Effects of red blood cell supernatants on hypoxia / reoxygenation injury in H 9 C 2 cells
Effects of red blood cell supernatants on hypoxia / reoxygenation injury in H 9 C 2 cells
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发表时间：
2018
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通讯作者：
Fengyan Fan;Liping Sun;Dongqing Zhang;Liguo Zhu;Shuying Wang;Deqing Wang
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作者：
Fengyan Fan;Liping Sun;Dongqing Zhang;Liguo Zhu;Shuying Wang;Deqing Wang
背景：红细胞(RBC)在储存过程中会发生形态、功能和代谢的变化。尽管多项研究发现，较长的储存时间与不良临床结局的风险增加有关，特别是在危重患者中，但这种关系尚未得到充分探索。此外，红细胞上清液对模拟危重病人的细胞模型的影响的研究很少。本研究旨在探讨红细胞培养上清对缺氧/复氧(H/R)损伤的H9C2细胞可能的细胞毒作用。方法：用保存0天和35天的红细胞上清液处理缺氧处理的H9C2细胞。细胞复氧孵育2小时。我们用细胞计数试剂盒(CCK-8)检测细胞存活率，用吖啶橙/溴化乙锭(AO/EB)染色和Annexin V-FITC/PI法检测细胞凋亡率，用JC-1染色检测线粒体膜电位，用Enzy light ATP检测试剂盒检测ATP水平。结果：与正常细胞相比，H/R损伤的H9C2细胞存活率显著降低，细胞凋亡率显著增加，基质金属蛋白酶显著降低。经DMEM处理的H9C6细胞与保存0天或35天的RBC上清液相比，细胞存活率、凋亡率、基质金属蛋白酶和三磷酸腺苷水平均无显著差异，0天组和35天组之间也无显著差异。结论：保存0天或35天的RBC上清液对H/R损伤的H9C2细胞的存活率、凋亡率、基质金属蛋白酶和三磷酸腺苷水平均无影响。
Background: During storage, Red Blood Cell (RBC) undergo changes in morphology, function, and metabolism. Although multiple studies found that longer duration of storage was associated with increased risks of adverse clinical outcomes, especially in critically ill patients, this relationship has not been fully explored. Moreover, the effects of RBC supernatants on cell models simulating critically ill patients have been studied rarely. This study aimed to investigate the possible cytotoxic effects of RBC supernatants on H9C2 cells subjected to Hypoxia/Reoxygenation (H/R) injury. Method: Hypoxia-treated H9C2 cells were treated with RBC supernatants that had been stored for 0 or 35 days. Cells were reoxygenated and incubated for 2 hours. We analyzed cell viability using the Cell Counting Kit-8 (CCK-8) assay, apoptosis using Acridine Orange/Ethidium Bromide (AO/EB) staining and an annexin V-FITC/PI assay, mitochondrial membrane potential (MMP) using a JC-1 staining assay, and ATP levels using the Enzy light ATP Assay kit. Results: H/R-injured H9C2 cells showed significantly reduced cell viability, increased apoptosis, and decreased MMP compared to normally cultured cells. There were no significant differences between H9C6 cells treated with DMEM vs. RBC supernatants stored for 0 or 35 days in terms of cell viability, apoptosis, MMP, or ATP level, nor were there any significant differences between the 0and 35-days groups. Conclusion: RBC supernatants stored for 0 or 35 days had no effects on H/R-injured H9C2 cells in terms of cell viability, apoptosis, MMP, or ATP level.