Diet-Derived Advanced Glycation End Products (dAGEs) Induce Proinflammatory Cytokine Expression in Cardiac and Renal Tissues of Experimental Mice: Protective Effect of Curcumin.

The beneficial effect of curcumin (CU) on dietary AGEs (dAGEs) involves blocking the overexpression of proinflammatory cytokine genes in the heart and kidney tissues of experimental mice. The animals were divided into six groups (n = 6/group) and were fed a heat-exposed diet (dAGEs) with or without CU for 6 months. Their blood pressure (BP) was monitored by a computerized tail-cuff BP-monitoring system. The mRNA and protein expression levels of proinflammatory genes were analyzed by RT-PCR and western blot, respectively. A marked increase in BP (108 ± 12 mmHg vs 149 ± 15 mmHg) accompanied by a marked increase in the heart and kidney weight ratio was noted in the dAGE-fed mice. Furthermore, the plasma levels of proinflammatory molecules (C5a, ICAM-1, IL-6, MCP-1, IL-1ß and TNF-α) were found to be elevated (3-fold) in dAGE-fed mice. mRNA expression analysis revealed a significant increase in the expression levels of inflammatory markers (Cox-2, iNOS, and NF-κB) (3-fold) in cardiac and renal tissues of dAGE-fed mice. Moreover, increased expression of RAGE and downregulation of AGER-1 (p < 0.001) were noticed in the heart and kidney tissues of dAGE-fed mice. Interestingly, the dAGE-induced proinflammatory genes and inflammatory responses were neutralized upon cotreatment with CU. The present study demonstrates that dietary supplementation with CU has the ability to neutralize dAGE-induced adverse effects and alleviate proinflammatory gene expression in the heart and kidney tissues of experimental mice.

Peanut skin polyphenols inhibit toxicity induced by advanced glycation end-products in RAW264.7 macrophages.

This is the first work to use a polyphenolic fraction derived from peanut skin to attenuate the toxicity induced by advanced glycation-end products (AGEs) in RAW264.7 macrophages. The RAW264.7 cells were stimulated by AGEs using the bovine serum albumin-fructose (BSA-FRU), bovine serum albumin-methylglyoxal (BSA-MGO) and arginine-methylglyoxal (ARG-MGO) models. The AGEs increased considerably the levels of reactive oxygen species and the gene expression of proinflammatory cytokines interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and nitric oxide. Twenty-eight polyphenols, including catechin, phenolic acids, and resveratrol were annotated in peanut skin extract (PSE) with the use of ultra-performance liquid chromatography coupled to quadrupole time of flight mass spectrometry (UPLC-QTOF/MSE) and to the UNIFI Scientific Information System. The administration of PSE at 100 and 150 µg/mL significantly inhibited oxidative stress, by suppressing the production of reactive oxygen species up to 70% and reducing the production of nitric oxide, IL-6 and TNF-α up to 1.7-, 10- and 107-fold, respectively.

Phytochemical-Rich Antioxidant Extracts of Vaccinium Vitis-idaea L. Leaves Inhibit the Formation of Toxic Maillard Reaction Products in Food Models.

Thermal treatment of proteinaceous foods generates heat-induced Maillard reaction substances including toxic advanced glycation end products (AGEs) and heterocyclic amines (HAs). It is known that plant phenolic compounds may influence Maillard reaction. This study investigated the impact of lingonberry leaf extracts on the formation of Nε -(carboxymethyl)lysine (CML) and Nε -(2-furoylmethyl)-L-lysine (furosine) in milk model system and HAs in meat-protein and meat model systems. In addition, lingonberry leaf extracts obtained by different solvents were characterized by radical scavenging, Folin-Ciocalteu assays and ultrahigh pressure liquid chromatography quadruple-time-of flight mass spectrometry (UPLC-qTOF-MS). Water extract (WE) stronger suppressed CML than furosine formation in milk model system: CML levels were reduced by nearly 40%. Moreover, quinic acid and catechin, which were abundant in WE, were effective in inhibiting CML and furosine formation. WE and acetone extract (AE) at 10 mg/mL significantly inhibited HAs formation in both model systems. However, higher suppressing effect on HAs formation showed AE, which had lower antioxidant capacity and total phenolic content values than WE. WE contained higher amounts of hydroxycinnamic acids, proanthocyanidins and flavonols, while AE was richer in flavan-3-ols and arbutin derivatives. It indicates that the composition of phenolics might be a major factor for explaining different effect of extracts from the same plant on HAs formation. In general, the results suggest that lingonberry leaves is a promising source of phytochemicals for inhibiting toxic Maillard reaction products and enriching foods with plant bioactive compounds. PRACTICAL APPLICATION: The increased consumption in processed foods has been linked with the increased risks of various diseases, while thermal food processing is required to develop flavor, insure safety, and extend shelf life. Therefore, developing effective technological means for inhibiting the formation of heat-induced toxic substances is an important task. This study showed a potential of lingonberry leaf extracts containing health beneficial phytochemicals to suppress the formation of toxic Maillard reaction products during heating of milk and meat.

Effect of osthole on advanced glycation end products-induced renal tubular hypertrophy and role of klotho in its mechanism of action.

BACKGROUND: Osthole has been widely reported to have pharmacological activities such as anti-cancer, anti-inflammation and anti-hyperlipidemic effects. Klotho was identified as an anti-senescence protein in a variety of tissues. Loss of klotho has been associated with chronic kidney disease. However, potential roles and molecular events for osthole and klotho in diabetic nephropathy remain unclear. PURPOSE: In the current study, we undertook to study the effect of osthole on klotho expression in advanced glycation end products (AGE)-cultured human renal proximal tubular cells, and to investigate the molecular mechanisms of osthole and exogenous klotho against AGE-induced renal tubular hypertrophy. METHODS: Cell viability was elucidated by MTT assay. Protein expression was measured by Western blotting. mRNA level was analyzed by real-time PCR. Cellular hypertrophy growth was evaluated by hypertrophy index. Relative cell size was detected by flow cytometry. RESULTS: We found that raising the ambient AGE concentration causes a dose-dependent decrease in klotho synthesis. Osthole significantly increased AGE-inhibited klotho mRNA and protein expression. Osthole and exogenous klotho treatments significantly attenuated AGE-induced Janus kinase 2 (JAK2)-signal transducers and activators of transcription 1 (STAT1) and STAT3 activation. Moreover, protein levels of suppressor of cytokine signaling 1 (SOCS1) and SOCS3 were augmented by osthole and exogenous klotho. The abilities of osthole and exogenous klotho to reverse AGE-induced cellular hypertrophy were verified by the observation that osthole and exogenous klotho inhibited p21Waf1/Cip1/collagen IV/RAGE expression, total protein content, and cell size. CONCLUSION: Consequently, we found that osthole attenuated AGE-induced renal tubular hypertrophy via induction of klotho expression and suppression of the JAK2-STAT1/STAT3 signaling. These results also showed that klotho might be used as a unique molecular target for the treatment of diabetic nephropathy.

Vitamin D Ameliorates Impaired Wound Healing in Streptozotocin-Induced Diabetic Mice by Suppressing Endoplasmic Reticulum Stress.

BACKGROUND: This study is designed to investigate whether vitamin D promotes diabetic wound healing and explore the potential mechanism which may be involved in the healing process. MATERIAL AND METHODS: Human umbilical vein endothelial cells (HUVECs) were treated with 200 µg/ml of advanced glycation end product-modified human serum albumin (AGE-HSA) and 250 mg/dl of glucose with vitamin D. Cell viability was analyzed using the CCK-8 assay, and the apoptosis rate was measured using flow cytometry. Endogenous markers of ER stress were quantified using Western blot and a real-time polymerase chain reaction. Diabetic mice were treated with vitamin D (100 ng/kg per day) for 14 days. The ulcer area and ulcerative histology were detected dynamically. RESULTS: Vitamin D administration not only decreased the apoptosis rate but also increased cell viability. Furthermore, the expression of endogenous markers of ER stress was downregulated as a result of vitamin D treatment. Vitamin D supplementation significantly accelerated wound healing of diabetic mice and improved the healing quality. Further studies showed that reduced ER stress was associated with the positive outcome. CONCLUSION: These results suggest that vitamin D may ameliorate impaired wound healing in diabetic mice by suppressing ER stress.

Matrine-Type Alkaloids Inhibit Advanced Glycation End Products Induced Reactive Oxygen Species-Mediated Apoptosis of Aortic Endothelial Cells In Vivo and In Vitro by Targeting MKK3 and p38MAPK Signaling.

BACKGROUND: The matrine-type alkaloids are bioactive components extracted from Sophora flavescens, which is used in treatment of diabetes mellitus in traditional Chinese medicine. Advanced glycation end products mediate diabetic vascular complications. This study was aimed to investigate the protective effects and molecular mechanisms of matrine-type alkaloids on advanced glycation end products-induced reactive oxygen species-mediated endothelial apoptosis. METHODS AND RESULTS: Rats aorta and cultured rat aortic endothelial cells were exposed to advanced glycation end products. Matrine-type alkaloids, p38 mitogen-activated protein kinase (MAPK) inhibitor, and small interference RNAs against p38 MAPK kinases MAPK kinase kinase (MKK)3 and MKK6 were administrated. Intracellular reactive oxygen species production, cell apoptosis, phosphorylation of MKKs/p38 MAPK, and expression levels of heme oxygenase/NADPH quinone oxidoreductase were assessed. The nuclear factor erythroid 2-related factor 2 nuclear translocation and the binding activity of nuclear factor erythroid 2-related factor 2 with antioxidant response element were also evaluated. Matrine-type alkaloids suppressed intracellular reactive oxygen species production and inhibited endothelial cell apoptosis in vivo and in vitro by recovering phosphorylation of MKK3/6 and p38 MAPK, nuclear factor erythroid 2-related factor 2 nuclear translocation, and antioxidant response element binding activity, as well as the expression levels of heme oxygenase/NADPH quinone oxidoreductase. p38 MAPK inhibitor treatment impaired the effects of matrine-type alkaloids in vivo and in vitro. MKK3/6 silencing impaired the effects of matrine-type alkaloids in vitro. CONCLUSIONS: Matrine-type alkaloids exert endothelial protective effects against advanced glycation end products induced reactive oxygen species-mediated apoptosis by targeting MKK3/6 and enhancing their phosphorylation.

The anti-inflammation effect of Moutan Cortex on advanced glycation end products-induced rat mesangial cells dysfunction and High-glucose-fat diet and streptozotocin-induced diabetic nephropathy rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Moutan Cortex (MC, family: Paeonia suffruticosa Andr.) is a well-known traditional herbal medicine that has been shown to hold a protective effect on inflammation in several diseases. However, its anti-inflammatory activity on diabetic nephropathy (DN) has been less reported. The present study was conducted to evaluate the potential attenuation activities of MC on inflammation in AGEs-induced rat mesangial cells dysfunction and high-glucose-fat diet and streptozotocin (STZ)-induced DN rats and explore the possible mechanism underlying its DN effect. MATERIALS AND METHODS: The inflammation in mesangial cells (HBZY-1) was induced by 200 µg/ml advanced glycation end products (AGEs). DN rats model was established by an administration high-glucose-fat diet and an intraperitoneal injection of STZ (30 mg/kg). Interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) level in cell supernatant and rats serum were detected by appropriate kits. A co-culture system of mesangial cells and macrophages was performed to evaluate the migration of macrophages. Immunohistochemical assay was applied to examine transforming growth factor beta1 (TGF-ß1), IL-6, MCP-1 and intercellular adhesion molecule-1 (ICAM-1) expression in kidney tissues of rats. Furthermore, western blot analysis was carried out to examine TGF-ß1, IL-6, MCP-1, ICAM-1 and RAGE protein expressions in mesangial cells. RESULTS: Pretreatment with MC could significantly inhibit AGEs-induced migration of macrophages in the co-culture system of mesangial cell and macrophage. MC could decrease IL-6 and MCP-1 levels in serum of DN rats in a dose-dependent manner. Furthermore, MC also improved the blood glucose, serum creatinine and urine protein levels. Both immunocytochemistry analysis and western blot analysis showed that MC decreased significantly the over-expression of IL-6, MCP-1, TGF-ß1, ICAM-1 and RAGE in mesangial cells or kidney tissues. Additionally, the protein expression of proinflammatory cytokine could also be down-regulated by the pretreatment of RAGE-Ab (5 µg/ml). CONCLUSION: These findings indicated that the extract of MC had an amelioration activity on the inflammation in AGEs-induced mesangial cells dysfunction and high-glucose-fat diet and STZ-induced DN rats. The protective effect might be associated with the intervention of MC via target of RAGE. These findings suggested that MC might be a benefit agent for the prevention and treatment of DN.

Effect of fresh apple extract on glycated protein/iron chelate-induced toxicity in human umbilical vein endothelial cells in vitro.

Consumption of fruits and vegetables has been associated with a low incidence of cardiovascular and other chronic diseases. The present study was aimed at evaluating the protective effects of fresh apple extract (AE) on human umbilical vein endothelial cells (HUVEC) exposed to cytotoxic glycated protein (GFBS)/iron (FeCl(3)) chelate. The experimental design comprised 10 groups with 5 flasks in each group. Group I was treated with 15% foetal bovine serum (FBS). Groups II, III and IV were treated with GFBS (70 microM), FBS + FeCl(3) (20 microM), and GFBS + FeCl(3), respectively. The other six groups were as follows: Group V, GFBS + AE (100 microg); Group VI, FBS + FeCl(3) + AE (100 microg); Group VII, GFBS + FeCl(3) + AE (100 microg); Group VIII, GFBS + AE (250 microg); Group IX, FBS + FeCl(3) + AE (250 microg); and Group X, GFBS + FeCl(3) + AE (250 microg). After 24 h incubation, cells were collected from all the experimental groups and assessed for lipid peroxidation (LPO) and activities of the antioxidant enzymes cytochrome c reductase and glutathione S-transferase (GST). HUVEC incubated with glycated protein (GFBS) either alone or combined with iron chelate showed a significant (p < 0.001) elevation of LPO accompanied by depletion of superoxide dismutase, catalase, glutathione peroxidase (GPx) and glutathione reductase (GR), in addition to increased microsomal cytochrome c reductase and decreased GST activities. Treatment of GFBS- or GFBS + FeCl(3)-exposed HUVEC with AE at 100 or 250 microg significantly decreased the level of LPO and returned the levels of antioxidants cytochrome c reductase and GST to near normal in a dose-dependent manner. The extracts recovered viability of HUVEC damaged by GFBS-iron treatment in a concentration-dependent manner. These findings suggest a protective effect of AE on HUVEC against glycated protein/iron chelate-induced toxicity, which suggests that AE could exert a beneficial effect by preventing diabetic angiopathies.

[Protective effects of extract of Ginkgo biloba on vascular endothelial dysfunction induced by AGEs-BSA in vivo].

OBJECTIVE: To explore the effect of extract of Ginkgo biloba (EGb) on vascular endothelial dysfunction induced by AGEs and to investigate the potential mechanisms. METHODS: Exogenous glycosylated bovine serum Albumin (AGEs-BSA) was prepared according to the methods of article. Vascular endothelial dysfunction was induced by tail vein injection of AGEs-BSA. The treatment group rats were given tail vein injections with AGEs-BSA followed by immediate intragastric of EGb (15,30 mg/kg/day, respectively) for 30 days. At the end of 30 days period, rats were anaesthetized with an intraperitoneal injection of sodium pentobarbital. Blood samples were collected from the carotid artery for biochemical assay of NO, MDA, SOD, DDAH, ADMA. The thoracic aorta was immediately isolated and cut into rings of 3 - 4 mm. Then ACh-induced EDR response and sodium SNP-induced endothelium-independent relaxation of aortic rings were examined. RESULTS: Results from in vivo experiments showed that the injection of AGEs-BSA significantly inhibited ACh-induced EDR response, but had no effect on SNP-induced endothelial-independent relaxation. The injection of AGEs-BSA decreased concentration of serum NO, activity of serum SOD and elevated serum MDA and ADMA level. Egb markedly attenuated AGEs-BSA induced inhibition of EDR response, increase of serum MDA and ADMA level, reduction of both NO level and activity of serum SOD.

Green tea polyphenols inhibit advanced glycation end product-induced rat vascular smooth muscle cell proliferation.

OBJECTIVE: To determine the effects of green tea polyphenols(GTP) on advanced glycation end products (AGEs)-induced proliferation and expression of p44/42 mitogen-activated protein kinase (MAPK) of rat vascular smooth muscle cells (VSMCs) METHODS: Rat aortic VSMCs isolated and cultured in vitro were stimulated with AGEs in the presence or absence of GTP at different concentrations, followed by quantitative analysis of the cell proliferation with colorimetric assay. The p44/42 MAPK activity was evaluated by immunoblotting technique using anti-p44/42 phospho-MAPK antibody. RESULTS: Compared with the control cells(without GTP treatment), GTP dose-dependently inhibited AGE-stimulated VSMC proliferation (P<0.05), and the p44/42 MAPK activity was significantly enhanced. The effects of AGEs were antagonized by GTP (372+/-41 vs 761+/-56, P<0.05). CONCLUSION: GTP can inhibit the AGE-induced proliferation and p44/42 MAPK expression of rat VSMCs.

Genotoxicity of advanced glycation end products in mammalian cells.

In patients with chronic renal failure, cancer incidence is enhanced. Since levels of advanced glycation end products (AGEs) are markedly elevated in renal insufficiency, we investigated potential effects of various AGEs on structural DNA integrity in tubule cells. The comet-assay was employed, a method based on the computer-aided microscopic analysis of single cells after electrophoretic separation of their nuclear DNA. Incubation of pig kidney LLC-PK1-cells for 24 h with AGE-BSA (AGE-bovine serum albumin), carboxymethyllysine-BSA as well as methylglyoxal-BSA resulted in a significant increase in DNA damage. Pretreatment of the cells with the proteases trypsin and bromelain abolished the AGE-induced comet-formation. This is in agreement with the idea that the observed genotoxicity of AGEs could be receptor-mediated and that proteases inactivate the extracellular domain of the receptor for AGEs. Binding of AGEs to the RAGE receptor leads to an increased intracellular formation of active oxygen species, which are known to induce DNA damage. It is concluded that AGEs induce genotoxicity in tubule cells, which may be involved in the enhanced cancer development in advanced kidney diseases.

Phytoestrogens attenuate oxidative DNA damage in vascular smooth muscle cells from stroke-prone spontaneously hypertensive rats.

OBJECTIVES: A recent study demonstrated that reactive oxygen species (ROS) were involved in the maintenance of hypertension in stroke-prone spontaneously hypertensive rats (SHRSP). However, the role of oxidative stress in hypertension and its related diseases in SHRSP remains unknown. To determine whether phytoestrogens attenuate oxidative DNA damage in vascular smooth muscle cells (VSMC) from SHRSP and Wistar-Kyoto (WKY) rats, we investigated the effect of daidzein, genistein and resveratrol on oxidative DNA damage in VSMC, induced by advanced glycation end-products (AGEs). METHODS: VSMC were treated with AGEs in the presence or absence of phytoestrogens for the indicated time. Cellular degeneration induced by AGEs was characterized in terms of intracellular oxidant levels, intracellular total glutathione (GSH) levels, mRNA expression for gamma-glutamylcysteine synthetase (GCS), and a new marker of oxidative stress, 8-hydroxy-2'-deoxyguanosine (8-OHdG) contents. RESULTS: AGEs stimulated 8-OHdG formation in VSMC in a time- and dose-dependent manner. We also confirmed that VSMC from SHRSP were more vulnerable to oxidative stress induced by AGEs, than VSMC from WKY rats. Daidzein, genistein or resveratrol reduced AGEs-induced 8-OHdG formation in a dose-dependent manner. The preventive effects of phytoestrogens on 8-OHdG formation remarkably paralleled changes in the intracellular oxidant levels in VSMC following AGEs treatment. We further demonstrated that phytoestrogens increase intracellular total GSH level in VSMC. Increased GSH synthesis was due to enhanced expression of the rate-limiting enzyme for GSH synthesis, GCS. Phytoestrogens-stimulated total GSH level in VSMC could lead to decreased intracellular oxidant levels, and thus prevent oxidative DNA damage, induced by AGEs. The phytoestrogens are powerful antioxidants able to interfere with AGEs-mediated oxidative DNA damage of VSMC, and are potentially useful against vascular diseases where ROS are involved in hypertension.