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Zhao S.,Shanghai JiaoTong University | Li J.,Lishui City Center Hospital | Wang N.,Shanghai JiaoTong University | Zheng B.,Shanghai JiaoTong University | And 4 more authors.
Molecular Medicine Reports | Year: 2015

Inflammation is a major contributing factor in the development of diabetic microvascular complications, regardless of whether improved glycaemic control is achieved. Studies have increasingly indicated that fenofibrate, a lipid-lowering therapeutic agent in clinical use, exerts a potential anti-inflammatory effect, which is mediated by sirtuin 1 (SIRT1; an NAD+-dependent deacetylase) in endothelial cells. The aim of the present study was to investigate the inhibitory effect of fenofibrate on metabolic memory (via the regulation of SIRT1), and inflflammatory responses in cell and animal models of diabetic retinopathy (DR). The data demonstrated that high glucose treatment in human retinal endothelial cells (HRECs) inhibited the expression and deacetylase activity of SIRT1. The reduction of SIRT1 expression and deacetylase activity persisted following a return to normal glucose levels. Furthermore, nuclear factor-.B expression was observed to be negatively correlated with SIRT1 expression and activity in HRECs under high glucose levels and the subsequent return to normal glucose levels. Fenofibrate treatment abrogated these changes. Knockdown of SIRT1 attenuated the effect of fenofibrate on high glucose-induced NF-.B expression. In addition, fenofibrate upregulated SIRT1 expression through peroxisome proliferator-activated receptor a in high glucose-induced metabolic memory. These findings indicate that fenofibrate is important in anti-inflflammatory processes and suppresses the cellular metabolic memory of high glucose-induced stress via the SIRT1-dependent signalling pathway. Thus, treatment with fenofifibrate may offer a promising therapeutic strategy for halting the development of DR and other complications of diabetes. Source


Zhao S.,Shanghai JiaoTong University | Li T.,Shanghai JiaoTong University | Li J.,Lishui City Center Hospital | Lu Q.,Shanghai JiaoTong University | And 7 more authors.
Diabetologia | Year: 2016

Aims/hypothesis: The mechanisms underlying the cellular metabolic memory induced by high glucose remain unclear. Here, we sought to determine the effects of microRNAs (miRNAs) on metabolic memory in diabetic retinopathy. Methods: The miRNA microarray was used to examine human retinal endothelial cells (HRECs) following exposure to normal glucose (N) or high glucose (H) for 1 week or transient H for 2 days followed by N for another 5 days (H→N). Levels of sirtuin 1 (SIRT1) and acetylated-nuclear factor κB (Ac-NF-κB) were examined following transfection with miR-23b-3p inhibitor or with SIRT1 small interfering (si)RNA in the H→N group, and the apoptotic HRECs were determined by flow cytometry. Retinal tissues from diabetic rats were similarly studied following intravitreal injection of miR-23b-3p inhibitor. Chromatin immunoprecipitation (ChIP) analysis was performed to detect binding of NF-κB p65 to the potential binding site of the miR-23b-27b-24-1 gene promoter in HRECs. Results: High glucose increased miR-23b-3p expression, even after the return to normal glucose. Luciferase assays identified SIRT1 as a target mRNA of miR-23b-3p. Reduced miR-23b-3p expression inhibited Ac-NF-κB expression by rescuing SIRT1 expression and also relieved the effect of metabolic memory induced by high glucose in HRECs. The results were confirmed in the retina using a diabetic rat model of metabolic memory. High glucose facilitated the recruitment of NF-κB p65 and promoted transcription of the miR-23b-27b-24-1 gene, which can be suppressed by decreasing miR-23b-3p expression. Conclusions/interpretation: These studies identify a novel mechanism whereby miR-23b-3p regulates high-glucose-induced cellular metabolic memory in diabetic retinopathy through a SIRT1-dependent signalling pathway. © 2015, Springer-Verlag Berlin Heidelberg. Source


Li J.,Shanghai JiaoTong University | Wang P.,Lishui City Center Hospital | Yu S.,Lishui City Center Hospital | Zheng Z.,Shanghai JiaoTong University | Xu X.,Shanghai JiaoTong University
Molecular Vision | Year: 2012

Purpose: Hyperglycemia-induced vascular cell apoptosis is a seminal early event in diabetic retinopathy. Prolonged hyperglycemia is known to increase intracellular cytosolic free calcium ([Ca2+]i) in retinal vascular endothelial cells (RECs), suggesting that [Ca2+]i is a critical trigger for microvascular degeneration. This study aims to elucidate Ca2+- dependent signaling mechanisms that mediate hyperglycemia-induced apoptosis in RECs. Methods: A cultured macaque choroid-retinal endothelial cell line (RF/6A) was incubated in normal glucose (NG), NG plus the Ca2+ entry blocker 2-aminoethoxydiphenyl borate (2-APB), high glucose (HG), or HG plus either 2-APB, the c-jun N-terminal kinase (JNK) inhibitor SP600125, or the calcium/calmodulin-dependent protein kinase II (CaMKII) inhibitor KN93. Changes in [Ca2+]i evoked by adenosine 5′-triphosphate (ATP) were measured in fluo-3/AM-loaded RF/6A cells by confocal microscopy. The mitochondrial membrane potential (ΔΨm) and apoptosis were assessed by flow cytometry. Expression levels of CaMKII, phosphorylated CaMKII (p-CaMKII), c-Jun N-terminal kinase (JNK), phosphorylated JNK (p-JNK), the death receptor (Fas), and cytochrome c were detected by western blotting analysis. Results: Prolonged exposure to HG (96 h) potentiated ATP-evoked Ca2+ entry as well as CaMKII phosphorylation and RF/6A cell apoptosis. Enhanced apoptosis was blocked by 2-APB and KN93. Furthermore, HG increased JNK phosphorylation and Fas expression, and both responses were partially blocked by 2-APB and KN93, while the JNK inhibitor SP600125 partially reduced HG-induced Fas expression. In addition, HG depolarized the ΔΨm and triggered the release of mitochondrial cytochrome c. These early signs of mitochondria-dependent apoptosis were partially reversed by 2-APB and KN93. Conclusions: HG-induced apoptosis in RF/6A cells depends on Ca2+ entry and CaMKII activation, leading to the activation of both Fas-dependent and mitochondria-dependent apoptosis pathways. The CaMKII-JNK-Fas pathway is involved in HG-evoked apoptosis of RECs. © 2012 Molecular Vision. Source


Li J.,Shanghai JiaoTong University | Zhao S.-Z.,Shanghai JiaoTong University | Wang P.-P.,Lishui City Center Hospital | Yu S.-P.,Lishui City Center Hospital | And 2 more authors.
Acta Pharmacologica Sinica | Year: 2012

Aim:To investigate the effects of high glucose (HG) medium on expression of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) in cultured rat retinal Müller cells and to determine the signaling pathways mediating the effects.Methods:Primary cultures of retinal Müller cells were prepared from Sprague-Dawley rats, and incubated in a medium containg HG (30 mmol/L) in the presence of the membrane-permeable Ca 2+ chelator BAPTA-AM (10 mol/L) or the CaMKII inhibitor KN93 (10 mol/L). The levels of CaMKII, p-CaMKII, CREB, p-CREB, HIF-1α, and VEGF proteins were measured with Western blotting, while HIF-1á and VEGF mRNA levels were determined using real-time RT-PCR.Results:The stimulation of retinal Müller cell with HG for 24 h remarkably increased the expression levels of HIF-1α and VEGF. These responses were significantly inhibited in the presence of BAPTA-AM or KN93. Both BAPTA-AM and KN93 also significantly inhibited HG-induced phosphorylation of CaMKII and CREB in the cultured retinal Müller cells. Transfection of the cultured retinal Müller cells with antisense CREB oligonucleotide (300 nmol/L) was similarly effective in blocking the HG-induced increase of HIF-1α and VEGF.Conclusion:HG-induced HIF-1α and VEGF expression in cultured rat retinal Müller cells depends on intracellular free Ca2+ and activation of CaMKII-CREB pathway. The activation of CaMKII-CREB pathway by HG may be a possible mechanism underlying the pathogenesis of diabetic retinopathy. © 2012 CPS and SIMM. All rights reserved. Source

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