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Occludin downregulation in high glucose is regulated by SSTR2 via the VEGF/NRP1/Akt signaling pathway in RF/6A cells.

ABSTRACT: Occludin is a tight junction protein that forms the permeability barrier, which is typically disturbed in ischemic associated diseases. The aim of the present study was to determine whether somatostatin receptor 2 (SSTR2) in RF/6A cells is involved in the modulation of the downregulation of occludin induced by high glucose, and to evaluate the implicated molecules. RF/6A cells were maintained in Dulbecco's modified Eagle medium and treated with 0 or 30 mM D-glucose. SSTR2 agonist octreotide (OCT), OCT with SSTR2 antagonist cycle-somatostatin (c-SOM) and neuropilin 1 (NRP1) inhibitor ATWLPPR, respectively, were administered to RF/6A cells under high glucose conditions. Cell apoptosis was evaluated by terminal deoxynucleotidyl transferase dUTP nick-end labeling. Western blot analysis was used to detect the protein expression level of SSTR2, occludin, vascular endothelial growth factor (VEGF), protein kinase B (Akt), phosphorylated Akt (p-Akt), extracellular signal-related kinases (ERK) and p-ERK proteins. The amount of VEGF released was determined by ELISA. Notably, the level of occludin reduced significantly under high glucose conditions. The results indicated that the administration of OCT prevented the reduction of occludin induced by high glucose, and co-administration with c-SOM reversed the effect of OCT. Increased VEGF secretion and expression of VEGF, p-Akt and p-ERK in RF/6A cells induced by high glucose were inhibited by OCT. ATWLPPR also prevented the downregulation of occludin, but did not inhibit p-Akt and p-ERK levels under high glucose conditions. The current study concluded that the activation of SSTR2 prevents high glucose-induced occludin downregulation in RF/6A cells, and VEGF, NRP1, p-Akt and p-ERK were implicated in this process. The pharmacological effects of SSTR2 targeting to endothelium may be used to assess the role of resistance of permeability and anti-inflammation.

SUBMITTER: Li M

PROVIDER: S-EPMC5526144 | biostudies-other | 2017 Aug

REPOSITORIES: biostudies-other

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Project description:Hyperglycaemia-induced retinal microvascular endothelial cell apoptosis is a critical and principle event in diabetic retinopathy (DR), which involves a series of complex processes such as mitochondrial dysfunction and oxidative stress. Ginsenoside Re (Re), a key ingredients of ginseng, is considered to have various pharmacologic functions, such as antioxidative, inhibition of inflammation and anti-apoptotic properties. However, the effects of Re in DR and the related mechanisms of endothelial cell injury induced by high glucose (HG) exposure remain unclear. The present study was designed to investigate and evaluate the ability of Re to ameliorate HG-induced retinal endothelial RF/6A cell injury and the potential mechanisms involved in the hypoxia-inducible factor-1-alpha (HIF-1?)/vascular endothelial growth factor (VEGF) signaling regulated by phosphoinositide 3-kinase (PI3K)/AKT pathway. Our results showed that preincubation with Re exerted cytoprotective effects by reversing the HG-induced decrease in RF/6A cell viability, downregulation of apoptosis rate and inhibition of oxidative-related enzymes, thereby reducing the excess intracellular reactive oxygen species (ROS) and HG-triggered RF/6A cell injury. In addition, Western blot analysis results showed ginsenoside Re significantly increased HIF-1? expression in the cytoplasm but decreased its expression in the nucleus, suggesting that it reduced the translocation of HIF-1? from the cytoplasm to the nucleus, and downregulated VEGF level. Moreover, this effect is involved in the activation of the PI3K/Akt pathway. LY294002, a PI3K inhibitor, was used to block the Akt pathway. Afterwards, the effects of Re on the regulation of apoptotic related proteins, VEGF and HIF-1? nuclear transcription was partially reversed. These findings suggested the exerting protective effects of ginsenoside Re were associated with regulating of PI3K/AKT and HIF-1?/VEGF signaling pathway, which indicates that ginsenoside Re may ameliorates HG-induced retinal angiogenesis and suggests the potential for the development of Re as a therapeutic for DR.

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Occludin downregulation in high glucose is regulated by SSTR2 via the VEGF/NRP1/Akt signaling pathway in RF/6A cells.

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