Nicotinamide adenine dinucleotide inhibits the production of reactive oxygen species and myocardial cell pyroptosis caused by hypoxia/re-oxygenation injury

Figure 1 Effects of nicotinamide adenine dinucleotide oxidized form on the viability of H9c2 cells following hypoxia/re-oxygenation treatment, with or without acetylcysteine or nicotinamide adenine dinucleotide oxidized form (NAD⁺) (n = 3). The normal group refers to H9c2 cells cultured in an incubator containing 21% oxygen and 5% CO₂, and serves as the control group; the hypoxia/re-oxygenation (H/R) group refers to H9c2 cells subjected to H/R; the H/R + acetyl group refers to H9c2 cells cultured under an H/R procedure with acetylcysteine treatment; the H/R + nicotinamide adenine dinucleotide group refers to H9c2 cells cultured under an H/R procedure with nicotinamide adenine dinucleotide oxidized. H/R: Hypoxia/re-oxygenation; NAD: Nicotinamide adenine dinucleotide; NAD⁺: Nicotinamide adenine dinucleotide, oxidized form.

Figure 2 Assessment of cell viability and apoptosis in H9c2 cells after hypoxia/re-oxygenation injury. A: Live/dead staining of H9c2 cells following hypoxia/re-oxygenation (H/R) treatment, with or without acetylcysteine or nicotinamide adenine dinucleotide oxidized form (NAD⁺) (n = 3); B: Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling assay results for H9c2 cells subjected to H/R treatment, with or without acetylcysteine or NAD+ (n = 3); C: Reactive oxygen species staining of H9c2 cells after H/R treatment with or without acetylcysteine or NAD⁺ (n = 3). H/R: Hypoxia/re-oxygenation; NAD: Nicotinamide adenine dinucleotide; NAD⁺: Nicotinamide adenine dinucleotide oxidized form.

Figure 3 Measurement of total cellular nicotinamide adenine dinucleotide levels and the nicotinamide adenine dinucleotide oxidized form/nicotinamide adenine dinucleotide reduced form ratio in H9c2 cells after hypoxia/re-oxygenation treatment, with or without acetylcysteine or nicotinamide adenine dinucleotide oxidized form (n = 4). A and B: The normal group refers to H9c2 cells cultured in an incubator containing 21% oxygen and 5% CO₂, and serves as the control group; the hypoxia/re-oxygenation (H/R) group refers to H9c2 cells subjected to H/R; the H/R + acetyl group refers to H9c2 cells cultured under an H/R procedure with acetylcysteine treatment; the H/R + nicotinamide adenine dinucleotide group refers to H9c2 cells cultured under an H/R procedure with nicotinamide adenine dinucleotide oxidized form (NAD⁺) treatment. H/R: Hypoxia/re-oxygenation; NAD: Nicotinamide adenine dinucleotide; NAD⁺: Nicotinamide adenine dinucleotide oxidized form; NADH: Nicotinamide adenine dinucleotide reduced form.

Figure 4 Effects of nicotinamide adenine dinucleotide oxidized form on the expression of NOD-like receptor pyrin domain-containing 3, associated speck-like protein containing a CARD, and caspase-1, reverse transcription polymerase chain reaction was used to analyze the gene expression of NOD-like receptor pyrin domain-containing 3, associated speck-like protein containing a CARD, and caspase-1 (n = 5). The normal group refers to H9c2 cells cultured in an incubator containing 21% oxygen and 5% CO₂, and serves as the control group; the hypoxia/re-oxygenation (H/R) group refers to H9c2 cells subjected to an H/R process; the H/R + acetyl group refers to H9c2 cells cultured under an H/R procedure with acetylcysteine treatment; the H/R + nicotinamide adenine dinucleotide group refers to H9c2 cells cultured under an H/R procedure with nicotinamide adenine dinucleotide oxidized form (NAD⁺) treatment. NLRP3: NOD-like receptor pyrin domain-containing 3; H/R: Hypoxia/re-oxygenation; ASC: Associated speck-like protein containing a CARD; NAD: Nicotinamide adenine dinucleotide; NAD⁺: Nicotinamide adenine dinucleotide oxidized form.

Figure 5 Western blot analysis of the protein levels of NOD-like receptor pyrin domain-containing 3, associated speck-like protein containing a CARD, and procaspase-1 in H9c2 cells after hypoxia/re-oxygenation treatment, with or without acetylcysteine or nicotinamide adenine dinucleotide oxidized form (NAD⁺) (n = 3). NLRP3: NOD-like receptor pyrin domain-containing 3; ASC: Associated speck-like protein containing a CARD; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; H/R: Hypoxia/re-oxygenation; NAD: Nicotinamide adenine dinucleotide; NAD⁺: Nicotinamide adenine dinucleotide oxidized form.