畜牧兽医学报  2023, Vol. 54 Issue (9): 3784-3792. DOI: 10.11843/j.issn.0366-6964.2023.09.018    PDF    
引用本文
韩坤良, 兰伟, 胡新, 崔亚东, 孔祥峰. 复方中药超微粉对蛋鸡抗氧化性能及相关基因表达的影响[J]. 畜牧兽医学报, 2023, 54(9): 3784-3792.  
HAN Kunliang, LAN Wei, HU Xin, CUI Yadong, KONG Xiangfeng. Effects of Compound Chinese Herb Ultrafine Powder on Antioxidant Ability and Related Gene Expression in Laying Hens[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(9): 3784-3792.  
复方中药超微粉对蛋鸡抗氧化性能及相关基因表达的影响
韩坤良1,2, 兰伟1, 胡新1, 崔亚东1, 孔祥峰1,2     
1. 阜阳师范大学生物与食品工程学院, 阜阳 236037;
2. 中国科学院亚热带农业生态研究所 动物营养生理与代谢过程湖南省重点实验室, 长沙 410125
收稿日期：2022-08-30
基金项目：阜阳师范大学承接阜阳市科技专项经费市校合作项目(SXHZ2020007);湖南省科技领军人才项目(2019RS3022)
作者简介：韩坤良(1990-), 男, 河南驻马店人, 博士生, 主要从事家禽营养调控研究, E-mail: 623757876@qq.com.
通信作者：孔祥峰, 主要从事畜禽肠道微生态调控及中药饲料添加剂研发, E-mail: nnkxf@isa.ac.cn.
摘要：本文旨在研究复方中药超微粉对产蛋后期蛋鸡抗氧化性能的影响。选取307日龄的新杨黑羽蛋鸡216只, 随机分为3组, 每组8个重复, 每个重复9只。对照组饲喂基础日粮, 试验组分别在基础饲粮中添加0.5%丹参+0.25%女贞子+0.25%蒲公英(复方1)、0.3%益母草+0.2%丹参+0.25%女贞子+0.25%蒲公英(复方2)的超微粉。于试验第60和120天采集蛋鸡血浆和肝组织, 测定氧化-抗氧化指标及相关基因表达。结果发现: 与对照组相比, 试验第60天, 复方1组蛋鸡血浆和肝组织MDA含量显著降低、肝NQO1和GST的mRNA表达量显著上调(P < 0.05), 复方2组蛋鸡肝组织GSH-Px2、GSH-Px3、GSH-Px4、SOD2、SOD3、TXNRD1、TXNRD3、NQO1和HO-1的mRNA表达量显著上调(P < 0.05), 复方1组和2组蛋鸡肝组织GSH-Px、SOD和CAT活性以及T-AOC显著增加(P < 0.05);试验第120天, 复方1组蛋鸡肝组织MDA含量显著降低(P < 0.05), 复方2组蛋鸡血浆CAT活性显著增加、肝组织Nrf2和GSH-Px2的mRNA表达量显著上调(P < 0.05), 复方1组和2组蛋鸡的肝组织GSH-Px、SOD和CAT活性显著增加(P < 0.05)。与复方1组相比, 试验第60天, 复方2组蛋鸡肝组织CAT和GST的mRNA表达量显著下调, GSH-Px2、GSH-Px3、GSH-Px4、SOD2、SOD3、TXNRD1、TXNRD3、NQO1和HO-1的mRNA表达量显著上调(P < 0.05);试验第120天, 复方2组蛋鸡血浆MDA含量显著增加, 肝组织Nrf2、GSH-Px2、TXNRD1和TXNRD3的mRNA表达量显著上调(P < 0.05)。综上所述, 饲粮添加由益母草、丹参、女贞子和蒲公英组成的复方中药超微粉可通过增加血浆和肝组织抗氧化酶活性、激活肝组织相关抗氧化信号通路来增强产蛋后期蛋鸡的抗氧化能力, 且两个复方对机体抗氧化能力的调控作用具有靶标特异性。
关键词：中药    超微粉    蛋鸡    抗氧化性能    信号通路    
Effects of Compound Chinese Herb Ultrafine Powder on Antioxidant Ability and Related Gene Expression in Laying Hens
HAN Kunliang1,2, LAN Wei1, HU Xin1, CUI Yadong1, KONG Xiangfeng1,2     
1. School of Biology and Food Engineering, Fuyang Normal University, Fuyang 236037, China;
2. Hunan Provincial Key Laboratory of Animal Nutrition Physiology and Metabolic Processes, Institute of Subtropical Agroecology, Chinese Academy of Sciences, Changsha 410125, China
Corresponding author: KONG Xiangfeng, E-mail: nnkxf@isa.ac.cn.
Abstract: This study was conducted to determine the effect of compound Chinese herb ultrafine powder on the antioxidant ability of laying hens during late laying period. A total of 216 Xinyang black-feather laying hens at 307-day-old were selected and randomly divided into three groups, with eight replicates per group and nine hens per replicate. The birds in control group was fed a basal diet, and the experimental groups were supplemented with 0.5% Salvia miltiorrhiza (SM) + 0.25% Ligustrum lucidum (LL) + 0.25% Dandelion (Compound 1) and 0.3% Motherwort + 0.2% SM + 0.25% LL + 0.25% Dandelion (Compound 2) ultrafine powder, respectively. Samples of plasma and liver tissue were collected on days 60 and 120 of the trial to measure oxidation-antioxidant indexes and related gene expression. The results showed that, compared with the control group, on day 60 of the trial, the plasma and liver MDA content were decreased and the mRNA expression levels of liver NQO1 and GST were upregulated in compound 1 group (P < 0.05), the mRNA expression levels of liver GSH-Px2, GSH-Px3, GSH-Px4, SOD2, SOD3, TXNRD1, TXNRD3, NQO1 and HO-1 were up-regulated (P < 0.05) in compound 2 group, while the liver activities of GSH-Px, SOD, CAT and T-AOC were increased (P < 0.05) in both compound 1 and 2 groups; On day 120 of the trial, the liver MDA content was decreased (P < 0.05) in compound group 1, the plasma CAT activity was increased (P < 0.05) in compound 2 group, the mRNA expression levels of liver Nrf2 and GSH-Px2 were increased (P < 0.05) in compound 2 group, and the liver GSH-Px, SOD and CAT activities were increased (P < 0.05) in compound 1 and 2 groups. Compared with the compound 1 group, on day 60 of the trial, the mRNA expressions of liver CAT and GST were decreased (P < 0.05) in compound 2 group, and the liver mRNA expression levels of GSH-Px2, GSH-Px3, GSH-Px4, SOD2, SOD3, TXNRD1, TXNRD3, NQO1 and HO-1 were increased (P < 0.05) in compound 2 group; On day 120 of the trial, plasma MDA content and mRNA expressions of liver Nrf2, GSH-Px2, TXNRD1 and TXNRD3 were increased (P < 0.05) in compound 2 group. In conclusion, these findings suggested that dietary addition of the compound Chinese herb ultrafine powder composed of motherwort, SM, LL and dandelion could enhance the anti-oxidative ability of laying hens during late laying period by increasing the activity of antioxidant enzymes in plasma and liver and activating liver antioxidant-related signaling pathways. In addition, these two compounds present target specificity for the regulation of anti-oxidative capacity in the body.
Key words: Chinese herb    ultrafine powder    laying hens    antioxidant properties    signaling pathway    

机体内氧化-还原水平失衡是众多疾病的病理生理基础[1]，家禽生产中的大部分问题均与氧化应激有关。随着产蛋日龄的增加，产蛋后期蛋鸡机体内发生代谢紊乱和氧化应激[2]，对其生产性能、机体健康和产品质量等均会产生不利影响[3]。因此，通过营养手段增强机体的抗氧化性能，对保障蛋鸡正常生产具有重要意义。中药的优点包括无有害残留、毒副作用小和不易产生耐药性等，可促进动物新陈代谢、增强机体抗氧化能力，是一类绿色饲料添加剂[4]。现有研究显示，饲粮添加蒲公英可增加蛋鸡血清谷胱甘肽过氧化物酶(GSH-Px)、过氧化氢酶(CAT)和超氧化物歧化酶(SOD)活性以及总抗氧化能力(T-AOC)，降低丙二醛(MDA)含量[5]；添加女贞子提取物可显著降低肉鸡肝MDA含量，提高肝SOD活性[6]；添加丹参可增加肉鸡血清T-AOC，降低MDA含量[7]。笔者以“凉血利尿、祛瘀调经”为组方原则，选用一定比例的益母草、女贞子、蒲公英和丹参等中药材组方后进行超微粉碎，发现饲粮添加0.5%丹参+0.25%女贞子+0.25%蒲公英的超微粉后，试验1~30 d蛋鸡产蛋率显著增加7.56%、料蛋比显著降低4.98%，31~60 d产蛋率显著增加4.00%，61~90 d平均日采食量显著增加6.37%，1~120 d产蛋率显著增加5.31%；饲粮添加0.3%益母草+0.2%丹参+0.25%女贞子+0.25%蒲公英后，试验1~30和61~90 d蛋鸡平均日采食量分别显著增加3.91%和6.56%，试验1~120 d产蛋率显著增加5.22%[8]。这些复方中药超微粉是否能够通过改善产蛋后期蛋鸡机体抗氧化性能来发挥上述效果尚不清楚。因此，本文研究了复方中药超微粉对产蛋后期蛋鸡机体抗氧化性能的影响，并从抗氧化信号通路方面探讨其作用机理，为其作为饲料添加剂用于蛋鸡生产提供依据。

1 材料与方法 1.1 试验动物、分组及饲养管理

试验选用216只307日龄的健康新杨黑羽蛋鸡随机分为3组，每组8个重复，每个重复9只。试验开始前各组产蛋率和体重均无显著差异。对照组饲喂基础饲粮，试验组分别在基础饲粮中添加复方1(0.5%丹参+0.25%女贞子+0.25%蒲公英)、复方2(0.3%益母草+0.2%丹参+0.25%女贞子+0.25%蒲公英)的超微粉。试验所用中药购自阜阳药材加工厂。中药复方制剂超微粉碎后过300目筛，其主要成分为多糖、多酚、黄酮、氨基酸、脂肪酸、微量元素和色素等物质[6, 9]。基础饲粮参照NRC(1994)蛋鸡营养需要配制，其组成及营养水平见表 1。预试期7 d，正试期120 d。试验期间每天光照16 h，自由饮水，每天喂食2次(09:00、15:00)。

表 1 基础饲粮组成及营养水平(风干基础) Table 1 Composition and nutrient levels of the basal diets (air-dry basis) 
1.2 样品采集与处理

分别于试验第60和120天，每组随机选取8只体重相近且健康状态良好的蛋鸡，禁饲12 h(不禁饮水)后称重，翅静脉采血5 mL，肝素抗凝，3 500 r ·min－1离心10 min分离血浆样品－20 ℃保存，用于氧化-抗氧化指标测定；颈动脉放血处死，采集肝组织样品，液氮速冻后－80 ℃保存，用于氧化-抗氧化指标测定及相关基因表达量的分析。

1.3 血浆与肝氧化-抗氧化指标测定

称取肝组织0.100 0 g，加入9倍冰冷的PBS溶液，置于EP管中研磨，2 500 r ·min－1离心10 min，取上清液。取血浆和肝组织匀浆上清液，采用ELISA试剂盒(上海沪宇生物科技有限公司)测定GSH-Px、CAT和SOD活性，采用生化试剂盒(南京建成生物工程研究所)测定MDA含量和T-AOC，按照试剂盒说明书在酶标仪(Infinite M200 PRO)上测定。

1.4 肝组织相关基因表达水平检测

取肝组织液氮研磨，TRIzol法提取总RNA，用Nanodrop 2000微量紫外分光光度计测定总RNA浓度，用Evo M-MLV反转录试剂盒(艾科瑞生物工程有限公司)将总RNA反转录为cDNA，以β-actin基因为内参，用PCR仪(LightCycler ® 480II，Roche)检测抗氧化相关基因的mRNA表达水平。20 μL反应体系包括：2×SYBR Green Pro Taq HS Premix 10 μL，上、下游引物各0.4 μL (10 μmol ·L－1)，cDNA 0.8 μL，ddH2O 8.4 μL。PCR反应条件为：95 ℃预变性30 s；95 ℃变性5 s，60 ℃退火/延伸30 s，40个循环。所用引物由生工生物工程(上海)股份有限公司合成，引物序列见表 2。用2-△△Ct法计算mRNA的相对表达量。

表 2 实时荧光定量PCR所用引物序列 Table 2 Primer sequences used for real-time fluorescence quantitative PCR
1.5 数据分析与处理

试验数据经Excel 2016整理后采用SPSS 26.0软件进行单因素方差分析(one-way ANOVA)，并采用Duncan氏法进行多重比较。结果以“平均值±标准差”表示。P＜0.05为差异显著，0.05≤P＜0.10为有变化趋势。

2 结果 2.1 复方中药超微粉对蛋鸡血浆氧化-抗氧化指标的影响

由表 3可知，试验第60天，复方1组和2组血浆SOD活性和MDA含量显著低于对照组(P＜0.05)；试验第120天，复方2组血浆CAT活性和MDA含量显著高于对照组(P＜0.05)，复方1组血浆CAT活性和MDA含量显著低于复方2组(P＜0.05)。

表 3 复方中药超微粉对蛋鸡血浆氧化-抗氧化指标的影响 Table 3 Effects of compound Chinese herb ultrafine powder on plasma oxidant-antioxidant indexes of laying hens
2.2 复方中药超微粉对蛋鸡肝组织氧化-抗氧化指标的影响

由表 4可知，试验第60天，与对照组相比，复方1组和2组肝组织GSH-Px、SOD和CAT活性以及T-AOC显著增加(P＜0.05)，复方1组肝组织MDA含量显著降低(P＜0.05)；与复方1组相比，复方2组肝组织GSH-Px活性和T-AOC显著增加、SOD和CAT活性显著降低(P＜0.05)。试验第120天，与对照组相比，复方1组和2组肝组织GSH-Px、SOD和CAT活性显著增加(P＜0.05)，复方1组MDA含量显著降低(P＜0.05)；与复方1组相比，复方2组肝组织GSH-Px活性显著增加、CAT活性显著降低(P＜0.05)。

表 4 复方中药超微粉对蛋鸡肝组织氧化-抗氧化指标的影响 Table 4 Effects of compound Chinese herb ultrafine powder on liver oxidant-antioxidant indexes of laying hens
2.3 复方中药超微粉对蛋鸡肝组织抗氧化相关基因mRNA表达量的影响

由图 1可知，试验第60天，与对照组相比，复方1组蛋鸡肝组织NQO1和GST的mRNA表达量显著上调(P＜0.05)，复方2组肝组织GSH-Px2、GSH-Px3、GSH-Px4、SOD2、SOD3、TXNRD1、TXNRD3、NQO1和HO-1的mRNA表达量显著上调、SOD1的mRNA表达量显著下调(P＜0.05)；与复方1组相比，复方2组肝组织GSH-Px2、GSH-Px3、GSH-Px4、SOD2、SOD3、TXNRD1、TXNRD3、NQO1和HO-1的mRNA表达量显著上调，CAT和GST的mRNA表达量显著下调(P＜0.05)。试验第120天，与对照组相比，复方2组肝组织Nrf2和GSH-Px2的mRNA表达量显著上调、GSH-Px1的mRNA表达量显著下调(P＜0.05)；与复方1组相比，复方2组肝组织Nrf2、GSH-Px2、TXNRD1和TXNRD3的mRNA表达量显著上调(P＜0.05)。

A. 试验第60天肝抗氧化相关基因mRNA表达量；B.试验第60天肝抗氧化相关信号通路基因mRNA表达量；C.试验第120天肝抗氧化相关基因mRNA表达量；D.试验第120天肝抗氧化相关信号通路基因mRNA表达量。柱上不同字母表示差异显著(P＜0.05) A. mRNA expression of liver antioxidant-related gene on day 60; B. mRNA expression of liver antioxidant related signaling pathway gene on day 60; C. mRNA expression of liver antioxidant related genes on day 120; D. mRNA expression of liver antioxidant related signaling pathway genes on day 120. Bars with different letters mean significant difference (P < 0.05) 图 1 复方中药超微粉对蛋鸡肝组织抗氧化相关基因mRNA表达量的影响 Fig. 1 Effect of compound Chinese herb ultrafine powder on the liver mRNA expression of antioxidant related genes in laying hens
3 讨论

MDA是氧化应激的标志物，其含量反映了机体氧化损伤的程度；GSH-Px、SOD和CAT等抗氧化酶是机体抗氧化损伤的第一道防线，它们可共同反映机体抗氧化的潜力[10]。本试验中，饲粮添加中药复方2后血浆CAT活性显著增加，添加中药复方1和2后血浆MDA含量呈先降低后升高趋势，说明试验前期机体抗氧化性能增强，试验后期蛋鸡自由基产生较多，可能与试验后期蛋鸡体内抗氧化酶类活性下降、自由基累积引起脂质过氧化[11]、损伤细胞功能[12]有关。有报道表明，饲粮添加蒲公英[5]和益母草[13]可增加蛋鸡血浆GSH-Px、CAT、SOD和T-AOC水平，降低MDA含量。这与本试验结果不尽一致，可能与中草药的配伍和添加剂量有关，具体原因还需要进一步研究。

肝组织的抗氧化指标是反映蛋鸡机体健康状况的重要指标[14-15]。本试验中，饲粮添加中药复方1和2后肝组织GSH-Px、SOD、CAT活性和T-AOC均有所增加、MDA含量有所降低，说明肝组织清除自由基的能力增强，可能是因为益母草[16]、女贞子[17]、蒲公英[18]和丹参[19]等中药中含有的多糖、黄酮类物质可有效提高动物机体的抗氧化水平。张金汉等[20]报道，饲粮添加女贞子等中药可显著提高产蛋后期蛋鸡肝组织T-AOC、SOD、GSH-Px和CAT活性，降低MDA含量；洪玲玲等[6]研究表明，饲粮添加女贞子可增加肉鸡肝组织SOD活性、降低肝组织MDA含量，这与本研究结果一致。

Kelch样环氧氯丙烷相关蛋白-1(Keap1)/核因子E2相关因子2(Nrf2)/抗氧化反应元件(ARE)信号通路可抵抗机体的氧化应激反应，正向调控下游相关抗氧化蛋白酶的合成，被认为是机体内最重要的内源性抗氧化信号通路[21]；GPXs和TXNRD是动物体内两种重要的抗氧化系统[22]，GST、HO-1和NQO 1均是必不可少的解毒酶，在清除体内ROS等有害物质中发挥重要作用[23]。本试验中，饲粮添加中药复方1第60天肝NQO1的mRNA表达量显著上调，添加中药复方2第60天肝GSH-Px2、GSH-Px3、GSH-Px4、SOD2、SOD3、TXNRD1、TXNRD3、NQO1和HO-1的mRNA表达量显著上调，与肝组织GSH-Px活性的增加趋势一致；添加中药复方2第120天蛋鸡肝组织Nrf2和GSH-Px2的mRNA表达量显著上调，与肝组织中T-AOC、GSH-Px和CAT等抗氧化酶活性的增加趋势一致，提示机体的抗氧化性能增强。这可能是因为益母草[16]、女贞子[17]、蒲公英[18]和丹参[19]等中药中的黄酮类物质能够激活Keap1/-Nrf2/ARE、Nrf2/HO-1信号通路[24]，增强动物机体的抗氧化能力[25]。

4 结论

综上所述，在本试验条件下，饲粮添加由益母草、丹参、女贞子和蒲公英组成的复方中药超微粉可通过增加血浆和肝组织抗氧化酶活性、激活肝组织相关抗氧化信号通路增强蛋鸡的抗氧化能力，且两个复方对机体抗氧化能力的调控作用具有靶标特异性。

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(编辑   范子娟)