研究论文 RESEARCH PAPER

竹叶黄酮对热应激诱导奶牛乳腺上皮细胞氧化损伤的保护作用

  • 李建凤 ,
  • 赵佳桢 ,
  • 沈义媛 ,
  • 关淑文 ,
  • 韩思雨 ,
  • 熊本海 ,
  • 王梦 ,
  • 楚康康 ,
  • 方洛云 ,
  • 童津津 ,
  • 蒋林树
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  • 1. 北京农学院动物科学技术学院, 奶牛营养学北京市重点实验室, 北京 102206;
    2. 中国农业科学院北京畜牧兽医研究所, 北京 100193;
    3. 首农集团, 北京 100192
李建凤(1999-),女,山西吕梁人,本科生,研究方向为反刍动物营养与免疫。E-mail:3307034271@qq.com

收稿日期: 2021-06-23

  网络出版日期: 2022-01-18

基金资助

2021年北京农学院本科生科研训练项目(KX2021015);北京市教委重点项目(20JF0008)

Protective Effects of Bamboo Leaf Flavonoids on Oxidative Damage of Bovine Mammary Epithelial Cells Induced by Heat Stress

  • LI Jianfeng ,
  • ZHAO Jiazhen ,
  • SHEN Yiyuan ,
  • GUAN Shuwen ,
  • HAN Siyu ,
  • XIONG Benhai ,
  • WANG Meng ,
  • CHU Kangkang ,
  • FANG Luoyun ,
  • TONG Jinjin ,
  • JIANG Linshu
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  • 1. Beijing Key Laboratory of Dairy Nutrition, School of Animal Science and Technology, Beijing Institute of Agriculture, Beijing 102206, China;
    2. Beijing Institute of Animal Husbandry and Veterinary Medicine, Chinese Academy of Agricultural Sciences, Beijing 100193, China;
    3. Beijing Capital Agribusiness Group, Beijing 100192, China

Received date: 2021-06-23

  Online published: 2022-01-18

摘要

本研究以竹叶黄酮(BLF)为添加剂,探究其对热应激诱导奶牛乳腺上皮细胞(BMECs)损伤的保护作用。将不添加BLF、37℃处理的BMECs设为对照组,不添加BLF、42℃处理的BMECs设为热应激组,以添加不同浓度(1、5、10 μg/mL)的BLF、42℃处理的BMECs设为试验组,每组3个重复。应用乳酸脱氢酶(LDH)法检测BLF对热应激诱导BMECs氧化损伤的影响以及流式细胞仪和试剂盒法检测BLF对热应激诱导BMECs氧化损伤后抗氧化指标的影响,并应用实时荧光定量PCR检测凋亡相关基因以及核因子红细胞相关因子2(Nrf2)信号通路中相关基因表达量的变化。结果表明:与对照组相比,热应激组BMECs活性显著下降(P<0.05),而添加1、5、10 μg/mL的BLF与热应激损伤的BMECs共培养12 h后,极显著降低了细胞毒性、早期凋亡率和总凋亡率(P<0.01),极显著降低了BMECs的线粒体膜电位(MMP)水平(P<0.01),极显著抑制了细胞内活性氧(ROS)的产生(P<0.01);与热应激组相比,BLF极显著提高了总超氧化物歧化酶(T-SOD)和谷胱甘肽过氧化物酶(GSH-Px)活性(P<0.01),而极显著降低了丙二醛(MDA)含量(P<0.01);实时荧光定量PCR结果表明,与热应激组相比,BLF极显著降低了热休克转录因子1(HSF1)、热休克蛋白70(HSP70)和热休克蛋白90(HSP90)、环氧化酶-2(COX-2)和半胱氨酸蛋白酶-3(Caspase-3)的mRNA相对表达量(P<0.01),极显著上调了Nrf2、血红素加氧酶-1(HO-1)、半胱氨酸摄取转运蛋白(xCT)、硫氧还蛋白还原1(Txnrd 1)和NADPH-醌氧化还原酶1(NQO 1)的mRNA相对表达量(P<0.01)。综上所述,BLF通过调控Nrf2信号通路中关键基因缓解热应激对BMECs的氧化损伤,且5 μg/mL BLF对热应激诱导BMECs氧化损伤的保护效果最好。

本文引用格式

李建凤 , 赵佳桢 , 沈义媛 , 关淑文 , 韩思雨 , 熊本海 , 王梦 , 楚康康 , 方洛云 , 童津津 , 蒋林树 . 竹叶黄酮对热应激诱导奶牛乳腺上皮细胞氧化损伤的保护作用[J]. 动物营养学报, 2022 , 34(1) : 626 -639 . DOI: 10.3969/j.issn.1006-267x.2022.01.057

Abstract

The objective of this study was to investigate the protective effects of bamboo leaf flavonoid (BLF) on bovine mammary epithelial cells (BMECs) response to heat stress. Experiments used BMECs treated without BLF, 37℃ as the control group and BMECs treated without BLF, 42℃ as the heat stress group, with different concentrations of BLF (1, 5, 10 g/mL), 42℃ treated BMECs as the experimental groups, with 3 replicates in each group. Lactate dehydrogenase (LDH) method was used to detect the effects of BLF on oxidative damage of BMECs induced by heat stress, as well as the reactive oxygen species (ROS) content, mitochondrial membrane potential (MMP) level and antioxidant indexes were detected. Quantitative real-time PCR was used to detect the gene expression related to apoptosis and nuclear factor erythroid 2 related factor 2 (Nrf2) signaling pathway. The results showed as follows:compared with the control group, BMECs activity decreased significantly in heat stress group (P<0.05). While the concentrations of 1、5、10 μg/mL BLF could significantly reduce the early apoptosis rate and total apoptosis rate and MMP level of BMECs (P<0.01), and the content of intracellular ROS significantly inhibited in BMECs (P<0.01). Moreover, compared with heat stress group, BLF significantly increased the activity of total superoxide disambiguase (T-SOD) and glutathione peroxidase (GSH-Px) while decreasing the content of malondialdehyde (MDA) (P<0.01). Real-time PCR showed that BLF significantly decreased the mRNA relative expression levels of heat shock transcription factor 1 (HSF1), heat shock protein 70 (HSP70), heat shock protein 90 (HSP90), cyclooxygenase-2 (COX-2) and cysteine protease-3 (Caspase-3) (P<0.01), and the mRNA relative expression levels of Nrf2, heme oxygenase-1 (HO-1), cysteine uptake transporter (xCT), thioredoxin reduction 1 (Txnrd1) and NADPH quinone oxidoreductase 1 (NQO1) significantly up-regulated compared with heat stress group (P<0.01). In conclusion, BLF alleviates the oxidative damage of BMECs induced by heat stress by regulating key genes in Nrf2 signaling pathway, and 5 μg/mL BLF has the best protective effect on the oxidative damage of BMECs induced by heat stress.

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