Cannabidiol Ameliorates Doxorubicin-Induced Myocardial Injury via Activating Hippo Pathway

Abstract

Background: Doxorubicin (DOX) is a chemotherapeutic agent widely used for cancer treatment and has non-negligible cardiotoxicity. Some previous studies have reported that cannabidiol (CBD) has cardioprotective effects. In this study, we evaluated the protective effects of CBD against DOX-induced cardiomyocyte injury, and explored the downstream molecular mechanism.

Methods and materials: GSE193861, containing healthy myocardial tissues and myocardial tissues with DOX-induced injury, was analyzed to screen for the involved proteins and pathways. Molecular docking was performed to identify candidate drugs. After H9c2 cells were treated with DOX and CBD, their viability, oxidative stress, and apoptosis were assessed. After YAP depletion, the role of the Hippo pathway in CBD function was investigated. C57BL/6 mice were treated with DOX to establish an in vivo model, and CBD and verteporfin (VP) were used to treat the mice. Histological analyses and immunofluorescence were used to evaluate myocardial tissue injury, and apoptosis and oxidative stress of the myocardial tissues were also analyzed. Western blotting was used to investigate the regulatory effects of CBD on the Hippo and apoptosis-related pathways.

Results: Bioinformatic analysis suggested that the Hippo pathway was a crucial pathway involved in DOX-induced myocardial injury. Molecular docking showed that CBD targeted multiple regulators of the Hippo pathway. CBD showed cardioprotective effects against DOX-induced myocardial injury both in vitro and in vivo and regulated Hippo pathway activity in cardiomyocytes. After inactivation of the Hippo pathway by YAP knockdown or VP intervention, the protective effects of CBD were reversed.

Conclusion: For the first time, we revealed that CBD is likely to reduce DOX-induced myocardial injury by regulating the Hippo signaling pathway.

Keywords: Hippo pathway; cannabidiol; doxorubicin; heart injury.

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.

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.

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.

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.

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.

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.

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.