Fisetin protects against streptozotocin-induced diabetic cardiomyopathy in rats by suppressing fatty acid oxidation and inhibiting protein kinase R

Abstract

This study examined if the Fisetin against streptozotocin-induced diabetic cardiomyopathy (DC) in rats involves regulating cardiac metabolism and suppressing protein kinase R (PKR). Male rats were divided (12/groups) as control (non-diabetic), control + Fisetin, T1DM, and T1DM + Fisetin. Fisetin was administered orally at a final dose of 2.5 mg/kg for 12 weeks. In T1DM1-induced rats, Fisetin prevented heart and final body weights loss, lowered circulatory levels troponin I and creatinine kinase-MB (CK-MB), increased fasting insulin levels, and improved ventricular systolic and diastolic functions. It also preserved the structure of the cardiomyocytes and reduced oxidative stress, fibrosis, protein levels of transforming growth factor-β1 (TGF-β1), collagenase 1A, caspase-3, and the activation of JNK, p53, and p38 MAPK. In the control and diabetic rats, Fisetin attenuated fasting hyperglycaemia, the increases in glucose levels after the oral and insulin tolerance tests, and HOMA-IR. It also increased cardiac glucose oxidation by increasing the activity of private dehydrogenase (PDH), phosphofructokinase (PFK), protein levels of PPAR-α and suppressed cardiac inflammation by inhibiting NF-κB. These effects were associated with a reduction in the activity of PKR and subsequent increase in the activity of eeukaryotic initiation factor 2 (eIF2) with a parallel increase in protein levels of p67, a cellular inhibitor of PKR. In cultured cardiomyocytes, Fisetin, prevented high glucose (HG)-induced activation of PKR and reduction in p67, in a dose-dependent manner. However, the effect of Fisetin on PKR was diminished in LG and HG-treated cardiomyocytes with p67-siRNA. In conclusion, Fisetin protects against DC in rats by improving cardiac glucose metabolism and suppressing PKR.

Keywords: Diabetic cardiomyopathy; Fisetin; Glucose; Protein Kinase R; Rats.

Graphical abstract

Fig. 1

Plasma glucose and its calculated area under the curve (UAC) after the oral glucose tolerance test (OGTT) (A & B) or intraperitoneal insulin tolerance test (IPITT) (C & D). Data are presented as mean ± SD. Data were analyzed by two-way ANOVA followed by Tukey's t-test as post-hoc for n = 12/group. *, **, ***: vs. control at p < 0.05, 0.01, and 0.001, respectively. ##, ###: vs. Control + Fisetin at p < 0.05, and 0.001, respectively. $$$: vs. T1DM at p < 0.001.

Fig. 2

Cardiac histological alterations (A-D) and serum levels of Troponin-I and Creatinine kinase-MB (CKMB) (F) in all experimental groups. Aand B: were taken from control and control + Fisetin-treated hearts and showed normal myofibrillar structure with normal striations and branches (long arrow) and continuity with normal nuclei located centrally. C: was taken from a T1DM-treated rat (T1DM) and showed damage in myofibrils (short arrow), loss of striations, congestion (short arrow), and presence of inflammatory cells (arrowhead). D: was taken from a T1DM + Fisetin-treated rat and showed almost normal structure like the control hearts. For E and F: Data are presented as mean ± SD. Data were analyzed by two-way ANOVA followed by Tukey's t-test as post-hoc for n = 6/group. *, **, ***: vs. control at p < 0.05, 0.01, and 0.001, respectively. #, ##, ###: vs. Control + Fisetin at p < 0.05, 0.01, and 0.001, respectively. $$$: vs. T1DM at p < 0.001.

Fig. 3

Cardiac ultrastructural changes (A-D) and cardiac levels of reactive oxygen species (ROS) (E), malondialdehyde (MDA) (F), reduced glutathione (GSH) (G) and superoxide dismutase (SOD) (H) in all experimental groups. A and B: were taken from control and control + Fisetin-treated hearts and showed normal myofibrillar structure (S), in longitudinal section, well-identified Z-line, and H bands with intact mitochondria (m) and nucleus (N). C: was taken from a T1DM rat and showed a reduction in muscle fiber mass with severe degeneration of myofibrils (S), abnormal nucleus (N), and degenerated blood vessels (BV). D: was taken from a T1DM + Fisetin-treated rat and showed improvement in the structure of the myofibrils and mitochondria. In E-H: Data are presented as mean ± SD. Data were analyzed by two-way ANOVA followed by Tukey's t-test as post-hoc for n = 6/group. *, **, ***: vs. control at p < 0.01, 0.05, and 0.001, respectively. #, ##, ###: vs. Control + Fisetin at p < 0.01, 0.05, and 0.001, respectively. $$$: vs. T1DM at p < 0.001.

Fig. 4

Collagen deposition (A-F) and protein levels of transforming growth factor-β-1 (TGF-β1) and collagenase 1A (COL1A) (G) in the hearts of all experimental groups. A and B: were taken from control and control + Fisetin-treated hearts and showed few filaments of collagen (blue color) (long arrow. C-F: were taken from T1DM-treated rats (T1DM) and showed increased collagen deposition between the myofibrils (periventricular) and around the blood vessels (perivascular). D: was taken form a T1DM + Fisetin-treated rat and showed almost normal collagen deposition like the control hearts. In G: Data are presented as mean ± SD. Data were analyzed by two-way ANOVA followed by Tukey's t-test as post-hoc for n = 6/group. **, ***: vs. control (lane 1) at p < 0.01, and 0.001, respectively. ###: vs. Control + Fisetin (lane 2) at p < 0 0.001. $$$: vs. T1DM (lane 3) at p < 0.001. Lane 4: T1DM + Fisetin. Lane 4: T1DM + Fisetin.

Fig. 5

Levels of tumor necrosis factor-alpha (TNF-α) (A), interleukin-6 (IL-6) (B), and nuclear activity and protein level of nuclear factor kappa Beta (NF-κB) (C and D, respectively) in the heart of hearts of all experimental groups. Data are presented as mean ± SD. Data were analyzed by two-way ANOVA followed by Tukey's t-test as post-hoc for n = 12/group. *, ***: vs. control (lane 1) at p < 0.05 and 0.001, respectively. ###: vs. Control + Fisetin (lane 2) at p < 0 0.001. $$$: vs. T1DM (lane 3) at p < 0.001. Lane 4: T1DM + Fisetin.

Fig. 6

Activity of pyruvate dehydrogenase kinase-4 (PDK-4) (A), levels of pyruvate dehydrogenase (B), the activity of medium-chain acyl-coenzyme A dehydrogenase (MCAD) (C), and protein levels of peroxisome proliferator-activated receptor-α (PPAR-α) in the left ventricles of all experimental groups. Data are presented as mean ± SD. Data were analyzed by two-way ANOVA followed by Tukey's t-test as post-hoc for n = 12/group. *, ***: vs. control (lane 1) at p < 0.05 and 0.001, respectively. #, ##, ###: vs. Control + Fisetin (lane 2) at p < 0.05, 0.01, and 0 0.001. $$$: vs. T1DM (lane 3) at p < 0.001. Lane 4: T1DM + Fisetin.

Fig. 7

Activity of protein kinase R (PKR) (A), as well as protein levels of PKR/phospho-PKR (B) and Eukaryotic initiation factor 2 (eIf-2)/p-eIF2 (C) in the heart of hearts of all experimental groups. Data are presented as mean ± SD. Data were analyzed by two-way ANOVA followed by Tukey's t-test as post-hoc for n = 12/group. *, ***: vs. control (lane 1) at p < 0.01 and 0.001, respectively. #, ###: vs. Control + Fisetin (lane 2) at p < 0.05 and 0.001, respectively. $$$: vs. T1DM (lane 3) at p < 0.001. Lane 4: T1DM + Fisetin.

Fig. 8

Protein levels of protein kinase R activating protein (PACT), p58IPK, and MetAP2 (p67) in the heart of all experimental groups. Data are presented as mean ± SD. Data were analyzed by two-way ANOVA followed by Tykey’s t test as post-hoc for n = 12/group. ***: vs. control (lane 1) at p < 0 0.001. ###: vs. Control + Fisetin (lane 2) at p < 0 0.001. $$$: vs. T1DM (lane 3) at p < 0.001. Lane 4: T1DM + Fisetin.

Fig. 9

Protein levels of NH2 -terminal Jun kinase (JNK)/phospho-JNK (A), p53 (B), p38 mitogen-activated protein kinase (p38 MAPK)/phospho-p38 MAPK (Thr¹⁸⁰/Tyr¹⁸²) (C), and cleaved caspase-3 (D) in the hearts of all experimental groups. Data are presented as mean ± SD. Data were analyzed by two-way ANOVA followed by Tukey's t-test as post-hoc for n = 12/group. ***: vs. control (lane 1) at p < 0 0.001. ###: vs. Control + Fisetin (lane 2) at p < 0 0.001. $$$: vs. T1DM (lane 3) at p < 0.001. Lane 4: T1DM + Fisetin.

Fig. 10

Cell survival (A) and levels of single-stranded DNA (ssDNA) (Cell apoptosis) (B) as well as protein levels of protein kinase R (C) and MetAP2 (p67) (D) in cultured cardiomyocytes of all treatments. Cells were the heart of all experimental groups. Cells (1X10⁴) were cultured in a humified atmosphere in DMEM (Dulbecco’s modified Eagle’s medium) in either low/high glucose media (5 µM or 25 µM glucose, respectively). In HG conditions, some cells were co-incubated with an increasing concentration of Fisetin (5, 10, 20 µM). Data are presented as mean ± SD. Data were analyzed by two-way ANOVA followed by Tukey's t-test as post-hoc for three trials each in duplicate for each treatment. **, ***: vs. LG-treated cells (lane 1) at p < 0.01 and 0 0.001, respectively. #, ##, ##: vs. HG-treated cells (lane 2) at p < 0.05, 0.01, and 0 0.001, respectively. $$, $$$: vs. HG + Fiestin (5 µM) (lane 3) at p < 0.01 and 0.001, respectively. &, &&: vs. HG + Fiestin (10 µM) (lane 4) at p < 0.05 and 0 0.001, respectively. Lane 5: HG + Fisetin (20 µM).

Fig. 11

Cell survival (A) and levels of single-stranded DNA (ssDNA) (Cell apoptosis) (B) as well as protein levels of protein kinase R (C) in cultured cardiomyocytes of all treatments. Cells (1X10⁴) were cultured in a humified atmosphere in DMEM (Dulbecco’s modified Eagle’s medium) in either low/high glucose media (5 µM or 25 µM glucose, respectively). In HG conditions, some cells were co-incubated with 20 µM Fisetin. Data are presented as mean ± SD. Data were analyzed by two-way ANOVA followed by Tukey's t-test as post-hoc for three trials each in duplicate for each treatment. ***: vs. LG-treated cells (lane 1) at p < 0 0.001. ###: vs. LG + Fisetin-teated cells (lane 2) at p < 0 0.001, $$$: vs. LG + SiRN p67-treated cells (lane 3) at p < 0.001. &, &&: vs. HG + SiRN p67-treated cells at p < 0.05 and 0.01, respectively. Lane 5: HG + SiRN p67 + Fiestin (20 µM).

Fig. 12

A graphical abstract presenting the possible mechanism of action of Fisetin to prevent diabetic cardiomyopathy in rats. These effects are shown in the left ventricles (LVs) of both the control and streptozotocin-induced type1 diabetes mellitus (T1DM). the anti-oxidant and anti-apoptotic effect of Fisetin is mediated by acting through different mechanisms including 1) upregulation of cardiac expression of peroxisome proliferator-activated receptor-α (PPAR-α) which leads to shifting the cardiac metabolism toward glucose oxidation mediated by the activation of pyruvate dehydrogenase (PDH) and phosphofructokinase (PFK) and suppression of pyruvate dehydrogenase kinase-4 (PDK-4) and medium-chain acyl-coenzyme A dehydrogenase (MCAD), 2) activating of p67 which in turn inhibit protein kinase-3. The suppression of PKR results in potent antioxidant, anti-inflammatory, and anti-apoptotic through inactivation of JNK, p53, and P38 mAPK, preventing inflammation by and NF-κB; and 3) stimulating peripheral insulin sensitivity which leads to a hypoglycemic effect, which may mediate all these events.

Supplementary Fig. 1