NOX4 aggravates doxorubicin-induced cardiomyocyte pyroptosis by increasing reactive oxygen species content and activating the NLRP3 inflammasome

Background: Nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4)-mediated reactive oxygen species (ROS) has been reported to induce cardiomyocyte apoptosis, but its effect on pyroptosis of cardiomyocytes has been rarely reported. This paper aimed to explore the effects of NOX4-mediated ROS production on doxorubicin (DOX)-induced myocardial injury and pyroptosis through nucleotide-binding and oligomerization domain-like receptor protein 3 (NLRP3) inflammasome. Methods: HL-1 cells were treated with DOX or mice (30 mice were divided into five groups with six mice/group) underwent intraperitoneal injection with DOX (5 mg/kg, once a week, five times) to induce myocardial injury, followed by assessment of NOX4 and NLRP3 expression in cell supernatant and myocardial tissues. In cardiomyocyte HL-1 cells, cell proliferation was tested by MTT assay and the activity of ROS by probes. The superoxide dismutase (SOD) activity, malondialdehyde (MDA) content, and glutathione (GSH) activity were evaluated by kits. The expression of pyroptosis proteins was assessed by western blotting. Subsequently, the expression of NOX4 or NLRP3 was altered to determine the effect of NOX4 or NLRP3 expression on cardiomyocyte injury and pyroptosis. The animal models were utilized to evaluate the changes in the cardiac function of mice using an echocardiographic system, with these parameters measured including left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), and left ventricular end-diastolic diameter (LVEDD). Furthermore, the content of myocardial injury markers and the protein expression of pyroptosis proteins were determined to evaluate myocardial injury in the mice. Results: DOX treatment led to cardiomyocyte injury and pyroptosis, as evidenced by weakened LVEF, LVFS, and cell proliferation (P<0.05), elevated LVEDD, ROS, and MDA (P<0.05), increased expression of pyroptosis proteins (P<0.05), and decreased SOD and GSH (P<0.05). Additionally, NOX4 and NLRP3 were highly-expressed (P<0.05) in cell supernatant and myocardial tissues. In DOX-induced HL-1 cells, the overexpression of NOX4 intensified ROS levels to aggravate cardiomyocyte injury and pyroptosis, which was reversed by treatment of the ROS scavenger N-acetyl-cysteine. Furthermore, it was revealed that the combination of short hairpin RNA (sh)-NOX4 and overexpressed (oe)-NLRP3 reversed the cardioprotective effects of sh-NOX4 and increased myocardial tissue or cell injury and pyroptosis in vitro and in vivo. No mice died during the animal experiments, and only two were ruled out due to a weight loss greater than 20%. Conclusions: NOX4-mediated ROS production activated NLRP3 inflammasome, thereby aggravating DOX-induced myocardial injury in vitro and in vivo.