Yangxin granules alleviate doxorubicin-induced cardiotoxicity by suppressing oxidative stress and apoptosis mediated by AKT/GSK3 β / β -catenin signaling

Figures

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  Figure 1.. Yangxin granules (YXC) promoted H9c2 cell proliferation and AKT/GSK3β/β-catenin pathway. (a) The MTT assay was performed to detect the viability of H9c2 cells following treatment with YXC (1, 5, 10, or 20 mg/mL) for 8, 12, or 24 hours. *P < 0.05, ***P < 0.001 versus 8 hours. (b) p-AKT, p-GSK3β, and total and nuclear β-catenin expression after treatment with YXC or an AKT inhibitor (MK-2206) was detected using western blotting. (c) The transcriptional activity of the downstream β-catenin pathway T-cell factor/lymphoid enhancer factor (TCF/LEF) was detected using the TOP/FOP Flash reporter assay. YXC+dox, treatment with YXC (5, 10, or 20 mg/mL) and subsequent co-treatment with 1 µM doxorubicin. 20 mg+dox+MK-2206, treatment with YXC (20 mg/mL) and MK-2206 (50 nM) and subsequent co-treatment with 1 µM doxorubicin. **P < 0.01, ***P < 0.001 versus control. #P < 0.05, ##P < 0.01, ###P < 0.001 versus doxorubicin. ΔΔΔP < 0.001 versus 20 mg YXC+dox.

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  Figure 2.. Yangxin granule (YXC) treatment suppressed doxorubicin-induced cytotoxicity and inflammation in H9c2 cells. (a) Cell viability was detected using the MTT assay. (b) Levels of the pro-inflammatory factors TNF-α, IL-1β, and IL-6 were detected using ELISA. (c) Phosphorylation of p65 after treatment with YXC or the AKT inhibitor MK-2206 was detected using western blotting. YXC+dox, treatment with YXC (5, 10, or 20 mg/mL) and subsequent co-treatment with 1 µM doxorubicin. 20 mg+dox+MK-2206, treatment with YXC (20 mg/mL) and MK-2206 (50 nM) and subsequent co-treatment with 1 µM doxorubicin. ***P < 0.001 versus control. #P < 0.05, ##P < 0.01, ###P < 0.001 versus dox. ΔΔP < 0.01, ΔΔΔP < 0.001 versus 20 mg YXC+dox.

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  Figure 3.. Yangxin granules (YXC) reduced doxorubicin-induced oxidative stress and cytotoxicity in H9c2 cells. (a) Reactive oxygen species (ROS), (b) malondialdehyde (MDA), (c) lactate dehydrogenase (LDH), and (d) superoxide dismutase (SOD) were detected using a commercial kit. YXC+dox, treatment with YXC (5, 10, or 20 mg/mL) and subsequent co-treatment with 1 µM doxorubicin. 20 mg+dox+MK-2206, treatment with YXC (20 mg/mL) and MK-2206 (50 nM) and subsequent co-treatment with 1 µM doxorubicin. ***P < 0.001 versus control. ##P < 0.01, ###P < 0.001 versus dox. ΔΔΔP < 0.001 versus 20 mg YXC+dox.

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  Figure 4.. Yangxin granules compound (YXC) reduced doxorubicin-induced apoptosis in H9c2 cells. (a and b) Flow cytometry analysis was performed to detect apoptotic cells. (c) The levels of apoptotic proteins including Bax, Bcl-2, cleaved caspase-3, and caspase 3 were assessed using western blotting. YXC+dox, treatment with YXC (5, 10, or 20 mg/mL) and subsequent co-treatment with 1 µM doxorubicin. 20 mg+dox+MK-2206, treatment with YXC (20 mg/mL) and MK-2206 (50 nM) and subsequent co-treatment with 1 µM doxorubicin. ***P < 0.001 versus control. #P < 0.05, ##P < 0.01, ###P < 0.001 versus dox. ΔP < 0.05, ΔΔP < 0.01, ΔΔΔP < 0.001 versus 20 mg YXC+dox.