Nicorandil alleviates apoptosis in diabetic cardiomyopathy through PI3K/Akt pathway

Abstract

Nicorandil exerts myocardial protection through its antihypoxia and antioxidant effects. Here, we investigated whether it plays an anti-apoptotic role in diabetic cardiomyopathy. Sprague-Dawley rats were fed with high-fat diet; then single intraperitoneal injection of streptozotocin was performed. Rats with fasting blood glucose (FBG) higher than 11.1 mmol/L were selected as models. Eight weeks after the models were built, rats were treated with nicorandil (7.5 mg/kg day and 15 mg/kg day respectively) for 4 weeks. H9c2 cardiomyocytes were treated with nicorandil and then stimulated with high glucose (33.3 mmol/L). TUNEL assay and level of bcl-2, bax and caspase-3 were measured. 5-HD was used to inhibit nicorandil. Also, PI3K inhibitor (Miltefosine) and mTOR inhibitor (rapamycin) were used to inhibit PI3K/Akt pathway. The results revealed that nicorandil (both 7.5 mg/kg day and 15mg/kg day) treatment can increase the level of NO in the serum and eNOS in the heart of diabetic rats compared with the untreated diabetic group. Nicorandil can also improve relieve cardiac dysfunction and reduce the level of apoptosis. In vitro experiments, nicorandil (100 µmol) can attenuate the level of apoptosis stimulated by high glucose significantly in H9C2 cardiomyocyte compared with the untreated group. The effect of nicorandil on apoptosis was blocked by 5-HD, and it was accompanied with inhibition of the phosphorylation of PI3K, Akt, eNOS, and mTOR. After inhibition of PI3K/Akt pathway, the protective effect of nicorandil is restrained. These results verified that as a NO donor, nicorandil can also inhibit apoptosis in diabetic cardiomyopathy which is mediated by PI3K/Akt pathway.

Keywords: PI3K/Akt signal; apoptosis; diabetic cardiomyopathy; nicorandil.

Figure 1

Characteristic of type 2 diabetic rats. IPGTT(A) and IPITT(B) were performed after 4 weeks of high‐fat diet. AUC were calculated in control and HF group. After nicorandil treatment for 4 weeks, IPGTT(C) and IPITT(D) were performed in four groups. AUC were calculated in 4 groups. *P < 0.05 compared with the control group

Figure 2

Nicorandil alleviates myocardial remodelling in diabetic rats. A: A1: gross morphology; A2: HE staining of cross shaft of musculi papillares in heart A3: HE staining longitudinal section; A4: HE staining of cross section; B: heart weight/bodyweight; C: cardiomyocyte cell diameter. DM: Diabetic mellitus. N7.5: nicorandil, 7.5 mg/kg·day; N15: nicorandil, 15 mg/kg·day. *P < 0.05 compared with control; ^# P < 0.05 compared with DM; Data are means ± SD

Figure 3

Nicorandil alleviates cardiac dysfunction in diabetic rats. A1: representive 2D echocardiograms. A2: representative M‐mode echocardiograms. A3: representative pulse‐wave Doppler echocardiograms of mitral inflow. A4: representative tissue Doppler echocardiograms. B1: Left ventricle ejection fraction (LVEF). B2: Fractional shortening (FS). B3: Early to late mitral flow (E/A). B4: Ratio of diastolic mitral annulus velocities (e’/a’). B5: E/e’. B6: Left ventricle end‐diastolic dimension (LVEDd). DM: Diabetic mellitus. N7.5: nicorandil, 7.5 mg/kg·day; N15: nicorandil, 15 mg/kg·day. *P < 0.05 compared with control; ^# P < 0.05 compared with DM; Data are means ± SD

Figure 4

Nicorandil alleviate cardiac fibrosis in type 2 diabetic rat. A: Masson's trichrome staining and Picorosirius Red staining of myocardium. Immunohistochemical staining of Collagen I and Collagen III; B: Western blot analysis of MMP2, MMP9, Collagen I and Collagen III. DM: Diabetic mellitus, N7.5: nicorandil, 7.5 mg/kg·day; N15: nicorandil, 15 mg/kg·day. *P < 0.05 compared with control; ^# P < 0.05 compared with DM; ^# P < 0.05 compared with HG + N, Data are means ± SD

Figure 5

Nicorandil alleviates cardiac apoptosis in type 2 diabetic rat. A: TUNEL staining and TUNEL‐positive cells rate. B: Western blot analysis of Bax/Bcl‐2 and cleaved caspase‐3. C: Level of nitric oxide and ADMA in serum. D: Western blot analysis of p‐eNOS. DM: Diabetic mellitus, N7.5: nicorandil, 7.5 mg/kg·day; N15: nicorandil, 15 mg/kg·day. *P < 0.05 compared with control; ^# P < 0.05 compared with DM; ^# P < 0.05 compared with HG + N, Data are means ± SD

Figure 6

Apoptosis level reduced after nicorandil treatment in high glucose‐induced H9c2 cardiomyocyte. A: Western blot analysis of bax and bcl‐2 in high glucose‐induced H9c2 cardiomyocyte after nicorandil treatment with different concentrations for 24 h. B: Western bolt analysis of cleaved caspase‐3 in high glucose‐induced H9c2 cardiomyocyte after nicorandil treatment. HG (33.3 mmol/L), NG (5.5 mmol/L), n1: nicorandil (10 µmol); n2: nicorandil (50 µmol); n3: nicorandil (100 µmol). ^# P < 0.05 compared with NG; *P < 0.05 compared with HG, Data are means ± SD

Figure 7

Nicorandil protects H9C2 cells from apoptosis through PI3K/AKT pathway. A: Western blot analysis of Bax/Bcl‐2 and cleaved caspase‐3 in high glucose‐induced H9c2 cardiomyocyte after nicorandil treatment or both nicorandil treatment and 5‐HD which is a inhibitor of nicorandil. B: TUNEL assay of apoptosis rate of high glucose‐induced H9c2 cardiomyocyte after nicorandil treatment or both nicorandil treatment and nicorandil inhibitor（5‐HD, 500 µmol) (scale bar: 20 µm). I:NG, II:HG, III:HG + N, IV:HG + N+5‐HD; C: Western blot analysis of phosphorylation level of PI3K, AKT, eNOS and mTOR in high glucose‐induced H9c2 cardiomyocyte after nicorandil treatment or both nicorandil treatment and nicorandil inhibitor（5‐HD). N: Nicorandil (100 µmol); NG: normal glucose (5.5 mmol/L); HG: high glucose (25 mmol/L). *P < 0.05 compared with NG; ^# P < 0.05 compared with HG; & P < 0.05 compared with HG + N, Data are means ± SD

Figure 8

PI3K/AKT pathway inhibition blocked the protection of nicorandil on H9c2 cardiomyocyte treated with high glucose. A: Western blot analysis of Bax/Bcl‐2 and cleaved caspase‐3 in high glucose‐induced H9c2 cardiomyocyte after nicorandil treatment or both nicorandil treatment and PI3K/mTOR inhibitors. B: TUNEL assay of apoptosis rate of high glucose‐induced H9c2 cardiomyocyte after nicorandil treatment or both nicorandil treatment and PI3K/mTOR inhibitors (scale bar: 20 µm). C: Western blot analysis of p‐eNOS in high glucose‐induced H9c2 cardiomyocyte after nicorandil treatment or both nicorandil treatment and PI3K/mTOR inhibitors. N: Nicorandil (100 µmol); MTF: miltefosine (100 µmol); Rapa: rapamycin (100 µmol) NG: normal glucose (5.5 mmol/L); HG: high glucose (25 mmol/L). *P < 0.05 compared with NG; ^# P < 0.05 compared with HG; &P < 0.05 compared with HG + N, Data are means ± SD

Figure A1

Nicorandil can increase the NO in serum in diabetic coronary heart disease (CHD) patients. A: Basic characteristic of the selected diabetic CHD patients. B: Level of nitric oxide in serum. Nicorandil (5mg tid); *P<0.05 compared with Control.

Figure A2

Nicorandil and L‐arginine can synergistically alleviate the eNOS mediated apoptosis in H9C2 cells treated with high glucose. A: ADMA level in medium. B: Western blot analysis of p‐eNOS/eNOS. C: Western blot analysis of caspase‐3. D: Western blot analysis of Bax/bcl‐2. HG: high glucose (33mol/l); N: Nicorandil (100umol); Arg: L‐arginine (75umol). *P<0.05 compared with HG.