NLRP3 and MCU are Essential for Ferulic Acid Alleviating Endoplasmic Reticulum- and Mitochondria-Dependent Oxidative Stress and Inflammation in Hyperglycemia-Induced Retinal Pigment Epithelium Cells

Abstract

Background: Oxidative stress and inflammation play the central roles in the pathophysiological process of diabetic retinopathy. Our purpose was to elaborate the effect and regulatory mechanism of ferulic acid (FA) in mitigating diabetic retinopathy as well as the functional roles of mitochondrial calcium uniporter (MCU) and NLR Family Pyrin Domain Containing 3 (NLRP3) inflammasome and their interplay. Methods: Human retinal pigment epithelial cells ARPE-19 were pre-treated with 10 mmol/L FA, or transfected with NLPR3 or MCU siRNA or overexpression plasmids. Afterwards, ARPE-19 cells were exposed to 30 mmol/L high glucose for simulating diabetic retinopathy. Intracellular reactive oxygen species (ROS) generation, cytosolic Ca²⁺ level, endoplasmic reticulum (ER) and mitochondrial stress, mitochondrial membrane potential were assayed with 2,7-dichlorofluorescein diacetate (DCFH-DA), Fluo-4 acetoxymethyl ester (Fluo-4 AM), ER-Tracker Red, Seahorse XFe96 Analyzer and 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimi-dazolylcarbocyanine iodide (JC-1) staining, respectively. MCU, NLRP3, Interleukin-1beta (IL-1β), toll-like receptor 4 (TLR4), nuclear factor κB (NFκB), AKT serine/threonine kinase1 (AKT1) and phosphorylated-Akt1 (p-AKT1) were measured with western blots or immunohistochemistry. Results: Both FA pre-treatment and blockage of NLRP3 mitigated apoptosis, intracellular ROS, cytosolic Ca²⁺ level as well as ER and mitochondria stress in hyperglycemia-induced ARPE-19 cells. Additionally, MCU-mediated mitochondrial oxidative stress and NLRP3 inflammasome- and TLR4-dependent AKT/NFκB oxidative and inflammatory signaling were alleviated. Blockage of MCU alleviated hyperglycemia-induced NLRP3 inflammasome activation. Conversely, up-regulation of MCU exacerbated hyperglycemia-induced NLRP3 inflammasome- and TLR4-mediated oxidative and inflammatory signaling, which can be attenuated by FA. Similarly, MCU-mediated mitochondrial oxidative stress was mitigated by FA through NLRP3 inhibition. Conclusion: Collectively, we present evidence that NLRP3 and MCU are essential for FA alleviating ER- and mitochondria-dependent oxidative stress and inflammation in hyperglycemia-induced ARPE-19 cells. Disturbance of NLRP3 and MCU transcription regulation opens novel avenues to mitigate diabetic retinopathy.