中国组织工程研究

中国组织工程研究 ›› 2024, Vol. 28 ›› Issue (13): 2054-2060.doi: 10.12307/2024.133

• 干细胞基础实验 basic experiments of stem cells • 上一篇    下一篇

缺氧后处理通过piRNA-005854调控衰老心肌细胞自噬发挥保护心肌作用

迟宏扬1，2，杨慧霞1，2，郝银菊3，杨安宁2，4，白志刚2，5，焦  运2，6，熊建团2，4，马胜超2，4，姜怡邓2，4   

  1. 宁夏医科大学，1临床医学院，2国家卫生健康委代谢性心血管疾病研究重点实验室，3药学院，4基础医学院，宁夏回族自治区银川市   750004；5宁夏回族自治区人民医院，宁夏回族自治区银川市   750004；6宁夏医科大学总医院感染科，宁夏回族自治区银川市   750004
  • 收稿日期:2023-01-28 接受日期:2023-04-07 出版日期:2024-05-08 发布日期:2023-08-28
  • 通讯作者: 姜怡邓，博士，教授，博士生导师，宁夏医科大学，基础医学院，国家卫生健康委代谢性心血管疾病研究重点实验室，宁夏回族自治区银川市 750004 马胜超，博士，副教授，硕士生导师，宁夏医科大学，基础医学院，国家卫生健康委代谢性心血管疾病研究重点实验室，宁夏回族自治区银川市 750004
  • 作者简介:迟宏扬，1998年生，汉族，宁夏医科大学在读硕士，主要从事代谢性心血管疾病方面的研究。
  • 基金资助:

    宁夏回族自治区重点研发计划重点项目(2022BFH02013)，项目负责人：郝银菊；宁夏回族自治区重点研发计划重点项目(2020BFH02003)，项目负责人：杨安宁；宁夏回族自治区重点研发计划重点项目(2021BEG02033)，项目负责人：熊建团；宁夏回族自治区重点研发计划重点项目(2020BEG03005)，项目负责人：焦运；宁夏回族自治区重点研发计划重点项目(2020BFH02001)，项目负责人：白志刚；国家自然科学基金项目(U21A20343)，项目负责人：姜怡邓；国家自然科学基金项目(82160049)，项目负责人：熊建团；国家自然科学基金项目(81900273)，项目负责人：马胜超；国家自然科学基金项目(82060412)，项目负责人：焦运；中国医学科学院中央级公益性科研院所基本科研业务费项目(2019PT330002)，项目负责人：姜怡邓

Hypoxic postconditioning protects myocardium by regulating autophagy in aging cardiomyocytes through piRNA-005854

Chi Hongyang1, 2, Yang Huixia1, 2, Hao Yinju3, Yang Anning2, 4, Bai Zhigang2, 5, Jiao Yun2, 6, Xiong Jiantuan2, 4, Ma Shengchao2, 4, Jiang Yideng2, 4   

  1. 1School of Clinical Medicine, 2National Health Commission Key Laboratory of Metabolic Cardiovascular Disease Research, 3School of Pharmacy, 4School of Basic Medicine, Ningxia Medical University, Yinchuan 750004, Ningxia Hui Autonomous Region, China; 5People’s Hospital of Ningxia Hui Autonomous Region, Yinchuan 750004, Ningxia Hui Autonomous Region, China; 6Department of Infection, General Hospital of Ningxia Medical University, Yinchuan 750004, Ningxia Hui Autonomous Region, China
  • Received:2023-01-28 Accepted:2023-04-07 Online:2024-05-08 Published:2023-08-28
  • Contact: Jiang Yideng, MD, Professor, Doctoral supervisor, National Health Commission Key Laboratory of Metabolic Cardiovascular Disease Research; School of Basic Medicine, Ningxia Medical University, Yinchuan 750004, Ningxia Hui Autonomous Region, China Ma Shengchao, MD, Associate professor, Master’s supervisor, National Health Commission Key Laboratory of Metabolic Cardiovascular Disease Research; School of Basic Medicine, Ningxia Medical University, Yinchuan 750004, Ningxia Hui Autonomous Region, China
  • About author:Chi Hongyang, Master candidate, School of Clinical Medicine; National Health Commission Key Laboratory of Metabolic Cardiovascular Diseases Research, Ningxia Medical University, Yinchuan 750004, Ningxia Hui Autonomous Region, China
  • Supported by:
    Key Project of Key Research and Development Program of Ningxia Hui Autonomous Region, No. 2022BFH02013 (to HYJ); Key Project of Key Research and Development Program of Ningxia Hui Autonomous Region, No. 2020BFH02003 (to YAN); Key Project of Key Research and Development Program of Ningxia Hui Autonomous Region, No. 2021BEG02033 (to XJT); Key Project of Key Research and Development Program of Ningxia Hui Autonomous Region, No. 2020BEG03005 (to JY); Key Project of Key Research and Development Program of Ningxia Hui Autonomous Region, No. 2020BFH02001 (to BZG); National Natural Science Foundation of China, No. U21A20343 (to JYD); National Natural Science Foundation of China, No. 82160049 (to XJT); National Natural Science Foundation of China, No. 81900273 (to MSC); National Natural Science Foundation of China, No. 82060412 (to JY); Fundamental Research Funds for Central Public Welfare Scientific Research Institutes of the Chinese Academy of Medical Sciences, No. 2019PT330002 (to JYD)

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摘要：


文题释义：

Piwi蛋白互作RNA(piRNA)：是一类可以沉默转座子及调控mRNA翻译的新型非编码小RNA。piRNA主要参与生殖细胞的发育和精子的发生，与肿瘤等疾病的发生、发展也密切相关。在特定疾病状态下，piRNA会出现差异表达，具有作为新型生物标志物的潜力。
自噬：是一种高度保守的细胞内成分降解过程，在维持细胞结构和功能稳定中具有重要作用。自噬参与缺血再灌注损伤的病理生理过程，但自噬过度可能导致代谢应激、细胞成分降解，甚至细胞死亡。


背景：缺血后处理是减轻缺血再灌注损伤的有效方式之一，近年来被越来越广泛地应用于临床实践，但其具体分子机制还有待研究。

目的：探讨piRNA-005854在衰老心肌细胞缺氧后处理中的作用及机制。
方法：体外给予心肌细胞8 mg/mL D-半乳糖9 d诱导其衰老，β-半乳糖苷酶染色观察心肌细胞的衰老情况；衰老后细胞给予缺氧/复氧处理和缺氧后处理，ELISA检测心肌损伤标志物肌酸激酶同工酶MB以及乳酸脱氢酶水平；Western blot检测衰老心肌细胞中自噬相关蛋白LC3Ⅱ、p62和ULK1及其磷酸化ULK1的表达；qRT-PCR检测piRNA-005854的表达水平；进一步用piRNA-005854 inhibitor及piRNA-005854 mimics转染衰老心肌细胞并进行缺氧后处理，Western blot检测LC3Ⅱ、p62和ULK1及其磷酸化ULK1的表达。

结果与结论：①D-半乳糖诱导9 d后心肌细胞出现明显衰老；②与正常氧组比较，缺氧/复氧组肌酸激酶同工酶MB以及乳酸脱氢酶水平增加(P < 0.01)；LC3Ⅱ/Ⅰ表达升高、p62表达降低、ULK1磷酸化水平升高、piRNA-005854表达升高(P < 0.01)；③与缺氧/复氧组比较，缺氧后处理组肌酸激酶同工酶MB以及乳酸脱氢酶水平明显减少(P < 0.01)；LC3Ⅱ/Ⅰ表达明显降低(P < 0.05)、p62表达升高(P < 0.01)、ULK1磷酸化水平降低(P < 0.05)、piRNA-005854表达降低(P < 0.01)；④转染piRNA-005854 inhibitor后，LC3Ⅱ/Ⅰ表达降低(P < 0.01)，p62表达明显升高(P < 0.05)，ULK1磷酸化水平明显降低(P < 0.01)；转染piRNA-005854 mimics后，LC3Ⅱ/Ⅰ表达显著升高，p62表达降低，ULK1磷酸化水平明显增加(P < 0.01)；⑤结果表明，piRNA-005854介导的ULK1依赖性自噬水平降低是衰老心肌细胞缺氧后处理发挥保护作用的可能机制。

https://orcid.org/0009-0002-2263-1199 (迟宏扬) 

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

关键词: piRNA, 自噬, 衰老心肌细胞, 缺血再灌注损伤, 缺氧/复氧, 缺氧后处理

Abstract: BACKGROUND: Ischemic postconditioning is one of the effective ways to reduce ischemia-reperfusion injury and has been more and more widely used in clinical practice in recent years, but its specific molecular mechanism has yet to be studied.  
OBJECTIVE: To investigate the role and mechanism of piRNA-005854 in the aging cardiomyocytes caused by hypoxic postconditioning.
METHODS: In vitro, cardiomyocytes were administered 8 mg/mL D-galactose for 9 days to induce their aging. β-Galactosidase staining was used to observe the aging of cardiomyocytes. Senescent cells were treated with hypoxia/reoxygenation and hypoxic postconditioning. ELISA was utilized to detect changes in myocardial injury markers creatine kinase isoenzyme MB and lactate dehydrogenase levels. Western blot assay was applied to detect the expression changes of autophagy-related proteins LC3II, p62, ULK1 and phosphorylated ULK1 in aging cardiomyocytes. qRT-PCR was employed to determine the expression level of piRNA-005854. piRNA-005854 inhibitor and piRNA-005854 mimics were transferred into aging cardiomyocytes and followed with hypoxic postconditioning. Western blot assay was used to examine the expression of LC3II, p62, ULK1 and phosphorylated ULK1.  
RESULTS AND CONCLUSION: (1) D-galactose induced obvious senescence of cardiomyocytes 9 days later. (2) Compared with the normoxia group, creatine kinase isoenzyme MB and lactate dehydrogenase levels increased in the hypoxia/reoxygenation group (P < 0.01); LC3 II/I expression was increased; p62 expression was decreased; ULK1 phosphorylation level was increased, and piRNA-005854 expression was increased (P < 0.01). (3) Compared with the hypoxia/reoxygenation group, creatine kinase isoenzyme MB and lactate dehydrogenase levels significantly reduced in the hypoxic postconditioning group (P < 0.01); LC3 II/I expression significantly decreased (P < 0.05); p62 expression increased (P < 0.01); ULK1 phosphorylation level decreased (P < 0.05), and piRNA-005854 expression decreased (P < 0.01). (4) After transfection of piRNA-005854 inhibitor, LC3II/I expression was decreased (P < 0.01); the expression of p62 was increased significantly (P < 0.05); the phosphorylation level of ULK1 was decreased significantly (P < 0.01). After transfection of piRNA-005854 mimics, LC3II/I expression was increased significantly; the expression of p62 was decreased, and the phosphorylation level of ULK1 was increased significantly (P < 0.01). (5) The results show that piRNA-005854-mediated reduction of ULK1-dependent autophagy level is a possible mechanism that hypoxic postconditioning exerts its protective effect on aging cardiomyocytes.

Key words: piRNA, autophagy, aging cardiomyocyte, ischemia-reperfusion injury, hypoxia/reoxygenation, hypoxic postconditioning

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