AZA Protects against Cardiomyocyte Injury by Ameliorating Mitochondrial Dysfunction Attenuating Oxidative Stress, Inflammation and Apoptosis via VDAC1

Abstract

Background: Azathioprine (AZA) is a purine-derived drug used for immunosuppression. The molecular mechanisms by which AZA protects cardiomyocytes remain unclear. This experiment will further elaborate, on the basis of the previous study, the mechanism of AZAs protection against hypoxia-induced cardiomyocyte injury in high glucose conditions. Methods: We utilized a high glucose (HG) and hypoxia/reoxygenation (H/R) cell model and a diabetic Sprague-Dawley (SD) rat ischaemia/reperfusion (I/R) model to detect cellular calcium ions, mitochondrial membrane potential, and reactive oxygen species (ROS) levels using Fluo-4 AM, MitoTracker Red, and ROS Assay Kit. Malondialdehyde (MDA), superoxide dismutase (SOD) and pro-inflammatory cytokines (interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α) levels were measured using the appropriate kits. Cellular energy metabolism was analyzed by oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). The expression of Voltage-dependent anion channel 1 (VDAC1), Nucleotide-binding oligomerization domain-like receptor protein 3 (NALP3), Nuclear factor-kappa B p65 (NF-κB p65), ATP synthase subunit alpha (ATP5A1), ATP synthase subunit beta (ATP5B), B-cell lymphoma 2 (Bcl-2), Caspase-3 and Bcl-2 associated X protein (Bax) was detected. Results: It was shown in the HG+H/R cell model that AZA up-regulated the levels of calcium ions (p < 0.05) and mitochondrial membrane potential (p < 0.05). AZA reduced ROS accumulation (p < 0.01) and oxidative stress marker MDA levels (p < 0.01), improved cellular energy metabolism, and increased expression of the antioxidant defense enzyme SOD (p < 0.05). AZA treatment inhibited VDAC1, NALP3, and NF-κB activation, upregulated ATP synthase (ATP5A1, ATP5B), and Bcl-2 expression, as well as inhibited apoptosis by downregulating Bax and Caspase3 expression in HG+H/R cells and I/R rat cardiomyocytes (p < 0.001). We constructed a VDAC1 siRNA cell model, and knockdown of VDAC1 significantly promoted the expression of ATP synthases (ATP5A1, ATP5B) under HG+H/R conditions (p < 0.001). Conclusion: Our data reveal the molecular mechanisms by which AZA protects cardiomyocytes from injury induced by HG+H/R and I/R in diabetic rats. We demonstrate that AZA ameliorates mitochondrial dysfunction, oxidative stress, inflammation, and apoptosis by modulating VDAC1 and ATP synthase expression. Our findings suggest that AZA may be a potential therapeutic agent for mitigating myocardial ischemic injury in diabetes.