Effect of advanced glycation end products on inflammation in cultured cardiomyocytes

AIM: To investigate the effect of advanced glycation end products on inflammation in cultured cardiomyocytes.METHODS: Primary cardiomyocytes were isolated from Sprague-Dawley neonatal (1 to 2 days old) rats ventricles.The insulin resistant cardiomyocyte model was established.Neonatal rat ventricular myocytes were exposed to AGEs for 24 hours.TNF-α mRNA and PPAR-γ mRNA expressions were determined by RT-PCR.Activation of NF-κB in the cells was examined by using immunocytochemistry.The ultrastructure of the cells was detected by transmission electron microscope.RESULTS: The exprssion of TNF-α mRNA and the activation of NF-κB increased,the expression of PPAR-γ mRNA decreased compared with control group (P<0.05).The differences among different AGE-BSA groups were significant (P<0.05).The numbers of chondriosome and smooth endoplasmic reticulum increased.CONCLUSION: AGEs significantly increase TNF-α mRNA expression and NF-κB activation,and restrain the expression of PPAR-γ mRNA.These data suggest that AGEs play an important role in the onset of diabetic cardiomyopathy.     

Activation of smad signaling and collagenⅠsynthesis in NRK52E cells induced by advanced glycation end products

AIM:To investigate the effects of advanced glycation end products on activation of Smad signaling pathway and collagenⅠ synthesis in proximal tubular epithelial cells. METHODS:Advanced glycation end products (AGE-BSA) were prepared by incubation of bovine serum albumin (BSA) with D-glucose. Normal rat proximal tubular epithelial (NRK52E) cells were cultured in RPMI-1640 medium with AGE-BSA. Phosphorylation and nuclear translocation of Smad2/3 were examined by immunocytochemistry. Levels of TGF-β1 in supernatant of cell culture were measured by enzyme-linked immunosorbent assay (ELISA). Expression of TGF-β1, Smad2, Smad3 and Smad7 mRNA were detected by RT-PCR. Expression of α-SMA , E-cadherin and collagenⅠproteins were detected by Western blotting.RESULTS:AGE-BSA induced Smad2/3 phosphorylation and nuclear translocation, two peaks occured at 30 min (68% vs 16%, P<0.05) and 24 h (76% vs 16%, P<0.05) compared to 0 min. The level of TGF-β1 markedly increased in supernatant of cell culture by induced AGE-BSA at 24 h and 48 h. The expression of TGF-β1 mRNA markedly increased at 24 h, and associated with high expression of Smad2, Smad3 and Smad7 mRNA at 48 h. AGE-BSA up-regulated significantly the expression of α-SMA and collagenⅠproteins, down-regulated the expression of E-cadherin protein. CONCLUSION:AGEs induces activation of Smad signaling, as well as transdifferentiation and collagenⅠ synthesis in proximal tubular epithelial cells.     

Advanced glycation end products change spontaneous calcium spark morphology in cardiomyocytes

AIM:To investigate the effect of advanced glycation end products (AGEs) on the activity of rynodine receptor (RyR) and calcium regulation in cardiomyocytes, in order to clarify the underlying mechanisms of cardiac dysfunction in diabetic cardiomyopathy. METHODS:The frequency and morphology of spontaneous calcium sparks were observed in cultured neonatal rat ventricular myocytes by the imaging technique of laser scanning confocal microscope. RESULTS:Compared with control group, AGEs increased the frequency of calcium sparks, where 10 mg/L of AGEs increased by 48% (P<0.01), 50 mg/L of AGEs increased by 200% (P<0.01) and 150 mg/L of AGEs increased by 110% (P<0.01). AGEs alter the morphology of spontaneous calcium sparks. AGEs decreased the amplitude and width of calcium sparks, and calcium spark duration. AGEs increased the level of cardiomyocyte resting Ca2+. CONCLUSION: AGEs increases the frequency of spontaneous calcium sparks, thereby enhancing calcium spark-mediated leaking of sarcoplasmic reticulum Ca2+, which may be the cause of diabetic cardiomyopathy systolic dysfunction.     

Advanced glycation end products induce rat chondrocyte injury by modulating oxidative stress

ATM: To explore the possibility that advanced glycation end products (AGEs) induces rat chondrocyte injury by modulating oxidative stress. METHODS: Primarily cultured rat chondrocytes were identified. The viability of the chondrocytes was measured by CCK-8 assay. The intracellular levels of reactive oxygen species (ROS) were detected by DCFH-DA staining. The number of apoptotic cells was determined by Hoechst 33342 nuclear staining and flow cytometry. RT-PCR was performed to measure the mRNA levels of Bax, Bcl-2, caspase-3, MMP3, MMP13 and COL2 in the chondrocytes. Western blotting was used to evaluate the protein levels of cleaved caspase-3, MMP3, MMP13 and COL2. RESULTS: Compared with control group, the intracellular levels of ROS in the chondrocytes treated with AGEs were significantly increased (P<0.05), and pretreatment with N-acetyl-L-cysteine (NAC) suppressed the formation of ROS (P<0.05). Besides, NAC inhibited AGEs-induced apoptosis of the chondrocytes, as indicated by reduceing the levels of Bax/Bcl-2 and caspase-3, decreased the expression of MMP3 and MMP13, and reduced the loss of COL2.CONCLUSION: AGEs induce chondrocyte injury by activating oxidative stress.     

Effect of advanced glycosylation end products on the expression of receptor for advanced glycosylation end products in human monocyte-derived dendritic cells

AIM: To investigate the effect of advanced glycosylation end products on the expression of receptor for advanced glycosylation end products in human monocyte-derived dendritic cells. METHODS: Monocytes were purified (over 98%) using anti-CD14＋ microbeads. After 8 d culture in RPMI-1640 medium containing rhGM-CSF (100 μg/L) and rhIL-4 (50 μg/L), immature MDCs were derived, then exposed to AGE-BSA (0 or 200 mg/L) for 24 h. Expression of RAGE was semi-quantified by RT-PCR and Western blotting. At the same time, supernatants were collected. IFN-γ and IL-12 were analyzed by ELISA. RESULTS: mRNA and protein of RAGE incubated by 200 mg/L AGE-BSA was higher than that in control at 24 h. Treatment of DCs with AGE-BSA resulted in about two-fold increase in the expression of RAGE (P<0.05). The concentrations of IFN-γ and IL-12 were both significantly higher than that in control (P<0.05). CONCLUSION: AGEs up-regulates the expression of RAGE and induces the secretion of IFN-γ and IL-12 by DCs. These findings may provide insight into the effect of DCs on the processes of atherosclerosis.     

Inhibitory effect of catalpol on inflammation and expression of RAGE in EA.hy926 cells induced by advanced glycation end products

AIM: To investigate the inhibitory effect of catalpol on inflammation in EA.hy926 cells induced by advanced glycation end products(AGEs) and to explore its antioxidant mechanisms.METHODS: Human endothelial cell line EA.hy926 was cultured and randomly divided into control group, catalpol(0.5 mmol/L) group, AGEs group, high-dose(0.5 mmol/L) catalpol+AGEs group, middle-dose(0.25 mmol/L) catalpol+AGEs group and low-dose(0.05 mmol/L) catalpol+AGEs group. Intracellular reative oxygen species(ROS) production was detected by laser scanning confocal microscopy. The levels of monocyte chemotactic protein-1(MCP-1), tumor necrosis factor-α(TNF-α) and vascular cell adhesion molecule-1(VCAM-1) in culture supernatant were detected by commercial ELISA kits. The expression of MCP-1, TNF-α, VCAM-1 and receptor for advanced glycation end products(RAGE) in the EA.hy926 cells were detected by Western blot.RESULTS: In high-dose catalpol+AGEs and middle-dose catalpol+AGEs groups, the generation of ROS was decreased significantly. The levels of MCP-1, TNF-α and VCAM-1, and protein expression of MCP-1, TNF-α and VCAM-1 were significantly lower. The expression of RAGE protein in EA.hy926 cells were significantly inhibited(P<0.05).CONCLUSION: Catalpol effectively inhibits the AGEs-induced oxidative stress and inflammation in EA.hy926 cells, which may be associated with a decrease in the expression of RAGE.     

Progress in advanced glycosylation end products and diabetic vascular complications

The formation of advanced glycosylation end products (AGEs) is enhanced in diabetes mellitus, closely associated with diabetic vascular complication.In this review, biochemical properties and structures of AGEs, AGEs receptors and binding proteins, pathogenic properties of AGEs, deposition and turnover of AGEs, inhibitors of AGEs were summarized.     

Effects of advanced glycation end products on protein and mRNA expression of macrophage inflammatory protein-1α in cultured human umbilical vein endothelial cells

AIM: To investigate the effects of advanced glycation end products (AGEs) on protein and mRNA expression of macrophage inflammatory protein-1α (MIP-1α) in cultured human umbilical vein endothelial cells(HUVECs). METHODS: HUVECs were cultured with AGEs at different concentrations for 24 h and at a concentration of 400 mg/L for different time.The levels of mRNA and protein expression of MIP-1α in cultured HUVEC were detected by in situ hybridization and Western blot, respectively. RESULTS: In situ hybridization showed that after exposure of HUVECs to AGEs at different concentrations (100 mg/L, 200 mg/L, 400 mg/L) for 24 h, the average integrated optical density values (18.76±3.17, 26.58±1.61, 34.23±2.25) of MIP-1α mRNA expression in HUVECs were higher than that in control group (13.83±1.24, P0.05). After exposure of HUVECs to AGEs at a concentration of 400 mg/L for 12 h, 24 h and 36 h, the average integrated optical density values of MIP-1α mRNA expression in HUVECs were 22.67±1.46, 34.23±2.25 and 42.28±3.14, higher than that in 0 h group (12.56±1.24, P0.05). Western Blot showed that exposure of HUVECs to AGEs at different concentrations(100 mg/L, 200 mg/L, 400 mg/L) for 24 h resulted in a 1.34-fold, 1.87-fold and 2.46-fold increase in the expression of MIP-1α protein in HUVECs compared with BSA control group (P<0.05). Meanwhile, exposure of HUVECs to AGEs at a concentration of 400 mg/L for 12 h, 24 h and 36 h resulted in a 1.82-fold, 2.71-fold and 3.34-fold increase in MIP-1α protein expression in HUVECs compared with 0 h group (P<0.05). CONCLUSION:These data suggest that AGEs could induce a high expression of MIP-1αmRNA and protein in cultured HUVECs in a dose-dependent and time-dependent manner.     

Effect of probucol and simvastatin on reactive oxygen species production and HO-1 expression induced by advanced glycation end products in rat renal microvascular endothelial cells

AIM: To investigate the effect of probucol and simvastatin on the production of reactive oxygen species (ROS) and heme oxygenase-1 (HO-1) expression induced by advanced glycation end products (AGEs) in rat renal microvascular endothelial cells (RMECs). METHODS: RMECs isolated and cultured from rat kidney were divided into 4 groups: normal control group, AGEs group, probucol group and simvastatin group. The levels of ROS were determined by the molecular probes of DCFH-DA. The expression of HO-1 at mRNA and protein levels was detected by RT-PCR and Western blotting, respectively. RESULTS: (1) AGEs up-regulated ROS production and HO-1 expression in RMECs. (2) Probucol up-regulated HO-1 expression in RMECs, and inhibited the increasing level of ROS and expression of HO-1 in RMECs induced by AGEs. (3) Simvastatin also inhibited the increasing level of ROS in RMECs induced by AGEs, but it had no effect on HO-1 expression in RMECs with or without AGEs.CONCLUSION: Protective effect of probucol on the dysfunction of RMECs induced by AGEs may be related with its effect on the expression of HO-1 at mRNA and protein levels. Simvastatin also plays roles in antioxidation and renal protection, but is ineffective in the modulation of HO-1 expression.     

Autophagy plays a protective role in advanced glycation end product-induced apoptosis of vascular endothelial cells

AIM: To investigate the effect of advanced glycation end products (AGEs) on autophagy in human umbilical endothelial cells (HUVECs) and to identify the role of autophagy in advanced glycation end product-induced cell apoptosis. METHODS: HUVECs were cultured and treated with AGEs or bovine serum albumin. The protein expression was detected by Western blotting. Autophagosomes were observed under electron microscope. The cell apoptotic rate was determined by flow cytometry. The cell viability was quantified by MTT assay. RESULTS: After treated with AGEs, the level of autophagy-associated protein LC3-Ⅱ in HUVECs was up-regulated, and the number of autophagosomes was increased. Compared with control group, the apoptotic rate of HUVECs increased and the viability of HUVECs was decreased in AGEs treatment group. Furthermore, pretreating the cells with an autophagy inhibitor 3-methyladenine aggravated these effects. The levels of phospho-protein kinase B(Akt) and phospho-mammalian target of rapamycin(mTOR) in HUVECs were also decreased by treatment with AGEs. Pretreatment with Akt activator insulin-like growth factor 1 (IGF-1) increased Akt phosphorylation and suppressed the AGE-induced LC3-Ⅱ expression. CONCLUSION: AGEs induce autophagy in HUVECs through PI3K/Akt/mTOR signal pathway. Autophagy plays a protective role in AGE-induced apoptosis in HUVECs.     

Advanced glycation end products induce apoptosis of human ovarian granulosa COV434 cells and up-regulate pro-inflammatory cytokine HMGB1 autocrine

AIM:To study whether advanced glycation end products (AGEs) induce the apoptosis of human ovarian granulosa COV434 cells, and to explore the possible mechanism. METHODS:Human ovarian granulosa COV434 cells were treated with AGEs at different concentrations. Flow cytometry was used to observe the apoptotic rate. The protein levels of caspase-3 and cleaved caspase-3 were determined by Western blot. The release of high mobility group box 1 protein (HMGB1) in the culture supernatant was measured by ELISA. RESULTS:Compared with control group, early apoptotic rate and late apoptotic rate in 100 mg/L AGEs group and 200 mg/L AGEs group were significantly increased (P<0.05). No obvious difference of caspase-3 protein level in each group was observed, while the protein levels of cleaved caspase-3 in 100 mg/L AGEs group and 200 mg/L AGEs group were significantly increased compared with control group (P<0.05). In addition, compared with control group, pro-inflammatory factor HMGB1 in the culture medium in 100 mg/L AGEs group and 200 mg/L AGEs group was significantly increased. CONCLUSION:The apoptosis of human ovarian granulosa COV434 cells induced by AGEs may be related to pro-inflammatory reaction.     

Effect of advanced glycosylation end products on function of human adipose tissue-derived stem cells in vitro

AIM：To explore the effect of advanced glycosylation end products (AGEs) on the function of human adipose-derived stem cells (hADSCs) in promoting wound healing. METHODS：hADSCs were isolated by conventional method in vitro and divided into control bovine serum albumin (BSA) group, low-dose AGE-BSA group and high-dose AGE-BSA group. The proliferation and migration of hADSCs with different treatments were determined by WST-8 assay and Transwell assay, respectively. In addition, the expression of vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and insulin-like growth factor-1(IGF-1) at mRNA and protein levels was determined by real-time PCR and ELISA analysis. RESULTS：Compared with control group, the proliferation and migration abilities were significantly inhibited in the hADSCs of AGE-BSA group. The mRNA expression of VEGF, HGF and IGF-1 in AGE-BSA group was obviously lower than that in control group. The contents of VEGF, HGF and IGF-1 in hADSCs-conditioned me-dium in AGE-BSA group were also obviously lower than those in control group. CONCLUSION：AGEs alter the intrinsic properties of hADSCs and impair their functions in promoting wound healing, thus affecting the therapeutic potential of hADSCs in the treatment of diabetic ulcers.     

The mechanism of intercellular adhesion molecule-1 expression in endothelial cells stimulated by advanced glycosylation end products

AIM: To explore the relationship between intercellular adhesion molecule-1(ICAM-1)expression in endothelial cells(EC) and advanced glycosylation end products(AGEs) stimulation. METHODS: Murine bone marrow derived ECs was stimulated by AGEs after pretreated with anti-AGEs, anti-IL-1β and N-acetylcysteine(NAC),then SOD activity and ICAM-1 concentration and adhesion rate(AR) were evaluated. RESULTS: ECs which expressed ICAM-1 induced by AGEs showed lower SOD activity . The ICAM-1 expression as well as the increase of AR caused by AGEs stimulation could be suppressed by anti-AGEs(0.12±0.01) and NAC(0.11±0.05). Anti-IL-1β had no influence on these changes. CONCLUSION: AGEs could induce endothelial cells to express ICAM-1 in vitro, most probably due to the formation of free radicals. Besides, AGEs may stimulate other cells to secrete cytokines resulting in ICAM-1 expression in endothelial cells.     

Effect of advanced glycosylation end products on level of nitric oxide and activity of nitric oxide synthase in cultured macrophages

AIM:To study the mechanism and effect of advanced glycosylation end products (AGEs) on NO pathway in cultured macrophages.METHODS:The level of NO and NOS activity were measured by NO and NOS kits in cultured macrophages. RESULTS:The results showed that AGEs induced decreases in NO level and NOS activity in a time and dose-related manner in interleukin-1 (IL-1)-stimulated macrophages. VitE can significantly inhibited effects of AGEs on IL-1-stimulated macrophages. CONCLUSION:AGEs can decrease NO production via inhibiting NOS activity in IL-1-stimulated macrophages. VitE can protect the cells from AGEs injury. It is an important theoretical basis for preventing chronic complication in diabetes mellitus.     

Preconditioning with 3-nitropropionic acid induces rat cerebral ischemic tolerance and increases expression of glucose transporter 1 and glucose transporter 3

AIM: To explore the mechanism of 3-nitropropionic acid (3-NPA) preconditioning that induces cerebral ischemic tolerance in rats by affecting the expression of brain-type glucose transporters (GLUT1 and GLUT3) at mRNA and protein levels in cerebral tissues.METHODS: The male SD rats were used in the experiments and divided randomly into sham operation group (sham group, n=4), control group of 3-NPA preconditioning (3-NPA group, n=4), cerebral ischemia group (M group, n=16) and 3-NPA preconditioning group (IPC group, n=16). M group and IPC group were further divided into 4 subgroups according to the different reperfusion time(4 h, 12 h, 24 h and 48 h). All rats were killed at the corresponding time points. The cerebral tissues in the ischemic side (left) and coronal intermediate 1/3 of cortex were collected. The protein levels and mRNA expression of GLUT1 and GLUT3 were determined by Western blotting and RT-PCR. RESULTS: Compared with M group, the ischemic reperfusion and 3-NPA preconditioning induced the upregulation of GLUT1 and GLUT3 at protein levels with significant differences (F=5.848, P<0.05 and F=6.295, P<0.05, respectively), especially after ischemia-reperfusion for 48 h. The mRNA expression of GLUT1 in IPC group began to increase at 4 h, peaked at 48 h after reperfusion, with significant difference as compared to M group at the corresponding reperfusion time points in each group or sham group. In contrast, the mRNA expression of GLUT3 in IPC group increased at 24 h, and was the highest at 48 h as compared to cerebral ischemia group at the corresponding reperfusion time points or sham group.CONCLUSION: 3-NPA preconditioning increases the expression of GLUT1 and GLUT3 at protein and mRNA levels to maintain the energy supply in brain tissues, indicating a cerebral protective mechanism.