏肿大现象得到了明显改善;从组织病理切片来看,LSPD组肉鸡的肝脏组织出现了显著的淋巴细胞灶性浸润、肝窦淤血以及肝细胞坏死等病理特征,而添加金荞麦提取物干预后肝脏病理变化有了一定程度的改善,减轻了肝脏中央静脉周围肝细胞坏死;此外,通过对肝脏超微结构观察发现,LSPD组肝脏细胞结构紊乱,线粒体出现扩张、肿胀,质网结构受损,添加金荞麦提取物干预后肝细胞中线粒体与内质网等细胞器损伤均有所缓解。以上结果表明,金荞麦提取物能改善LPS/D-GalN诱导的肉鸡肝脏损伤。

3.3 金荞麦提取物对LPS/D-GalN诱导的肝脏损伤肉鸡肝脏功能的影响

ALT、AST、TBIL、DBIL都是最常见的肝脏损伤生化标志物,是反映肝脏功能状态最直观指标[15]。其中,血清ALT和AST是肝细胞损伤的敏感指标,当肝脏受到刺激发生病变时,肝细胞膜的通透性增加,AST、ALT会释放到血液中引起机体发生一系列病理性反应[16],血清ALT和AST活性同时出现异常上升时,预示着肝脏功能受到破坏,肝脏受到损伤[17]。本试验中,LSPD组肉鸡血清ALT、AST活性显著升高,镜下观察显示肝窦淤血,大量炎性细胞浸润,提示LPS/D-GalN诱导造成肝脏损伤,进一步说明LPS/D-GalN诱导的肉鸡肝脏损伤模型构建成功。相反,饲粮中添加金荞麦提取物后,肉鸡血清中各项肝脏功能指标较LSPD组均有一定程度的恢复,而且显著降低了肉鸡血清中ALT活性,此试验结果证实了金荞麦提取物对肝脏功能的恢复作用。有研究发现,饲粮中添加金荞麦提取物可使蛋鸡血清ALT、AST活性降低,缓解因热应激反应造成的肝脏损伤[4],这与本研究结果基本一致。

3.4 金荞麦提取物对LPS/D-GalN诱导的肝脏损伤肉鸡血清炎症因子含量的影响

LPS与D-GalN联合作用会导致肝细胞损伤,使细胞内容物大量释放,引起炎症反应[18]。TNF-α、IL-6和IL-1β等炎症细胞因子在肝脏损伤的发生发展中起着关键作用[19]。有研究表明,LPS/D-GalN诱导的肝脏损伤的发展往往伴随着促炎细胞因子TNF-α、IL-6和IL-1β含量的上升[20]。谭娅等[21]指出,在腹腔注射LPS的小鼠饮水中添加金荞麦可以显著下调小肠中TNF-α和IL-1β的表达水平。韩芳芳等[22]研究表明,金荞麦提取物可以显著下调猪肠道上皮细胞中TNF-α等炎症细胞因子的含量。本试验结果显示,LPSD组肉鸡血清中TNF-α、IL-1β和IL-6含量显著增加,这与肝脏组织病理变化中有大量炎性细胞产生一致;而饲粮中添加金荞麦提取物则在一定程度上降低了血清中TNF-α、IL-1β和IL-6的含量,说明金荞麦提取物可以抑制LPS/D-GalN诱导的肉鸡肝脏炎症产生,从而保护肝脏。

3.5 金荞麦提取物对LPS/D-GalN诱导的肝脏损伤肉鸡肠道菌群组成的影响

Firmicutes和Bacteroidota是家禽盲肠菌群中的优势群落,能够抵抗外来细菌的定植,在能量代谢中起重要作用[23]。本试验中,Firmicutes和Bacteroidota是肉鸡肠道内相对丰度较高的菌门,相对丰度占96%以上,以JQM组最高,相对丰度占98%以上,是绝对优势菌群,其他菌属相对丰度各组间无显著差异。研究报道,适当的Firmicutes与Bacteroidota比值有助于维持肠道内的免疫平衡,减少炎症反应,通过产生短链脂肪酸和其他代谢产物调节免疫细胞的活性,从而抑制过度的炎症反应[24]。本试验中,给予LPS/D-GalN刺激会改变肉鸡肠道中Firmicutes与Bacteroidota比值,而金荞麦提取物提升了肠道菌群稳态;各组肉鸡盲肠菌群中Bacteroides、粪杆菌属(Faecalibacterium)、瘤胃球菌属torques群(Ruminococcus_torques_group)、梭菌纲UCG-014(Clostridia_UCG-014)等所占比例较高,其中以Bacteroides占比最高,相对丰度在30%~50%。肠道菌群与肝脏疾病的发生密切相关,肝脏与肠道能通过门静脉血液循环形成功能双向联通网络。在不同药物导致的肝脏损伤中,肠道菌群的变化并不完全相同,但一般都存在多样性降低、有益菌减少和致病菌增加的现象。研究表明,健康个体与非酒精性肝炎患者肠道中Bacteroidota和Firmicutes数量有显著差异,糖尿病患者肠道中Firmicutes与Bacteroidota的比值显著增高[25]。本研究也发现,与LPSD组比较,JQM组盲肠菌群中Bacteroides相对丰度显著降低,表明金荞麦提取物能调节肠道有益菌群数量,改善肉鸡肠道微生态平衡,缓解肝脏炎症,从而起到保肝护肝作用。

4 结论

本试验通过腹腔注射LPS/D-GalN成功建立了肉鸡肝脏损伤模型,并发现金荞麦提取物具有抗炎、改善肝脏功能、缓解肝脏组织病变以及调节肠道菌群结构的作用,从而缓解LPS/D-GalN诱导的肉鸡肝脏损伤。

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