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摘要：
目的 探究睡眠剥夺引起心肌损伤的可能机制及内质网应激通过线粒体相关内质网膜（Mitochondria-associated endoplasmic reticulum membranes，MAMs）导致线粒体钙超载在其中的作用。 方法 取6-8周雄性C57BL/6小鼠27只，按随机数字表法分为对照组(NC组)、睡眠剥夺组(SD组)、睡眠剥夺+内质网应激抑制剂4-苯基丁酸（4-Phenylbutyric acid，4-PBA）组(SD+4-PBA组)。采用改良多平台水环境法构建小鼠睡眠剥夺模型；SD+4-PBA组小鼠在睡眠剥夺前腹腔注射20mg/kg 4-PBA；NC组及SD组注射同等剂量生理盐水。造模成功后,取小鼠心脏切片进行苏木精-伊红（H-E）染色观察小鼠心肌损伤情况、马森（Masson）染色和天狼猩红染色观察心肌纤维化情况，透射电子显微镜观察心肌MAMs和内质网的超微结构，钙离子荧光染色检测线粒体钙水平，免疫印迹法（Western blot）检测心肌内质网应激相关的C/EBP同源蛋白（C/EBP homologous protein，CHOP）、葡萄糖调节蛋白78（glucose-regulated protein 78，GRP78）及MAMs钙转运相关的电压依赖性阴离子选择性通道1（voltage-dependent anion channel 1，VDAC1）、葡萄糖调节蛋白75（glucose-regulated protein 75，GRP75）、肌醇1,4,5-三磷酸受体（inositol 1,4,5-trisphosphate receptor，IP3R）的相对表达水平。 结果 与NC组比较：SD组心肌损伤情况、纤维化程度、MAMs及线粒体钙负荷明显加重，CHOP、GRP78、VDAC1、GRP75、IP3R蛋白表达水平明显升高（P0.05）。与SD组相比：SD+4-PBA组心肌损伤情况、纤维化程度、MAMs及线粒体钙负荷明显减轻，CHOP、GRP78、VDAC1、GRP75、IP3R蛋白表达水平明显下降（P0.05）。结论 睡眠剥夺会引起心肌细胞发生内质网应激，并通过MAMs导致线粒体钙超载进而引起心肌损伤。
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Abstract: Objective To explore the possible mechanism of myocardial injury caused by sleep deprivation and the role of endoplasmic reticulum stress in causing mitochondrial calcium overload through mitochondria-associated endoplasmic reticulum membranes (MAMs) in this process. Methods According to the random number table method, twenty-seven male C57BL/6 mice aged 6 to 8 weeks were selected and divided into the control group (NC group), sleep deprivation group (SD group), sleep deprivation + 4-phenylbutyric acid group (SD+4-PBA group). The sleep deprivation model was induced by modified multi-platform water environment method. The SD+4-PBA group received 4-PBA at 20 mg/kg body weight intraperitoneally before sleep deprivation, while the NC group and SD group received the same dose of saline. The hematoxylin-eosin (H-E) staining was used to observe the histopathological changes of myocardium in mice, while the Masson staining and Sirius red staining were used to observe myocardial fibrosis. The transmission electron microscopy was used to observe the ultrastructure of MAMs and endoplasmic reticulum in the myocardium. The levels of mitochondrial calcium were detected by calcium ion fluorescence staining. Western blot was used to detect the relative expression levels of C/EBP homologous protein (CHOP), glucose-regulated protein 78 (GRP78), voltage-dependent anion channel 1 (VDAC1), glucose-regulated protein 75 (GRP75), and inositol 1,4,5-trisphosphate receptor (IP3R). Results Compared with the NC group, the SD group showed increased myocardial damage, fibrosis degree, MAMs and mitochondrial calcium load (P0.05), increased levels of CHOP, GRP78, VDAC1, GRP75 and IP3R (all P0.05). Compared with the SD group, the SD+4-PBA group showed reduced myocardial damage, fibrosis degree, MAMs and mitochondrial calcium load (P0.05), decreased levels of CHOP, GRP78, VDAC1, GRP75 and IP3R (all P0.05). Conclusion Sleep deprivation can cause myocardial endoplasmic reticulum stress, which leads to mitochondrial calcium overload through MAMs, thereby causing myocardial injury.
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