SYKT Alleviates Doxorubicin‐Induced Cardiotoxicity via Modulating ROS‐Mediated p53 and MAPK Signal Pathways

Figures

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  Figure 1. Culture and identification of mouse primary cardiomyocytes. (a) After 48 h of incubation, adherent cardiomyocytes accounted for approximately 90% of the cells. The cells exhibited various morphologies, such as spindle and polygonal shapes. After 72 h of incubation, the cells fused together (10×). (b) Cardiomyocytes identification using cTnI immunofluorescent staining (10×).

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  Figure 2. SYKT inhibits DOX-induced p53 activation. (A) DOX induces p53 expression in a time-dependent manner. Cardiomyocytes were treated with DOX for the indicated periods, and the p53 levels were analyzed by immunoblotting. β-Actin served as a loading control. (B) Effects of DOX (1 μM for 3 h), SYKT (30 mg/ml), and PFT-α (40 μM) on p53 expression. ap < 0.05 versus control; bp < 0.05 versus DOX; n = 6.

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  Figure 3. SYKT inhibits DOX-induced MAPK activation. (A) DOX induces p38/JNK MAPK activation in a time-dependent manner. (B) DOX-mediated p38/JNK MAPK activation is inhibited by SB 203580/SP600125 and by SYKT. The cardiomyocytes were treated with SB 203580/SP600125 prior to DOX (1 μM for 30 min) addition. β-Actin was used as an internal control. ap < 0.05 versus control; bp < 0.05 versus DOX; n = 6.

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  Figure 4. Influence of DOX on cytoplasmic and mitochondrial Bax and cytochrome c expression is time-dependent. Mouse primary cardiomyocytes were treated with 1 μM DOX for 1-8 h. Over time, cytoplasmic Bax expression gradually decreased and cytochrome c expression gradually increased, whereas mitochondrial Bax expression gradually increased and cytochrome c expression gradually decreased. β-Actin was used as the internal control, and Prohibitin was used as a mitochondrial marker.

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  Figure 5. Effects of SYKT on DOX-induced cardiomyocyte apoptosis-related proteins at 8 h after DOX processing. (a) The Bax levels were analyzed in the mitochondrial extracts by immunoblotting. (b) Effects of DOX and SYKT on cytoplasmic cytochrome c release in cardiomyocytes. (c) Western blotting analysis of the effects of DOX, SYKT, and DOX+SYKT on caspase-3 activation and PARP cleavage in cardiomyocytes. β-Actin was used as the internal control, and Prohibitin was used as a mitochondrial marker.

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  Figure 6. Determination of the MMP of cardiomyocytes from each group (n = 6 rats/group) at 8 h after DOX processing. (a) Histograms of the MMP of cardiomyocytes detected by Rhodamine 123 staining by FCM. (b) Photos of Rhodamine 123 staining of MMP in all groups by CLSM (100×).

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  Figure 7. Percent distribution of apoptotic and necrotic cells from each group (n = 6 rats/group) at 8 h after DOX processing. The cell distribution was analyzed using Annexin V binding and PI uptake. The FITC and PI fluorescence was measured using a FCM with FL-1 and FL-2 filters, respectively.