Astragalus polysaccharide restores autophagic flux and improves cardiomyocyte function in doxorubicin-induced cardiotoxicity

Figure 1. Doxorubicin induces cardiotoxicity by disturbing normal autophagy in cardiomyocytes A. LC3B I/II expression, caspase-3, cleaved caspase-3 (c-caspase-3), Beclin1 and p62/SQSTM1, as assayed by Western blots, in NRVMs treated with doxorubicin at 0.1-5.0 μM for 24 h (n = 5). B. Cardiomyocytes were transfected with GFP-LC3 adenovirus for 24 h prior to doxorubicin treatment to label autophagic vesicles. An M.O.I. of 30 was used. The transfection efficiency of the GFP-LC3 adenovirus after 24 h was above 95% (scale bar: 100 μM). C. Co-localization efficiency of mRFP with GFP signals of tfLC3 puncta in control (Con) and 0.5 μM doxorubicin-treated H9c2 cells (n = 4, scale bar: 50 μM). Transfection efficiency of tfLC3 after 24 h was approximately 70% (*P <0.05, **P< 0.01, ***P <0.001 vs. Con).

Figure 2. APS restores autophagy in doxorubicin-treated cardiomyocytes A. Primary neonatal rat ventricular myocytes were transfected with GFP-LC3 adenovirus for 24 h prior to doxorubicin and APS treatment (scale bar: 100 μM), GFP-LC3-positive dots were measured by ImageJ software (n = 4, scale bar: 100 μM). B. H9c2 cells were transfected with a plasmid expressing mRFP-GFP-LC3 for 24 h prior to doxorubicin and APS treatment. The cells were viewed and imaged with an inverted fluorescence microscope. Co-localization efficiency of mRFP with GFP signals of tfLC3 puncta was measured using ImageJ software, and the percentage of total number of GFP puncta is shown (n = 4, scale bar: 50 μM). Transfection efficiency of tfLC3 after 24 h was approximately 70% C. LC3B expression in Con, DOX-treated and DOX+APS-treated H9c2 cells with or without bafilomycin (BFA) pretreatment (n = 5). (*P<0.05, **P < 0.01).

Figure 3. The protective role of APS in doxorubicin-induced cardiomyocyte injury A. APS reversed doxorubicin-induced increased LC3B II/I and p62/SQSTM1 and activated of caspase-3 in a concentration-dependent manner in NRVMs, as assayed by Western blots (n = 5, *P < 0.05, **P<0.01 vs. Con group; # P< 0.05,## P<0.01 vs. DOX group). B. Pretreatment of 100 nM rapamycin effectively reversed the APS protective effects in cardiomyocytes, as assessed by Western blots. The NRVMs were pretreated for 1 h with 100 nM rapamycin before APS (50 μg/ml) and DOX (0.5 μM) treatment. (n = 5, *P < 0.05, **P<0.01)

Figure 4. APS ameliorates doxorubicin-induced heart failure by restoring normal autophagy and decreasing apoptosis in vivo A. The average data of mouse body weight and heart weight/tibia length rate (g/mm) in sham, DOX, and DOX + APS groups 5 days after doxorubicin treatment (n = 8). B-C. Echocardiographic M-mode tracings and measurements of fractional shortening % (FS %) and ejection fraction % (EF %) 5 days after doxorubicin treatment (n = 8). D. Heart H&E staining of sham mice, doxorubicin-induced mice (DOX), and mice with APS pretreatment followed by doxorubicin treatment (DOX + APS) (n = 5, scale bar: 100 μM). E. TUNEL staining of apoptotic cells in the Sham, DOX and DOX + APS groups (n = 5, scale bar: 100 μM). F. Western blot and average data for LC3B II/I, Caspase-3, Bcl-2 in the sham, DOX, and DOX + APS groups (n = 5). (*P<0.05, **P<0.01, ***P<0.001).

Figure 5. APS attenuates doxorubicin-induced heart injury by regulation of the phosphorylation of AMPK/mTOR A. APS (50 μg/ml) attenuateddoxorubicin (0.5 μM)-induced increased AMPK phosphorylation and decreased mTOR phosphorylation, as assayed by Western blots (n = 5). B. Western blot and average data for AMPK/mTOR phosphorylation in the hearts of sham, DOX, and DOX+APS pretreatment groups (n = 8). C. The protective effect of APS was reversed by the mTOR inhibitor rapamycin. The NRVMs were pretreated with 100 nM rapamycin for 1 h before APS (50 μg/ml) and DOX (0.5 μM) treatment. mTOR was analyzed by Western blots (n = 5). (*P<0.05, **P <0.01, ***P<0.001).