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Journal of Zhejiang University SCIENCE B
ISSN 1673-1581(Print), 1862-1783(Online), Monthly
2016 Vol.17 No.6 P.413-424
Magnolol pretreatment attenuates heat stress-induced IEC-6 cell injury
Abstract: Objective: Heat stress (HS) is an important environmental stressor that adversely influences livestock during the summer. The aim of this study was to investigate whether magnolol protects against HS-induced intestinal epithelial cell injury. Materials and methods: An intestinal epithelial cell line (IEC-6) was subjected to HS at 42 °C, with and without magnolol pretreatment. Cell injury was detected by monitoring lactate dehydrogenase (LDH) release. MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay was used to assess cell proliferation and viability, including identifying effective concentrations of magnolol. Flow cytometry confirmed G1-phase cell-cycle arrest and its alleviation by magnolol. Active DNA synthesis was measured by incorporation of nucleic acid 5-ethynyl-2'-deoxyuridine (EdU). G1-phase cell-cycle-related gene expression was assessed by real-time reverse transcription polymerase chain reaction (RT-PCR) and levels of G1-phase-related proteins by Western blotting. Results: HS induced IEC-6 cell injury and decreased cell viability, as demonstrated by data from LDH and MTS assays, respectively. Based on a number of criteria, IEC-6 cells subjected to HS were arrested in the G1 phase of the cell cycle. Magnolol pretreatment decreased HS-induced cell injury through relief of this cell-cycle arrest. Conclusions: Magnolol pretreatment attenuates HS-induced injury in IEC-6 cells. Magnolol is potentially promising as a protective strategy for HS in livestock.
Key words: Cell-cycle arrest, Cell injury, Heat stress, IEC-6 cell line, Magnolol
创新点：首次在大鼠小肠上皮细胞热应激模型中证明厚朴酚可明显降低细胞损伤程度，且此作用与细胞周期G1期相关。
方法：本试验采用大鼠小肠上皮细胞（IEC-6）为研究对象，将其分为五组：对照组（37 °C，5% CO2），热应激组（42 °C，3 h），热应激+厚朴酚低浓度组（5 µmol/L），热应激+厚朴酚中浓度组（10 µmol/L），热应激+厚朴酚高浓度组（20 µmol/L）。采用MTS法复制热应激模型及厚朴酚药物浓度筛选；采用流式细胞术检测热应激造成大鼠小肠上皮细胞细胞周期阻滞及不同浓度厚朴酚缓解细胞周期阻滞情况；利用电子显微镜观察热应激造成的细胞损伤情况；利用荧光电子显微镜观察EdU染色后，热应激对细胞增殖情况的影响及不同浓度厚朴酚的修复作用；采用荧光定量聚合酶链反应（PCR）技术检测热应激对细胞周期基因表达影响及不同浓度厚朴酚对细胞周期基因调节作用，采用蛋白质免疫印迹（Western blot）技术检测热应激对细胞周期蛋白表达的影响及不同浓度厚朴酚对细胞周期蛋白修复作用。
结论：流式细胞术显示热应激造成IEC-6细胞周期阻滞在G1期（96.5%），而三种浓度厚朴酚均可以缓解细胞周期阻滞现象且呈剂量依赖性（5 µmol/L 88.8%，10 µmol/L 81.0%，20 µmol/L 73.5%）。热应激导致阻滞细胞分裂的G1期基因p21、p27和Rb显著上调（P<0.01），显示细胞周期阻滞，不同浓度厚朴酚下调这三个基因表达且呈剂量依赖性；促进细胞分裂的G1期基因E2F1、CDK4和cyclin D1表达显著下调（P<0.01），显示细胞周期阻滞不能正常增值，厚朴酚有效上调这三个基因表达水平。热应激导致G1期阻滞细胞分裂蛋白p21及p27上调显著（P<0.01），厚朴酚有效下调p21及p27蛋白表达，且在20 µmol/L浓度时效果最佳；而G1期促进细胞分裂蛋白pRb、E2F1、CDK4和cyclin D1（CCND1）在热应激下表达显著下调（P<0.01），厚朴酚具有一定上调以上四个蛋白的作用，且在20 µmol/L浓度时效果最佳。厚朴酚作为天然成分药物，通过缓解热应激造成的IEC-6细胞G1期细胞周期阻滞，具有缓解热应激造成的细胞损伤能力，有望作为预防畜禽热应激的饲料添加剂。
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DOI:
10.1631/jzus.B1500261
CLC number:
S85
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On-line Access:
2024-08-27
Received:
2023-10-17
Revision Accepted:
2024-05-08
Crosschecked:
2016-05-12