Potential roles of 4HNE-adducted protein in serum extracellular vesicles as an early indicator of oxidative response against doxorubicin-induced cardiomyopathy in rats

Figures

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  Fig. 1. Protocol of the animal treatment (A). dynamic changes of NT-proBNP levels (B). and survival curves of rats in NSS and DOXO groups (C). Syringes pictured in A represent time points of intraperitoneal injections of NSS or DOXO 3 mg/kg. Droplets of blood represent time points of blood collection. * p < 0.05 vs day 0 (baseline), † p < 0.05 vs NSS group at the same time point.

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  Fig. 2. Levels of 4HNE-adducted protein (μg/ 1 mg protein) in heart tissue of NSS-treated rats (NSS_Heart) and DOXO-treated rats (DOXO_Heart) (A). Levels of 4HNE-adducted protein in brain tissues of NSS-treated rats (NSS_Brain) and DOXO-treated rats (DOXO_Brain) (D). Copy number of catalase gene expression relative to beta-actin gene expression in heart tissues (B). Copy number of catalase gene expression relative to beta-actin gene expression in brain tissues (E). Copy number of glutathione peroxidase-1 gene expression relative to beta-actin gene expression in heart tissues (C). Copy number of glutathione peroxidase-1 gene expression relative to beta-actin gene expression in brain tissues (F). * p < 0.05 vs NSS_Heart.

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  Fig. 3. Western blot of EV markers including Hsp70, flotillin-1 and CD81, as well as beta actin in serum EVs of rats treated with NSS or DOXO (A). A representative size distribution bar graph of EVs isolated from serum of rats at baseline. Each bar represents the concentration of particles per milliliter in each size range. The error bars represent the standard deviation (SD) for each concentration (B). Bar graph representing mean size of serum EVs at five time points comparing EVs from NSS-treated (NSS_EVs) and EVs from DOXO-treated (DOXO_EVs) rats (C). Bar graph representing concentrations (x 1010 particles/ml of serum EVs) at each time point comparing between NSS_EVs and DOXO_EVs) (D). * p < 0.05 vs baseline, †p < 0.05 vs NSS_EVs.

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  Fig. 4. Levels of 4HNE-adducted protein (μg/ 1 mg EV protein) in NSS_EVs and DOXO-EVs collected at each time point (A). Levels of 4HNE-adducted protein (fold change relative to baseline) at baseline, on day 1 and day 9 of DOXO-treated rats that survive until the endpoint (DOXO_Survive) and did not (DOXO_Not survive) (B). Levels of 4HNE-adducted protein (fold change relative to baseline) on day 30 comparing the DOXO_Survive and DOXO_Not survive groups (C). * p < 0.05 vs baseline, † p < 0.05 vs NSS_EVs at the same time point, ‡ p < 0.05 vs DOXO_EVs on day 9, # p < 0.05 vs DOXO_Survive.

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  Fig. 5. Correlation graph of 4HNE-adducted protein levels in serum EVs at day 9 and 4HNE adducted protein levels in heart tissues (A). Correlation graph of 4HNE-adducted protein level in serum EVs at day 9 and Cat mRNA copy number relative to Actb in heart tissues (B). Correlation graph of 4HNE-adducted protein levels in serum EVs at day 9 and Gpx1 mRNA copy number relative to Actb in heart tissues (C). Correlation graph of 4HNE-adducted protein level in serum EVs at the endpoint and left ventricular ejection fraction (%LVEF) at endpoint (D). Correlation graph of 4HNE-adducted protein level in serum EVs at endpoint and left ventricular fractional shortening (%LVFS) at endpoint (E). R = Pearson correlation coefficient, P = p value, * p < 0.05.