Spinach Methanolic Extract Attenuates the Retinal Degeneration in Diabetic Rats.
ABSTRACT: It has been suggested that spinach methanolic extract (SME) inhibits the formation of advanced glycation end products (AGEs), which are increased during diabetes progression, so it is important to know if SME has beneficial effects in the diabetic retina. In this study, in vitro assays showed that SME inhibits glycation, carbonyl groups formation, and reduced-thiol groups depletion in bovine serum albumin incubated either reducing sugars or methylglyoxal. The SME effect in retinas of streptozotocin-induced diabetic rats (STZ) was also studied (n = 10) in the normoglycemic group, STZ, STZ rats treated with SME, and STZ rats treated with aminoguanidine (anti-AGEs reference group) during 12 weeks. The retina was sectioned and immunostained for Nε-carboxymethyl lysine (CML), receptor RAGE, NADPH-Nox4, inducible nitric oxide synthase (iNOS), 3-nitrotyrosine (NT), nuclear NF-κB, vascular endothelial growth factor (VEGF), glial fibrillary acidic protein (GFAP), S100B protein, and TUNEL assay. Lipid peroxidation was determined in the whole retina by malondialdehyde (MDA) levels. The results showed that in the diabetic retina, SME reduced the CML-RAGE co-localization, oxidative stress (NOX4, iNOS, NT, MDA), inflammation (NF-κB, VEGF, S100B, GFAP), and apoptosis (p < 0.05). Therefore, SME could attenuate the retinal degeneration by inhibition of CML-RAGE interaction.
Project description:BACKGROUND:Extensive intracellular and extracellular formation of advanced glycation end-products (AGEs) is considered a causative factor for vascular injury triggered by hyperglycemia in diabetes. The hyperglycemia will cause accumulation of AGEs, damage to pericytes, nerve growth factor (NGF), glial acid fibrillary protein (GFAP) and increase in vascular endothelial growth factor (VEGF). AIM:This study aimed to assess the efficacy of RAGE inhibition in suppressing the development and progression of diabetic retinopathy through modulation of the inflammatory pathway involving NGF, GFAP, and VEGF. METHODS:The design was in vivo experimental study. Thirty white rats were induced with Alloxan monohydrate. Rats were divided into 5 groups, normal, negative control, groups with an anti-RAGE dose of 1 ?g/uL, the dose of 10 ?g/uL and 100 ?g/uL. After 4 weeks of treatment, HbA1c, NGF, and GFAP levels were measured using ELISA. Quantification of VEGF expression was done using the ImageJ® application. Data was expressed with mean ± SD. Independent T-test with ANOVA and Tukey's post hoc was done. RESULTS:RAGE inhibitors yielded a significant decrease in blood glucose and HbA1c levels. VEGF and RAGE expression were reduced in anti-RAGE groups in various doses. Inhibition of RAGE reduced the damage of retinal pericytes, by reducing GFAP and increasing NGF, and reduced the formation of new blood vessels, by decreasing VEGF expression, in diabetic retinopathy. CONCLUSION:Inhibition of receptor for advanced glycation end-products (RAGE) was effective in suppressing the development and progression of diabetic retinopathy.
Project description:Aminoguanidine (AG) inhibits advanced glycation end products (AGEs) and advanced oxidation protein products (AOPP) accumulated as a result of excessive oxidative stress in diabetes. However, the molecular mechanism by which AG reduces AGE-associated damage in diabetes is not well understood. Thus, we investigated whether AG supplementation mitigates oxidative-associated cardiac fibrosis in rats with type 2 diabetes mellitus (T2DM). Forty-five male Wistar rats were divided into three groups: Control, T2DM and T2DM+AG. Rats were fed with a high-fat, high-carbohydrate diet (HFCD) for 2 weeks and rendered diabetic using low-dose streptozotocin (STZ) (20 mg/kg), and one group was treated with AG (20 mg/kg) up to 25 weeks. In vitro experiments were performed in primary rat myofibroblasts to confirm the antioxidant and antifibrotic effects of AG and to determine if blocking the receptor for AGEs (RAGE) prevents the fibrogenic response in myofibroblasts. Diabetic rats exhibited an increase in cardiac fibrosis resulting from HFCD and STZ injections. By contrast, AG treatment significantly reduced cardiac fibrosis, ?-smooth muscle actin (?SMA) and oxidative-associated Nox4 and Nos2 mRNA expression. In vitro challenge of myofibroblasts with AG under T2DM conditions reduced intra- and extracellular collagen type I expression and Pdgfb, Tgf?1 and Col1a1 mRNAs, albeit with similar expression of Tnf? and Il6 mRNAs. This was accompanied by reduced phosphorylation of ERK1/2 and SMAD2/3 but not of AKT1/2/3 and STAT pathways. RAGE blockade further attenuated collagen type I expression in AG-treated myofibroblasts. Thus, AG reduces oxidative stress-associated cardiac fibrosis by reducing pERK1/2, pSMAD2/3 and collagen type I expression via AGE/RAGE signaling in T2DM.
Project description:The receptor for advanced glycation end products (RAGE) recognizes damage-associated molecular patterns (DAMPs) and plays a critical role for the innate immune response and sterile tissue inflammation. RAGE overexpression is associated with diabetic complications, neurodegenerative diseases and certain cancers. Yet, the molecular mechanism of ligand recognition by RAGE is insufficiently understood to rationalize the binding of diverse ligands. The N-terminal V-type Ig-domain of RAGE contains a triad of tryptophan residue; Trp51, Trp61 and Trp72. The role of these three Trp residues for domain folding, stability and binding of the RAGE ligand S100B was investigated through site-directed mutagenesis, UV/VIS, CD and fluorescence spectrometry, protein-protein interaction studies, and X-ray crystallography. The data show that the Trp triad stabilizes the folded V-domain by maintaining a short helix in the structure. Mutation of any Trp residue increases the structural plasticity of the domain. Residues Trp61 and Trp72 are involved in the binding of S100B, yet they are not strictly required for S100B binding. The crystal structure of the RAGE-derived peptide W72 in complex with S100B showed that Trp72 is deeply buried in a hydrophobic depression on the S100B surface. The studies suggest that multiple binding modes between RAGE and S100B exist and point toward a not previously recognized role of the Trp residues for RAGE-ligand binding. The Trp triad of the V-domain appears to be a suitable target for novel RAGE inhibitors, either in the form of monoclonal antibodies targeting this epitope, or small organic molecules.
Project description:The worldwide prevalence of type 2 diabetes (T2D) is increasing. Despite normal to higher bone density, patients with T2D paradoxically have elevated fracture risk resulting, in part, from poor bone quality. Advanced glycation endproducts (AGEs) and inflammation as a consequence of enhanced receptor for AGE (RAGE) signaling are hypothesized culprits, although the exact mechanisms underlying skeletal dysfunction in T2D are unclear. Lack of inducible models that permit environmental (in obesity) and temporal (after skeletal maturity) control of T2D onset has hampered progress. Here, we show in C57BL/6 mice that a onetime pharmacological intervention (streptozotocin, STZ) initiated in adulthood combined with high-fat diet-induced (HFD-induced) obesity caused hallmark features of human adult-onset T2D, including prolonged hyperglycemia, insulin resistance, and pancreatic ? cell dysfunction, but not complete destruction. In addition, HFD/STZ (i.e., T2D) resulted in several changes in bone quality that closely mirror those observed in humans, including compromised bone microarchitecture, reduced biomechanical strength, impaired bone material properties, altered bone turnover, and elevated levels of the AGE CML in bone and blood. Furthermore, T2D led to the premature accumulation of senescent osteocytes with a unique proinflammatory signature. These findings highlight the RAGE pathway and senescent cells as potential targets to treat diabetic skeletal fragility.
Project description:Background:Advanced glycation end products play an important role in diabetic atherosclerosis. The effects of advanced glycation end products (AGEs) on vascular smooth muscle cell- (VSMC-) derived foam cell formation and phenotypic transformation are unknown. Methods:Serological and histological samples were obtained from diabetic amputation patients and accident amputation patients from the Affiliated Hospital of Jiangsu University. CD68/Actin Alpha 2 (ACTA2) coimmunofluorescence sections were used to quantify the number of VSMCs with macrophage-like phenotypes. Western blotting was used to detect the expression of the receptor of advanced glycation end products in vascular samples. Enzyme-linked immunosorbent assay (ELISA) was used to evaluate the level of serum N?-carboxymethyl-lysine (CML). In vitro oil red O staining was used to examine lipid accumulation in VSMCs stimulated by CML. The expression of VSMCs and macrophage markers was measured by western blotting and quantitative real-time PCR. Furthermore, changes in VSMC migration and secretion were detected by the Transwell assay and ELISA. Results:In the arterial plaque sections of diabetic patients, VSMCs transformed to a macrophage-like phenotype. The serum CML and RAGE levels in the plaques were significantly higher in the diabetes group than those in the healthy control group and were significantly related to the number of macrophage-like VSMCs. CML stimulation promoted intracellular lipid accumulation. However, CML stimulation decreased the expression of VSMC markers and increased the expression of macrophage phenotype markers. Finally, CML promoted smooth muscle cell migration and the secretion of proinflammatory-related factors. Conclusions:CML induces VSMC-derived foam cell formation, and VSMCs transdifferentiate to a macrophage-like state, which may be mediated by the activation of RAGE.
Project description:The receptor for advanced glycation end products (RAGE) is a pattern recognition receptor involved in inflammatory processes and is associated with diabetic complications, tumor outgrowth, and neurodegenerative disorders. RAGE induces cellular signaling events upon binding of a variety of ligands, such as glycated proteins, amyloid-?, HMGB1, and S100 proteins. The X-ray crystal structure of the VC1 ligand-binding region of the human RAGE ectodomain was determined at 1.85 Å resolution. The VC1 ligand-binding surface was mapped onto the structure from titrations with S100B monitored by heteronuclear NMR spectroscopy. These NMR chemical shift perturbations were used as input for restrained docking calculations to generate a model for the VC1-S100B complex. Together, the arrangement of VC1 molecules in the crystal and complementary biochemical studies suggest a role for self-association in RAGE function. Our results enhance understanding of the functional outcomes of S100 protein binding to RAGE and provide insight into mechanistic models for how the receptor is activated.
Project description:<b>Purpose:</b> The present study is aimed to explore whether the aqueous extract of <i>Mori Folium</i> (MF) exhibits bone protective effect by regulating calcium and redox homeostasis in diabetic rats, and to identify the signaling pathways involved in this process. <b>Methods:</b> Diabetic rats were established using high-sugar and high-fat diet and streptozotocin (STZ) (30 mg/kg for 3 consecutive days). The serum levels of osteocalcin (OC), insulin-like growth factor-1 (IGF-1), tartrate-resistant acid phosphatase (TRAP), phosphorus (P), calcium (Ca), 1,25-dihydroxyvitamin D<sub>3</sub> [1,25(OH)<sub>2</sub>D<sub>3</sub>], parathormone (PTH), advanced glycation end products (AGEs), superoxide dismutase (SOD), and malondialdehyde (MDA), total antioxidant capacity (TAC), 8-hydroxy-2'-deoxyguanosine (8-OH-dG), and interleukin 6 (IL-6) were determined by ELISA or biochemical assays. Histopathological alterations in the femurs were evaluated by the stainings of hematoxylin-eosin (H&E) and alizarin red S. In addition, femoral strength was detected by a three-point bending assay, bone microstructure was detected with micro-computer tomography. Bone material properties were examined by Fourier-transform infrared spectroscopy. Furthermore, the expressions of IGF-1, runt-related transcription factor 2 (Runx2), osteoprotegerin (OPG), receptor activator of nuclear factor kappa-B ligand (RANKL), cathepsin K, AGEs, receptor of advanced glycation end products (RAGE), NADPH oxidase 4 (Nox4), and nuclear factor kappa-B (NF-?B) in the femurs and tibias, and the alterations in the levels of calcium-binding protein-28k (CaBP-28k), transient receptor potential V6 (TRPV6), and vitamin D receptor (VDR) in the kidneys and duodenums were determined by western blot and immunohistochemical analysis. <b>Results:</b> Treatment of diabetic rats with MF aqueous extract induces an increase in the levels of OC and IGF-1 as well as a decrease in TRAP level in serum. MF treatment also upregulates the expression of OPG, downregulates the expressions of AGEs, RAGE, Nox4, NF-?B, and RANKL, which leads to improve bone microstructure and strength exhibited by an increase in cortical area ratio, cortical thickness, and trabecular area ratio as well as ultimate load, elastic modulus, and bending stress in the femurs and tibias of diabetic rats. In addition, MF aqueous extract preserves bone material properties by decreasing the ratio of fatty acid/collagen and increasing the ratio of mineral/matrix in the femurs of diabetic rats. Moreover, MF treatment increases the levels of P, Ca, and 1,25(OH)<sub>2</sub>D<sub>3</sub>, and decreases the level of PTH in the serum, as well as upregulates the expressions of TRPV6 and VDR in the duodenums and CaBP-28k in the kidneys of diabetic rats. Additionally, MF has ability of rebuilding redox homeostasis and eliminating inflammatory stress by increasing the levels of SOD and TAC as well as decreasing the levels of IL-6, AGEs, MDA, and 8-OH-dG. <b>Conclusions:</b> MF treatment may improve bone quality through maintenance of calcium homeostasis via regulating the PTH/VDR/CaBP signaling, and elimination of oxidative stress via regulating the AGEs/RAGE/Nox4/NF-?B signaling. These results may suggest the potential of MF in preventing the development of diabetic osteoporosis.
Project description:Advanced glycation end products play major roles in diabetic complications. They act via their receptor RAGE to induce inflammatory genes such as cyclooxygenase-2 (COX-2). We examined the molecular mechanisms by which the RAGE ligand, S100b, induces COX-2 in monocytes. S100b significantly increased COX-2 mRNA accumulation in THP-1 monocytes at 2 h via mRNA stability. This was further confirmed by showing that S100b increased stability of luciferase-COX-2 3'-UTR mRNA. Chromatin immunoprecipitation and RNA immunoprecipitation revealed that S100b decreased occupancy of the DNA/RNA-binding protein, heterogeneous nuclear ribonuclear protein K (hnRNPK), at the COX-2 promoter but simultaneously increased its binding to the COX-2 3'-UTR. S100b treatment promoted the translocation of nuclear hnRNPK to cytoplasm, whereas a cytoplasmic translocation-deficient hnRNPK mutant inhibited S100b-induced COX-2 mRNA stability. Small interfering RNA-mediated specific knockdown of hnRNPK blocked S100b-induced COX-2 mRNA stability, whereas on the other hand, overexpression of hnRNPK increased S100b-induced COX-2 mRNA stability. S100b promoted the release of entrapped COX-2 mRNA from cytoplasmic processing bodies, sites of mRNA degradation. Furthermore, S100b significantly down-regulated the expression of a key microRNA, miR-16, which can destabilize COX-2 mRNA by binding to its 3'-UTR. MiR-16 inhibitor oligonucleotides increased, whereas, conversely, miR-16 mimic oligonucleotides decreased COX-2 mRNA stability in monocytes, further supporting the inhibitory effects of miR-16. Interestingly, hnRNPK knockdown increased miR-16 binding to COX-2 3'-UTR, indicating a cross-talk between them. These new results demonstrate that diabetic stimuli can efficiently stabilize inflammatory genes via opposing actions of key RNA-binding proteins and miRs.
Project description:The non-enzymatic reaction between glucose and protein can be chemically reversed by transglycation. Here we report the transglycation activity of hydralazine using a newly developed MALDI-TOF-MS based assay. Hydralazine mediated transglycation of HbA1c, plasma proteins and kidney proteins was demonstrated in streptozotocin (STZ) induced diabetic mice, as evidenced by decrease in protein glycation, as well as presence of hydralazine-glucose conjugate in urine of diabetic mice treated with hydralazine. Hydralazine down regulated the expression of Receptor for Advanced Glycation End products (RAGE), NADPH oxidase (NOX), and super oxide dismutase (SOD). These findings will provide a new dimension for developing intervention strategies for the treatment of glycation associated diseases such as diabetes complications, atherosclerosis, and aging.
Project description:Six-transmembrane epithelial antigen of prostate 4 (Steap4)-knockout mice develop hyperglycaemia and inflammation whereas Steap4 overexpression attenuates atherosclerosis in diabetic mice. Thus, we studied the roles of Steap4 in high glucose (HG, 27.5 mM) or S100B (1 ?M, a ligand for the receptor for advanced glycation end-product or RAGE)-induced effects in mouse mesangial (MES13) cells. We found that HG-induced Steap4 protein expression was dependent on S100B. HG increased cell membrane, but not cytosolic, Steap4 protein expression. HG increased protein-protein interaction between Steap4 and S100B, which was confirmed by mass spectrometry of immunoprecipitated S100B. SP600125, LY294002 and AG490 attenuated S100B-induced Steap4 protein expression or gene transcriptional activity. A mutation in signal transducer and activator of transcription 3 (Stat3) site 2 of the Steap4 promoter constructs resulted in a marked decrease in HG or S100B-induced activation of Steap4 gene transcription. Overexpression of Steap4 attenuates HG or S100B-induced collagen IV, fibronectin and cyclooxygenase 2 protein expression. Overexpression of Steap4 attenuates HG or S100B-induced transforming growth factor-? (TGF-?). Moreover, overexpression of Steap4 attenuates S100B-induced signalling. Finally, overexpressing Steap4 attenuated renal expression of fibronectin, S100B, TGF-?, type IV collagen, p-Akt, p-extracellular signal regulated kinase 1/2 and p-Stat3 in streptozotocin-diabetic mice. Thus, overexpression of Steap4 attenuated HG or S100B-induced effects in MES13 cells and attenuated some of S100B-induced effects in diabetic mouse kidneys.