动物营养学报 >

2020 , Vol. 32 >Issue 4: 1867 - 1874

DOI: https://doi.org/10.3969/j.issn.1006-267x.2020.04.046

分子与细胞营养 MOLECULAR AND CELLULAR NUTRITION

热休克蛋白70上调表达对热应激猪小肠上皮细胞凋亡的影响

  • 孙汇蕾 ,
  • 仲庆振 ,
  • 李明晔 ,
  • MOLAWA M N
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  • 1. 吉林农业大学动物科技学院, 长春 130118;
    2. 动物生产及产品质量安全教育部重点实验室, 长春 130118;
    3. 吉林省动物营养与饲料科学重点实验室, 长春 130118
孙汇蕾(1995-),女,河北石家庄人,硕士研究生,动物营养与饲料科学专业。E-mail:1160869483@qq.com

收稿日期: 2019-09-30

  网络出版日期: 2020-04-16

基金资助

国家自然科学基金青年基金项目（31601982）

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Effects of Up-Regulated Expression of Heat Shock Protein 70 on Apoptosis in Intestinal Epithelial Cells of Heat-Stressed Piglets

  • SUN Huilei ,
  • ZHONG Qingzhen ,
  • LI Mingye ,
  • MOLAWA M N
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  • 1. College of Animal Science and Technology, Jilin Agricultural University, Changchun 130118, China;
    2. Key Laboratory of Animal Production and Product Quality and Safety, Ministry of Education, Changchun 130118, China;
    3. Jilin Province Key Laboratory of Animal Nutrition and Feed Science, Changchun 130118, China

Received date: 2019-09-30

  Online published: 2020-04-16

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摘要

本试验以谷氨酰胺作为热休克蛋白70（HSP70）诱导剂，从细胞凋亡线粒体信号转导途径探讨HSP70对热应激猪小肠上皮细胞（IPEC-J2）凋亡相关因子的影响。试验以体外培养的猪小肠上皮细胞为模型，分别加入不同浓度（1、2、4、6、8、10 mmol/L）谷氨酰胺的培养基中培养24 h，提取总RNA和总蛋白，用实时荧光定量PCR和Western blot检测HSP70 mRNA和蛋白的相对表达量，筛选出谷氨酰胺诱导HSP70表达的最佳浓度。取对数生长期的细胞分为3组，分别为对照组（Con组，37℃培养12 h）、热应激组（Hs组，42℃培养12 h）、谷氨酰胺组（Gln组，6 mmol/L谷氨酰胺、42℃培养12 h）。采用Annexin V/PI双染法检测细胞凋亡水平，实时荧光定量PCR和Western blot分别检测HSP70、凋亡酶激活因子-1（Apaf-1）、半胱氨酸天冬氨酸蛋白水解酶-9（Caspase-9）、半胱氨酸天冬氨酸蛋白水解酶-3（Caspase-3）、B淋巴细胞瘤-2（Bcl-2）的mRNA和蛋白的相对表达量。结果表明：谷氨酰胺对HSP70上调表达呈先上升后下降的趋势，6 mmol/L的谷氨酰胺诱导时HSP70 mRNA和蛋白相对表达量最高，显著高于其他浓度（P<0.05），作为后续试验中Gln组的诱导条件。与Con组相比，Hs组细胞凋亡率极显著增加（P<0.01），HSP70、Apaf-1、Caspase-3、Caspase-9 mRNA和蛋白的相对表达量极显著升高（P<0.01），而Bcl-2 mRNA和蛋白的相对表达量极显著降低（P<0.01）。与Hs组相比，Gln组细胞凋亡率极显著降低（P<0.01），但仍极显著高于Con组（P<0.01）；Gln组HSP70 mRNA和蛋白的相对表达量极显著升高（P<0.01）；Gln组Apaf-1、Caspase-3、Caspase-9 mRNA和蛋白的相对表达量极显著降低（P<0.01），但极显著高于Con组（P<0.01）；Gln组Bcl-2 mRNA和蛋白的相对表达量极显著升高（P<0.01），但极显著低于Con组（P<0.01）。由此可见，上调HSP70的表达可有效缓解热应激导致的猪小肠上皮细胞凋亡。

关键词： 热休克蛋白70; 猪小肠上皮细胞; 凋亡; 线粒体通路; 半胱氨酸天冬氨酸蛋白水解酶

本文引用格式

孙汇蕾 , 仲庆振 , 李明晔 , MOLAWA M N . 热休克蛋白70上调表达对热应激猪小肠上皮细胞凋亡的影响[J]. 动物营养学报, 2020 , 32(4) : 1867 -1874 . DOI: 10.3969/j.issn.1006-267x.2020.04.046

Abstract

This experiment was used glutamine as a heat shock protein 70 (HSP70) inducer, to investigate the effects of HSP70 on intestinal epithelial cells (IPEC-J2) of heat-stressed piglets through mitochondrial signal transduction pathway of apoptosis. In this experiment, IPEC-J2 cells cultured in vitro were used as the model, and different concentrations (1, 2, 4, 6, 8, 10 mmol/L) of glutamine were added to the culture medium for 24 h, the total RNA and total protein were extracted. The relative expressions of HSP70 mRNA and protein were detected using real-time fluorescent quantitative PCR and Western blot, and the optimal concentration of glutamine induced HSP70 expression was screened. The cells in logarithmic growth period were divided into three groups:control group (Con group, 37℃ cultured 12 h), heat stress group (Hs group, 42℃ cultured 12 h) and glutamine group (Gln group, 6 mmol/L glutamine and 42℃ cultured 12 h). Annexin V/PI double staining was used to detect the level of apoptosis, real-time fluorescence quantitative PCR and Western blot were used to detect the relative expressions of HSP70, apoptotic protease activating facter-1 (Apaf-1), cysteinyl aspartate specific proteinase-9 (Caspase-9), cysteinyl aspartate specific proteinase-3 (Caspase-3), B-cell lymphoma-2 (Bcl-2) mRNA and protein. The results showed that the up-regulated expression of HSP70 by glutamine firstly increased and then decreased, the relative expression of HSP70 mRNA and protein induced by 6 mmol/L glutamine was the highest, and significantly higher than that of other concentration (P<0.05), which served as the induction condition of Gln group in the follow-up experiment. Compared with the Con group, the apoptosis rate of Hs group was significantly increased (P<0.01), the relative expressions of HSP70, Apaf-1, Caspase-3, Caspase-9 mRNA and protein were significantly increased (P<0.01), while the relative expression of Bcl-2 mRNA and protein was significantly decreased (P<0.01). Compared with the Hs group, the apoptosis rate of Gln group was significantly decreased (P<0.01), and still significantly higher than that of the Con group (P<0.01); the relative expression of HSP70 mRNA and protein of Gln group was significantly increased (P<0.01); the relative expressions of Apaf-1, Caspase-3, Caspase-9 mRNA and protein of Gln group were significantly decreased (P<0.01), but significantly higher than those of the Con group (P<0.01); the relative expression of Bcl-2 mRNA and protein of Gln group was significantly increased (P<0.01), but significantly lower than that of the Con group (P<0.01). In conclusion, the up-regulated expression of HSP70 can effectively alleviate the apoptosis of intestinal epithelial cells of piglets induced by heat stress.

Key words： heat shock protein 70; IPEC-J2 cells; apoptosis; mitochondrial pathway; Caspase

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