中国组织工程研究

中国组织工程研究 ›› 2021, Vol. 25 ›› Issue (5): 723-728.doi: 10.3969/j.issn.2095-4344.3006

• 组织构建细胞学实验 cytology experiments in tissue construction • 上一篇    下一篇

复方中药稳斑汤减少同型半胱氨酸诱导大鼠心肌微血管内皮细胞凋亡的作用机制

张雯雯1，2，金颂峰1，赵国梁1，宮丽鸿3   

  1. 1辽宁中医药大学，辽宁省沈阳市   110032；2辽宁中医药大学附属第三医院，辽宁省沈阳市   110032；3辽宁中医药大学附属医院，辽宁省沈阳市   110032
  • 收稿日期:2020-01-21 修回日期:2020-02-10 接受日期:2020-03-14 出版日期:2021-02-18 发布日期:2020-11-28
  • 通讯作者: 宮丽鸿，博士，教授，主任医师，博士生导师，辽宁中医药大学附属医院，辽宁省沈阳市 110032
  • 作者简介:张雯雯，女，1986年生，山东省日照市人，汉族，辽宁中医药大学在读博士，主治中医师，主要从事中西医结合防治心血管疾病研究。
  • 基金资助:
    辽宁省科学技术基金项目(20180530016)；辽宁特聘教授课题项目(辽委发[2017]3号)

Mechanism by which Wenban Decoction reduces homocysteine-induced apoptosis of myocardial microvascular endothelial cells in rats

Zhang Wenwen1, 2, Jin Songfeng1, Zhao Guoliang1, Gong Lihong3   

  1. 1Liaoning University of Traditional Chinese Medicine, Shenyang 110032, Liaoning Province, China; 2Third Affiliated Hospital of Liaoning University of Traditional Chinese Medicine, Shenyang 110032, Liaoning Province, China; 3First Affiliated Hospital of Liaoning University of Traditional Chinese Medicine, Shenyang 110032, Liaoning Province, China
  • Received:2020-01-21 Revised:2020-02-10 Accepted:2020-03-14 Online:2021-02-18 Published:2020-11-28
  • Contact: Gong Lihong, MD, Professor, Chief physician, Doctoral supervisor, First Affiliated Hospital of Liaoning University of Traditional Chinese Medicine, Shenyang 110032, Liaoning Province, China
  • About author:Zhang Wenwen, MD candidate, Attending physician, Liaoning University of Traditional Chinese Medicine, Shenyang 110032, Liaoning Province, China; Third Affiliated Hospital of Liaoning University of Traditional Chinese Medicine, Shenyang 110032, Liaoning Province, China
  • Supported by:
    the Science and Technology Research Foundation of Liaoning Province, No. 20180530016; Liaoning Provincial Distinguished Professor Project, No. [2017]3

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摘要：

文题释义：
PI3K/AKT信号通路：磷脂酰肌醇3-激酶(PI3Ks)信号参与增殖、分化、凋亡和葡萄糖转运等多种细胞功能的调节。IA型PI3K和其下游分子蛋白激酶B(PKB或Akt)所组成的信号通路与人类肿瘤的发生发展密切相关，该通路调节肿瘤细胞的增殖和存活，其活性异常不仅能导致细胞恶性转化，而且与肿瘤细胞的迁移、黏附、肿瘤血管生成以及细胞外基质的降解等相关，目前以PI3K-Akt信号通路关键分子为靶点的肿瘤治疗策略正在发展中。 
心脏微血管内皮细胞：是组成心脏微血管腔面单层扁平上皮样细胞，它所产生和分泌的生物活性物质对维持血管张力、调节血压、抗血栓形成等有重要作用。近年来大量研究表明，心肌微血管内皮细胞的功能和病理改变直接影响心肌细胞功能，也是诸多毒素、炎症因子及病毒等重要靶位，其作为体外研究的细胞模型在心肌缺血-再灌注发病机制和细胞间旁分泌细胞生长因子研究中起着重要作用。

背景：关于超敏C-反应蛋白水平正常者血清同型半胱氨酸浓度与冠状动脉病变的关系研究显示，在超敏C-反应蛋白水平正常者中，血清同型半胱氨酸浓度是冠状动脉疾病患病率和严重程度的独立预测因子。
目的：探讨稳斑汤调控PI3K/Akt信号通路对同型半胱氨酸诱导大鼠心脏微血管内皮细胞凋亡的保护作用。
方法：体外原代培养大鼠心脏微血管内皮细胞，分为对照组、模型组(用10 mmol/L 同型半胱氨酸诱导细胞损伤)、稳斑汤组(同型半胱氨酸+ 50 mg/L 稳斑汤给药)。CCK-8检测各组细胞存活能力；ELISA检测各组细胞上清中乳酸脱氢酶、丙二醛、全血氧化氢酶、超氧化物歧化酶、谷胱甘肽过氧化物酶、白细胞介素6、细胞间黏附分子1、白细胞介素1β、肿瘤坏死因子α含量；并在稳斑汤治疗基础上加入了PI3K抑制剂LY294002，流式细胞术检测各组细胞凋亡情况；Western blot法检测各组细胞中PI3K、Akt、p-Akt、Bax、Bcl-2和caspase3蛋白水平的表达。实验获得辽宁中医药大学动物实验伦理委员会批准(批准号为21000092018010)。
结果与结论：①与对照组相比，模型组心脏微血管内皮细胞存活能力显著降低，乳酸脱氢酶的渗漏量显著增加，引起氧化应激以及炎症因子的释放，最终导致细胞大量凋亡，差异有显著性意义；与模型组相比，稳斑汤组心脏微血管内皮细胞存活能力增加，乳酸脱氢酶的渗漏量降低，细胞中白细胞介素1β、细胞间黏附分子1、白细胞介素6和肿瘤坏死因子α的浓度降低(P < 0.05)，细胞凋亡数目减少；②PI3K抑制剂能够逆转稳斑汤对同型半胱氨酸诱导的心脏微血管内皮细胞凋亡的抑制作用；③结果说明，稳斑汤可显著提高同型半胱氨酸损伤的心脏微血管内皮细胞存活能力，降低乳酸脱氢酶的渗漏量，抑制氧化应激水平以及炎症因子的释放，最终减少细胞凋亡数，这种作用与抑制PI3K/Akt信号通路有关。 
https://orcid.org/0000-0002-2021-4618 (张雯雯) 

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

关键词: 冠心病, 心脏, 微血管, 内皮细胞, 细胞凋亡, 通路, 中药, 实验

Abstract: BACKGROUND: Studies have shown that serum homocysteine concentration is an independent predictor of the prevalence and severity of coronary artery disease for patients with normal hypersensitivity C-reactive protein levels. 
OBJECTIVE: To investigate the protective effect of Wenban Decoction on the apoptosis of rat cardiac microvascular endothelial cells (CMECs) induced by homocysteine by regulating PI3K/Akt signaling pathway. 
METHODS: Rat CMECs were primarily cultured in vitro, and the cells were randomly divided into control group, model group and Wenban Decoction group (50 mg/L Wenban Decoction). The cells in the latter two groups were injured by 10 mmol/L homocysteine prior to the treatment. Cell counting kit-8 was used to detect the cell viability of each group. ELISA was used to determine serum lactate dehydrogenase, malondialdehyde, whole blood catalase, superoxide dismutase, glutathione peroxidase, interleukin 6, intercellular adhesion molecule 1, interleukin 1β, and tumor necrosis factor α. Flow cytometry was used to detect cell apoptosis after addition of LY294002 based on the treatment with Wenban Decoction. Western blot was used to detect the expression of PI3K, Akt, p-Akt, Bax, Bcl-2 and caspase3 protein in the cells. An ethic approval was given by the Animal Experiment Ethics Committee of Liaoning University of Traditional Chinese Medicine (approval No. 21000092018010).
RESULTS AND CONCLUSION: Compared with the control group, the survival ability of CMECs in the model group was significantly reduced, the leakage of lactate dehydrogenase was significantly increased, which caused oxidative stress and the release of inflammatory factors, and finally led to a large number of apoptosis. Compared with the model group, Wenban Decoction improved the survival ability of CMECs, reduced the leakage of lactate dehydrogenase, significantly decreased the intracellular levels of interleukin 1β, intercellular adhesion molecule 1, interleukin 6 and tumor necrosis factor α (P < 0.05), as well as reduced the number of apoptotic cells. PI3K inhibitor reversed the inhibitory effect of Wenban Decoction on homocysteine-induced apoptosis of CMECs. To conclude, Wenban Decoction can significantly improve the survival ability of CMECs, reduce the leakage of lactate dehydrogenase, inhibit the level of oxidative stress and the release of inflammatory factors, and ultimately reduce the number of apoptotic cells, which is related to the inhibition of PI3K/Akt signaling pathway.

Key words: coronary heart disease, heart, microvessel, endothelial cell, apoptosis, pathway, Chinese herb, experiment

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