Wei Xiao-Hong, Chen Jie, Wu Xue-Fen, Zhang Qian, Xia Gui-Yang, Chu Xin-Yu, Xia Huan, Lin Sheng, Shang Hong-Cai
Key Laboratory of Chinese Internal Medicine of Ministry of Education and Beijing, Dongzhimen Hospital, Beijing University of Chinese Medicine, 5 Haiyuncang Hutong, Dongcheng District, Beijing, 100700, China.
Key Laboratory of Chinese Internal Medicine of Ministry of Education and Beijing, Dongzhimen Hospital, Beijing University of Chinese Medicine, 5 Haiyuncang Hutong, Dongcheng District, Beijing, 100700, China.
Phytomedicine. 2025 Jan;136:156260. doi: 10.1016/j.phymed.2024.156260. Epub 2024 Nov 14.
Mitochondrial ROS (mtROS) accumulation and NLRP3 inflammasome activation are critical in the pathogenesis of myocardial ischemia-reperfusion injury (MIRI). However, their upstream regulatory mechanisms and interaction remain inadequately understood.
The study aims to investigate the therapeutic effect of Salvianolic acid B (Sal B) on MIRI and elucidate its potential molecular mechanism, mainly focusing on the role of SIRT3.
SIRT3 was knocked down (SIRT3) and overexpressed (SIRT3) using small interfering RNA and plasmid, respectively. The role of SIRT3 in the cardioprotective effect of Sal B was explored using MIRI rat models and H9c2 cell hypoxia/reoxygenation (H/R) models. SIRT3, NLRP3 inflammasome proteins, and MnSOD expression were analyzed by Western blot and immunofluorescence staining. MtROS levels were assessed with mitochondrial superoxide indicators (MitoSOX™ Red). ELISA was utilized to measure the levels of LDH, CK-MB, cTnT, and markers of inflammation and oxidative stress. The interaction between SIRT3 and Sal B was studied through biolayer interferometry, cellular thermal shift assay and molecular docking.
Our findings revealed significantly decreased SIRT3 level, enhanced MnSOD acetylation, and activated NLRP3 inflammasome in myocardium after MIRI and H9c2 cardiomyocytes exposed to H/R conditions. SIRT3 promoted MnSOD acetylation and NLRP3 expression, aggravating mtROS accumulation and inflammation. Conversely, SIRT3 significantly inhibited MnSOD acetylation and NLRP3 inflammasome activation. In vitro studies confirmed the crosstalk between mtROS and NLRP3, demonstrating that mtROS scavenger inhibited NLRP3 inflammasome activation induced by H/R and SIRT3, and the NLRP3 inhibitor suppressed MnSOD acetylation in H/R and SIRT3 cardiomyocytes. Interestingly, Sal B was found to bind and upregulate SIRT3, reduce the expression of Acy-MnSOD, NLRP3, ASC, Caspase-1, and GSDMD, inhibit oxidative stress and inflammatory response, decrease myocardial infarct size and ST-segment elevation, and restore myocardial morphology. However, the protective effect of Sal B against MIRI was nullified by a specific SIRT3 inhibitor.
This study unveils that the SIRT3-mediated interplay between mtROS and the NLRP3 inflammasome is pivotal in the pathogenesis of MIRI. Furthermore, it highlights Sal B as a novel therapeutic agent that alleviates MIRI by targeting SIRT3, offering new insights into MIRI treatment.
线粒体活性氧（mtROS）积累和NLRP3炎性小体激活在心肌缺血再灌注损伤（MIRI）的发病机制中起关键作用。然而，它们的上游调控机制及其相互作用仍未得到充分了解。
本研究旨在探讨丹酚酸B（Sal B）对MIRI的治疗作用，并阐明其潜在的分子机制，主要聚焦于SIRT3的作用。
分别使用小干扰RNA和质粒敲低（SIRT3）和过表达（SIRT3）SIRT3。使用MIRI大鼠模型和H9c2细胞缺氧/复氧（H/R）模型探讨SIRT3在Sal B心脏保护作用中的作用。通过蛋白质免疫印迹和免疫荧光染色分析SIRT3、NLRP3炎性小体蛋白和MnSOD的表达。使用线粒体超氧化物指示剂（MitoSOX™ Red）评估mtROS水平。采用酶联免疫吸附测定法测量乳酸脱氢酶、肌酸激酶同工酶、心肌肌钙蛋白T以及炎症和氧化应激标志物的水平。通过生物膜干涉技术、细胞热位移分析和分子对接研究SIRT3与Sal B之间的相互作用。
我们的研究结果显示，在MIRI后心肌以及暴露于H/R条件的H9c2心肌细胞中，SIRT3水平显著降低，MnSOD乙酰化增强，NLRP3炎性小体激活。SIRT3促进MnSOD乙酰化和NLRP3表达，加重mtROS积累和炎症。相反，SIRT3显著抑制MnSOD乙酰化和NLRP3炎性小体激活。体外研究证实了mtROS与NLRP3之间的相互作用，表明mtROS清除剂抑制H/R和SIRT3诱导的NLRP3炎性小体激活，NLRP3抑制剂抑制H/R和SIRT3心肌细胞中的MnSOD乙酰化。有趣的是，发现Sal B与SIRT3结合并上调SIRT3，降低Acy-MnSOD、NLRP3、ASC、Caspase-1和GSDMD的表达，抑制氧化应激和炎症反应，减小心肌梗死面积和ST段抬高，并恢复心肌形态。然而，Sal B对MIRI的保护作用被一种特异性SIRT3抑制剂抵消。
本研究揭示SIRT3介导的mtROS与NLRP3炎性小体之间的相互作用在MIRI发病机制中起关键作用。此外，该研究突出了Sal B作为一种新型治疗药物，通过靶向SIRT3减轻MIRI，为MIRI治疗提供了新的见解。
