脂多糖调控 miR-211／Sirt1在加重缺氧诱导心肌细胞凋亡中的作用价值

The modulation effects of Lipopolysaccharide on miR-211/Siert1 to enhance cardiomyocytes apoptosis induced by hypoxia

目的：研究显示 Sirtuin1（ Sirt1）可通过去乙酰化酶活性调控 L 脂多糖（ lipopolysaccharide， LPS）病理过程，进而改善临床急诊脓毒血症患者的预后。通过数据库检索显示microRNA-211（miR-211）与Sirt1具有靶向匹配，然而其生物学关联意义尚无数据。本研究旨在探讨miR-211与Sirt1间的关联，以及它们是否参与LPS加重缺氧对心肌细胞损伤作用。方法原代培养大鼠（ SD）乳鼠心肌细胞，应用 qRT-PCR 法检测 LPS 处理4 h后对大鼠乳鼠心肌细胞（ neonatal nat cardiomyocytes， NRC）与H9c2心肌细胞中miR-211表达的变化，用Western blot法检测LPS处理后对NRC与H9c2心肌细胞中Sirt1蛋白表达的影响；同时应用CCK8法评价LPS对心肌细胞H9c2的细胞增殖情况；以及TUNEL法定量检测NRC与H9c2心肌细胞凋亡活性的影响。结果与对照组相比，20μg／mL、40μg／mL浓度的LPS处理4 h后， H9 c2心肌细胞在24-72 h时间段的增殖能力没有发生改变。然而， LPS预处理后NRC与H9c2在缺氧条件下细胞凋亡明显增加（较LPS未处理NRC与H9 c2组凋亡率增加均超过了100％， P＜0.05）；同时LPS处理后NRC与H9c2细胞中miR-211表达显著上调，且伴随其靶蛋白Sirt1表达明显下降（P分别＜0.05）。结论LPS可以增加缺氧诱导的心肌细胞凋亡，这与LPS通过上调miR-211水平进而抑制Sirt1表达这一作用机制密切相关。

Objective To study the effect of Sirtuin1 （Sirt1） on the pathological process through its activity of deacetylation to improve the clinical outcome of acute sepsis. After searching data base, microRNA-211 （miR-211） was found to have action potentially targeting at Sirt1.The present study aimed to find the interaction between miR-211 and Sirt1 in the pathogenesis of hypoxic injury to cardiomyocytes in the presence of lipopolysaccharide （ LPS ） . Methods Primary neonatal rat cardiomyocytes （ NRC ） isolated from neonatal SD rats and H9c2 （ cardiomyocytes after culture with 10% fetal serum of cattle and DMEM under 37 ℃ and 5% CO2 ） cell line were used in the experiments.The miR-211 expression was quantified by qRT-PCR after LPS exposure for 4 hours, and the changes in Sirt1 protein level were also detected in both NRC and H9c2 by western blot.At the same time, CCK-8 assay and TUNEL staining were also performed to measure cell proliferation and apoptosis activation when either treated with LPS alone or followed by exposure to hypoxia.Results Compared to the control group, the doses of 20μg/mL, 40μg/mL LPS treatment for 4 hours had no significant effects on H9c2 cell proliferation at 24 h, 48 h, 72 h, but it could significantly induce the cell apoptosis of neonatal rat cardiomyocytes and H9c2 cells after hypoxia, and the apoptosis rate increased all over 100% in both NRC and H9c2.At the same time, LPS treatment could significant up-regulate miR-211 expression which was closely associated with decrease in Sirt1 protein levels.Conclusions LPS enhanced cardiomyocytes injury after exposure to hypoxia which was closely associated with up-regulating miR-211 expression and in turn to suppress Sirt1 expression.