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

Jierong Luo,Fei Zeng,Zhengyuan Xia,Hong Liu,Michael G Irwin,Shiming Liu,Sisi Li,Huansen Huang,Chi Wai Cheung,Dan Yan

doi:10.1111/jcmm.14870

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.

Highlights

Diabetic cardiomyopathy (DCM), a common complication of diabetes mellitus, is the greatest mortality risk for patients with diabetes.[1]
In the presence of oxidative stress, Nrf[2] confers protective effects via translocation into the nuclear and subsequently activate antioxidant response elements (ARE) to induce antioxidant genes, such as NQO1, GCLC, heme oxygenase-1 (HO-1) and SQSTM1.8,9 We recently showed that cardiac Nrf[2] was reduced in streptozotocin (STZ)-induced diabetic rats and strategies that enhanced cardiac Nrf[2] could activate myocardial HO-1 and attenuate cardiac hypertrophy and cardiac dysfunction in diabetic rats.[10]
We suggested that the hyperglycaemia-induced oxidative stress and the over-activation of myocardial autophagy are key mechanisms that cause the imbalance of nuclear factor erythroid 2-related factor 2 (Nrf2)-Kelch-like epichlorohydrin-associated protein 1 (Keap1)-p62 and the development of DCM

Summary

| INTRODUCTION

Diabetic cardiomyopathy (DCM), a common complication of diabetes mellitus, is the greatest mortality risk for patients with diabetes.[1]. Allopurinol, a competitive inhibitor of xanthine oxidase enzyme, has dose-dependent free radical scavenging property.[18] It attenuates the diabetes-induced increase of myocardial xanthine oxidase activity,[19,20] reduces ROS production and improves diabetes-induced cardiac dysfunction.[21] Our previous studies demonstrated that ALP could ameliorate diabetic myocardial ischaemia/reperfusion (MI/R) injury by lowering ROS production.[22] Demirel et al[23] showed that ALP could alleviate acute liver failure via regulating cellular redox-sensitive transcription factor Nrf[2], indicating that ALP conferred protection through interacting with Nrf[2] signalling pathway. This hypothesis was tested both in type 1 diabetic rats and in H9C2 cardiomyocytes exposed to high glucose

| MATERIALS AND METHODS

Findings

| DISCUSSION

| CONCLUSION