畜牧兽医学报  2023, Vol. 54 Issue (1): 239-251. DOI: 10.11843/j.issn.0366-6964.2023.01.022    PDF    
引用本文
周婉婷, 杨晨, 彭翠甜, 付新亮, 钟焯华, 许丹宁, 黄运茂, 田允波, 刘文俊. 白藜芦醇对急性热应激条件下鸭肝抗氧化能力和细胞凋亡的影响[J]. 畜牧兽医学报, 2023, 54(1): 239-251.  
ZHOU Wanting, YANG Chen, PENG Cuitian, FU Xinliang, ZHONG Zhuohua, XU Danning, HUANG Yunmao, TIAN Yunbo, LIU Wenjun. Effects of Resveratrol on Anti-oxidation and Anti-apoptosis of Hepatocytes of Ducks on Exposure to Acute Heat Stress[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(1): 239-251.  
白藜芦醇对急性热应激条件下鸭肝抗氧化能力和细胞凋亡的影响
周婉婷1,2, 杨晨1,2, 彭翠甜1,2, 付新亮1,2, 钟焯华3, 许丹宁1,2, 黄运茂1,2, 田允波1,2, 刘文俊1,2     
1. 仲恺农业工程学院, 广州 510225;
2. 广东省水禽健康养殖重点实验室, 广州 510225;
3. 广州市番禺区畜牧兽医站, 广州 511495
收稿日期：2022-06-23
基金项目：国家重点研发计划(2018YFE0128200);广东省现代农业水禽产业技术体系创新团队(2022KJ137)
作者简介：周婉婷(2001-), 女, 广东汕头人, 硕士生, 主要从事环境应激与动物健康养殖研究, E-mail: 2608169899@qq.com.
通信作者：刘文俊, 主要从事兽医免疫学研究, E-mail: lwjhero123@126.com.
摘要：旨在探索饲粮中添加白藜芦醇对急性热应激条件下山麻鸭肝抗氧化能力和细胞凋亡的影响，确定添加白藜芦醇对山麻鸭肝组织SIRT1信号通路、抗氧化酶系统及凋亡相关基因的作用。试验选取120只60日龄体重相近且健康的雌性山麻鸭，随机分为2组，每组60只，既对照组(C，饲喂基础饲粮)和白藜芦醇组(RES，饲喂添加400mg·kg-1白藜芦醇的基础饲粮)。两组动物均在常温(24℃±2℃)下饲养，15d后，两组动物体重无明显差异。随后分别将每组动物随机分为3组，每组20只，在39℃环控仓中分别放置0、30、60min。热应激处理后，采集鸭血清样本及肝组织，应用qRT-PCR、Western blot、TUNEL技术对SIRT1信号通路及凋亡相关基因的mRNA、蛋白水平和细胞凋亡数进行检测。采用生化法检测血清及肝中的氧化指标。结果显示，与对照组相比，白藜芦醇组HSP70和HSP90 mRNA表达有所升高。同时，白藜芦醇激活了SIRT1途径，增加了下游PGC-1α、TFAM mRNA表达，显著增加了热应激60min条件下Nrf1、Nrf2核蛋白的表达。白藜芦醇提高了血清中T-AOC水平，在热应激60min时，白藜芦醇显著提高了血清中SOD、CAT、AIHR的水平并显著降低MDA的含量。在同一热应激条件下，RES组肝中T-AOC、CAT、GSH-Px均高于C组，RES组MDA的含量显著低于C组。在急性热应激条件下，RES组的肝细胞凋亡数显著低于C组，且白藜芦醇降低了Bax、Bak1、Bax/Bcl-2 mRNA的表达量，同时增加了Bcl-2蛋白的表达量，降低了Bax、Caspase3、Bax/Bcl-2蛋白的表达量。综上所述，饲料中添加400mg·kg-1白藜芦醇可以激活鸭肝SIRT1信号通路，使促凋亡基因的表达量下降，抗凋亡基因的表达量增加。白藜芦醇可提高鸭肝的抗氧化能力和抗细胞凋亡的能力，能够改善急性热应激对鸭肝的氧化损伤。
关键词：白藜芦醇    热应激    抗氧化    凋亡    
Effects of Resveratrol on Anti-oxidation and Anti-apoptosis of Hepatocytes of Ducks on Exposure to Acute Heat Stress
ZHOU Wanting1,2, YANG Chen1,2, PENG Cuitian1,2, FU Xinliang1,2, ZHONG Zhuohua3, XU Danning1,2, HUANG Yunmao1,2, TIAN Yunbo1,2, LIU Wenjun1,2     
1. Zhongkai University of Agriculture and Engineering, Guangzhou 510225, China;
2. Guangdong Key Laboratory of Waterfowl Health Breeding, Guangzhou 510225, China;
3. Animal Husbandry and Veterinary Station, Panyu District, Guangzhou 511495, China
Corresponding author: LIU Wenjun, E-mail: lwjhero123@126.com.
Abstract: This study was conducted to explore the effects of dietary resveratrol supplementation on liver anti-oxidant capacity and anti-apoptosis of Shanma ducks under acute heat stress, and to determine the effects of resveratrol supplementation on SIRT1 signaling pathway, antioxidant enzyme system and apoptosis related genes in the liver of Shanma ducks. A total of 120 healthy female Shanma ducks with similar weight at 60 days old were randomly divided into two groups, 60 in each group. The control (C) group was fed a basic diet, and the resveratrol (RES) group was fed a basic diet supplemented with 400 mg·kg-1 resveratrol. Animals in two groups were kept at a temperature of (24±2) ℃. After 15 days, there was no significant difference in the weight of the animals between the two groups. Then the animals in each group were randomly divided into 3 groups, 20 in each group, and placed in an artificial climate room at 39 ℃ for 0, 30, and 60 min, respectively. After heat stress treatment, duck serum samples and liver tissues were collected, and the mRNA, protein level and apoptosis number of SIRT1 signal pathway and apoptosis related genes were detected using qRT-PCR, Western blot and TUNEL techniques. Biochemical method was adopted to detect the oxidation index in serum and liver. The results showed that the mRNA expression of HSP70 and HSP90 in RES group was higher than those in control group. At the same time, resveratrol activated the SIRT1 pathway and increased the mRNA expressions of downstream PGC-1α and TFAM, and significantly increased the nuclear proteins expression of Nrf1 and Nrf2 under heat stress for 60 min. Resveratrol increased the levels of T-AOC in serum. Resveratrol significantly increased the levels of SOD, CAT, AIHR and decreased the content of MDA under heat stress for 60 min, and under the same heat stress condition, the T-AOC, CAT and GSH-Px in liver of RES group were higher than those of C group, while MDA content in RES group was lower than that in C group. Under the condition of acute heat stress, the number of hepatocyte apoptosis in RES group was significantly lower than that in C group, and resveratrol decreased the mRNA expression of Bax, Bak1, Bax/Bcl-2, increased the protein expression of Bcl-2, and decreased the protein expression of Bax, Caspase3, Bax/Bcl-2. In conclusion, the addition of 400 mg·kg-1 resveratrol to the diet can activate the SIRT1 signaling pathway in duck liver, reduce the expression of pro-apoptotic genes and increase the expression of anti-apoptotic genes. Resveratrol can improve the anti-oxidant capacity and anti-apoptosis ability of duck liver, and can improve the oxidative damage of duck liver caused by acute heat stress.
Key words: resveratrol    heat stress    antioxidant    apoptosis    

热应激导致家禽生产性能、采食量和日增重下降，严重时可导致动物的死亡[1-3]。肝是重要的新陈代谢器官，调节脂质和碳水化合物的代谢[4]。肝受外界压力的影响会破坏整个系统的代谢稳态[5]。研究证实，热应激会加速脂肪生成并增加肝系统的负担，显著增加肝重量和甘油三酯含量[6-8]。研究表明，急性热应激会导致肝抗氧化酶系统紊乱[3, 9-11]，长期热应激会降低抗氧化酶活性[12-13]。热应激导致的细胞氧化应激在促进肝细胞凋亡和坏死中发挥着重要作用。提高肝抗氧化能力、减缓肝细胞的凋亡是缓解家禽养殖过程中热应激问题的重要研究目标。

白藜芦醇具有抗炎、抗氧化、抗衰老、抗癌等多种功能，受到广泛关注[14-18]。在减抗、替抗的趋势下，白藜芦醇具有良好的应用前景。白藜芦醇是一种天然的沉默信息调节因子1(SIRT1)信号通路激活剂[19]。SIRT1信号通路可直接或间接影响细胞的氧化还原功能，SIRT1的激活可以促使过氧化物酶体增殖物激活受体γ共激活因子1α (PGC-1α) 去乙酰化[20]。PGC-1α作为一种细胞内重要的核受体转录辅激活因子，通过激活核呼吸因子1(Nrf1)和结合线粒体转录因子A(TFAM)启动子，调节细胞线粒体生物合成及氧化代谢[21]。研究表明，饲粮中添加白藜芦醇可以有效改善家禽日增重下降，提高肉品质[22]。白藜芦醇可使促凋亡基因表达量下降，抗凋亡基因表达量上升，继而缓解细胞凋亡的发生，同时，白藜芦醇具有缓解氧化损伤的作用，可提高机体抗氧化酶SOD、CAT、GSH-Px的活性，保护肠道健康[22-23]。

本试验研究了白藜芦醇对急性热应激蛋鸭肝中热休克蛋白70(HSP70)和热休克蛋白90(HSP90)表达的影响，并进一步检测了SIRT1信号通路基因的表达水平、血清和肝的抗氧化酶活性及凋亡相关基因的表达水平。本研究结果有助于了解急性热应激对肝的影响，明确饲粮中添加白藜芦醇对山麻鸭的作用效果，为白藜芦醇应用于缓解家禽热应激问题提供理论依据。

1 材料与方法 1.1 动物与试验分组

试验动物购自广东省农业科学院动物科学研究所，饲养在仲恺教学科研基地。选择120只健康的60日龄雌性山麻鸭，随机分为2组，每组60只置于常温((24±2) ℃)下饲养。对照组(C组)饲喂基础饲粮，白藜芦醇组(RES组)饲喂添加400 mg·kg-1白藜芦醇的试验饲粮。分开饲喂15 d后，测得C组体重平均值为(1.03±0.04) kg，RES组体重平均值为(1.13±0.04) kg，两组鸭体重无明显差异，随后将两组鸭随机分为3组(每组20只)分别放置于39 ℃环控仓中0、30、60 min。热应激处理后采集血清样本，按试验动物的原则处死后，屠宰，解剖后采集肝组织，-80 ℃保存，用于后续试验。

1.2 试验试剂与仪器

主要试剂：白藜芦醇购自阿拉丁(R107315)；BCA蛋白浓度测定试剂盒、总抗氧化能力检测试剂盒(ABTS快速法)(T-AOC)、总SOD活性检测试剂盒(WST-8法)(SOD)、过氧化氢酶检测试剂盒(CAT)、脂质氧化检测试剂盒(MDA)、TUNEL细胞凋亡检测试剂盒(绿色荧光)均购自碧云天；谷胱甘肽过氧化物酶测试盒(GSH-Px)和羟自由基测定试剂盒(AIHR)均购自南京建成有限公司；RNAiso Plus、PrimeScriptTMRT reagent Kit with gDNA Eraser (Perfect Real Time) 均购自TaKaRa；2×RealStar Green Fast Mixture with ROX Ⅱ购自GenStar；MinuteTM新鲜/冷冻组织胞质胞核分离试剂盒、Bcl-2 Rabbit pAb、Caspase-3 Rabbit pAb、Nrf2 Rabbit pAb、Nrf2 Rabbit pAb、PCNA Rabbit pAb、Donkey Anti-Rabbit IgG(H+L)、Donkey Antin-Mouse IgG(H+L)均购自莱恩生物科技有限公司。Bax Rabbit pAb购自万类生物科技有限公司。

主要仪器：Thermo ScientificTM MultiskanTM FC酶标仪、QuantStudioTM 7 Flex实时荧光定量PCR系统、SorvallTM LegendTM Micro 21R微量离心机、VeritiTM 96孔热循环仪均购自赛默飞世尔科技有限公司。

1.3 抗氧化能力和MDA水平检测

使用血清样品和肝组织匀浆上清液检测抗氧化指标T-AOC、SOD、CAT、GSH-Px、AIHR和MDA。检测操作严格按照试剂盒说明书进行。

1.4 RNA提取和qRT-PCR分析

采用RNAiso Plus从肝提取总RNA，使用PrimeScriptTM RT reagent Kit with gDNA Eraser (Perfect Real Time)将RNA逆转录为cDNA。所有引物由生工(北京)合成纯化(表 1)。反应体积为20 μL：10 μL 2×RealStar Green Fast Mixture with ROX Ⅱ，上、下游引物各0.4 μL，1 μL的cDNA模板和8.2 μL的DEPC水。使用QuantStudioTM 7 Flex实时荧光定量PCR系统进行qRT-PCR检测。使用β-actin作为内参基因，通过2-ΔΔCT法比较组间mRNA相对表达水平。

表 1 引物序列 Table 1 Primer sequence
1.5 蛋白表达水平的检测

提取肝组织的总蛋白，利用MinuteTM新鲜/冷冻组织胞质胞核分离试剂盒提取肝组织核蛋白Nrf1及Nrf2，用BCA蛋白浓度测定试剂盒测定蛋白浓度，SDS变性后，将蛋白上样于SDS-PAGE胶，进行电泳、转膜、封闭、一抗孵育、二抗孵育、ECL试剂盒显色，并在化学发光仪中进行曝光，拍照并保存，利用Image J软件对蛋白条带进行灰度值分析。

1.6 TUNEL染色观察肝细胞凋亡

用碧云天生产的免疫染色固定液(P0098)或4%多聚甲醛固定细胞30~60 min；PBS洗涤2次，每次10 min；加入含碧云天生产的免疫染色强力通透液(P0097)，室温孵育5 min；用PBS清洗后加入TUNEL检测液，37 ℃避光孵育60 min；PBS清洗3次，用抗荧光淬灭封片液封片后荧光显微镜下观察。

1.7 统计分析

采用GraphPad Prism 7.1 (GraphPad Software Inc.，USA)进行统计分析。采用双因素方差分析。所有试验数据均采用“平均值±标准差(S.E.M.)”进行分析。*表示P < 0.05，**表示P < 0.01，***表示P < 0.001，差异有统计学意义。

2 结果 2.1 白藜芦醇对热应激鸭肝HSP70和HSP90 mRNA表达的影响

热休克蛋白是评价动物热应激状态的重要指标，为了验证鸭急性热应激模型的建立和白藜芦醇在此过程中的作用，本研究用RT-qPCR检测了肝中HSP70和HSP90 mRNA的表达。如图 1所示，急性热应激刺激了肝组织中HSP70和HSP90 mRNA的表达。RES组中肝HSP70和HSP90 mRNA的表达水平相较于对照组均有所升高，且在热应激60 min时，RES组中HSP70和HSP90 mRNA的表达水平相较于对照组具有极显著的升高(P < 0.001)。

A、B.白藜芦醇对热休克蛋白HSP70、HSP90基因表达的RT-qPCR结果。*. P＜0.05，**. P＜0.01，***. P＜0.001, n=6，下同 A, B. RT-qPCR results of resveratrol on the expression of HSP70 and HSP90 genes. *. P < 0.05, **. P < 0.01, ***. P < 0.001, n=6, the same as below 图 1 白藜芦醇对HSP70、HSP90 mRNA的影响 Fig. 1 Effect of resveratrol on HSP70 and HSP90 mRNA levels
2.2 白藜芦醇对热应激鸭肝SIRT1通路相关基因mRNA表达的影响

为明确添加白藜芦醇是否能活化肝细胞SIRT1途径，采用RT-qPCR方法检测了肝中SIRT1通路相关基因mRNA的表达水平。研究发现，在热处理时间相同的情况下，RES组的SIRT1、PGC-1α、Nrf1、TFAM mRNA的表达量均高于对照组。热应激增强了SIRT1、PGC-1α、TFAM mRNA的表达。在热应激30 min时，RES组中SIRT1 mRNA表达水平显著高于对照组(P < 0.05)，RES组中Nrf1 mRNA极显著高于对照组(P < 0.01)。但是，荧光定量结果显示，Nrf2 mRNA变化无显著性差异，在热应激条件下，RES组较对照组的表达水平低(图 2)。

A、B、C、D、E. 白藜芦醇对SIRT1、PGC-1α、Nrf1、TFAM、Nrf2基因表达的RT-qPCR结果 A, B, C, D, E. RT-PCR results of resveratrol on SIRT1, PGC-1α, Nrf1, TFAM, Nrf2 genes expression 图 2 白藜芦醇对SIRT1通路相关基因mRNA的影响 Fig. 2 Effect of resveratrol on related genes mRNA in SIRT1 pathway
2.3 白藜芦醇对Nrf1、Nrf2核蛋白表达的影响

Nrf1、Nrf2活化后，进入细胞核促进一系列基因的转录。为了进一步确认通路的活化，提取了核蛋白，并通过Western blot检测了Nrf1、Nrf2的核蛋白水平。如图 3所示，以PCNA为内参蛋白，在热应激60 min条件下，RES组的Nrf1核蛋白水平显著高于对照组，RES组的Nrf2核蛋白水平极显著高于对照组。

A.白藜芦醇对Nrf2、Nrf1蛋白表达的Western blot结果；B、C.白藜芦醇对NRF2、NRF1蛋白表达影响的分析结果 A.Western blot results of resveratrol on the Nrf2 and Nrf1 proteins expression; B, C. Effect of resveratrol on the expression of Nrf2 and Nrf1 proteins 图 3 白藜芦醇对Nrf1、Nrf2核蛋白表达的影响 Fig. 3 Effects of resveratrol on the expression of Nrf1 and Nrf2 nuclear proteins
2.4 白藜芦醇对热应激鸭血清抗氧化能力的影响

血清抗氧化酶活性是判断白藜芦醇对鸭抗氧化能力影响的重要指标，使用ELISA检测了血清中抗氧化酶活性和MDA的含量。研究结果发现，热应激增加了血清中T-AOC；同一热应激条件下，RES组T-AOC、SOD酶活性、AIHR均高于对照组；热应激60 min时，RES组SOD酶活性、CAT酶活性、AIHR显著高于对照组；对照组和RES组的GSH-Px酶活性在热应激条件下并无显著变化；急性热应激会造成对照组MDA含量增加，但同一热应激条件下，RES组MDA含量比对照组低，特别是热应激60 min RES组MDA含量显著低于对照组(图 4)。

A.血清中的总抗氧化能力；B.血清中的超氧化物歧化酶活性；C.血清中过氧化氢酶活性；D.血清中谷胱氨肽过氧化物酶活性；E.血清中抑制羟自由基能力；F.血清中丙二醛含量 A. The T-AOC level in serum; B. The SOD activity in serum; C. The CAT activity in serum; D. The GSH-Px activity in serum; E. The AIHR activity in serum; F. The MDA content in serum 图 4 白藜芦醇对不同时长急性热应激血清氧化指标的影响 Fig. 4 Effect of resveratrol on serum oxidative indices under different duration of acute heat stress
2.5 白藜芦醇对热应激鸭肝抗氧化能力的影响

为了进一步探究白藜芦醇对鸭肝抗氧化能力的影响，检测了肝匀浆的抗氧化酶活性和MDA含量。结果如图 5所示，热应激会降低对照组肝中T-AOC含量和CAT酶活性。SOD的活性在热应激30 min有所增加，但在热应激60 min时降低。研究发现，白藜芦醇可以提高肝GSH-Px酶活性。在热应激30 min时，白藜芦醇显著提高了AIHR。此外，相比对照组，RES组T-AOC、CAT的水平在热应激状态下恢复更为迅速。急性热应激条件下，白藜芦醇降低了鸭肝中MDA的含量。

A.肝中的总抗氧化能力；B.肝中的超氧化物歧化酶活性；C.肝中过氧化氢酶活性；D.肝中谷胱氨肽过氧化物酶活性；E.肝中抑制羟自由基能力；F.肝中丙二醛含量 A. The T-AOC level in the liver; B. The SOD activity in the liver; C. The CAT activity in the liver; D. The GSH-Px activity in the liver; E. The AIHR activity in the liver; F. The MDA content in the liver 图 5 白藜芦醇对急性热应激肝氧化指标的影响 Fig. 5 Effect of resveratrol on liver oxidative indices under different duration of acute heat stress
2.6 白藜芦醇对热应激条件下蛋鸭肝细胞凋亡的影响

为了验证白藜芦醇在急性热应激条件下缓解肝细胞凋亡的作用，采用TUNEL法检测肝细胞凋亡的情况。如图 6所示，在对照组中，急性热应激组较无热应激组肝细胞凋亡数有极显著增加；在急性热应激条件下，与对照组相比，白藜芦醇组的肝细胞凋亡数极显著下降。

A.肝细胞TUNEL检测结果(50 μm), 绿色荧光表示细胞凋亡，蓝色荧光表示正常细胞; B.TUNEL染色细胞与正常细胞比例 A. Liver cell TUNEL assay results(50 μm), green fluorescence indicates cell apoptosis, blue fluorescence indicates normal cells; B. Proportion of TUNEL-stained cells to normal cells 图 6 白藜芦醇对热应激诱导的肝细胞凋亡的影响 Fig. 6 Effect of resveratrol on hepatocyte apoptosis induced by heat stress
2.7 白藜芦醇对蛋鸭肝细胞凋亡相关基因mRNA表达的影响

为进一步探究急性热应激及白藜芦醇对肝细胞凋亡的影响，采用RT-qPCR检测了肝中Bcl-2、Bax、Caspase3、Bak1、Cyt c、Bax/Bcl-2mRNA的表达。如图 7所示，热应激促进了Caspase-3的mRNA的表达；在同一热应激条件下，RES组的Bax、Bak1和Bax/Bcl-2的mRNA的表达量低于对照组；在急性热应激条件下，RES组Bcl-2和Caspase3 mRNA的表达量高于对照组。

A、B、C、D、E. 白藜芦醇对凋亡相关基因表达的RT-qPCR结果; F. 白藜芦醇对Bax/Bcl-2基因表达的影响 A, B, C, D, E. RT-qPCR results of resveratrol on the expression of apoptosis related genes; F. Effect of resveratrol on Bax/Bcl-2 gene expression 图 7 白藜芦醇对肝细胞凋亡相关基因mRNA表达的影响 Fig. 7 Effect of resveratrol on mRNA expression of apoptosis related genes in hepatocytes
2.8 白藜芦醇对热应激蛋鸭肝细胞凋亡相关蛋白表达的影响

本研究用Western blot检测了肝组织细胞凋亡相关蛋白的表达量。如图 8所示，同一热应激条件下，RES组Bax、Caspase3、Bax/Bcl-2的蛋白表达量均低于对照组；热应激30及60 min条件下，RES组Bcl-2蛋白表达量高于对照组。

A. 白藜芦醇对Bax、Bcl-2、caspase3蛋白表达的Western blot结果；B、C、D.白藜芦醇对Bcl-2、Bax、caspase3蛋白表达的影响；E. 白藜芦醇对Bax/Bcl-2蛋白表达的影响 A.Western blot results of resveratrol on the Bax, Bcl-2 and caspase3 proteins expression; B, C, D. Effect of resveratrol on the expression of Bcl-2, Bax and caspase3 proteins; E. Effect of resveratrol on Bax/bcl-2 protein expression 图 8 白藜芦醇对热应激蛋鸭肝细胞凋亡相关蛋白表达的影响 Fig. 8 Effect of resveratrol on the expression of apoptosis related proteins in heat stressed duck hepatocytes
3 讨论

热应激造成细胞氧化应激导致组织损伤，致使家禽生产性能下降[24-29]。白藜芦醇具有极强的抗氧化和抗炎能力，是具有潜力的抗热应激药物。本试验以雌性山麻鸭为研究对象，旨在揭示白藜芦醇对鸭急性热应激状态下抗氧化能力及抗细胞凋亡的影响和调控机制，为白藜芦醇缓解家禽热应激的应用提供理论依据。

热休克蛋白是热应激的关键指标，并在缓解压力中起重要作用[30]。HSP70和HSP90是重要的分子伴侣，前者在蛋白稳态中起着重要作用，后者参与促进几种受体和激酶的成熟[31]。白藜芦醇显著增加了HSP70和HSP90 mRNA的表达[12]，这提示白藜芦醇可能通过诱导HSPs的表达保护家禽因急性热应激引起的肝损伤。本研究发现，白藜芦醇显著升高了急性热应激下肝中SIRT1、PGC-1α、Nrf1和TFAM mRNA的表达水平，也显著升高了Nrf1、Nrf2的核蛋白水平。白藜芦醇被认为是SIRT1的天然化学激活剂。有研究表明，白藜芦醇可以提高家禽肾SIRT1、PGC-1α、Nrf1和TFAM的表达，调节和保护线粒体功能[32]。TFAM的表达可以增加在抗氧化机制中至关重要的mtDNA[33]。此外，SIRT1具有调控HSF1的作用，研究发现抑制SIRT1还可以降低HSP70和HSP90的表达[34]。这些结果说明，白藜芦醇可以通过促使SIRT1通路活化增强肝抗氧化能力，缓解热应激导致的氧化应激。

家禽暴露在高温环境会导致线粒体损伤产生过量活性氧，引起脂质、蛋白质和DNA氧化损伤，导致氧化应激破坏细胞稳态[9, 35-36]。肝是家禽氧化代谢和脂肪合成的重要器官[1-8]。SOD、CAT和GSH-Px是重要的抗氧化系统，它们可以将自由基和过氧化氢最终分解为水和分子氧[37-38]。本研究发现，急性热应激可以造成血清T-AOC、GSH-Px活性增加和和肝T-AOC、SOD、CAT、GSH-Px活性降低。MDA含量在受到热应激后均增加。这说明急性热应激会破坏自由基和过氧化氢产生和清除间的平衡。有趣的是，白藜芦醇可以有效改善热应激导致的家禽氧化应激，提高T-AOC、GSH-Px、CAT和AIHR，并降低MDA[9, 22-25, 30-35, 39]。有研究表明，白藜芦醇可以上调HO-1表达，激活Nrf-2，从而减轻心肌缺血再灌注和缺氧缺血性脑损伤引起的氧化应激和炎症[40]。因此，将白藜芦醇作为饲料添加剂具有显著的抗氧化效果。

内源性细胞凋亡的过程是由Bax等线粒体蛋白介导的，有研究表明，Bax/Bcl-2两蛋白的比例决定了细胞凋亡的强弱[39, 41]，本研究发现，饲料中添加白藜芦醇会使Bax/Bcl-2蛋白的表达量下降，同时也会使促凋亡蛋白Bak1、Cyt c、Caspase3 mRNA的表达量有所减少，这表明白藜芦醇对热应激条件下的肝细胞具有一定的保护作用。

4 结论

综上所述，急性热应激对山麻鸭肝的氧化-抗氧化系统产生负面影响，引起氧化应激。饲粮中添加400 mg·kg-1白藜芦醇可以通过上调HSP70和HSP90 mRNA，激活SIRT1信号通路，减少促凋亡基因，增加抗凋亡基因表达，增强肝的抗氧化及抗细胞凋亡的能力。研究结果说明，白藜芦醇可以提高鸭肝的抗氧化能力和抗细胞凋亡的能力，有效改善急性热应激条件下鸭肝的氧化损伤。

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(编辑   郭云雁)