Fig 1.
(A) and (B) Cell viability determined by CCK-8 assays after treatment with DOX at different concentrations for 24 h and treatment with 1 μmol/L DOX for different times (n = 4 or 5); (C) Representative DCFH-DA images and statistical results (n = 5); (D) SOD, GSH-Px, MDA, and NADPH levels in H9c2 cells (n = 4); (E) Apoptosis rate measured by flow cytometry (n = 3). (F) Representative TUNEL staining images and statistical results (n = 3). Values represent the mean±SD.*p<0.05 vs. Con group,**p<0.01 vs. Con group.
Fig 2.
DOX induces mitochondrial damage in H9c2 cells.
(A) Western blot detection of apoptosis-related protein levels and statistical results (n = 3); (B) Representative JC-1 images and quantification of fluorescence intensity for JC-1 monomers/aggregates (n = 4); (C) ATP level (n = 4); (D) Representative images of mitochondria in H9c2 cells observed by transmission electron microscopy; (E) Western blot detection of p-AMPK, AMPK, and UCP2 levels and statistical results (n = 3). Values are presented as the mean ± SD. *p<0.05 vs. Con group, **p<0.01 vs. Con group.
Fig 3.
AP39 ameliorates DOX-induced myocardial injury.
(A)-(C) Cell viability determined by CCK-8 assays after H9c2 cells were treated with different concentrations of AP39 for 24 h,1 μmol/L DOX and different concentrations of AP39 for 24 h (n = 4); (D) H2S content in cells of each group (n = 4); (E) Representative DCFH-DA images and statistical results (n = 5); (F) SOD, GSH-Px, MDA, and NADPH levels in H9c2 cells (n = 4); (G) Apoptosis rate measured by flow cytometry (n = 3). (H) Representative TUNEL staining images and statistical results (n = 3). Values are presented as the mean±SD. *p<0.05 vs. Con group, **p<0.01 vs. Con group. #p<0.05 vs. DOX group, ##p<0.01 vs. DOX group.
Fig 4.
AP39 ameliorates DOX-induced mitochondrial damage.
(A) Western blot detection of apoptosis-related protein levels and statistical results (n = 3 or 4); (B) Representative JC-1 images and quantification of fluorescence intensity for JC-1 monomers/aggregates (n = 4); (C) ATP level (n = 4); (D) Representative images of mitochondria in H9c2 cells observed by transmission electron microscopy; (E) Western blot detection of p-AMPK, AMPK, and UCP2 levels and statistical results (n = 3 or 4). Values are presented as the mean ± SD. *p < 0.05 vs. Con group, **p < 0.01 vs. Con group. #p < 0.05 vs. DOX group, ##p < 0.01 vs. DOX group. NS indicates no significant difference vs. Con group.
Fig 5.
Inhibition of AMPK expression limits the beneficial effect of AP39 on DOX-induced cardiotoxicity.
(A) Western blot detection of p-AMPK and AMPK levels and statistical results (n = 3); (B) CCK-8 assay of cell viability (n = 4); (C) Representative DCFH-DA images and statistical results (n = 5); (D) SOD, GSH-Px. MDA, and NADPH levels in H9c2 cells (n = 4); (E) Apoptosis rate measured by flow cytometry (n = 3); (F) Representative TUNEL staining images and statistical results (n = 3). (G) Western blot detection of apoptosis-related protein levels and statistical results (n = 3 or 4); (H) Representative JC-1 images and quantification of fluorescence intensity for JC-1 monomers/aggregates (n = 4); (I) ATP level (n = 4); (J) Western blot detection of UCP2 levels and statistical results (n = 3). Values are presented as the mean±SD.*p<0.05,**p<0.01. NS indicates no significant difference vs. Con group.
Fig 6.
AP39 improves DOX-induced cardiotoxicity by preventing the down-regulation of UCP2.
(A) Western blot analysis of UCP2 (n = 3) and qRT-PCR for UCP2 mRNA levels (n = 3); (B) CCK-8 assay of cell viability (n = 4); (C) Representative DCFH-DA images and statistical results (n = 5); (D) SOD, GSH-Px, MDA, and NADPH levels in H9c2 cells (n = 4); (E) Apoptosis rate measured by flow cytometry (n = 3); (F) Representative TUNEL staining images and statistical results (n = 3). (G) Western blot detection of apoptosis-related protein levels and statistical results (n = 3 or 4); (H) Representative JC-1 images and quantification offluorescence intensity for JC-1 monomers/aggregates (n = 4); (I) ATP levels (n = 4); (J) Western blot detection of p-AMPK and AMPK levels and statistical results (n = 3). Values are presented as the mean±SD.*p<0.05,**p<0.01. NS indicates no significant difference vs. Con group.
Fig 7.
AP39 ameliorates DOX-induced cardiotoxicity in rats by regulating the AMPK/UCP2 pathway.
(A) Body weight in different groups of rats (n = 10); (B) Heart/body weight ratio in different groups of rats (n = 10); (C) Representative echocardiographic images and quantitative analysis of EF%, FS%, and E/A (n = 7); (D) Serum TNNT2, CK-MB, LDH, and BNP levels in rats (n = 10); (E) Representative HE, Masson and TUNEL staining images of the rat myocardium;(F) Representative images of mitochondria in rat cardiomyocytes observed by transmission electron microscopy; (G) SOD, GSH-Px, MDA, and NADPH levels in rat serum (n = 10); (H) Westen blot detection of apoptosis-related protein, p-AMPK, AMPK and UCP2 levels and statistical results (n = 3 or 4). Values are presented as the mean±SD.*p<0.05,**p<0.01. NS indicates no significant difference vs. Con group.