Journal: Redox Report : Communications in Free Radical Research

Article Title: FOXO1-NMNAT3 axis dysregulation promotes doxorubicin cardiotoxicity: NAD + replenishment as a redox-targeted antioxidant therapy

doi: 10.1080/13510002.2025.2565033

Figure Lengend Snippet: Doxorubicin (DOX) induced cardiotoxicity and nicotinamide adenine dinucleotide (NAD + ) deficiency. (A) Viability of HL-1 and AC16 cells after treatment with 0.3125, 0.625, 1.25, 2.5 and 5 μM DOX for 24 and 48 h ( n = 3). (B) Lactate dehydrogenase (LDH) activity in culture supernatants of DOX-treated HL-1 cells and AC16 cells ( n = 3). (C-D) DOX induced body (C) and heart weight (D) loss in mice ( n = 10). (E) No significant change in heart-to-weight ratio in mice ( n = 10). (F-H) DOX induced elevated serum levels of brain natriuretic peptide (BNP) (F), cardiac troponin I (cTnI) (G), and creatine kinase-MB isoenzyme (CK-MB) (H) in mice ( n = 10). (I) Echocardiography in mice; DOX induced reduction in the left ventricular ejection fraction (EF%) and fractional shortening rate (FS%) of mice ( n = 10). (J) HE staining of heart tissue in mice; DOX induced significant cardiac atrophy in mice, with disorganised myocardial fibre arrangement, consolidation and wave-like degeneration ( n = 10); the arrows indicated key morphological changes. (K) Semi-quantitative scoring of myocardial injury in mice ( n = 10). (L) Wheat germ agglutinin (WGA) staining of heart tissue in mice; DOX induced a decrease in the cross-sectional area of cardiomyocytes in mice ( n = 10). (M-N) DOX induced reduction in NAD + levels in HL-1 cells, AC16 cells (M), and mouse heart tissues (N) ( n = 10). Data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 compared to the Control group; ns indicates no statistically significant difference between groups.

Article Snippet: Part 2: Mice were divided into six groups ( n = 5/group): (a) control group; (b) DOX group; (c) NAD + (#HY-B0445, MedChemExpress, Monmouth Junction, NJ, USA) group; (d) NAD + +DOX group; (e) nicotinamide mononucleotide (NMN) (#1094-61-7, BONTAC Bioengineering, Shenzhen, China) group; (f) NMN+DOX group.

Techniques: Activity Assay, Staining, Control

Journal: Redox Report : Communications in Free Radical Research

Article Title: FOXO1-NMNAT3 axis dysregulation promotes doxorubicin cardiotoxicity: NAD + replenishment as a redox-targeted antioxidant therapy

doi: 10.1080/13510002.2025.2565033

Figure Lengend Snippet: Nicotinamide adenine dinucleotide (NAD + ) is involved in doxorubicin (DOX)-induced oxidative stress in vitro ( n = 3). (A–B) Pretreatment with NAD + 1 h prior to DOX administration attenuated DOX-induced loss of the viability at 24 h in both HL-1 (A) and AC16 (B) cells. (C–D) Administration of NAD + 1 h post-DOX treatment attenuated DOX-induced loss of the viability at 24 h in HL-1 (C) and AC16 (D) cells. (E-F) Pretreatment with NAD + 1 h prior to DOX administration attenuated DOX-induced loss of the viability at 48 h in both HL-1 (E) and AC16 (F) cells. (G–H) Administration of NAD + 1 h post-DOX treatment attenuated DOX-induced loss of the viability at 48 h in HL-1 (G) and AC16 (H) cells. (I–K) Pretreatment of HL-1 and AC16 cells with NAD + (10 and 20 μM) 1 h prior to DOX administration attenuated DOX-induced elevation of malondialdehyde (MDA) levels (I), reduction in superoxide dismutase (SOD) activity (J), and decline in glutathione (GSH) levels (K). Data are presented as mean ± SD. ## P < 0.01, ### P < 0.001 and #### P < 0.0001 compared to the PBS or Control group, * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 compared to the DOX group; ns indicates no statistically significant difference compared to the DOX group.

Article Snippet: Part 2: Mice were divided into six groups ( n = 5/group): (a) control group; (b) DOX group; (c) NAD + (#HY-B0445, MedChemExpress, Monmouth Junction, NJ, USA) group; (d) NAD + +DOX group; (e) nicotinamide mononucleotide (NMN) (#1094-61-7, BONTAC Bioengineering, Shenzhen, China) group; (f) NMN+DOX group.

Techniques: In Vitro, Activity Assay, Control

Journal: Redox Report : Communications in Free Radical Research

Article Title: FOXO1-NMNAT3 axis dysregulation promotes doxorubicin cardiotoxicity: NAD + replenishment as a redox-targeted antioxidant therapy

doi: 10.1080/13510002.2025.2565033

Figure Lengend Snippet: Nicotinamide adenine dinucleotide (NAD + ) is involved in doxorubicin (DOX)-induced oxidative stress in vivo ( n = 5). (A) Schematic of NAD + or nicotinamide mononucleotide (NMN) supplementation administration in mice. (B) Effect of NAD + or NMN supplementation on NAD + levels in cardiac tissues of DOX-treated mice. (C) NAD + supplementation reduced DOX-induced increase in malondialdehyde (MDA) levels in mouse heart tissues. (D–E) NAD + supplementation improved DOX-induced decrease in superoxide dismutase (SOD) activity (D) and glutathione (GSH) levels (E) in mouse heart tissues. (F–H) Supplementation with NAD + or NMN had no significant effect on DOX-induced weight loss (F), heart weight loss (G) and heart-to-weight ratio (H) in mice. (I) Echocardiograms of DOX-treated mice after supplementation with NAD + or NMN. (J-K) NAD + supplementation alleviated the DOX-induced decrease in ejection fraction (EF%) (J) and fractional shortening rate (FS%) (K) of mice. (L) HE staining plots of cardiac tissues of DOX-treated mice after supplementation with NAD + or NMN; the arrows indicated key morphological changes. (M) Myocardial injury scores in DOX-treated mice after supplementation with NAD + or NMN. (N-P) Supplementation with NAD + attenuated DOX-induced elevation of serum brain natriuretic peptide (BNP) (N), cardiac troponin I (cTnI) (O) and creatine kinase-MB isoenzyme (CK-MB) (P) in mice. Data are presented as mean ± SD. ## P < 0.01, ### P < 0.001 and #### P < 0.0001 compared to the Control group, * P < 0.05, ** P < 0.01 and *** P < 0.001 compared to the DOX group, ns indicates no statistically significant difference.

Article Snippet: Part 2: Mice were divided into six groups ( n = 5/group): (a) control group; (b) DOX group; (c) NAD + (#HY-B0445, MedChemExpress, Monmouth Junction, NJ, USA) group; (d) NAD + +DOX group; (e) nicotinamide mononucleotide (NMN) (#1094-61-7, BONTAC Bioengineering, Shenzhen, China) group; (f) NMN+DOX group.

Techniques: In Vivo, Activity Assay, Staining, Control

Journal: Redox Report : Communications in Free Radical Research

Article Title: FOXO1-NMNAT3 axis dysregulation promotes doxorubicin cardiotoxicity: NAD + replenishment as a redox-targeted antioxidant therapy

doi: 10.1080/13510002.2025.2565033

Figure Lengend Snippet: Effects of doxorubicin (DOX) on the protein and gene expression of nicotinamide adenine dinucleotide (NAD + ) synthases and consuming enzymes. (A-C) DOX induced a decrease in nicotinamide mononucleotide adenylyl transferase 2 (NMNAT2) and NMNAT3 protein expression in HL-1 cells (A) ( n = 3), AC16 cells (B) ( n = 3) and mouse heart tissues (C) ( n = 5); quantitative analysis results of grey value of western blot bands. (D-F) DOX induced elevated cluster of differentiation 38 (CD38) protein expression and decreased sirtuin 1 (SIRT1), SIRT3, and poly-ADP-ribose polymerase 1 (PARP1) protein expression in HL-1 cells (D) ( n = 3), AC16 cells (E) ( n = 3) and mouse heart tissues (F) ( n = 5); quantitative analysis results of grey value of western blot bands. (G-I) DOX induced a decrease in the mRNA levels of NMNAT2 and NMNAT3 in HL-1 cells (G) ( n = 3), AC16 cells (H) ( n = 3) and mouse heart tissues (I) ( n = 5). (J-M) DOX induced an increase in mRNA levels of CD38 and a decrease in mRNA levels of SIRT1, SIRT3 and PARP1 in HL-1 cells (J) ( n = 3), AC16 cells (K) ( n = 3) and mouse heart tissues (M) ( n = 5). Data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 compared to the Control group; ns indicates no statistically significant difference.

Article Snippet: Part 2: Mice were divided into six groups ( n = 5/group): (a) control group; (b) DOX group; (c) NAD + (#HY-B0445, MedChemExpress, Monmouth Junction, NJ, USA) group; (d) NAD + +DOX group; (e) nicotinamide mononucleotide (NMN) (#1094-61-7, BONTAC Bioengineering, Shenzhen, China) group; (f) NMN+DOX group.

Techniques: Gene Expression, Expressing, Western Blot, Control

Journal: Redox Report : Communications in Free Radical Research

Article Title: FOXO1-NMNAT3 axis dysregulation promotes doxorubicin cardiotoxicity: NAD + replenishment as a redox-targeted antioxidant therapy

doi: 10.1080/13510002.2025.2565033

Figure Lengend Snippet: Overexpression of nicotinamide mononucleotide adenylyltransferase 3 (NMNAT3) and supplementation of nicotinamide mononucleotide (NMN) ameliorated the doxorubicin (DOX)-induced decrease in the viability, decrease in nicotinamide adenine dinucleotide (NAD + ) levels and oxidative stress ( n = 3). (A-B) Overexpression of NMNAT3 ameliorated DOX-induced decrease in NMNAT3 protein expression in HL-1 cells (A) and AC16 cells (B); quantitative analysis results of grey value of western blot bands. (C-D) Overexpression of NMNAT3 and supplementation of NMN ameliorated the decrease in the viability of DOX-treated HL-1 (C) and AC16 cells (D) for 24 h. (E-F) Overexpression of NMNAT3 and supplementation of NMN ameliorated the decrease in the viability of DOX-treated HL-1 (E) and AC16 cells (F) for 48 h. (G-H) Overexpression of NMNAT3 and supplementation with NMN ameliorated the DOX-induced decrease in NAD + levels in HL-1 (G) and AC16 cells (H). (I-J) Overexpression of NMNAT3 and supplementation with NMN ameliorated DOX-induced increase in malondialdehyde (MDA) levels in HL-1 (I) and AC16 cells (J). (K-L) Overexpression of NMNAT3 and supplementation of NMN improved DOX-induced decrease in superoxide dismutase (SOD) activity in HL-1 (K) and AC16 cells (L). (M-N) Overexpression of NMNAT3 and supplementation with NMN improved DOX-induced decrease in glutathione (GSH) levels in HL-1 (M) and AC16 cells (N). Data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 compared to the Lv-Control group; ns indicates no statistically significant difference.

Article Snippet: Part 2: Mice were divided into six groups ( n = 5/group): (a) control group; (b) DOX group; (c) NAD + (#HY-B0445, MedChemExpress, Monmouth Junction, NJ, USA) group; (d) NAD + +DOX group; (e) nicotinamide mononucleotide (NMN) (#1094-61-7, BONTAC Bioengineering, Shenzhen, China) group; (f) NMN+DOX group.

Techniques: Over Expression, Expressing, Western Blot, Activity Assay, Control

Journal: Redox Report : Communications in Free Radical Research

Article Title: FOXO1-NMNAT3 axis dysregulation promotes doxorubicin cardiotoxicity: NAD + replenishment as a redox-targeted antioxidant therapy

doi: 10.1080/13510002.2025.2565033

Figure Lengend Snippet: Effects of overexpression of nicotinamide mononucleotide adenylyltransferase 3 (NMNAT3) on nuclear factor erythroid-2-related factor 2 (NRF2), NAD(P)H: quinone oxidoreductase 1 (NQO1) and heme oxygenase-1 (HO-1) protein expression in doxorubicin (DOX)-treated HL-1 and AC16 cells ( n = 3). (A) Overexpression of NMNAT3 ameliorated DOX-induced reduction of NRF2 nuclear translocation in HL-1 cells compared with that in the Lv-Control group; quantitative analysis results of grey value of western blot bands. (B) Overexpression of NMNAT3 ameliorates DOX-induced decrease in NQO1 and HO-1 protein expression in HL-1 cells compared with that in the Lv-Control group; quantitative analysis results of grey value of western blot bands. (C) Overexpression of NMNAT3 ameliorated DOX-induced reduction of NRF2 nuclear translocation in AC16 cells compared with that in the Lv-Control group; quantitative analysis results of grey value of western blot bands. (D) Overexpression of NMNAT3 ameliorates DOX-induced decrease in NQO1 and HO-1 protein expression in AC16 cells compared with that in the Lv-Control group; quantitative analysis results of grey value of western blot bands. Data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 compared to the Lv- Control group; ns indicates no statistically significant difference.

Article Snippet: Part 2: Mice were divided into six groups ( n = 5/group): (a) control group; (b) DOX group; (c) NAD + (#HY-B0445, MedChemExpress, Monmouth Junction, NJ, USA) group; (d) NAD + +DOX group; (e) nicotinamide mononucleotide (NMN) (#1094-61-7, BONTAC Bioengineering, Shenzhen, China) group; (f) NMN+DOX group.

Techniques: Over Expression, Expressing, Translocation Assay, Control, Western Blot

Journal: Redox Report : Communications in Free Radical Research

Article Title: FOXO1-NMNAT3 axis dysregulation promotes doxorubicin cardiotoxicity: NAD + replenishment as a redox-targeted antioxidant therapy

doi: 10.1080/13510002.2025.2565033

Figure Lengend Snippet: Effects of overexpression of nicotinamide mononucleotide adenylyltransferase 3 (NMNAT3) and supplementation of nicotinamide mononucleotide (NMN) on nuclear factor erythroid-2-related factor 2 (NRF2), NAD(P)H: quinone oxidoreductase 1 (NQO1) and heme oxygenase-1 (HO-1) protein expression in doxorubicin (DOX)-treated HL-1 and AC16 cells ( n = 3). (A) Supplementation of NMN had no significant effect on DOX-induced reduction of NRF2 nuclear translocation in HL-1 cells not overexpressing NMNAT3 compared with the DOX group; quantitative analysis results of grey value of western blot bands. (B) Supplementation of NMN ameliorated DOX-induced reduction of NRF2 nuclear translocation in HL-1 cells overexpressing NMNAT3 compared with the DOX group; quantitative analysis results of grey value of western blot bands. (C) Supplementation of NMN had no significant effect on DOX-induced reduction of NRF2 nuclear translocation in AC16 cells not over-expressing NMNAT3 compared with the DOX group; quantitative analysis results of grey value of western blot bands. (D) Supplementation of NMN ameliorated DOX-induced reduction of NRF2 nuclear translocation in AC16 cells overexpressing NMNAT3 compared with that in the DOX group; quantitative analysis results of grey value of western blot bands. (E–F) Supplementation of NMN further ameliorated the DOX-induced decrease in NQO1 and HO-1 protein expression in HL-1 (E) and AC16 cells (F) overexpressing NMNAT3 compared with that in the Lv-Control group; quantitative analysis results of grey value of western blot bands. Data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 compared to the Lv-Control group; ns indicates no statistically significant difference.

Article Snippet: Part 2: Mice were divided into six groups ( n = 5/group): (a) control group; (b) DOX group; (c) NAD + (#HY-B0445, MedChemExpress, Monmouth Junction, NJ, USA) group; (d) NAD + +DOX group; (e) nicotinamide mononucleotide (NMN) (#1094-61-7, BONTAC Bioengineering, Shenzhen, China) group; (f) NMN+DOX group.

Techniques: Over Expression, Expressing, Translocation Assay, Western Blot, Control

Journal: Redox Report : Communications in Free Radical Research

Article Title: FOXO1-NMNAT3 axis dysregulation promotes doxorubicin cardiotoxicity: NAD + replenishment as a redox-targeted antioxidant therapy

doi: 10.1080/13510002.2025.2565033

Figure Lengend Snippet: Effects of overexpression of nicotinamide mononucleotide adenylyltransferase 3 (NMNAT3) and supplementation of nicotinamide mononucleotide (NMN) on protein expression of nicotinamide adenine dinucleotide (NAD + )-consuming enzymes in doxorubicin (DOX)-treated HL-1 and AC16 cells ( n = 3). (A-B) Overexpression of NMNAT3 ameliorated DOX-induced decrease in sirtuin 1 (SIRT1), SIRT3, and poly-ADP-ribose polymerase 1 (PARP1) protein expression in HL-1 (A) and AC16 cells (B); quantitative analysis results of grey value of western blot bands. (C–D) Supplementation of NMN ameliorated DOX-induced reduction in SIRT1, SIRT3 and PARP1 protein expression in HL-1 (C) and AC16 cells (D) overexpressing NMNAT3; quantitative analysis results of grey value of western blot bands. Data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 compared to the Lv-Control group; ns indicates no statistically significant difference.

Article Snippet: Part 2: Mice were divided into six groups ( n = 5/group): (a) control group; (b) DOX group; (c) NAD + (#HY-B0445, MedChemExpress, Monmouth Junction, NJ, USA) group; (d) NAD + +DOX group; (e) nicotinamide mononucleotide (NMN) (#1094-61-7, BONTAC Bioengineering, Shenzhen, China) group; (f) NMN+DOX group.

Techniques: Over Expression, Expressing, Western Blot, Control

Journal: Redox Report : Communications in Free Radical Research

Article Title: FOXO1-NMNAT3 axis dysregulation promotes doxorubicin cardiotoxicity: NAD + replenishment as a redox-targeted antioxidant therapy

doi: 10.1080/13510002.2025.2565033

Figure Lengend Snippet: Mechanism model diagram of DIC. DOX causes a decrease in phosphorylation at the FOXO1 Ser319 site, an increase in FOXO1 transcriptional activity, and inhibition of NMNAT3 transcription, leading to a decrease in intracellular NAD + levels. Decreased NAD + levels directly affect NRF2 or modulate NRF2 by influencing SIRT1 expression, leading to oxidative stress and damage to cardiomyocytes.

Article Snippet: Part 2: Mice were divided into six groups ( n = 5/group): (a) control group; (b) DOX group; (c) NAD + (#HY-B0445, MedChemExpress, Monmouth Junction, NJ, USA) group; (d) NAD + +DOX group; (e) nicotinamide mononucleotide (NMN) (#1094-61-7, BONTAC Bioengineering, Shenzhen, China) group; (f) NMN+DOX group.

Techniques: Phospho-proteomics, Activity Assay, Inhibition, Expressing