Soluble Epoxide Hydrolase Inhibition Protected against Diabetic Cardiomyopathy through Inducing Autophagy and Reducing Apoptosis Relying on Nrf2 Upregulation and Transcription Activation

Figures

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  Figure 1. AUDA administration alleviated cardiac dysfunction and reduced apoptosis in db/db mice heart. (a) Representative immunoblots and quantitation of soluble epoxide hydrolase (sEH) expression, (b) +dp/dt max, (c) -dp/dt min, (d) left ventricular ejection fraction (LVEF), (e) left ventricular internal diameter end systole (LVID(s)), (f) left ventricular internal diameter end diastole (LVID(d)), and (g) E/A ratio. (h and i) Representative immunoblots and quantitation of protein expression of cleaved caspase-3, Bax, Bcl-2, NOX4, NOX2, and SOD1 in the myocardia of mice treated with or without the sEH inhibitor AUDA. (j and k) Representative immunoblots and quantitation of protein expression of cleaved caspase-3, Bax, Bcl-2, NOX4, NOX2, and SOD1 in HGHF-stimulated cardiomyocytes pretreated with or without AUDA. (l–o) Histological analysis and quantitation of DHE, TUNEL, and 3-NT staining in the myocardia in different groups. Data were expressed as mean ± SEM (n = 8 mice per group). ∗p < 0.05 vs. Con and #p < 0.05 vs. DM or HGHF. Con: db/m+vehicle; AUDA: db/m+AUDA; DM: db/db+vehicle; MABSA: mannitol+ BSA; DHE: dihydroethidium; HGHF: high glucose and high fat.

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  Figure 2. AUDA administration increased autophagy in diabetic mice heart and promoted autophagic flux under HGHF-treated cardiomyocytes in vitro. (a and b) Representative immunoblots and quantitation of protein level of Beclin 1, Atg3, and LC3II in the myocardia of mice from different groups. (c) Representative autophagic vacuoles from cardiac tissues visualized via transmission electron microscopy. (d and e) Representative immunoblots and quantitation of protein level of Beclin 1, Atg3, and LC3II in HGHF-stimulated cardiomyocytes in different groups. (f) LC3-II/β-actin ratio with or without bafilomycin A1 treatment during culture in HGHF conditions. (g and h) Representative images of fluorescent LC3 puncta and quantitation of cardiomyocytes with different treatments. Data were expressed as mean ± SEM (n ≥ 3 per group). ∗p < 0.05 vs. Con or BafA1-free and #p < 0.05 vs. DM or HGHF. Con: db/m+vehicle or control; AUDA: db/m+AUDA or AUDA; DM: db/db+vehicle; MABSA: mannitol+BSA; HGHF: high glucose and high fat.

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  Figure 3. AUDA administration upregulated the expression of Nrf2 and promoted its nuclear translocation rely on p-Akt. (a) Representative immunoblots and quantitation of protein expression of Nrf2, p-Akt, Akt, p-GSK3β, and GSK3β in the myocardia of mice from different groups. (b) Confocal observation of immunofluorescent staining for Nrf2 in the myocardia of mice from different groups. (c) Representative immunoblots of nuclear Nrf2 expression in HGHF-stimulated cardiomyocytes at different time points. (d) Representative immunoblots of nuclear Nrf2 expression in the HGHF-stimulated cardiomyocytes pretreated with AUDA at different time points. (e) Representative immunoblots of nuclear Nrf2 expression in the HGHF- and AUDA-stimulated cardiomyocytes pretreated with 50 μM LY294002 at different time points. (f) Representative images of fluorescent Nrf2 expression in the HGHF-stimulated cardiomyocytes in different groups. Data were expressed as mean ± SEM (n ≥ 3 per group). ∗p < 0.05 vs. Con or 0 h and #p < 0.05 vs. DM. Con: db/m+vehicle or control; AUDA: db/m+AUDA or AUDA; DM: db/db+vehicle; MABSA: mannitol+BSA; HGHF: high glucose and high fat.

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  Figure 4. Small interfering RNA of Nrf2 specifically reduced the protective effects of AUDA on cardiomyocytes autophagy and apoptosis. (a and b) Representative immunoblots and quantitation of protein expression of Beclin 1, Atg3, and LC3II in HGHF-stimulated cardiomyocytes in different groups. (c) LC3-II/β-actin ratio with or without bafilomycin A1 treatment under HGHF culture conditions. (d and e) Representative images of fluorescent LC3 puncta and quantitation of cardiomyocytes with different treatments. (f and g) Representative immunoblots and quantitation of protein expression of cleaved caspase-3, Bax, Bcl-2, NOX4, NOX2, and SOD1 in the HGHF-stimulated cardiomyocytes pretreated with or without AUDA. (h and i) Representative images and quantitation of cardiomyocyte apoptosis via the Annexin-V/PI apoptotic assay. (j and k) Representative images and quantitation of cardiomyocyte ROS levels via DCFH-DA. Data were expressed as mean ± SEM (n ≥ 3 per group). ∗p < 0.05 vs. si-Con+Con and #p < 0.05 vs. si-Nrf2+Con. HGHF: high glucose and high fat.

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  Figure 5. Virus carrying code for Nrf2 shRNA specifically reduced the protective effects of AUDA on cardiac function and autophagy level in db/db mice. (a) +dp/dt max, (b) -dp/dt min, (c) LVEF, (d) LVID(s), (e) LVID(d), and (f) E/A ratio. (g and h) Representative immunoblots and quantitation of protein expression of Beclin 1, Atg3, and LC3II in the myocardia of mice from different groups. (i) Representative images of autophagic vacuoles in cardiac tissues by transmission electron microscopy. Data were expressed as mean ± SEM (n = 8 per group). ∗p < 0.05 vs. ScshRNA+Con, #p < 0.05 vs. Nrf2shRNA+Con, and &p < 0.05 vs. Nrf2shRNA+DM. ScshRNA: AAV-ScshRNA; Nrf2shRNA: AAV-Nrf2shRNA; Con: db/m+vehicle; AUDA: db/m+AUDA; DM: db/db+vehicle.

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  Figure 6. Virus carrying code for Nrf2 shRNA specifically reduced the protective effects of AUDA on apoptosis in db/db mice heart. (a and b) Representative immunoblots and quantitation of protein expression of cleaved caspase-3, Bax, Bcl-2, NOX4, NOX2, and SOD1 in the myocardia of mice from different groups. (c–f) Histological analysis and quantitation of DHE, TUNEL, and 3-NT staining in the myocardia from different groups. (g) Mechanism diagram of full text. Data were expressed as mean ± SEM (n = 8 per group). ∗p < 0.05 vs. ScshRNA +Con, #p < 0.05 vs. Nrf2shRNA+Con, and &p < 0.05 vs. Nrf2shRNA+DM. ScshRNA: AAV-ScshRNA; Nrf2shRNA: AAV-Nrf2shRNA; Con: db/m+vehicle; AUDA: db/m+AUDA; DM: db/db+vehicle; DHE: dihydroethidium.