Department of Pharmacy, Peking Union Medical College Hospital, No.1 Shuaifuyuan, Wangfujing, Dongcheng District, Beijing, 100730, China.
Department of Cardiology, Peking Union Medical College Hospital, No.1 Shuaifuyuan, Wangfujing, Dongcheng District, Beijing, 100730, China.
Cardiovasc Drugs Ther. 2021 Aug;35(4):691-705. doi: 10.1007/s10557-020-07104-8. Epub 2020 Nov 2.
BACKGROUND/AIMS: The Nod-like receptor protein-3 (NLRP3) inflammasome signalling pathway is involved in the inflammatory reaction of myocardial ischaemia-reperfusion (I/R) injury. Our previous study showed that miR-330-5p was differentially expressed in both cerebral and myocardial I/R injury, and thus might be a biomarker for I/R injury-related diseases. Another study also indicated that miR-330-5p could promote NLRP3 inflammasome activation in renal IRI. However, the role of miR-330-5p in myocardial I/R injury-induced inflammatory responses is unknown. This study aimed to investigate the role of miR-330-5p in NLRP3 inflammasome-mediated myocardial I/R injury.
Myocardial I/R injury was induced in mice by occlusion of the left anterior descending coronary artery for 45 min followed by reperfusion. For NLRP3 inflammasome stimulation in vitro, cardiomyocytes were treated with 2 h of oxygen and glucose deprivation (OGD) or LPS (100 ng/ml). Myocardial miR-330-5p expression was examined by PCR at different treatment times. A miR-330-5p antagomir and an agomir were used to regulate miR-330-5p expression. To evaluate the role of miR-330-5p in myocardial I/R injury, 2,3,5-triphenyltetrazolium chloride (TTC) staining, echocardiography, and immunoblotting were used to assess infarct volume, cardiac function, and NLRP3 inflammasome activation respectively. A luciferase binding assay was used to examine whether miR-330-5p could directly bind to the T cell immunoglobulin domain and mucin domain-containing molecule-3 (TIM3). Finally, the role of the miR-330-5p/TIM3 axis in regulating apoptosis and NLRP3 inflammasome formation was evaluated with flow cytometry assays and immunofluorescence staining.
Compared to that in the model group, the inhibition of miR-330-5p significantly aggravated myocardial I/R injury, resulting in increased infarct volume and more severe cardiac dysfunction. Moreover, inhibition of miR-330-5p significantly increased the levels of NLRP3 inflammasome-related proteins, including caspase-1, IL-1β, IL-18 and TNF-α, in both in-vivo and in-vitro models. Furthermore, TIM3 was confirmed as a potential target of miR-330-5p. As predicted, suppression of TIM3 by siRNA ameliorated the anti-miR-330-5p-mediated activation of the NLRP3 inflammasome induced by OGD and LPS, thus decreasing cardiomyocyte apoptosis.
Our study indicated that the miR-330-5p/TIM3 axis was involved in the regulatory mechanism of NLRP3 inflammasome-mediated myocardial inflammation.
背景/目的：核苷酸结合寡聚化结构域样受体蛋白 3（NLRP3）炎症小体信号通路参与心肌缺血再灌注（I/R）损伤的炎症反应。我们之前的研究表明，miR-330-5p 在脑和心肌 I/R 损伤中均有差异表达，因此可能是 I/R 损伤相关疾病的生物标志物。另一项研究还表明，miR-330-5p 可以促进肾 IRI 中 NLRP3 炎症小体的激活。然而，miR-330-5p 在心肌 I/R 损伤诱导的炎症反应中的作用尚不清楚。本研究旨在探讨 miR-330-5p 在 NLRP3 炎症小体介导的心肌 I/R 损伤中的作用。
通过结扎左前降支冠状动脉 45 分钟后再灌注的方法诱导小鼠心肌 I/R 损伤。为了在体外刺激 NLRP3 炎症小体，用 2 小时的氧葡萄糖剥夺（OGD）或 LPS（100ng/ml）处理心肌细胞。在不同的处理时间用 PCR 检测心肌 miR-330-5p 的表达。用 miR-330-5p 反义寡核苷酸和激动剂来调节 miR-330-5p 的表达。为了评估 miR-330-5p 在心肌 I/R 损伤中的作用，用 2,3,5-氯化三苯基四氮唑（TTC）染色、超声心动图和免疫印迹分别评估梗死面积、心功能和 NLRP3 炎症小体的激活。用荧光素酶结合实验检测 miR-330-5p 是否可以直接与 T 细胞免疫球蛋白结构域和粘蛋白结构域包含分子-3（TIM3）结合。最后，用流式细胞术和免疫荧光染色评估 miR-330-5p/TIM3 轴在调节细胞凋亡和 NLRP3 炎症小体形成中的作用。
与模型组相比，抑制 miR-330-5p 显著加重心肌 I/R 损伤，导致梗死面积增加，心功能更严重。此外，在体内和体外模型中，抑制 miR-330-5p 均显著增加了 NLRP3 炎症小体相关蛋白，包括半胱氨酸天冬氨酸蛋白酶-1、白细胞介素-1β、白细胞介素-18 和肿瘤坏死因子-α的水平。此外，TIM3 被确认为 miR-330-5p 的一个潜在靶点。正如预期的那样，siRNA 抑制 TIM3 可改善 OGD 和 LPS 诱导的抗 miR-330-5p 介导的 NLRP3 炎症小体的激活，从而减少心肌细胞凋亡。
本研究表明，miR-330-5p/TIM3 轴参与了 NLRP3 炎症小体介导的心肌炎症的调节机制。
