Abstract
Microglial activation and melatonin protection have been reported in diabetic retinopathy (DR). Whether melatonin could regulate microglia to protect the inner blood–retinal barrier (iBRB) remains unknown. In this study, the role of microglia in iBRB breakdown and the mechanisms of melatonin’s regulation on microglia were explored. In diabetic rat retinas, activated microglia proliferated and migrated from the inner retina to the outer retina, accompanied by the obvious morphological changes. Meanwhile, significant leakage of albumin was evidenced at the site of close interaction between activated microglia and the damaged pericytes and endothelial cells. In vitro, inflammation-related cytokines, such as tumor necrosis factor-α (TNF-α), inducible nitric oxide synthase (iNOS), interleukin (IL)-1β, and arginase-1 (Arg-1), were increased significantly in CoCl2-treated BV2 cells. The supernatant derived from CoCl2-treated BV2 cells significantly decreased the cell viability and disrupted the junctional proteins in both pericytes and endothelial cells, resulting in severe leakage. Melatonin suppressed the microglial overactivation, i.e., decreasing the cell number and promoting its anti-inflammatory properties in diabetic rat retinas. Moreover, the leakage of iBRB was alleviated and the pericyte coverage was restored after melatonin treatment. In vitro, when treated with melatonin in CoCl2-treated BV2 cells, the inflammatory factors were decreased, while the anti-inflammatory factors were increased, further reducing the pericyte loss and increasing the tight junctions. Melatonin deactivated microglia via inhibition of PI3K/Akt/Stat3/NF-κB signaling pathways, thus maintaining the integrity of iBRB. The present data support a causal role for activated microglia in iBRB breakdown and highlight the therapeutic potential of melatonin in the treatment of DR by regulating microglia.
Highlights
Diabetic retinopathy (DR) is the leading cause of the visual impairment in working-age people worldwide, which has been characterized as a neurovascular disease
The microglia were mainly distributed in the inner retina, e.g., from the nerve fiber layer (NFL) to the inner plexiform layer (IPL), while they migrated to the outer retina, e.g., the outer plexiform layer (OPL), and even the subretinal space and retinal pigment epithelial layer, in the diabetic group
The results showed that the Gene Ontology (GO) terms were primarily enriched in NF-kB-mediated regulation, inflammatory response, cellular response to growth factor stimulus, and kinase activity (Figure 6C), and Kyoto Encyclopedia of Genes and Genomes (KEGG) indicated that the microglia-related genes were mainly enriched in the PI3K-Akt signaling pathway, chemokine signaling pathway, and cytokine–cytokine receptor interaction (Figure 6D)
Summary
Diabetic retinopathy (DR) is the leading cause of the visual impairment in working-age people worldwide, which has been characterized as a neurovascular disease. Microglia interact with other neurons, glial cells, and endothelial cells by producing growth factors and neuroprotective mediators [4], to maintain the homeostasis of retina. Noxious insults such as inflammation, hypoxia, or oxidative stress trigger microglial activation, which is manifested by the amoeboid morphology, increased proliferation, and enhanced migration to the sites of injury [5, 6]. The activated microglia secrete pro-inflammatory cytokines, such as tumor necrosis factor-a (TNF-a), interleukin (IL)-1b, IL-6, and chemotactic factors, as well as high levels of anti-inflammatory cytokines [7]. The balance between pro-inflammatory and anti-inflammatory effects is hazy, and usually microglia can adapt their phenotypes to meet the demands [8]
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