Salvianolic acid B alleviated myocardial ischemia-reperfusion injury via modulating SIRT3-mediated crosstalk between mitochondrial ROS and NLRP3

Abstract

Background: 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.

Purpose: 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.

Methods: SIRT3 was knocked down (SIRT3^KD) and overexpressed (SIRT3^OE) 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.

Results: 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^KD promoted MnSOD acetylation and NLRP3 expression, aggravating mtROS accumulation and inflammation. Conversely, SIRT3^OE 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^KD, and the NLRP3 inhibitor suppressed MnSOD acetylation in H/R and SIRT3^KD 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.

Conclusion: 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.

Keywords: Acetylation; Myocardial ischemia reperfusion injury; NLRP3; Oxidative stress; SIRT3; Salvianolic acid B.