An Aza resveratrol–chalcone derivative 6b protects mice against diabetic cardiomyopathy by alleviating inflammation and oxidative stress

Figures

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  Figure 1. 6b attenuates HG‐induced cardiac hypertrophy, fibrosis and apoptosis in H9C2 cells. A. The chemical structures of Aza resveratrol–chalcone derivative 6b. B. H9c2 cells were pre‐treated with indicated dose of 6b for 1 hr and then incubated with HG (33 mM) for 36 hrs. Cells were harvested, and total lysates were immunoblotted. C–F. H9c2 cells were pre‐treated with indicated dose of 6b for 1 hr, following HG (33 m) incubation for 12 hrs. mRNA was extracted and subjected to real‐time qPCR assay as described in Materials and methods. Normalized values are plotted for COL‐1, TGF‐β, CTGF and MyHC, respectively. G and H. Rhodamine–phalloidin staining assay was performed as described in Materials and methods. The images were quantified (G). Normalized values are plotted (H). *P < 0.05, **P < 0.01, ***P < 0.001 versus CON; # P < 0.05, ## P < 0.01, ### P < 0.001 versus HG.

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  Figure 2. 6b mitigates HG‐induced NF‐κB‐mediated inflammatory response in H9c2 cells. A–B. H9c2 cells were pre‐treated with indicated dose of 6b for 1 hr and then incubated with HG (33 mM) for 8 hrs. The cell lysates were made, following nucleus protein extraction and immunoblotted (A). Immunofluorescence assay was performed as described in Materials and methods (B). The mRNA expression levels of VCAM‐1 (C), IL‐6 (D) and TNF‐α (E) were determined. The normalized values are plotted. F. H9c2 cells were pre‐treated with 6b for 1 hr, incubated with HG (33 mM) for 24 hrs as followed. TNF‐α secretion was subjected to ELISA as described in Materials and methods. Values detected were normalized with total protein. *P < 0.05, **P < 0.01, ***P < 0.001 versus CON; # P < 0.05, ## P < 0.01, ### P < 0.001 versus HG.

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  Figure 3. 6b attenuates HG‐induced myocardial oxidative stress in H9c2 cells. H9c2 cells were pre‐treated with indicated dose of 6b for 1 hr and then incubated with HG (33 mM) for 12 hrs. The cell lysates were immunoblotted for Nrf2, HO‐1 and NQO‐1, respectively (A). The mRNA expression levels of Nrf2 (B), NQO‐1 (C) and HO‐1 (D) were determined. Cells were subjected to immunofluorescence assay as described in Materials and methods. Images were acquired (E) and quantified (F). Normalized values are plotted. *P < 0.05, **P < 0.01 versus CON; ## P < 0.01, ### P < 0.001 versus HG.

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  Figure 4. 6b mitigates cardiac hypertrophy and apoptosis in diabetic myocardium. A. Representative histomorphometric images for the haematoxylin and eosin staining (H&E) of formalin‐fixed myocardial tissues from each group (400× magnification). B and C. The mRNA expression levels of ANP and BNP were subjected to real‐time qPCR assay as described in Materials and methods. D. Total tissue lysates were immunoblotted with anti‐MyHC and anti‐BAX antibodies. E and F. Heart tissues were subjected to TUNEL assay as described in Materials and methods. TUNEL images (400× magnification) were acquired (E) and quantified (F). G. Serum CK‐MB was determined as described in Materials and methods. **P < 0.001 versus Vehicle; # P < 0.05, ## P < 0.01, ### P < 0.001 versus STZ‐DM1.

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  Figure 5. 6b attenuates diabetes‐induced myocardial fibrosis. A–C. Myocardial tissues from each group were subjected to Masson Trichrome‐ and Sirius red staining assay as described in Materials and methods. Images were acquired (A) and quantified accordingly (B and C). D–E. The mRNA expression levels of TGF‐β and collagen I (COL‐1) in myocardial tissues of each group were determined by real‐time qPCR. F. The heart tissue lysates were immunoblotted with anti‐TGF‐β and anti‐COL‐1 antibodies, respectively. *P < 0.05, **P < 0.01, ***P < 0.001 versus Vehicle; # P < 0.05, ## P < 0.01, ### P < 0.001 versus STZ‐DM1.

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  Figure 6. 6b inhibits NFκB‐mediated myocardial inflammation in diabetic myocardium. A–D. Heart tissues were subjected to IHC analysis with anti‐VCAM‐1, anti‐CD68 and anti‐TNF‐α antibodies as described in Materials and methods (400× magnification). Images were acquired (A) and quantified accordingly (B–D). E and F. The mRNA expression levels of IL‐6 and IL‐1β in myocardial tissues were determined using real‐time qPCR. G and H. Heart tissues were subjected to immunofluorescence with anti‐P65 antibody as described in Materials and methods (400 × magnification). The images were quantified, and the normalized values are plotted (H). **P < 0.01, ***P < 0.001 versus Vehicle; # P < 0.05, ## P < 0.01, ### P < 0.001 versus STZ‐DM1.

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  Figure 7. 6b attenuates diabetes‐induced myocardial oxidative mechanistic through Nrf2. A–C. Myocardial tissues from STZ‐induced diabetic mice were subjected to immunostaining with anti‐DHE and anti 3‐NT antibodies as described in Materials and methods. The images were acquired (A) and quantified (B and C). D and E. Heart tissues from STZ‐induced diabetic mice were subjected to immunofluorescence with anti‐Nrf2 antibody. The images were acquired (D) and quantified (E). **P < 0.01, ***P < 0.001 versus Vehicle; ## P < 0.01, ### P < 0.001 versus STZ‐DM1.

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  Figure 8. A schematic illustration for the prevention of compound 6b from diabetes/HG‐induced injury in cardiomyocytes and heart.