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Jing Zhao, Sylvia Smith, Kathryn Bollinger; Suppression of microglial inflammatory response by sigma receptor 1 ligand (+)-Pentazocine. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1595.
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© ARVO (1962-2015); The Authors (2016-present)
Sigma Receptor 1 (σR1) is a member of a family of membrane-associated proteins that are expressed throughout the mammalian nervous system and visceral organs. The endogenous function of σR1is not known. However, ligands for this receptor have shown robust neuroprotective effects in brain and retina. In addition, σR1 activation suppresses aspects of brain-derived microglial inflammatory response. The goal of this work is to determine whether (+)-PTZ can suppress inflammatory responses of retina-derived microglia.
Primary cultures of microglia were prepared from postnatal (1-5 days) of Sprague-Dawley rat pups. Purity of cultured microglia was confirmed by specific expression of microglia marker Iba-1 using immunocytochemistry. Purified microglia were subjected to LPS (1μg/ml) in the presence/absence of (+)-PTZ (3μM) for 1, 3, 6 or 24 hours. Supernatants were collected and TNF-α, IL-10 and MCP-1 release level were detected using ELISA assay. Nitric oxide level in the supernatant was determined using Griess assay. Intracellular ROS was detected by incubation cells with 5μM CellROX green dye followed by observation under fluorescence microscope. Immunocytochemistry was performed for detection of any morphology change. Westerns were performed to detect MAPK and NF-κB expression after the cells were treated with LPS in the presence/absence of (+)-PTZ.
Our data showed that σR1 ligand (+)-PTZ suppressed microglia morphology change in response to LPS stimulation at the 6 hours time point. LPS stimulated primary retinal microglia to secret TNF-α, IL-10, MCP-1 and nitric oxide, and secretion levels were significantly decreased when the cells were pre-treated with (+)-PTZ for 30 min. LPS also stimulated intracellular ROS generation, and ROS generation was significantly inhibited by (+)-PTZ. Our western results showed that ERK, JNK and p38 MAPKs were activated when microglia were treated with LPS. (+)-PTZ inhibited MAPK phosphorylation.
σR1 ligand (+)-PTZ suppresses inflammatory responses of retina-derived microglia and decreases MAPK activation.
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