Melatonin protects RPE cells from necroptosis and NLRP3 activation via promoting SERCA2-related intracellular Ca2+ homeostasis

Abstract

Background: Melatonin is an antioxidant that also has anti-inflammatory effects. It has been reported to delay the progression of age-related macular degeneration (AMD), however, the mechanism has not been fully recognized.

Purpose: The aim of the present study was to investigate the effects of melatonin on sodium iodate (SI)-induced retinal degeneration and elucidate the specific mechanisms, then, provide novel targets in AMD treatment.

Methods: Retinal degeneration mouse model and in vitro retinal pigment epithelium (RPE) death model were established by SI treatment. Melatonin was administrated intraperitoneally at a concentration of 20, 40 or 80 mg/kg for in vivo study or treated at 48 h before SI treatment. To confirm the therapeutic effects of melatonin on mouse, the retinal structure and visual function were evaluated. The specific cell death rates were determined by CCK-8 assay, PI staining and protein level of RIPK3. The cytosolic or mitochondrial calcium levels were determined by Fluo-4AM or Rhod-2AM staining. Mitochondrial functions including mitochondrial dynamics, mitochondrial membrane potential, or mitochondrial permeability pore opening were evaluated. The proteins involved in endoplasmic reticulum (ER) stress were measured by western blot assay while the genes expression in calcium signaling pathway were measured by RT-qPCR.

Results: We show that melatonin protects RPE cells from necroptosis and NLRP3 inflammasome activation induced by SI. Mechanistically, melatonin suppresses ER stress and intracellular calcium overload triggered by SI through restoring the function of SERCA2. Silencing of SERCA2 or blocking of melatonin receptors inhibit the protective effects of melatonin. Melatonin reduces mitochondrial Ca²⁺ levels and restores mitochondrial membrane potential. Constant mitochondrial Ca²⁺ overload directly promote cell necroptosis through mitochondrial fission. Inhibition of mitochondrial fission by Mdivi-1 prevent necroptosis induced by SI without altering the level of mitochondrial Ca²⁺.

Conclusions: The results confirmed that melatonin protects RPE cells from SI-induced injury by regulates MT2/SERCA2/Ca²⁺ axis. This study highlighted the potential of melatonin in the treatment of AMD and elucidated the mechanism and signaling pathway that mediate the protective effects.

Keywords: Calcium signaling; ER-stress; Inflammation; Melatonin; Necroptosis; Retinal degeneration.