Signaling mechanisms of growth hormone-releasing hormone receptor in LPS-induced acute ocular inflammation.

Ocular inflammation is a major cause of visual impairment attributed to dysregulation of the immune system. Previously, we have shown that the receptor for growth-hormone-releasing hormone (GHRH-R) affects multiple inflammatory processes. To clarify the pathological roles of GHRH-R in acute ocular inflammation, we investigated the inflammatory cascades mediated by this receptor. In human ciliary epithelial cells, the NF-κB subunit p65 was phosphorylated in response to stimulation with lipopolysaccharide (LPS), resulting in transcriptional up-regulation of GHRH-R. Bioinformatics analysis and coimmunoprecipitation showed that GHRH-R had a direct interaction with JAK2. JAK2, but not JAK1, JAK3, and TYK2, was elevated in ciliary body and iris after treatment with LPS in a rat model of endotoxin-induced uveitis. This elevation augmented the phosphorylation of STAT3 and production of proinflammatory factors, including IL-6, IL-17A, COX2, and iNOS. In explants of iris and ciliary body, the GHRH-R antagonist, MIA-602, suppressed phosphorylation of STAT3 and attenuated expression of downstream proinflammatory factors after LPS treatment. A similar suppression of STAT3 phosphorylation was observed in human ciliary epithelial cells. In vivo studies showed that blocking of the GHRH-R/JAK2/STAT3 axis with the JAK inhibitor Ruxolitinib alleviated partially the LPS-induced acute ocular inflammation by reducing inflammatory cells and protein leakage in the aqueous humor and by repressing expression of STAT3 target genes in rat ciliary body and iris and in human ciliary epithelial cells. Our findings indicate a functional role of the GHRH-R/JAK2/STAT3-signaling axis in acute anterior uveitis and suggest a therapeutic strategy based on treatment with antagonists targeting this signaling pathway.

Growth hormone-releasing hormone receptor mediates cytokine production in ciliary and iris epithelial cells during LPS-induced ocular inflammation.

The receptor for growth hormone-releasing hormone (GHRH-R) has been shown to upregulate specifically in the ciliary and iris epithelial cells and infiltrating cells in the aqueous humor in a rat model of acute anterior uveitis. Treatment with GHRHR-R antagonist alleviates significantly these inflammatory responses. Herein we investigated whether the ciliary and iris epithelial cells can respond directly to lipopolysaccharide (LPS) without the influences of circulating leukocytes to produce inflammatory mediators through a GHRH-R mediated mechanism. In explant cultures of rat ciliary body and iris, LPS caused a substantial increase of GHRH-R in 24 h. Immunohistochemistry showed a localization of TLR4, the receptor for LPS, and an elevated expression of IL-6 and IL-1ß in ciliary and iris epithelial cells after LPS treatment. LPS also elevated the level of IL-1ß, IL-6, and iNOS and increased secretion of IL-1ß and IL-6 from the explants. The GHRH-R antagonist, MIA-602, suppressed the elevated expression of IL-1ß and IL-6, and reduced the release of IL-6. Such effects were not seen for the GHRHR agonist, MR-409. When co-cultured with leukocytes, expression of GHRH-R in the ocular explants was further enhanced during LPS treatment. Our results demonstrate a direct action of LPS on ciliary and iris epithelial cells to produce pro-inflammatory factors through a GHRH-R mediated mechanism, and suggest a role of these epithelial cells, in addition to the resident antigen presenting cells, in immune surveillance of the eye. Infiltrating leukocytes may enhance these inflammatory responses by regulating GHRH-R in ciliary and iris epithelial cells, in addition to their functions of synthesizing proinflammatory cytokines.

Green tea extract attenuates LPS-induced retinal inflammation in rats.

Inflammation is in a wide spectrum of retinal diseases, causing irreversible blindness and visual impairment. We have previously demonstrated that Green Tea Extract (GTE) is a potent anti-inflammatory agent for anterior uveitis. Here we investigated the anti-inflammatory effect of GTE on lipopolysaccharides (LPS)-induced retinal inflammation in rats and explored the underlying mechanism. Adult rats were injected with LPS and GTE was administered intra-gastrically at 2, 8, 26 and 32 hours post-injection. Staining of whole-mount retina showed that the number of activated microglia cells was significantly increased at 48 hours post-injection, which was suppressed after GTE treatment in a dose-dependent manner. Activation of astrocytes and Müller glia in the retina was also suppressed after GTE treatment. Meanwhile, GTE reduced the expression of pro-inflammatory cytokines including IL-1ß, TNF-α and IL-6 in retina and vitreous humor. These anti-inflammatory effects were associated with a reduced phosphorylation of STAT3 and NF-κB in the retina. Furthermore, the surface receptor of EGCG, 67LR, was localized on the neurons and glia in the retina. These findings demonstrate that GTE is an effective agent in suppressing LPS-induced retinal inflammation, probably through its potent anti-oxidative property and a receptor-mediated action on transcription factors that regulate production of pro-inflammatory cytokines.

LncRNA-NEF antagonized epithelial to mesenchymal transition and cancer metastasis via cis-regulating FOXA2 and inactivating Wnt/ß-catenin signaling.

Emerging evidence indicates that the long noncoding RNAs extensively participate in cancer progression. Nevertheless, the molecular pathogenesis of how these lncRNAs regulate tumorigenesis has not been fully elucidated especially in hepatocellular carcinoma (HCC). Here, we sought to define the role of a novel lncRNA named lncRNA-NEF in modulating epithelial to mesenchymal transition (EMT) in HCC. It was found that the lncRNA-NEF was transcriptionally activated by EMT suppressor FOXA2 and frequently downregulated in HCC cell lines as well as clinical specimens. Although enhanced expression of lncRNA-NEF did not affect tumor cell growth, ectopic expression of lncRNA-NEF significantly suppressed EMT program and cell migration. Animal studies validated that lncRNA-NEF alleviated in vivo tumor metastasis and protected mice from tumor-induced mortality. Interestingly, we verified that lncRNA-NEF acted as a novel activator of its neighbor gene FOXA2, which formed a positive feedback loop. Subsequent studies revealed that lncRNA-NEF physically interacted with ß-catenin to increase the binding of GSK3ß with ß-catenin and therefore promoted the inhibitory phosphorylation of ß-catenin, leading to the suppression on Wnt/ß-catenin signaling and activation of FOXA2 expression. Hence, our findings illustrated a novel feedback loop including FOXA2 and its neighboring gene lncRNA-NEF, which might provide mechanistic insights into the metastatic progress of HCC.

Green tea extract treatment alleviates ocular inflammation in a rat model of endotoxin-induced uveitis.

Green tea extract (GTE) ingested by rats exerted anti-oxidative activities in various ocular tissues as shown in our previous studies. The present work investigated anti-inflammatory effects of GTE on endotoxin-induced uveitis (EIU). EIU was generated in adult rats by a footpad injection of 1 mg/kg lipopolysaccharide (LPS). Oral administration of GTE (550 mg/kg) was given one, two or four times after LPS injection. Twenty-four hours later, LPS produced severe hyperemia and edema in the iris. Immunocytochemical examinations showed an accumulation of infiltrating cells in the aqueous humor that were immunopositive for cluster of differentiation 43 (CD43) and CD68, markers for leucocytes and macrophages, respectively. Analyses of the aqueous humor showed an increase in pro-inflammatory mediators including tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1). GTE treatments improved the clinical manifestations and reduced infiltrating cells and protein exudation in the aqueous humor, which were not observed under half dose of GTE (275 mg/kg). The number of CD68 positive macrophages residing in the iris and ciliary was also reduced. GTE suppressed production of TNF-α, IL-6 and MCP-1 in the aqueous humor, which was associated with a down-regulation of LPS receptor complex subunits, Toll-like receptor 4 (TLR-4) and CD14, and suppression of nuclear factor-kappa Bp65 (NF-κBp65) in the iris and ciliary body. Our findings show that GTE is a potent anti-inflammatory agent against the inflammation of EIU, and suggest a potential use in treatment of acute uveitis.