Allopurinol reduces oxidative stress and activates Nrf2/p62 to attenuate diabetic cardiomyopathy in rats

Abstract

Allopurinol (ALP) attenuates oxidative stress and diabetic cardiomyopathy (DCM), but the mechanism is unclear. Activation of nuclear factor erythroid 2-related factor 2 (Nrf2) following the disassociation with its repressor Keap1 under oxidative stress can maintain inner redox homeostasis and attenuate DCM with concomitant attenuation of autophagy. We postulated that ALP treatment may activate Nrf2 to mitigate autophagy over-activation and consequently attenuate DCM. Streptozotocin-induced type 1 diabetic rats were untreated or treated with ALP (100 mg/kg/d) for 4 weeks and terminated after heart function measurements by echocardiography and pressure-volume conductance system. Cardiomyocyte H9C2 cells infected with Nrf2 siRNA or not were incubated with high glucose (HG, 25 mmol/L) concomitantly with ALP treatment. Cell viability, lactate dehydrogenase, 15-F2t-Isoprostane and superoxide dismutase (SOD) were measured with colorimetric enzyme-linked immunosorbent assays. ROS, apoptosis, was assessed by dihydroethidium staining and TUNEL, respectively. The Western blot and qRT-PCR were used to assess protein and mRNA variations. Diabetic rats showed significant reductions in heart rate (HR), left ventricular eject fraction (LVEF), stroke work (SW) and cardiac output (CO), left ventricular end-systolic volume (LVVs) as compared to non-diabetic control and ALP improved or normalized HR, LVEF, SW, CO and LVVs in diabetic rats (all P < .05). Hearts of diabetic rats displayed excessive oxidative stress manifested as increased levels of 15-F2t-Isoprostane and superoxide anion production, increased apoptotic cell death and cardiomyocytes autophagy that were concomitant with reduced expressions of Nrf2, heme oxygenase-1 (HO-1) and Keap1. ALP reverted all the above-mentioned diabetes-induced biochemical changes except that it did not affect the levels of Keap1. In vitro, ALP increased Nrf2 and reduced the hyperglycaemia-induced increases of H9C2 cardiomyocyte hypertrophy, oxidative stress, apoptosis and autophagy, and enhanced cellular viability. Nrf2 gene silence cancelled these protective effects of ALP in H9C2 cells. Activation of Nrf2 subsequent to the suppression of Keap1 and the mitigation of autophagy over-activation may represent major mechanisms whereby ALP attenuates DCM.

Keywords: Nrf2; allopurinol; autophagy; diabetic cardiomyopathy; oxidative stress.

Figure 1

Allopurinol reduced myocardial oxidative stress and apoptosis cell death in 5‐wk streptozotocin‐induced diabetic rats. A, B, Free 15‐F2t‐isoprostane (15‐F2t‐IsoP) in heart tissue was measured by suing an enzyme‐linked immunoassay kit. C, D, SOD activity was detected in cardiac tissue homogenates and plasma, respectively, using commercially available kits (Cayman Chemical). E, F, Myocardial O₂‐ production assessed by dihydroethidium (DHE) staining (stained in red) and quantification in (C, D) and (DA). G‐I, Protein expressions of cardiac Bax, Bcl2 and cleaved caspase 3. J, K, Myocardial cell apoptosis assessed by terminal deoxynucleotidyl transferase dUTP nick‐end labelling (TUNEL). Data are shown as means ± SEM, with n = 6 animals per group. *P < .05 vs C; ^# P < .05 vs DA

Figure 2

Allopurinol increased the cardiac expression and nuclear translocation of Nrf2 and its down ARE (HO‐1, p62). A‐C, Cardiac expression of total, nuclear and cytosolic Nrf2. D, Cardiac Keap1 protein expression. E, F, Cardiac HO‐1 and p62 protein expression. G, The ratio of LC3 II/LC3 I in the myocardium. H‐J, Cardiac mRNA expression of Nrf2, Keap1 and p62, respectively, calculated against the housekeeping gene GAPDH. Data are shown as mean ± SEM, with n = 6 animals per group. *P < .05 vs C; ^# P < .05 vs DA; H3, histone H3

Figure 3

Allopurinol treatment prevented HG‐induced increased cell size and elevated oxidative stress, which can be cancelled by gene silence of Nrf2 in H9C2 cells. H9C2 cardiomyocytes were pretreated with Nrf2 siRNA and control siRNA and then treated with control medium with normal glucose (NG, 5.5 mmol/L glucose) or HG (25 mmol/L glucose) for 48 h; subgroups were treated with ALP for 48 h at the meantime before sample collection. A, B, H9C2 cell size visualization (400 magnification, 50 µm) and quantitation. D, E, Cellular reactive oxygen species production assessed by dihydroethidium (DHE) staining in H9C2 cell (The scale bar in the figure represents 400 µm). C, H9C2 cardiomyocyte viability assessed by CCK8 agent. F, Cardiomyocyte death assessed by lactate dehydrogenase (LDH) release. G, H, Apoptosis of H9C2 myocytes assessed by TUNEL staining (The scale bar in the figure represents 400 µm). Data are mean ± SEM of two independent experiments each performed in triplicate, *P < .05. vs NG; ^# P < .05 vs HG; ^$ P < .05 vs HA; ^& P < .05 vs Nrf2

Figure 4

Nrf2/p62 signalling pathway played the key role in the ALP protection to high glucose‐induced cardiomyopathy. A, B, Protein and mRNA expression of Nrf2 after siRNA treatment in the normal glucose. C, Protein expression of Nrf2. D, Protein expression of Keap1. E, Protein expression of p62. F, The ratio of LC3 II/LC3 I in the H9C2 cells. G‐I, The mRNA expressions of Nrf2, p62 and Keap1, respectively, in H9C2 cells, calculated against the housekeeping gene GAPDH. J, Nuclear Nrf2 expression in high glucose cultured H9C2 with or without ALP. K, Treatment with Bafilomycin (Baf), a lysosomal inhibitor that can impair autophagosome‐lysosome fusion and thus track autophagic flux, induced higher P62 protein level than NG group, whereas HG group displayed decreased P62 protein level than NG group. Data are mean ± SEM of two independent experiments each performed in triplicate, *P < .05. vs NG; ^# P < .05 vs HG; ^$ P < .05 vs HA; ^& P < .05 vs Nrf2

Figure 5

Schematic of proposed signalling involved in ALP attenuates DCM and cardiac dysfunction via activating Nrf2 pathway. Hyperglycaemia‐induced oxidative stress destructs Nrf2 signalling by inhibiting its nuclear translocation, which concomitantly with activated autophagy and subsequently reduced HO‐1 and p62 expression, resulting in cardiomyocytes apoptosis and cardiac hypertrophy. ALP activates Nrf2 by increasing its nuclear translocation following with target on ARE elements (including p62 and HO‐1) and concomitantly normalizes autophagy, which in turns reduces myocardial oxidative stress, attenuates cardiac hypertrophy and apoptosis and eventually improves cardiac function