Carvacrol Attenuates Diabetic Cardiomyopathy by Modulating the PI3K/AKT/GLUT4 Pathway in Diabetic Mice

Figure 1. Chemical structure of carvacrol.

Figure 2. Carvacrol protects against hyperglycemia-induced cardiac remodeling in mice with type 1 diabetes mellitus (T1DM). (A) Gross view of whole hearts (upper); hematoxylin and eosin (H&E) staining of longitudinal sections of the heart in control mice or mice with STZ-induced T1DM after carvacrol or vehicle treatment for 6 weeks (lower); scale bar, 2.5 mm. (B) Heart weight/body weight (HW/BW) ratio of mice from the study groups. (C) Fold changes in natriuretic peptide A (Nppa, white rectangle) and β-myosin heavy chain (Myh7, black rectangle) mRNA levels determined by quantitative polymerase chain reaction (qPCR) in mouse hearts from the indicated study groups. (D) Representative images of H&E staining of control and diabetic mouse hearts after carvacrol or vehicle administration; scale bar, 50 μm. (E) Representative images of Masson’s trichrome staining for interstitial fibrosis of the myocardium (upper) and perivascular fibrosis (bottom) in five sets of mouse hearts from each group; scale bar, 50 μm. (F) Chamber sizes of left ventricle and right ventricle. (G) Quantification of cardiomyocytes area. (H) Quantification of relative fibrotic area. CON, control mice treated with vehicle…

Figure 3. Echocardiographic examination indicated that carvacrol mitigated myocardial dysfunction in STZ-induced type 1 diabetic mice. (A) Representative M-mode images (left) and PW Doppler mode waveform images of mitral valve flow (right) in control mice or mice with STZ-induced T1DM of carvacrol or vehicle treatment for 6 weeks. (B–H) Cardiac parameters in the indicated groups were measured: left ventricular ejection fractions (LVEF%, (B) and left ventricular fractional shortening (LVFS%, (B), early diastolic filling to atrial filling velocity ratio of mitral flow (E/A ratio, (C), mitral valve descending time (DT, D), anterior left ventricular wall (LVAW, E), left ventricular posterior wall (LVPW, F), left ventricular internal diameter (LVID, G), and heart rate (HR, H). CON, control mice treated with vehicle (0.1% DMSO); CON20, control mice treated with 20 mg/kg/day carvacrol; T1DM, mice with STZ-induced type 1 diabetes mellitus with vehicle (0.1% DMSO); T1DM+CAR10, diabetic mice treated with 10 mg/kg/day carvacrol; T1DM+CAR20, diabetic mice treated with 20 mg/kg/day carvacrol. Data are presented as mean ± SEM of 8–10 mice per group. *P < 0.05 and **P < 0.01 vs. CON group; # P < 0.05, ##…

Figure 4. Carvacrol protected against hyperglycemia-induced cardiac remodeling in db/db mice. (A) Gross view of whole hearts (upper); hematoxylin and eosin (H&E) staining of longitudinal sections of the heart in control or db/db mice after carvacrol, metformin, or vehicle treatment for 6 weeks (bottom); scale bar, 2.5 mm. (B) Heart weight/body weight (HW/BW) ratio in mice from the indicated groups. (C) Fold changes in Nppa and Myh7 mRNA levels, determined by qPCR, in murine hearts from the indicated groups. (D) Representative images of H&E staining of control and diabetic murine hearts after administration of carvacrol, metformin, or vehicle; scale bar, 50 μm. (E) Representative images of Masson’s trichrome staining for interstitial fibrosis of the myocardium (top) and perivascular fibrosis (bottom) in murine hearts from each group; scale bar, 50 μm. (F) Chamber sizes of the left and right ventricles. (G) Quantification of the area of cardiomyocytes. (H) Quantification of the relative fibrotic area. CON, control mice treated with vehicle (0.1% DMSO); T2DM, db/db mice treated with vehicle (0.1% DMSO); T2DM + MET, db/db mice treated with 100 mg/kg/day metformin; T2DM+CAR, db/db mice treated…

Figure 5. Carvacrol improved myocardial dysfunction, as indicated by echocardiographic examination in db/db mice. (A) Representative M-mode images (left) and PW Doppler mode waveform images of mitral valve flow (right) in control mice or diabetic db/db mice after treatment with carvacrol, metformin, or vehicle for 6 weeks. (B–H) Cardiac parameters were measured in the indicated groups and included: left ventricular ejection fractions (LVEF%, B) and left ventricular fractional shortening (LVFS%, B), ratio of early diastolic filling to atrial filling velocity of mitral flow (E/A ratio, C), mitral valve descending time (DT, D), anterior left ventricular wall (LVAW, E), left ventricular posterior wall (LVPW, F), left ventricular internal diameter (LVID, G), and heart rate (HR, H). CON, control mice treated with vehicle (0.1% DMSO); T2DM, db/db mice treated with vehicle (0.1% DMSO); T2DM+MET, db/db mice treated with 100 mg/kg/day metformin; T2DM+CAR, db/db mice treated with 20 mg/kg/day carvacrol. Data are presented as means ± SEM; 8–10 mice in each group. *P < 0.05, **P < 0.01 vs. CON group; # P < 0.05, ## P < 0.01 vs. T2DM group.

Figure 6. Carvacrol attenuates diabetic myocardial remodeling by restoring the PI3K/AKT pathway in diabetic mice. (A and B) Representative Western blotting images of whole lysate (A) and fold changes (B) in relative densitometric values of p-PI3K p85, p-PDK1, p-PTEN, and p-AKT in hearts from mice with STZ-induced type 1 diabetes treated with 10 mg/kg/day carvacrol, 20 mg/kg/day carvacrol, or vehicle. CON, control mice treated with vehicle (0.1% DMSO); CON20, control mice treated with 20 mg/kg/day carvacrol; T1DM, mice with STZ-induced type 1 diabetes mellitus with vehicle (0.1% DMSO); T1DM+CAR10, diabetic mice treated with 10 mg/kg/day carvacrol; T1DM+CAR20, diabetic mice treated with 20 mg/kg/day carvacrol. Data are presented as means ± SEM; 8–10 mice per group. *P < 0.05 and **P < 0.01 vs. CON group; # P < 0.05, ## P < 0.01 vs. T1DM group. (C and D) Representative Western blotting images of whole lysate (A) and fold changes (B) in relative densitometric values of phospho-PI3K p85, p-PDK1, p-PTEN, and p-AKT in murine hearts from the indicated groups. CON, control mice treated with vehicle (0.1% DMSO); T2DM, db/db mice treated with vehicle (0.1% DMSO); T2DM+MET, db/db mice treated with 100…

Figure 7. Carvacrol stimulated GLUT4 translocation by activating the AKT/AS160 pathway in the hearts of mice with STZ-induced diabetes. (A and B) Representative Western blotting images of whole lysate (A) and fold changes (B) in relative densitometric values of p-AKT and p-AS160 in the hearts of mice with STZ-induced type 1 diabetes treated with 10 mg/kg/day carvacrol, 20 mg/kg/day carvacrol, or vehicle. (C and D) Representative Western blotting images (C) and fold changes (D) of GLUT4 protein levels by relative densitometric values in membrane, cytoplasm, and whole lysates from murine hearts of the indicated groups. (E) Representative confocal microscopy of triple stains of GLUT4 (red), wheat germ agglutinin (WGA, green), and 4’,6-diamidino-2-phenylindole (DAPI, blue) in murine hearts of the indicated groups. Representative images from five sets of staining are shown. CON, control mice treated with vehicle (0.1% DMSO); CON20, control mice treated with 20 mg/kg/day carvacrol; T1DM, mice with STZ-induced type 1 diabetes mellitus with vehicle treatment (0.1% DMSO); T1DM+CAR10, diabetic mice treated with 10 mg/kg/day carvacrol; T1DM+CAR20, diabetic mice treated with 20 mg/kg/day carvacrol.…

Figure 8. Carvacrol enhanced membrane translocation of GLUT4 by upregulating the AKT/AS160 pathway in the hearts of db/db mice. (A and B) Representative Western blotting images of whole lysate (A) and fold changes (B) in relative densitometric values of p-AKT and p-AS160 in murine hearts from the indicated groups. (C and D) Representative Western blotting images (C) and fold changes (D) of GLUT4 protein levels by relative densitometric values in membrane, cytoplasm, and whole lysates from murine hearts in each study group. (E) Representative confocal images of triple stains of GLUT4 (red), wheat germ agglutinin (WGA, green), and 4’,6-diamidino-2-phenylindole (DAPI, blue) in the murine hearts of the indicated groups. Representative images from five sets of staining are shown. CON, control mice treated with vehicle (0.1% DMSO); T2DM, db/db mice treated with vehicle (0.1% DMSO); T2DM+MET, db/db mice treated with 100 mg/kg/day metformin; T2DM+CAR, db/db mice treated with 20 mg/kg/day carvacrol. Data are presented as means ± SEM; 8–10 mice in each group. *P < 0.05, **P < 0.01 vs. CON group; # P < 0.05, ## P < 0.01 vs. T2DM group.

Figure 9. Diagram of the signaling mechanism involved in the myocardial protection mediated by carvacrol.