诊断学理论与实践

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2026 , Vol. 25 >Issue 03: 346 - 353

DOI: https://doi.org/10.16150/j.1671-2870.2026.03.011

论著

RyR2敲低引起心肌细胞钙稳态失衡和凋亡可被咖啡因缓解

  • 叶远征 ,
  • 付晓晓 ,
  • 门莉 ,
  • 马前程 ,
  • 章安迪 ,
  • 范平
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  • 1 新疆医科大学第一附属医院心脏中心心功能科， 新疆 乌鲁木齐市 830000
    2 乌鲁木齐市中医医院药学部， 新疆 乌鲁木齐市 830000
    3 新疆医科大学第一临床医学院， 新疆 乌鲁木齐市 830000
    4 上海交通大学医学院附属瑞金医院心内科， 上海 200025
作者贡献/Authors’ Contributions

叶远征负责课题设计、实验方案制定、论文撰写与修改，对研究工作的整体构思与学术内容负责。付晓晓主要完成细胞实验操作，参与数据采集与整理。门莉负责基因与蛋白表达检测及相关数据分析。马前程完成流式细胞术、钙稳态指标等测定。章安迪提供技术指导，参与课题思路讨论、论文审阅与学术修改。范平参与数据统计分析及图表制作，协助论文初稿整理与格式规范。

章安迪 E-mail：xlyouxiangzad@sina.com

收稿日期: 2025-10-09

  修回日期: 2026-03-06

  录用日期: 2026-03-06

  网络出版日期: 2026-06-27

基金资助

新疆维吾尔自治区卫生健康青年医学科技人才专项科研项目(WJWY-202338);国家自然科学基金(82460097)

收起

Caffeine-mediated alleviation of imbalance of calcium homeostasis and apoptosis in cardiomyocytes caused by RyR2 knockdown

  • YE Yuanzheng ,
  • FU Xiaoxiao ,
  • MEN Li ,
  • MA Qiancheng ,
  • ZHANG Andi ,
  • FAN Ping
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  • 1 Department of Cardiac Function， Cardiac Center， The First Affiliated Hospital of Xinjiang Medical University， Xinjiang Urumqi 830000， China
    2 Department of Pharmacy， Urumqi Hospital of Traditional Chinese Medicine， Xinjiang Urumqi 830000， China
    3 First Clinical Medical College， Xinjiang Medical University， Xinjiang Urumqi 830000， China
    4 Department of Cardiology， Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine， Shanghai 200025， China

Received date: 2025-10-09

  Revised date: 2026-03-06

  Accepted date: 2026-03-06

  Online published: 2026-06-27

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摘要

目的：探讨低浓度咖啡因对兰尼碱受体2（ryanodine receptor 2， RyR2）敲低的大鼠心肌细胞钙稳态及凋亡的影响。方法：采用SPF级新生SD大鼠，分离提取新生大鼠心室肌细胞（newborn rat ventricular myocytes， NRVMs），分别将合成的转染RyR2小干扰RNA（small interfering RNA， siRNA）和阴性对照（negative control，NC）转染至NRVMs细胞，并将细胞分为NC组（转染siRNA NC）、siRYR2组（转染RyR2 siRNA）、NC+咖啡因组（转染siRNA后，添加0.2 mmol/L咖啡因），及siRYR2+咖啡因组（转染RyR2 siRNA后，添加0.2 mmol/L咖啡因）4组。采用实时荧光定量聚合酶链反应（quantitative real-time polymerase chain reaction，qRT-PCR）和蛋白印迹法检测RyR2的mRNA和蛋白表达量，用流式细胞仪检测细胞凋亡和钙离子，用共聚焦钙离子成像检测游离钙离子浓度。对NC组、siRYR2、NC+咖啡因组、siRYR2+咖啡因组的数据进行单因素方差分析。结果：与NC组相比，siRyR2组的RyR2 mRNA和蛋白相对表达量显著降低（mRNA：0.41±0.04比1.00±0.09，P=0.000 3；蛋白：0.34±0.04比0.72±0.02，P=0.000 1），钙离子浓度显著降低（流式细胞仪：0.80±0.02比1.00±0.02，P<0.000 1；共聚焦钙离子成像仪：61.08%±0.75%比100.00%±2.33%，P<0.000 1），细胞凋亡率显著增加（9.21%±0.07%比4.19%±0.07%，P<0.000 1），而NC+咖啡因组的RyR2 mRNA和蛋白表达量显著增加（mRNA：1.27±0.15比1.00±0.09，P=0.033 6；蛋白：0.92±0.10比0.72±0.02，P=0.006 5），钙离子浓度显著增加（流式细胞仪：1.33±0.04比1.00±0.02，P<0.000 1；共聚焦钙离子成像仪：116.21%±1.51%比100.00%±2.33%，P=0.000 6），细胞凋亡率显著降低（2.97%±0.13%比4.19%±0.07%，P<0.000 1）。与siRyR2组相比，si-RyR2+咖啡因组的RyR2 mRNA和蛋白相对表达量显著增加（mRNA：0.67±0.08比0.41±0.04，P=0.044 7；蛋白：0.54±0.01比0.34±0.04，P=0.009 1），钙离子浓度显著增加（流式细胞仪：0.87±0.02比0.80±0.02，P=0.026 3；共聚焦钙离子成像仪：79.14%±5.02%比61.08%±0.75%，P=0.000 3），细胞凋亡率显著降低（7.59%±0.10%比9.21%±0.07%，P<0.000 1）。与NC+咖啡因组相比，si-RyR2+咖啡因组的RYR2 mRNA和蛋白表达量显著降低（mRNA：0.67±0.08比1.27±0.15，P=0.000 2；蛋白：0.54±0.01比0.92±0.10，P<0.000 1），钙离子浓度显著降低（流式细胞仪：0.87±0.02比1.33±0.04，P<0.000 1；共聚焦钙离子成像仪：79.14%±5.02%比116.21%±1.51%，P<0.000 1），细胞凋亡率显著增加（7.59%±0.10%比2.97%±0.13%，P<0.000 1）。结论：低浓度咖啡因可以通过上调心肌细胞RyR2，促进钙离子释放，恢复钙稳态，减少细胞凋亡。这种咖啡因与RyR2的相互作用机制，可为开发由RyR2表达降低引起的心律失常等心脏疾病的治疗药物提供参考依据。

关键词： 心血管疾病; 兰尼碱受体2; 咖啡因; 钙稳态; 凋亡

本文引用格式

叶远征 , 付晓晓 , 门莉 , 马前程 , 章安迪 , 范平 . RyR2敲低引起心肌细胞钙稳态失衡和凋亡可被咖啡因缓解[J]. 诊断学理论与实践, 2026 , 25(03) : 346 -353 . DOI: 10.16150/j.1671-2870.2026.03.011

Abstract

Objective This study aims to investigate the effects of low-concentration caffeine on calcium homeostasis and apoptosis in rat cardiomyocytes with ryanodine receptor 2 (RyR2) knockdown. Methods Neonatal Sprague-Dawley (SD) rats of specific pathogen-free (SPF) grade were used, and neonatal rat ventricular myocytes (NRVMs) were isolated and extracted. Synthetic small interfering RNA (siRNA) targeting RyR2 and the negative control (NC) were transfected into NRVMs, and the cells were divided into four groups: NC group (transfected with siRNA NC), siRyR2 group (transfected with RyR2 siRNA), NC+caffeine group (0.2 mmol/L caffeine added after siRNA NC transfection), and siRyR2+caffeine group (0.2 mmol/L caffeine added after RyR2 siRNA transfection). The mRNA and protein expression levels of RyR2 were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. Cell apoptosis and calcium ions were detected by flow cytometry, and free calcium ion concentration was detected by confocal calcium ion imaging. Data from the NC, siRyR2, NC+caffeine, and siRyR2+caffeine groups were analyzed by one-way analysis of variance. Results Compared with the NC group, the mRNA and protein expression levels of RyR2 in the siRyR2 group were significantly decreased (mRNA: 0.41±0.04 vs 1.00±0.09, P=0.000 3; protein: 0.34±0.04 vs 0.72±0.02, P=0.000 1), calcium ion concentration was significantly decreased (flow cytometry: 0.80±0.02 vs 1.00±0.02, P<0.000 1; confocal: 61.08%±0.75% vs 100.00%±2.33%, P<0.000 1), and the apoptosis rate was significantly increased (9.21%±0.07% vs 4.19%±0.07%, P<0.000 1). In the NC+caffeine group, the mRNA and protein expression levels of RyR2 were significantly increased (mRNA: 1.27±0.15 vs 1.00±0.09, P=0.033 6; protein: 0.92±0.10 vs 0.72±0.02, P=0.006 5), calcium ion concentration was significantly increased (flow cytometry: 01.33±0.04 vs 1.00±0.02, P<0.000 1; confocal: 116.21%±1.51% vs 100.00%±2.33%, P=0.000 6), and the apoptosis rate was significantly decreased (2.97%±0.13% vs 4.19%±0.07%, P<0.000 1). Compared with the siRyR2 group, the mRNA and protein expression levels of RyR2 in the siRyR2+caffeine group were significantly increased (mRNA: 0.67±0.08 vs 0.41±0.04, P=0.044 7; protein: 0.54±0.01 vs 0.34±0.04, P=0.009 1), calcium ion concentration was significantly increased (flow cytometry: 0.87±0.02 vs 0.80±0.02, P=0.026 3; confocal: 79.14%±5.02% vs 61.08%±0.75%, P=0.000 3), and the apoptosis rate was significantly decreased (7.59%±0.10% vs 9.21%±0.07%, P<0.000 1). Compared with the NC+caffeine group, the mRNA and protein expression levels of RyR2 in the siRyR2+caffeine group were significantly decreased (mRNA: 0.67±0.08 vs 1.27±0.15, P=0.000 2; protein: 0.54±0.01 vs 0.92±0.10, P<0.000 1), calcium ion concentration was significantly decreased (flow cytometry: 0.87±0.02 vs 1.33±0.04, P<0.000 1; confocal: 79.14%±5.02% vs 116.21%±1.51%, P<0.000 1), and the apoptosis rate was significantly increased (7.59%±0.10% vs 2.97%±0.13%, P<0.000 1). Conclusions Low-concentration caffeine can promote calcium ion release, restore calcium homeostasis, and reduce apoptosis by upregulating RyR2 in cardiomyocytes. This interaction mechanism between caffeine and RyR2 can provide a reference for the development of therapeutic drugs for heart diseases such as arrhythmias caused by decreased RyR2 expression.

Key words： Cardiovascular diseases; Ryanodine receptor 2; Caffeine; Calcium homeostasis; Apoptosis

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