Long-Acting Thioredoxin Ameliorates Doxorubicin-Induced Cardiomyopathy via Its Anti-Oxidative and Anti-Inflammatory Action

Figures

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  Figure 1. Effect of HSA-Trx on myocardial atrophy and fibrosis in the heart of Dox-induced cardiomyopathy model mice. (A) Experimental protocol used for the evaluation of HSA-Trx on Dox-induced cardiomyopathy model mice: Dox-induced heart failure was induced by an intraperitoneal injection of 3 mg/kg Dox three times per week for 4 weeks. HSA-Trx (400 nmol/kg) was administered i.v. three times a week at 2 weeks after the first injection of Dox. An equivalent amount of PBS (10 mL/kg) was administered to the saline injection group (normal mice) and the Dox injection group. (B) Heart weight at 4 weeks after the first injection of Dox. Change in the level of (C) creatine phosphokinase (CPK) and (D) lactate dehydrogenase (LDH) at 4 weeks after the first injection of Dox. (E) Representative photomicrographs of Sirius red-stained heart sections at 4 weeks after the first injection of Dox. Original magnification: ×200. Scale bars represent 100 mm. Image analysis was performed to quantify the area of Sirius red staining. (F) α-SMA and (G) TGF-β mRNA expression in the heart at 4 weeks after the first injection of Dox were determined by real-time qPCR. Results are the means ± S.E. (n = 4). * p < 0.05,…

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  Figure 2. The effect of HSA-Trx on myocardial oxidative stress and inflammation in the hearts of Dox-induced cardiomyopathy model mice. (A) Representative photomicrographs of immunostaining for myocardial 8-OHdG (8-hydroxy-2′-deoxygenase) are shown at 4 weeks after the first injection of Dox. Original magnification: ×400. Scale bars represent 100 mm. Image analysis was performed to quantify the extent and intensity of 8-OhdG staining. (B) Trx and (C) MIF expression in the heart at 4 weeks after the first injection of Dox were assessed by Western blotting. (D) TNF-α, (E) I IL-6, (F) IL-10, (G) iNOS and (H) CD206 mRNA expression in the heart at 4 weeks after the first injection of Dox were determined by real-time qPCR. PBS or HSA-Trx (400 nmol/kg) was administered i.v. three times a week from 2 weeks after the first injection of Dox. Results are the means ± S.E. (n = 4). * p < 0.05, ** p < 0.01 compared with the saline injection group (normal mice).

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  Figure 3. Effect of HSA-Trx on myocardial atrophy-related genes in the heart of Dox-induced cardiomyopathy model mice. (A) Atrogin-1, (B) myostatin and (C) PGC-1a mRNA expression in the heart at 4 weeks after the first injection of Dox were determined by real-time qPCR. PBS or HSA-Trx (400 nmol/kg) was administered i.v. three times a week from 2 weeks after the first injection of Dox. Results are the means ± S.E. (n = 4). * p < 0.05 compared with the saline injection group (normal mice).

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  Figure 4. Effect of HSA-Trx on cell shrinkage and cellular ROS level in Dox-treated NRCMs: (A) Dox-induced cell shrinkage was determined by phalloidin staining in NRCMs. The phalloidin fluorescence was excited by illumination at 488 nm and then observed at room temperature using a confocal laser microscopy. NRCMs were also treated with DAPI (blue). Original magnification: ×25. Scale bar represents 100 mm. (B) Production of ROS in NRCMs was measured using CM-H2DCFDA, an ROS-sensitive fluorescent dye. Image analysis was performed to quantify the extent and intensity of DCF staining. Original magnification: ×10. Scale bar represents 100 µm. Results are the means ± S.E. (n = 9). ** p < 0.01 compared with control.

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  Figure 5. Schematic diagram showing the effect of HSA-Trx on Dox-induced myocardial atrophy.