A Synthesized Glucocorticoid- Induced Leucine Zipper Peptide Inhibits Retinal Müller Cell Gliosis

Abstract

Purpose: The anti-inflammatory activities of protein glucocorticoid-induced leucine zipper (GILZ) have been demonstrated in vivo and in vitro. Here, we examined the potential effect of a synthetic peptide derived from the leucine zipper motif and proline-rich region of GILZ on suppressing inflammatory responses in primary cultured rat Müller cells.Methods: Peptides were selected from amino acids 98–134 of the GILZ protein (GILZ-p). Solid-phase peptide synthesis was used to generate the cell-penetrating peptide TAT, which was bound to the amino terminus of GILZ-p. Primary cultured retinal Müller cells were stimulated with lipopolysaccharide (LPS) alone or in combination with different concentrations of GILZ-p, and the interaction of GILZ-p with nuclear factor (NF)-κB p65 in Müller cells was investigated by western blotting, immunoprecipitation, and immunofluorescence. The expression of the Müller cell gliosis marker glial fibrillary acidic protein (GFAP), functional protein aquaporin (AQP)-4, and the inflammatory cytokines interleukin (IL)-1β, tumor necrosis factor (TNF) α, intercellular adhesion molecule (ICAM)-1, and monocyte chemoattractant protein (MCP)-1 was measured by Western Blotting. The concentration of those cytokines in culture medium was measured by using Enzyme-Linked Immunosorbent Assay.Results: The synthesized GILZ-p, which was water-soluble, entered cells and bound with NF-κB p65, inhibiting p65 nuclear translocation. GILZ-p inhibited the LPS-induced expression of GFAP, IL-1β, TNFα, ICAM-1, and MCP-1 in Müller cells and prevented the LPS-induced downregulation of AQP4.Conclusions: These results indicate that GILZ-p interacted with NF-κB p65 and suppressed p65 nuclear translocation, thereby inhibiting inflammatory cytokine release and Müller cell gliosis.

Synthetic Glucocorticoid-Induced Leucine Zipper Peptide Inhibits Lipopolysaccharide-Induced Ocular Inflammation in Rats

“…In our previous research, the GILZ 98-134 peptide from the Cterminus of GILZ was synthesized and purified with solid-phase peptide synthesis in vitro. The intracellular delivery of the synthetic GILZ 98-134 peptide was achieved in primary cultured retinal Müller cells in vitro and in the retina in vivo [13] by attaching it to the cell-penetrating peptide TAT (YGRKKRRQRRR), derived from human immunodeficiency virus [11]. The biological activity of the synthetic GILZ 98-134 peptide was tested in vitro in cultured rat Müller cells [11] and was shown to interact with and suppress the nuclear translocation of NF-κB p65, thereby inhibiting inflammatory cytokine release and Müller cell gliosis.…”

“…The intracellular delivery of the synthetic GILZ 98-134 peptide was achieved in primary cultured retinal Müller cells in vitro and in the retina in vivo [13] by attaching it to the cell-penetrating peptide TAT (YGRKKRRQRRR), derived from human immunodeficiency virus [11]. The biological activity of the synthetic GILZ 98-134 peptide was tested in vitro in cultured rat Müller cells [11] and was shown to interact with and suppress the nuclear translocation of NF-κB p65, thereby inhibiting inflammatory cytokine release and Müller cell gliosis. In this study, we demonstrate that the synthetic GILZ 98-134 peptide inhibited the inflammatory reaction in a rat model of EIU and may therefore have utility as a drug for the treatment of ocular inflammatory diseases.…”

“…This whole process was performed on an automatic peptide synthesizer (Symphony, Protein Technologies, Tucson, AZ, USA) by China Peptides (Shanghai, China). To achieve the efficient intracellular delivery of GILZ-p, the cell-penetrating TAT (YGRKKRRQRRR) was added to the amino terminus of the GILZ 98-134 peptide (YGRK-KRRQRRR-KTLASPEQLEKFQSRLSPEEPAPEAPETPEAP-GGSAV) [11]. The TAT peptide alone was used as the control peptide (Control-p).…”

“…By overexpressing retinal GILZ, we demonstrated [10] that GILZ attenuates the inflammatory response in rats with endotoxin-induced uveitis (EIU). Furthermore, a synthetic GILZ peptide derived from the C-terminal domain of GILZ, which includes 8 proline residues, 8 glutamic acid residues, and 5 PxxP sequences, prevents Müller cell gliosis and inflammatory cytokine secretion [11]. The aim of this study was to further examine the effects of the synthetic peptide GILZ 98-134 on the inflammatory responses in vivo in a rat model of EIU.…”

Protection of the Retinal Ganglion Cells: Intravitreal Injection of Resveratrol in Mouse Model of Ocular Hypertension

“…The viability of MGCs was determined using a Cell Counting Kit-8 (CCK-8) assay (Dojindo Laboratories, Kumamoto, Japan), as in previous studies. 66 , 67 The isolated and cultured mouse MGCs were plated in 96-well plates (5 × 10 3 cells per well) and incubated for 24 hours. RSV was diluted in DMEM with 0.2% dimethyl sulfoxide (Sigma-Aldrich), and the vehicle was used as control.…”