Fig 1.
Protective effect of GB against DOX-induced cardiac cell death.
(A) Time and dose-dependent DOX-induced cardiac cell death was determined by MTT assay. (B) The safety dosage of GB on the cardiomyocytes viability was assessed by MTT assay. (C) Cardiomyocytes were treated with low (LG, 1μM) medium (MG, 5 μM) and high (HG, 50 μM) of GB after DOX treatment for 3 h and no improvement was found on the cardiomyocytes viability in DOX induced cardiotoxicity. (D) Pretreatment with low (LG), medium (MG) and high (HG) doses of GB for 30 min prior to DOX-induced cardiotoxicity increased cardiomyocytes viability. (E) Representative images of the effects of DOX and pretreatment with GB for 30 min on cardiomyocytes proliferation. ****p<0.0001 when compared to control group, #p<0.01, ##p<0.001 when compared to DOX.
Fig 2.
Protective effect of GB against DOX induced cardiomyocytes apoptosis.
(A) Representative images of Hoechst nuclear staining in each group. The extent of nuclear damage was decreased in the group of GB pretreatment for 30 min followed by DOX-induced cardiotoxicity comparatively to the DOX group. (B) Flow cytometry analysis of the cardiomyocytes apoptosis. Protective effects of GB pretreatment on the apoptosis of cardiomyocytes were demonstrated by Annexin V/PI staining. (C) Quantitative representation of the flow cytometry analysis. Cardiomyocytes apoptosis was inhibited following GB pretreatment for 30 min before DOX-induced cardiotoxicity. (D) and (E) Western-blot analysis of apoptosis related proteins (cleaved caspase 3, Bcl-2 and Bax) in cardiomyocytes after GB pretreatment for 30 min prior to DOX-induced cardiotoxicity. ****p<0.0001 when compared to control group, #p<0.05 ##p<0.01, ###p<0.001 when compared to the DOX group.
Fig 3.
GB pretreatment decreased the level of reactive oxygen species (ROS) in cardiomyocytes treated with DOX.
(A) Flow cytometry analysis of ROS level in cardiomyocytes treated with 5 μM GB (MG) before subjection to DOX induced cardiotoxicity. (B) Quantification of the mean fluorescence intensity (MFI) obtained from Flow cytometry. ****p<0.0001 when compared to control group, #p<0.0001 when compared to DOX.
Fig 4.
Intracellular calcium level and CaMKII phosphorylation was involved in the protective effect of GB in DOX induced cardiotoxicity.
(A) Intracellular calcium level was determined by Fura-2/AM probe. Increased intracellular calcium level was found after DOX treatment while GB pretreatment could significantly decrease the calcium level (B). Western blot analysis of CaMKII phosphorylation. Increased CaMKII phosphorylation was found after DOX treatment while GB pretreatment could significantly decrease the CaMKII phosphorylation. ****p<0.0001 when compared to control group, #p<0.0001 when compared to DOX.
Fig 5.
Akt phosphorylation was involved in the protective effect of GB in DOX induced cardiotoxicity.
(A) Immunofluorescence analysis of Akt phosphorylation. Decreased Akt phosphorylation was found after DOX treatment but pretreatment with GB restored this effect. (B) Western-blot analysis of Akt phosphorylation. Significantly decreased Akt phosphorylation was found after DOX treatment but GB treatment significantly activated the Akt phosphorylation. (C) Phosphorylation of p38, JNK, Erk, mTOR and GSK3β was not involved in the protective effect of GB in DOX induced cardiotoxicity. No significant difference was found on p38, p-JNK, p-Erk, p-mTOR and p-GSK3β as detected by western-blot analysis. ****p<0.0001when compared to control group, #p<0.0001 when compared to DOX.
Fig 6.
GB exert its protective effect by activating the PI3K/Akt pathway and inhibiting the CaMKII pathway.
Cardiomyocytes cell viability analysis was determined by MTT assay. PI3K inhibitor LY294002 (10 μM) pretreatment inhibited the protective effect of GB pretreatment in DOX induced cardiotoxicity while CaMKII inhibitor KN62 (10 μM) pretreatment significantly increased cell viability. ****p<0.0001 when compared to control group, #p<0.01 when compared to DOX group.
Fig 7.
Protective effect of GB on cardiac function in DOX induced cardiotoxic model in vivo.
(A) No significant difference was recorded regarding mouse weight in mice treated with DOX, GB or both. (B) Significant improvement of the left ventricle ejection fraction (LVEF) and decreased left ventricle mass (LVM) were found in mice treated with GB in DOX-induced cardiotoxic model. ****p<0.0001 when compared to control group, #p<0.05 when compared to DOX.