中国组织工程研究

中国组织工程研究 ›› 2023, Vol. 27 ›› Issue (28): 4519-4524.doi: 10.12307/2023.568

• 血管组织构建 vascular tissue construction • 上一篇    下一篇

松弛素保护心肌微血管内皮细胞缺氧复氧损伤的机制

魏  琴1，2，阿曼古丽·如则2，3，陈冰心4，赵  翎2，3，赵帮豪2，3，姜  涛2，5，张  春2，5，李志强2，5，高晓明2，3，6，段明军2，5   

  1. 新疆医科大学，1中心实验室，5动物实验中心，6临床医学研究院，新疆维吾尔自治区乌鲁木齐市  830054；2新疆医学动物模型研究重点实验室，新疆维吾尔自治区乌鲁木齐市  830054；3省部共建中亚高发病成因与防治国家重点实验室，新疆维吾尔自治区乌鲁木齐市  830054；4新疆医科大学第一附属医院心功能科，新疆维吾尔自治区乌鲁木齐市  830054
  • 收稿日期:2022-08-16 接受日期:2022-09-24 出版日期:2023-10-08 发布日期:2023-01-29
  • 通讯作者: 高晓明，博士，教授，研究员，新疆医学动物模型研究重点实验室，新疆维吾尔自治区乌鲁木齐市 830054；省部共建中亚高发病成因与防治国家重点实验室，新疆维吾尔自治区乌鲁木齐市 830054；新疆医科大学临床医学研究院，新疆维吾尔自治区乌鲁木齐市 830054 段明军，硕士，助理研究员，新疆医学动物模型研究重点实验室，新疆维吾尔自治区乌鲁木齐市 830054；新疆医科大学动物实验中心，新疆维吾尔自治区乌鲁木齐市 830054
  • 作者简介:魏琴，女，1980年生，汉族，硕士，实验师，主要从事心血管疾病基础研究。 阿曼古丽·如则，女，1988年生，维吾尔族，博士，助理研究员，主要从事心血管疾病基础研究。
  • 基金资助:
    省部共建中亚高发病成因与防治国家重点实验室开放课题(SKL-HIDCA-2017-Y11)，项目负责人：段明军；省部共建中亚高发病成因与防治国家重点实验室开放课题(SKL-HIDCA-2021-XXG1)项目负责人：阿曼古丽·如则；新疆维吾尔自治区自然科学基金(2018D01C197)，项目负责人：段明军；新疆维吾尔自治区重点实验室开放课题(2021D04020)，项目负责人：高晓明

Relaxin protects myocardial microvascular endothelial cells from hypoxia-reoxygenation injury

Wei Qin1, 2, Amanguli·Ruze2, 3, Chen Bingxin4, Zhao Ling2, 3, Zhao Banghao2, 3, Jiang Tao2, 5, Zhang Chun2, 5, Li Zhiqiang2, 5, Gao Xiaoming2, 3, 6, Duan Mingjun2, 5   

  1. 1Center Laboratory, 5Animal Laboratory Center, 6Institute of Clinical Medicine, Xinjiang Medical University, Urumqi 830054, Xinjiang Uygur Autonomous Region, China; 2Xinjiang Key Laboratory of Medical Animal Model Research, Urumqi 830054, Xinjiang Uygur Autonomous Region, China; 3Key Laboratory of Pathogenesis, Prevention and Treatment of High Incidence Diseases in Central Asia, Urumqi 830054, Xinjiang Uygur Autonomous Region, China; 4Department of Cardiac Function, The First Affiliated Hospital of Xinjiang Medical University, Urumqi 830054, Xinjiang Uygur Autonomous Region, China
  • Received:2022-08-16 Accepted:2022-09-24 Online:2023-10-08 Published:2023-01-29
  • Contact: Gao Xiaoming, MD, Professor, Researcher, Xinjiang Key Laboratory of Medical Animal Model Research, Urumqi 830054, Xinjiang Uygur Autonomous Region, China; Key Laboratory of Pathogenesis, Prevention and Treatment of High Incidence Diseases in Central Asia, Urumqi 830054, Xinjiang Uygur Autonomous Region, China; Institute of Clinical Medicine, Xinjiang Medical University, Urumqi 830054, Xinjiang Uygur Autonomous Region, China Duan Mingjun, Master, Assistant researcher, Xinjiang Key Laboratory of Medical Animal Model Research, Urumqi 830054, Xinjiang Uygur Autonomous Region, China; Animal Laboratory Center, Xinjiang Medical University, Urumqi 830054, Xinjiang Uygur Autonomous Region, China
  • About author:Wei Qin, Master, Experimentalist, Center Laboratory, Xinjiang Medical University, Urumqi 830054, Xinjiang Uygur Autonomous Region, China; Xinjiang Key Laboratory of Medical Animal Model Research, Urumqi 830054, Xinjiang Uygur Autonomous Region, China Amanguli·Ruze, MD, Assistant researcher, Xinjiang Key Laboratory of Medical Animal Model Research, Urumqi 830054, Xinjiang Uygur Autonomous Region, China; Key Laboratory of Pathogenesis, Prevention and Treatment of High Incidence Diseases in Central Asia, Urumqi 830054, Xinjiang Uygur Autonomous Region, China
  • Supported by:
    the Open Projects of Key Laboratory of Pathogenesis, Prevention and Treatment of High Incidence Diseases in Central Asia, Nos. SKL-HIDCA-2017-Y11 (to DMJ) and SKL-HIDCA-2021-XXG1 (to ARZ); Natural Science Foundation of Xinjiang Uygur Autonomous Region, No. 2018D01C197 (to DMJ); the Open Project of the Key Laboratory of Xinjiang Uygur Autonomous Region, No 2021D04020 (to GXM)

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摘要：

文题释义：

松弛素：作为一种妊娠激素，对心肌功能障碍具有保护作用，临床实验证明其具有正性肌力、扩血管、改善呼吸困难等作用。松弛素因具有抗炎和抗纤维化作用，在临床上对于心血管和炎症相关疾病(如缺血再灌注、心衰和糖尿病等)治疗具有较大应用潜力。
Akt/GSK-3β信号通路：Akt/GSK-3β是介导细胞信号传导的重要信号通路，Akt激活后可作用于下游的主要效应分子GSK-3β，GSK-3β激活可以减少线粒体通透性转换孔开放，发挥保护心肌的作用。

背景：研究报道松弛素可显著改善病理因素导致的心肾功能障碍，抑制心肌肥厚、抗纤维化以及改善缺血再灌注损伤等，但其对内皮细胞缺氧复氧损伤的保护机制尚不明确。
目的：探讨松弛素保护心肌微血管内皮细胞缺氧复氧损伤的机制。
方法：取生长状态良好的小鼠心肌微血管内皮细胞系(H5V)，分3组处理：对照组常规培养33 h，模型组常规培养24 h后给予6 h缺氧+3 h复氧模拟心肌缺氧再灌注损伤，松弛素组常规培养(加180 ng/mL松弛素)24 h后给予6 h缺氧+3 h复氧(加180 ng/mL松弛素)模拟心肌缺氧再灌注损伤。检测细胞通透性与Caspase-3的表达，细胞上清中肿瘤坏死因子α、白细胞介素1β和白细胞介素6水平，RT-PCR检测细胞中钙黏蛋白、Akt1、GSK-3β mRNA表达；Western blot检测细胞中钙黏蛋白、Akt、GSK-3β蛋白表达。

结果与结论：①与对照组比较，模型组细胞通透性增强(P < 0.05)，细胞内Caspase-3表达升高(P < 0.05)；与模型组比较，松弛素组细胞通透性降低(P < 0.05)，细胞内Caspase-3表达降低(P < 0.05)；②与对照组比较，模型组上清中肿瘤坏死因子α、白细胞介素1β和白细胞介素6水平升高(P < 0.05)；与模型组比较，松弛素组3种炎症因子水平降低(P < 0.05)；③3组间细胞中钙黏蛋白、Akt1、GSK-3β mRNA表达比较差异无显著性意义(P > 0.05)；④3组间细胞中钙黏蛋白、Akt、GSK-3β总蛋白表达比较差异无显著性意义(P > 0.05)；与对照组比较，模型组磷酸化钙黏蛋白、p-Akt、p-GSK-3β蛋白表达降低(P < 0.05)；与模型组比较，松弛素组磷酸化钙黏蛋白、p-Akt、p-GSK-3β蛋白表达升高(P < 0.05)；⑤结果表明，松弛素治疗通过增强钙黏蛋白表达，减少缺氧复氧引起的小鼠心肌微血管内皮细胞损伤，抑制炎症因子的释放，减轻细胞凋亡，该作用可能与激活Akt/GSK-3β信号通路有关。

https://orcid.org/0000-0003-0344-8305(魏琴)

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: 松弛素, 心血管疾病, Akt/GSK-3β, 内皮细胞通透性, 细胞凋亡, 炎症因子

Abstract: BACKGROUND: Relaxin can significantly improve cardiac and renal dysfuction caused by pathological factors, inhibit myocardial hypertrophy, have an anti-fibrosis effect, and improve ischemia-reperfusion injury. However, its protective mechanism against hypoxia-reoxygenation injury of endothelial cells remains unclear.
OBJECTIVE: To investigate the protective mechanism of relaxin against hypoxia-reoxygenation injury of myocardial microvascular endothelial cells.
METHODS: Mouse myocardial microvascular endothelial cell line (H5V cells) was used for the experiment. Cells were treated by three different interventions: in control group, cells were normally cultured for 33 hours; in model group, cells were treated by 6-hour hypoxia followed by 3-hour reoxygenation); and in relaxin group, 24 hours of routine culture (180 ng/mL relaxin), 6 hours of hypoxia and 3 hours of reoxygenation (180 ng/mL relaxin) were given to simulate myocardial hypoxia-reperfusion injury. Cell permeability and Caspase-3 activity were then detected. Levels of tumor necrosis factor-α, interleukin-1β and interleukin-6 in cell supernatants were detected by ELISA. Expressions of VE-cadherin, Akt, and GSK-3β at mRNA and protein levels were detected by RT-PCR and western blot, respectively.
RESULTS AND CONCLUSION: Compared with the control group, the cell permeability and expression of caspase-3 increased significantly in the model group (P < 0.05). Compared with the model group, the cell permeability and expression of caspase-3 decreased in the relaxin group (P < 0.05). Moreover, the levels of tumor necrosis factor-α, interleukin-1β and interleukin-6 were elevated in the model group, while the levels were significantly decreased after relaxin treatment (all P < 0.05). There were no significantly changes in the mRNA and protein expressions of VE-cadherin, Akt1, and GSK-3β mRNA among three groups (all P > 0.05). Compared with the control group, the expression of phosphorylated VE-cadherin, Akt1 and GSK-3β were decreased in the model group (P < 0.05), and relaxin treatment reversed these changes to the control levels (P < 0.05). To conclude, relaxin treatment could enhance VE-cadherin expression, reduce hypoxia-reoxygenation-induced microvascular endothelial cell damage, inhibit inflammatory cytokine release, and reduce cell apoptosis, which may be related to the activation of the Akt/GSK-3β signaling pathway.

Key words: relaxin, cardiovascular disease, Akt/GSK-3β, endothelial cell permeability, apoptosis, inflammatory cytokine

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