Cannabidiol Ameliorates Doxorubicin-Induced Myocardial Injury via Activating Hippo Pathway

Figures

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  Figure 1. DOX shows cardiotoxicity effects, and CBD shows no cardiotoxicity effects on H9c2 cells. (A and B) H9c2 cardiomyocytes were treated with different concentrations of DOX (A) and CBD (B) for 12 h or 24 h, and CCK-8 was used to detect cell viability. All of the experiments were repeated independently for 3 times. *, **, *** represent P<0.05, P<0.01, P<0.001, vs 0 μM group, respectively.

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  Figure 2. CBD shows cardioprotective effects against DOX treatment via ameliorating oxidative stress. (A) H9c2 cells were divided into 5 groups: control group, DOX group, 5μM CBD group, 10μM CBD group, 20μM CBD group. Then the morphology of the cells was observed under a microscope. (B) Immunofluorescence was applied to observe ROS level in different groups of H9ce cells (n=6 in each group). All of the experiments were repeated independently for 3 times. ### represents P<0.001 vs the control group. **, *** represent P<0.01, P<0.001, vs DOX group, respectively.

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  Figure 3. CBD shows cardioprotective effects against DOX treatment via repressing apoptosis and modulating Hippo pathway. (A) TUNEL assay was used to evaluate the apoptosis of H9c2 cells in different groups (n=6 in each group). (B) Western blot was used to detect apoptosis-related proteins including Bcl2, Bax and cleaved caspase-3 (n=3 in each group). (C) Western blotting was used to detect the levels of p-MST1, p-LATS1, p-YAP, MST1, LATS1, YAP, and TEAD4 in H9c2 cells from different groups (n=3 in each group). All of the experiments were repeated independently for 3 times. ### represents P<0.001 vs the control group. *** represents P<0.001, vs DOX group.

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  Figure 4. Depletion of YAP reverses the effects of CBD on the oxidative stress of cardiomyocytes in vitro. (A) H9c2 cells were divided into 5 groups: control group, DOX group, CBD treatment group, CBD treatment + YAP siRNA group, CBD treatment + control siRNA group. Immunofluorescence was applied to detect the expression and subcellular location of YAP (n=6 in each group). (B and C) The levels of SOD (B) and MDA (C) in H9c2 cells were detected to evaluate the oxidative stress of the cells (Figure 5B and C) (n=6 in each group). All of the experiments were repeated independently for 3 times. ## represents P<0.01 vs the control group. ** represents P<0.01, vs DOX group. ^^ represents P<0.01, vs CBD group.

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  Figure 5. Depletion of YAP reverses the effects of CBD on the apoptosis of cardiomyocytes in vitro. (A) TUNEL assay was applied to detect the apoptosis of H9c2 cells after DOX treatment, CBD treatment and YAP knockdown (n=6 in each group). (B) Flow cytometry was applied to detect the apoptosis of H9c2 cells after DOX treatment, CBD treatment and YAP knockdown (n=6 in each group). (C) Western blotting was used to detect apoptosis-related proteins including Bcl2, Bax, cleaved caspase-3, p53, PI3K, p-PI3K, Akt, and p-Akt (n=3 in each group). All of the experiments were repeated independently for 3 times. ### represents P<0.01 vs the control group. *** represents P<0.001, vs DOX group. ^^^ represents P<0.001, vs CBD group.

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  Figure 6. CBD shows cardioprotective effects against DOX-induced myocardial injury in mice via Hippo pathway. (A) The mice were divided into 4 groups. The control group, DOX treatment group, DOX treatment + CBD treatment group, DOX treatment + CBD treatment + VP treatment group (n=10 in each group). HE staining was applied to detect the injury of myocardial tissues (n =6 in each group). (B) WGA staining was applied to detect the injury of myocardial tissues in the mice of different groups (n =6 in each group). (C) Western blotting was performed to detect the expression levels of Hippo pathway-related proteins, including p-MST1, p-LATS1, p-YAP, MST1, LATS1, YAP and TEAD4, in the myocardial tissues of mice (n=3 in each group). ### represents P<0.001 vs the control group. *** represents P<0.001, vs DOX group. ^^^ represents P<0.001, respectively, vs DOX+CBD group.

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  Figure 7. CBD regulates apoptosis-related pathways in vivo via a Hippo pathway-dependent manner. (A) TUNEL assay was used to detect the apoptosis of cardiomyocytes in myocardial tissues of the mice in different groups (n=6 in each group). (B) Western blotting was used to detect apoptosis-related proteins including Bcl2, Bax, cleaved caspase-3, p53, PI3K, p-PI3K, Akt, and p-Akt, in the myocardial tissues of mice in different groups (n=3 in each group). ### represents P<0.001 vs the control group. *** represents P<0.001, vs DOX group. ^^^ represents P<0.001, respectively, vs DOX+CBD group.