Myricetin Possesses Potential Protective Effects on Diabetic Cardiomyopathy through Inhibiting IκBα/NFκB and Enhancing Nrf2/HO‐1

Figure 1. Myricetin prevented the mouse heart from STZ-induced remodeling. (a) Hematoxylin and eosin (HE) staining presented the morphological changes of myocardial tissue, black stars showed the disorder of striated muscle and interstitial fibrosis (n = 8). (b) The cross-sectional area of cardiomyocytes (CSA) (n = 8). (c) and (d) The biomarkers of cardiac remodeling, atrial natriuretic peptide (ANP), and B-type natriuretic peptide (BNP) (n = 4); data were presented as means ± SEM. ∗P < 0.05 versus CON or M and #P < 0.05 versus STZ group.

Figure 2. Myricetin inhibited the cardiac fibrosis of DCM induced by treatment of STZ. (a) Histological sections of mouse hearts stained by picrosirius red (PSR) for fibrosis and immunohistochemistry for collagen I/III (n = 8). (b) The calculation of PSR positive area. (c) and (d) The calculation of collagen I/III positive area (n = 8). (e) and (f) The relative expression of biomarkers of cardiac fibrosis, fibronectin (Fn), and connective tissue growth factor (CTGF) (n = 4). (g) Representative blots of transforming growth factor (TGFβ1), T-Smad3, p-Smad3, and glyceraldehyde-3-phoshate dehydrogenase (GAPDH), (n = 6). (h) and (i) Histogram indicated the expression changes among different groups (n = 6). ∗P < 0.05 as compared with the corresponding CON or M group. #P < 0.05 as compared with STZ group, GAPDH was selected as the internal reference, and all of the proteins were normalized to GAPDH before quantitative statistics.

Figure 3. Myricetin inhibited the nuclear translocation of p-P65 and secretion of inflammatory cytokines. (a) Frozen section for the detection of p-P65 by immunofluorescence, white arrows showed the merging of p-P65 and nuclei (n = 6). (b) The calculation of p-P65-positive nuclei among different groups (n = 6). (c), (d), and (e) The relative mRNA expression of interleukin 6 (IL-6) and IL-1β and tumor necrosis factor (TNF-α) (n = 4). GAPDH was selected as the internal reference, all of the mRNA was normalized to GAPDH before relative quantitative statistics. Data were presented as means ± SEM. ∗P < 0.05 versus CON or M and #P < 0.05 versus STZ.

Figure 4. Myricetin inhibited IκBa/P65 signaling pathway. (a) Representative blots of p-P65, T-P65, GAPDH, Nuc-P65, histone 3, and p-IκBa (n = 6). (b) Histogram showed the quantitative statistics of the Western blots (n = 6); phosphorylation (p), total (T), nuclei (Nuc), and glyceraldehyde-3-phoshate dehydrogenase (GAPDH); GAPDH and histone 3 were selected for internal reference, the phosphorylated and total protein were normalized to GAPDH, and the nuclei protein was normalized to histone 3. Data were presented as means ± SEM. ∗P < 0.05 versus CON or M and #P < 0.05 versus STZ group.

Figure 5. Myricetin promoted the expression and nuclei translocation of Nrf2. (a) Frozen section for detecting the expression and location of Nrf2 by immunofluorescence (n = 6). (b) and (c) Representative blots and histogram of total expression of Nrf2 in the indicated groups (n = 6). (d) and (e) Representative blots and histogram of Nrf2 expression in the nuclei (n = 6); GAPDH and histone 3 were selected for internal reference, the total protein were normalized to GAPDH, and the nuclei protein was normalized to histone 3. Data were presented as means ± SEM. ∗P < 0.05 versus CON or M and #P < 0.05 versus STZ group.

Figure 6. Myricetin promoted the production of Nrf2 downstream genes and inhibited the expression of 4-HEN in vivo. (a) Representative blots of heme oxygenase 1 (HO-1) and GAPDH (n = 6). (b) Histogram presented the calculation of Western blots of HO-1 (n = 6). (c), (d), and (e) The relative mRNA expression of NQO1, SOD1, and SOD2 (n = 4). (f) Representative blots showed the expression of 4-hydroxynonenal (4-HEN). (g) Histogram presented the calculation of Western blots of 4-HEN; all of the proteins and mRNA expression were normalized to GAPDH before relative quantitative analysis. Data were presented as means ± SEM. ∗P < 0.05 versus CON and #P < 0.05 as compared with STZ group.

Figure 7. Myricetin attenuated STZ-induced apoptosis in vivo. (a) TUNEL staining of frozen section, white arrows showed the TUNEL-positive nuclei (n = 6). (b) The calculation of TUNEL-positive nuclei among different groups (n = 6). (c) Representative blots of c-caspase 3 and Bax (n = 6). (d) and (e) Histogram presented the fold change of c-caspase 3 and Bax; all of the proteins were normalized to GAPDH before relative quantitative analysis. Data were presented as means ± SD. ∗P < 0.05 as compared with CON and #P < 0.05 as compared with STZ group.

Figure 8. Myricetin regulated the expression of Nrf2 and HO-1 in neonatal rat cardiomyocytes (NRCM). (a) Myricetin enhanced the expression and nuclei translocation of Nrf2 after 36 h treatment with or without high glucose. (b) Representative blots showed the expression of Nrf2 and HO-1. (c), (d), and (e) Histogram showed the fold change of T-Nrf2, Nuc-Nrf2, and HO-1; all of the proteins were normalized to GAPDH or histone 3 before relative quantitative analysis, and all experiments were repeated 3 times independently. Data were presented as means ± SD. ∗P < 0.05 as compared with CON and #P < 0.05 as compared with HG group.

Figure 9. Myricetin blocked nuclei translocation of p65 and apoptosis in NRCM. (a) After 36 h of high glucose treatment, p65 were phosphorylated and translocated into the nuclei. (b) After 72 h of high glucose treatment, apoptosis-associated proteins (Bax and c-caspase 3) were accumulated in NRCM, which could be blocked by treatment of myricetin. (c), (d), (e), and (f) Histogram showed the fold change of p-P65, Nuc-p65, Bax, and c-caspase 3; all of the proteins were normalized to GAPDH or histone 3 before relative quantitative statistics, and all experiments were repeated 3 times independently. Data were presented as means ± SD. ∗P < 0.05 as compared with CON or M group and #P < 0.05 as compared with HG group.

Figure 10. Myricetin inhibited activity of IκBα/NF-κB pathway independent on Nrf2 in high glucose-treated NRCM. (a) NRCM were transfected with siRNA for Nrf2 for 24 h, followed by treatment with high glucose (HG) or M for another 36 h; the total protein were extracted for detecting the expression of Nrf2, p-IκBα, and p-P65. (b), (c), and (d) Histogram showed the fold change of Nrf2, p-P65, and p-IκBα; all of proteins were normalized to GAPDH before relative quantitative analysis, and all experiments were repeated 3 times independently. Data were presented as means ± SD. ∗P < 0.05 as compared with CON, #P < 0.05 as compared with CON or siRNA group, and &P < 0.05 as compared with LPS + siRNA group.

Figure 11. Myricetin decreased the expression of downstream inflammatory cytokines of IκBα/NF-κB pathway independent on Nrf2 in lipopolysaccharide- (LPS-) treated NRCM. (a), (b), and (c) NRCM were transfected with siRNA of Nrf2 for 24 h, followed by treatment with LPS (1 μg/ml) or M (40 μmol) for another 24 h; RT-PCR was performed to detect the expression of IL-1β, IL-6, and TNF-α; all of the mRNA expressions were normalized to GAPDH before relative quantitative analysis, and all experiments were repeated 3 times independently. Data were presented as means ± SD. ∗P < 0.05 as compared with CON, #P < 0.05 as compared with CON or siRNA group, and &P < 0.05 as compared with LPS + siRNA group.