NF-κB-Gasdermin D (GSDMD) Axis Couples Oxidative Stress and NACHT, LRR and PYD Domains-Containing Protein 3 (NLRP3) Inflammasome-Mediated Cardiomyocyte Pyroptosis Following Myocardial Infarction
Qian Lei, Tao Yi, Can Chen
Department of Cardiovascular Medicine, Affiliated Hospital of Guangdong Medical University, Guangdong Medical University, Zhanjiang, Guangdong, China (mainland)
Med Sci Monit 2018; 24: ANS6044-6052
Pyroptosis and oxidative stress play pivotal roles in cardiomyocyte loss after myocardial infarction. NF-κB is associated with oxidative stress and gasdermin D (GSDMD), the effector molecule of pyroptosis. However, the exact relationship between oxidative stress and cardiomyocyte pyroptosis remains unknown.
MATERIAL AND METHODS: We measured inflammasome-mediated cardiomyocyte pyroptosis in vivo via membrane pore formation, lactate dehydrogenase (LDH) release, and expression of caspase-1, cleaved caspase-1, NACHT, LRR and PYD domains-containing protein 3 (NLRP3), and apoptosis-associated speck-like protein containing a CARD (ASC). Furthermore, we induced pyroptosis in vitro by oxygen-glucose deprivation (OGD) in H9C2 cells. NLRP3 inflammasome-mediated pyroptosis was confirmed by LDH assay kit and Western blot. Oxidative stress was evaluated by reactive oxygen species (ROS) and superoxide dismutase (SOD) activity. We suppressed oxidative stress with N-acetyl-cysteine (NAC) and measured subsequent changes to the NF-κB-GSDMD axis and pyroptosis by LDH assay kit and Western blot. Then, we inhibited NF-κB activation with pyrrolidine dithiocarbamate (PDTC) and measured changes to GSDMD activity and pyroptosis by qRT-PCR, Western blot, and LDH assay kit.
RESULTS: Suppression of oxidative stress by NAC reduced NF-κB and GSDMD activation and increased pyroptosis, characterized by LDH release and NLRP3 inflammasome activation in H9C2 cells under OGD. Moreover, inhibition of NF-κB activation reduced GSDMD transcription and activation and NLRP3 inflammasome-mediated pyroptosis of H9C2 cells under OGD.
CONCLUSIONS: We demonstrated that the NF-κB-GSDMD axis functioned as a bridge between oxidative stress and NLRP3 inflammasome-mediated cardiomyocyte pyroptosis. Our findings provide important insight into the mechanism of myocardial infarction-related ventricular remodeling.
Keywords: Inflammasomes, NF-kappa B, Oxidative Stress