TLR9 deficiency alleviates doxorubicin‐induced cardiotoxicity via the regulation of autophagy

Figures

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  Figure 1. TLR9 deficiency protects against doxorubicin‐induced cardiotoxicity. (A) Myocardial damage markers in serum were detected on the 3rd day after the first DOX injection (n = 6). (B) Body weight change of mice during the first week (n = 6). (C‐F) Cardiac function and hemodynamic parameters of mice on the fourth week after the first DOX administration (n = 6‐9). (G) H&E staining and the cross‐sectional area analysis (n = 6‐9, 10 fields per coverslip). (H) PSR staining and collagen density measurement (n = 6‐9, 10 fields per coverslip). (I) Relative expression of mRNA (normalized to WT CTL) is presented in bar graphs (n = 6). Results are presented as mean ± SEM. *P < .05 versus corresponding group

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  Figure 2. TLR9 deficiency alleviates DOX‐induced cardiomyocyte apoptosis and oxidative stress. (A) Western blot analysis of apoptosis‐related proteins and oxidative stress‐related proteins in the hearts (n = 6). (B) Relative expression of proteins is presented in bar graphs (n = 6). (C) Images of TUNEL and the quantitative results (n = 6, 10 fields per coverslip). (D) Images of DHE and the quantitative results (n = 6, 10 fields per coverslip). Results are presented as mean ± SEM. *P < .05 vs corresponding group

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  Figure 3. TLR9 deficiency enhances autophagy in vivo and in vitro. (A) Western blot analysis of autophagy‐related proteins in the heart (n = 6). (B‐C) Relative expression of proteins is showed (n = 6). (D) Cells were treated with 1 μm DOX or PBS for 24 h, alone or with the TLR9 inhibitor ODN2088 (0.2 μmol/L), or with agonist ODN1826 (0.2 μM). Cell Immunofluorescence pictures, showing the differences of LC3 signal intensity among groups (n = 6, 10 fields per coverslip). (E‐F) Relative expression of autophagy‐related proteins in cell models (n = 6). Results are presented as mean ± SEM. *P < .05 vs corresponding group

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  Figure 4. Inhibition of autophagy in vitro abolishes the protective effect of TLR9 deficiency. (A) H9C2 cells treated with DOX or PBS were treated in combination with ODN2088 and 3‐MA (10 mmol/L). Images of TUNEL and the quantitative results (n = 6, 10 fields per coverslip). (B‐C) Relative expression of autophagy‐related genes in cell models (n = 6). (D) Images of DHE and the quantitative results of each cell group (n = 6, 10 fields per coverslip). Results are presented as mean ± SEM. *P < .05 vs corresponding group

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  Figure 5. Inhibition of autophagy in TLR9 knockout mice abrogates the protection against DOX‐ induced cardiac injury and dysfunction. (A‐B) Four groups of mice were treated with 3‐MA (10 mg/kg/d, i.p,) to determine the role of autophagy in DOX model. CK‐MB and LDH in serum were detected on the 3rd day after the first DOX injection (n = 6). (C‐F) Cardiac function and hemodynamic parameters of mice on the 4th week after the first DOX administration (n = 6‐8). Results are presented as mean ± SEM. *P < .05 vs corresponding group

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  Figure 6. Inhibition of autophagy abrogates the protection of TLR9 knockout in DOX‐ induced cardiotoxicity. (A) H&E staining and the cross‐sectional area analysis(n = 6‐8, 10 fields per coverslip). (B) PSR staining and collagen density measurement (n = 6‐8, 10 fields per coverslip). (C) Images of TUNEL and the quantitative results (n = 6, 10 fields per coverslip). (D) Images of DHE and the quantitative results (n = 6, 10 fields per coverslip). Results are presented as mean ± SEM. *P < .05 vs corresponding group

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  Figure 7. TLR9 promotes DOX‐related oxidative stress and apoptosis via p38 MAPK‐dependent autophagy in vitro. (A‐B) H9C2 cells were treated in combination with ODN1826 (0.2 μmol/L) with or without SB203580 (10 μmol/L) before DOX administration. Representative western blot and analysis of protein levels (n = 6). Results are presented as mean ± SEM. *P < .05 vs corresponding group

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  Figure 8. Illustrative diagram of the protection of TLR9 deficiency against DOX‐induced cardiotoxicity. TLR9 promoted DOX‐related oxidative stress and apoptosis via p38 MAPK‐dependent autophagy