丹酚酸A对脂多糖诱导人肺微血管内皮细胞线粒体自噬和细胞损伤的影响

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摘要目的：探讨丹酚酸A（Sal A）对脂多糖（LPS）诱导的人肺微血管内皮细胞（HPMVECs）线粒体自噬和细胞损伤的影响。方法：采用终浓度为10 μg/mL LPS构建细胞损伤模型，并将实验分为对照组、Sal A组、LPS组、LPS+Sal A组。采用噻唑蓝染色法分析细胞增殖情况，流式细胞技术检测细胞内活性氧（ROS）含量和线粒体膜电位水平，蛋白免疫印迹法检测微管相关蛋白轻链3（LC3）-II/I、Beclin1、PTEN介导的假定激酶蛋白1（PINK1）和Parkin蛋白相对表达量。结果：10 μg/mL LPS呈时间依赖性抑制HPMVECs的增殖；LPS+Sal A组细胞相对存活率高于LPS组，低于对照组（P＜0.05）。LPS组ROS含量（283.96%±10.88%）增高，膜电位水平（38.25%±5.69%）降低，与对照组（100%）相比差异有统计学意义（P＜0.05）；LPS+Sal A组ROS含量（138.76%±11.82%）低于LPS组，线粒体膜电位水平（76.56%±6.22%）高于LPS组（P＜0.05）。LPS组细胞LC3-II/I、Beclin1、PINK1和Parkin蛋白表达量较对照组增高（P＜0.05），LPS+Sal A组LC3-II/I、Beclin1、PINK1和Parkin蛋白相对表达量低于LPS组（P＜0.05）；LPS组线粒体PINK1和Parkin蛋白表达量与对照组比较增高（P＜0.05），LPS+Sal A组明显低于LPS组（P＜0.05）。结论：Sal A减轻LPS诱导的HPMVECs细胞细胞损伤作用，其可能机制与其降低细胞内ROS，稳定线粒体膜电位，抑制线粒体自噬有关。

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关键词 ：丹酚酸A, 线粒体自噬, 脂多糖, 人肺微血管内皮细胞, 细胞损伤

Abstract：Objective: To investigate the effect of salvianolic acid A (Sal A) on mitochondrial autophagy and cell injury in human pulmonary microvascular endothelial cells (HPMVECs) injury induced by lipopolysaccharide (LPS). Methods: LPS 10 μg/mL was used to establish cell injury in vitro. The cells were divided into four groups: control group, Sal A group, LPS group, LPS+Sal A group. MTT was used to analyze cell proliferation. The reactive oxygen species (ROS) content and mitochondrial membrane potential were detected by flow cytometry. Western blot was used to analyze the expression of microtubules associated protein light chain 3 (LC3)-II/I, Beclin1, PTEN mediated putative kinase protein 1 (PINK1) and Parkin. Results: LPS decreased the cell viability in time-dependent manner. The viability in LPS+Sal A group was higher than the LPS group, but lower than the control group (P<0.05). Compared with the control group (100%), the ROS content (283.96%±10.88%) increased and the level of membrane (38.25%±5.69%) potential decreased in LPS group (P<0.05). Compared with LPS group, ROS content (138.76%±11.82%) decreased, and the mitochondrial membrane potential (76.56%±6.22%) increased in LPS+Sal A group (P<0.05). The expression of LC-3II/I, Beclin1, PINK1 and Parkin in LPS group was higher than the control group. The expression of LC-3II/I, Beclin1, PINK1 and Parkin in LPS+Sal A group was lower than LPS group. Conclusion: Sal A alleviates the proliferation inhibition by LPS, which may be related with decreased intracellular ROS content, stabilization of mitochondrial membrane potential and mitochondrial autophagy.

Key words： salvianolic acid A mitochondrial autophagy lipopolysaccharide human pulmonary microvascular endothelial cells cell injury

收稿日期: 2017-11-21

基金资助:浙江省公益性（实验动物）计划项目（2016C37100）。

作者简介: 李伟文（1979-），男，浙江丽水人，副主任医师。

链接本文:

https://xb.wmu.edu.cn/CN/ 或 https://xb.wmu.edu.cn/CN/Y2018/V48/I4/267

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