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.     

Early treatment with aminoguanidine on level of plasma and renal AngⅡ in diabetic rats

AIM: To investigate the effect of aminoguanidine (AG) on plasma and renal levels of angiogenesis Ⅱ (AngⅡ), and to identify the relationship of AGEs with AngⅡ in STZ-induced diabetic rats. METHODS: Wistar rats were randomly assigned to three groups. Diabetes was induced, rats were then received AG in treatment group. At the end of 12th week, urine albumin excretion rate (UAER) and calculate creatinine clearance (Ccr) were detected. Periodic acid-Schiff reagent was used to evaluate renal pathology. Plasma and renal AngⅡ were analyzed by radioimmunoassay and immunohistochemistry, respectively. RESULTS: AG treatment significantly prevented the increase in UAER (P<0.01), renal pathology (P<0.01), and level of renal AngⅡ (P<0.01). However, plasma concentration of AngⅡ was higher than that in diabetic rats without AG treatment (P<0.01). CONCLUSION: AG down-regulates renal Ang Ⅱ level, probably by reducing the formation of AGEs, which may be one of the renoprotective factors in diabetic nephropathy. More proofs are needed to identify the result that plasma AngⅡ concentration increases in DMA group.     

Influences of Vit-E on the mtDNA damage and Ca2+ homostasis of hippocampus and antioxidative ability in brain of aging mice induced by D-galactose

AIM: To study the influences of vitamin E (Vit-E) on the mtDNA damage and Ca²⁺ homeostasis in hippocampus and antioxidative ability in aging brain induced by D-galactose.METHODS: D-galactose (1 000 mg·k^-1·d^-1 ) was injected into mice hypodermically for 8 weeks to induce aging animal model, and Vit-E (100 mg·kg^-1; 250 mg·kg^-1) was administered for 6 weeks by ig at the 3rd week of making model. After Vit-E treatment for 8 weeks, water maze test was used to determine the ability of mice’s learning and memory. The activities of glutathione peroxidase (GSH-Px) and succinate dehydrogenase (SDH), the content of nitric oxide (NO) and activity of nitric oxide synthase (NOS) in the brain tissue were detected separately. Fura-2/AM, double-wave-length fluorospectrophotometer and PCR method were used to measure the concentration of calcium ion and mtDNA mutation in the hippocampus cells.RESULTS: Administration of Vit-E improved significantly the ability of learning and memory in model mice, inhibited the activity of NOS and decreased the amount of NO, and increased the activities of GSH-Px and SDH respectively in brain tissues, decreased the concentration of calcium ion (P<0.01, P<0.05), and prevented the damage of mtDNA in hippocampus.CONCLUSION: Vit-E can enhance the antioxidative ability, regulate the homeostasis of Ca²⁺ and inhibit the damage of mtDNA caused by oxidative stress in aging brain, and improve the ability of learning and memory in aging mice.     

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.     

Effect of acarbose on the aortic collagen nonenzymatic glycosylation in diabetic rats

AIM: To observe the effect of α-glucosindase inhibitor, acarbose on the aortic collagen nonenzymatic glycosylation in streptozotocin-induced diabetic rats . METHODS: The treated group (DM+A) was given acarbose (1 mg·kg^-1). The aortic collagen and its AGEs concentration were measured at the scheduled periods (1, 3 and 6 months ). RESULTS: During the observed period , the aortic collagen and its AGEs concentration were higher than that of control group in a time-dependent manner (P＜0.01 ), but decreased in acarbose-treated rats as compared with untreated group (P＜0.01 ). CONCLUTION:Acarbose can prevent the aortic vascular collagen nonenzymatic glycosylation in diabetic rats.     

Oxidative stress induces apoptosis in HepG2 cells

AIM: Direct exposure of cells to reactive oxygen species can induce apoptosis. In this study we investigate how oxidative stress induces cell death in HepG2 cells and characterize the molecular events involved.METHODS: Oxidative stress was created by exposing HepG2 cells to 2 mmol/L H2O2. Apoptosis was determined by analysis of DNA fragmentation by agarose gel electorphoresis. The mitochondrial membrane potential was analyzed using DePsipher fluorescent staining and the expression of cytochrome c in the cytosolic fraction was measured by Western blotting analysis. The caspase activity was detected using fluorometric assay kit by a fluorescence microplate reader.RESULTS: When HepG2 cells were treated with 2 mmol/L H2O2, the cells displayed DNA fragmentation, a typical feature of apoptosis, after 12 h. The mitochondrial membrane potential appeared different in two group of cells. H2O2-treated cells appeared green fluorescence as early as 4 h, which represents de-energized mitochondria, the untreated cells appeared red fluorescence, a feature of mitochondria with intact membrane potential. In treated cells, the expression of cytochrome c increased and accumulated in cytosolic fraction with treatment time, caspase-3 activity increased by 6.7-fold (P<0.01) at 8 h and caspase-9 activity increased by 3.6-fold (P<0.01) at 12 h, respectively, however, the activity of caspase-8 remained unchanged.CONCLUSION: These findings suggest that oxidative stress can induce apoptotic cell death in HepG2 cells, and the mechanism is related to mitochondrial pathway, which activates caspase-9 and-3, but not caspase-8.     

Expression of SR-A II and CD36 in white blood cells correlate with plasma AGEs levels and diabetic complications

AIM: To investigate the correlation of the expression of scavenger receptors SR-A II and CD36 in white blood cells (WBCs) with the plasma levels of advanced glycosylation end products (AGEs) and different diabetic complications.METHODS: Blood samples were collected from 78 patients with diabetic complications. The levels of plasma AGEs were determined by using spectrofluorimetry. RNA in WBCs was extracted with Trizol reagent and the mRNA levels of SR-A II and CD36 were determined by RT-PCR. RESULTS: In the patients tested, the mRNA level of SR-A Ⅱ was found to be the highest in those with diabetic nephropathy, and lowest in those with fatty liver. The expression of CD36 was found to be the highest in diabetic patients with fatty liver and lowest in those with coronary heart disease. The expression of both receptors in WBCs showed significantly higher levels in diabetic patients with hypertension, and lower in those with cataract. The plasma levels of AGEs negatively correlated with mRNA levels of CD36 (r=-0.89,P<0.01), while positively correlated with SR-A II mRNA levels (r=-0.82, P< 0.05).CONCLUSION: The increased plasma levels of AGEs may stimulate the expression of SR-A II in WBCs, and higher expression of SR-A Ⅱ and CD36 was significantly related to diabetic complications, nephropathy and fatty liver, respectively. However, low expression of CD36 in some diabetic patients with complications might be important causes for their high plasma AGEs levels.     

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.     

Effects of sodium ferulate on the nonenzymatic glycation and oxidation in kidneys of diabetic rats

AIM: To investigate the blocking effects of sodium ferulate (SF) on the nonenzymatic glycation and oxidation in kidneys of diabetic rats. METHODS: The diabetic rats induced by streptozotocin (STZ) were treated with SF (110 mg/kg) per day for 8 weeks. The renal weight/body weight, clearance rate of creatinine (Ccr), proteinuria, advanced glycation end products (AGEs) in renal cortex, and the malonyldialdehyde (MDA), fructosamine (FMN), antioxidant enzymes in serum and renal cortex were measured. The pathologic changes of kidneys were also observed. RESULTS: The levels of Ccr, proteinuria, FMN and AGEs in renal cortex, FMN in serum, and renal weight/body weight in diabetic control group were significantly higher than those in normal control group. In the diabetic control group, there was a highter level of MDA and a lower activity of superoxide dismutase (SOD) and catalase (CAT) in serum and renal cortex than those in normal control group. Except for FMN in renal cortex, the abnormalities were significantly ameliorated in the treatment group. The pathologic changes was significant in the diabetic control group, which was significantly ameliorated with the treatment of SF. CONCLUSIONS: SF protects the activity of antioxidant enzymes, clears away oxygen radicals and inhibits the deposit of AGEs in kindey, which may be the mechanisms of protecting effects of SF on kindey in diabetic rats.     

Effects of glycated serum albumin on expression of MCP-1 in cultured human umbilical vein endothelial cells

AIM: To investigate the effects of glycated serum albumin (GSA) on the expression of monocyte chemoattratant protein-1 (MCP-1) in endothelial cells.METHODS: Human umbilical vein endothelial cells (HUVECs) were cultured with GSA at different concentrations in the presence or absence of glycosylation-product inhibitor aminoguanidine (AG) and anti-oxidant N-acetylcysteine (NAC). The expression of MCP-1 was evaluated by the methods of immunocytochemistry and sandwich ELISA.Malondialdehyde(MDA) content and superoxide dismutase(SOD) activity were determined by the technique of thiobarbituric acid(TBA) and xanthine oxidase(XOD),respectively. RESULTS: GSA stimulated HUVECs to produce and release MCP-1. After HUVECs were treated with 50 mg/L GSA, the expression of MCP-1 at 4 h, 8 h and 12 h was 1.3, 1.9 and 2.8 folds higher than that in control group (P<0.01), respectiuely.The significant difference among the experiment groups (P<0.01) was observed, indicating that GSA took effect in a concentration-dependent manner. The release of MCP-1 in cultured supernatants in the experiment groups with 3 different concentrations of GSA was 1.6, 2.4 and 3.0 folds as much as that in control group (P<0.01), and the significant difference among the experiment groups (P<0.01) was also observed. GSA decreased the activity of SOD (P<0.05) and increased the content of MDA (P<0.01). AG and NAC obviously inhibited the upregulation of MCP-1 expression in HUVECs by GSA (P<0.01). NAC also inhibited the effect of GSA on SOD activity and MDA content in HUVECs (P<0.05). CONCLUSION: GSA stimulates the expression of MCP-1 by inducing oxidative stress in endothelial cells.     

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.     

Effect of adipokine chemerin on mitochondrial dysfunction in cardiac H9C2 cells with hypertrophy

AIM To study the regulation of adipokine chemerin on mitochondrial function of rat cardiac H9C2 cells with hypertrophy, and to explore its effect on mitochondrial dysfunction in the H9C2 cells. METHODS In vitro cell experiments were performed, and the H9C2 cells were divided into normal group, chemerin group, angiotensin Ⅱ (Ang Ⅱ) model group and Ang Ⅱ+chemerin group. Immunohistochemistry and qPCR were used to identify whether the model was successfully constructed. The morphological changes of mitochondria in the H9C2 cells were observed under electron microscope. The mitochondrial membrane potential and membrane permeability were analyzed by flow cytometry. The activity of cytochrome C oxidase (COX) and succinate dehydrogenase (SDH) was measured by enzyme activity kit. RESULTS Compared with normal group, the mitochondrial structure in Ang Ⅱ group and chemerin group was seriously damaged, the permeability of mitochondrial membrane was significantly increased (P<0.01), and the mitochondrial membrane potential and the activity of COX and SDH were significantly reduced (P<0.01). Meanwhile, the mitochondrial damage in the H9C2 cells was more serious in Ang Ⅱ+chemirin group (P<0.05). CONCLUSION Chemerin stimulation not only induces cardiomyocyte hypertrophy, but also promote the pathological process of mitochondrial dysfunction in the myocardial cells with hypertrophy.     

Effect of mitochondrial aconitase activity on energy synthesis during aging

AIM：To investigate the effect of mitochondrial aconitase (ACO2) on energy synthesis during aging in male SD rats and D-galactose-induced cell aging model. METHODS：D-galactose at concentration of 55 mmol/L was used to establish MRC-5 cell aging model. Intact mitochondria in the rat brain and MRC-5 cells were isolated by a sucrose density gradient centrifugation. The formation of NADPH was used to represent the ACO2 activity and determined by observation of the absorbance at 340 nm. Fluorescence quantitative PCR and Western blotting were applied to detected ACO2 expression. Superoxide dismutase (SOD) activity and malondialdehyde (MDA) content were measured by the commercial assay kits. The tissue iron content was detected by ferrozine method. Mitochondrial membrane potential was detected by JC-1 mitochondrial membrane potential detection kit. The content of ATP, ADP and AMP was measured by HPLC analysis, and the energy charge was then calculated by the formula. RESULTS：ACO2 activity and iron content presented age-related decline and increase, respectively, while the expression level of ACO2 kept stable. ACO2 activity significantly declined when the cells were treated with hydrogen peroxide at different concentrations. In the aging cells, SOD activity and ACO2 activity were decreased and MDA content was increased significantly, while the expression level of ACO2 was unchanged. During aging, mitochondrial membrane potential, ATP synthesis and energy charge presented significant reduction. CONCLUSION：The activity of ACO2, which is sensitive to oxidative stress, declines during aging, and may affect the efficiency of the Krebs cycle. At the same time, mitochondrial membrane potential decreases, thus reducing the energy synthesis in mitochondria.     

Knock-down of TLR4 expression reduces secretion of inflammatory factors in LPS-induced pancreatic acinar AR42J cells

AIM:To study the effect of Toll-like receptor 4 (TLR4) on the secretion of inflammatory factors in the pancreatic acinar AR42J cells induced by lipopolysaccharides (LPS). METHODS:The rat pancreatic acinar AR42J cells were treated with LPS. The expression of TLR4 at mRNA and protein levels was determined by real-time PCR and Western blot. The lentivirus carrying TLR4 small interfering RNA (siRNA) was used to infect the AR42J cells. Under LPS stimulation, the interference efficacy was measured by real-time PCR and Western blot. The cell viability was measured by MTT assay, and the leakage rate of lactate dehydrogenase (LDH) was examined by dinitrophenylhydrazine method. The releases of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the cell culture medium were detected by ELISA, and the malondialdehyed (MDA) content in supernatant was measured by thiobarbituric acid method. The activity of superoxide dismutase (SOD) in the supernatant was determined by xanthine oxidation, and the activity of glutathione peroxidase (GSH-Px) and catalase (CAT) was detected by colorimetry. RESULTS:The expression of TLR4 at mRNA and protein levels in LPS-treated AR42J cells was significantly increased (P<0.05). Infection with TLR4 siRNA-carrying lentivirus significantly inhibited the expression of TLR4 at mRNA and protein levels in the AR42J cells under LPS stimulation(P<0.05). The viability of AR42J cells was decreased after LPS treatment. The leakage rate of LDH was increased, the levels of IL-1β and TNF-α secreted by the AR42J cells were increased, the content of MDA was increased in the supernatant, and the activity of SOD, GSH-Px and CAT was reduced (P<0.05). After knock-down of TLR4 expression, the viability of AR42J cells was increased under LPS stimulation, the LDH leakage rate, secreted levels of IL-1β and TNF-α, and the content of MDA in cell culture medium were decreased, and the SOD, GSH-Px and CAT levels were increased (P<0.05). CONCLUSION:LPS induces the expression of TLR4 in the pancreatic acinar AR42J cells. Knock-down of TLR4 expression reduces the secretion of inflammatory factors IL-1β and TNF-α, and attenuates the oxidative damage in pancreatic acinar AR42J cells induced by LPS.     

Effect of RYR1 gene expression knock-down by siRNA on mitochondria in C2C12 cells

AIM: To investigate the effect of ryanodine receptor 1 (RYR1) down-regulation on mitochondria in mouse myoblast C2C12 cell line and to explore the possible mechanism. METHODS: The expression of RYR1 in the C2C12 cells was knocked down by the targeted small interfering RNA (siRNA). The mitochondrial number and morpholo-gical changes were evaluated by transmission electron microscopy and the stereoscopic analysis. Real-time PCR and Western blot were used to determine the mRNA and protein levels of mitofusin 2 (Mfn2), peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) and extracellular signal-regulated kinase 1/2 (ERK1/2), respectively. RESULTS: The expression of RYR1 was significantly down-regulated by siRNA transfection (P<0.01), with fragmentized mitochondria in the C2C12 cells in knock-down (KD) group. Although no statistical difference of the mitochondrial number was observed, the mitochondria area and circumference were significantly lowered in KD group (P<0.05). In KD group, the mRNA and protein expression of Mfn2 was significantly reduced (P<0.01). The mRNA level of PGC-1α was also reduced (P<0.01), but no significant change at protein level was observed. No change of ERK1/2 expression and phosphorylated ERK1/2 level was detected. CONCLUSION: Knock-down of RYR1 expression leads to morphological changes of mitochondria, and down-regulation of Mfn2 expression may be involved in the underlying mechanism. While PGC-1α and ERK1/2-associated oxidative stress pathway may not play an important role in the process.     

Abietic acid alleviates AGEs-induced apoptosis and endoplasmic reticulum stress in cardiomyocytes via inducing autophagy

AIM: This study aims to explore the effect of abietic acid (AA) on advanced glycosylation end products (AGEs)-induced apoptosis and endoplasmic reticulum stress in H9c2 cardiomyocytes. METHODS: H9c2 cells were divided into 5 groups. The cells in control group were treated with saline for 24 h. The cells in AGEs treatment group were treated with AGEs (100 mg/L) for 24 h. The cells in AGEs+AA (10, 25 and 50 μmol/L) groups were simulta-neously treated with AGEs (100 mg/L) and AA (10, 25 and 50 μmol/L) for 24 h. The cell viability was measured by MTT assay. The protein levels of myoglobin (Mb), creatine kinase MB isoenzyme (CK-MB), cardiac troponin I (cTnI), C/EBP homologous protein (CHOP), cleaved caspase-12, GADD34, BiP, LC3, P62 and beclin 1 were determined by Western blot. The levels of lactate dehydrogenase (LDH) were measured by ELASA. The apoptosis was analyzed by flow cytometry. RESULTS: The low concentration (<50 μmol/L) of abietic acid had no obvious effect on the viability of H9c2 cells. The high concentration (>50 μmol/L) of abietic acid decreased the viability of H9c2 cells. The levels of Mb, CK-MB, cTnI and LDH in AGEs group were higher than those in control group (P<0.05). Compared with AGEs group, the levels of Mb, CK-MB, cTnI and LDH in AGEs+AA (10, 25 and 50 μmol/L) groups were obviously reduced (P<0.05). Abietic acid at concentrations of 10, 25 and 50 μmol/L inhibited AGEs-induced apoptosis, elevated the protein levels of CHOP and cleaved caspase-12, and attenuated expression of GADD34 and BiP (P<0.05). Moreover, abietic acid at concentrations of 10, 25 and 50 μmol/L suppressed AGEs-induced decreased ratio of LC3-Ⅱ/LC3-Ⅰ and expression of beclin 1, and enhanced the expression of P62 (P<0.05). 3-Methyladenine, an inhibitor of autophagy, reversed the effect of abietic acid on the protein levels of LC3, Mb, cleaved caspase-12 and BiP (P<0.05). CONCLUSION: Abietic acid alleviates AGEs-induced apoptosis and endoplasmic reticulum stress in H9c2 cardiomyocytes via inducing autophagy.