This study investigated the effect of advanced glycation end products (AGEs) on differentiation of naïve CD4(+) T cells and the role of the receptor of AGEs (RAGE) and peroxisome proliferator-activated receptors (PPARs) activity in the process in order to gain insight into the mechanism of immunological disorders in diabetes. AGEs were prepared by the reaction of bovine serum albumin (BSA) with glucose. Human naïve CD4(+) T cells, enriched from blood of healthy adult volunteers with negative selection assay, were cultured in vitro and treated with various agents including AGEs, BSA, high glucose, PGJ2 and PD68235 for indicated time. In short hairpin (sh) RNA knock-down experiment, naïve CD4(+) T cells were transduced with media containing shRNA-lentivirus generated from lentiviral packaging cell line, Lent-X(TM) 293 T cells. Surface and intracellular cytokine stainings were used for examination of CD4(+) T cell phenotypes, and real-time PCR and Western blotting for detection of transcription factor mRNA and protein expression, respectively. The suppressive function of regulatory T (Treg) cells was determined by a [(3)H]-thymidine incorporation assay. The results showed that AGEs induced higher pro-inflammatory Th1/Th17 cells differentiated from naïve CD4(+) T cells than the controls, whereas did not affect anti-inflammatory Treg cells. However, AGEs eliminated suppressive function of Treg cells. In addition, AGEs increased RAGE mRNA expression in naïve CD4(+) T cells, and RAGE knock-down by shRNA eliminated the effect of AGEs on the differentiation of CD4(+) T cells and the reduction of suppressive function of Treg cells. Furthermore, AGEs inhibited the mRNA expression of PPARγ, not PPARα PPARγ agonist, PGJ2, inhibited the effect of AGEs on naïve CD4(+) T cell differentiation and reversed the AGE-reduced suppressive function of Treg cells; on the other hand, PPARγ antagonist, PD68235, attenuated the blocking effect of RAGE shRNA on the role of AGEs. It was concluded that AGEs may promote CD4(+) T cells development toward pro-inflammatory state, which is associated with increased RAGE mRNA expression and reduced PPARγ activity.
Adult ; Animals ; Blotting, Western ; CD4-Positive T-Lymphocytes ; drug effects ; metabolism ; Cattle ; Cell Differentiation ; drug effects ; Cells, Cultured ; Glucose ; pharmacology ; Glycation End Products, Advanced ; pharmacology ; HEK293 Cells ; Humans ; Interferon-gamma ; metabolism ; Interleukin-17 ; metabolism ; PPAR gamma ; agonists ; genetics ; metabolism ; Prostaglandin D2 ; analogs & derivatives ; pharmacology ; RNA Interference ; Receptor for Advanced Glycation End Products ; Receptors, Immunologic ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Serum Albumin, Bovine ; pharmacology ; T-Lymphocytes, Regulatory ; drug effects ; metabolism ; Th1 Cells ; drug effects ; metabolism ; Th17 Cells ; drug effects ; metabolism

OBJECTIVETo investigate the effects of advanced glycosylation end products (AGEs) modified bovine serum albumin (AGE-BSA) on the expression of reactive oxygen species (ROS) and monocyte chemoattractant protein-1 (MCP-1) in human renal mesangial cells (HRMCs).

METHODSHRMCs were cultured in vitro with medium containing different doses of AGE-BSA or BSA (50,100, 200, 400 mg/L) for 48 hours, or with AGE-BSA (200 mg/L) for different times (12, 24, 48, 72 h). Immunocytochemistry assay was used to estimate the protein level of RAGE. The ROS in cells were measured by flow cytometry and the mRNA expression of MCP-1 were analyzed by semi-quantiative reverse transcription-polymerase chain reaction (RT-PCR) after treatment with AGE-BSA or BSA.

RESULTSThe protein level of RAGE was upregulated in the HRMCs with AGE-BSA. The expression of ROS and MCP-1 significantly enhanced by incubation of AGE-BSA in a time- and dose-dependent manner. The effects of AGE-BSA-induced up-regulation of ROS and MCP-1 level was significantly blocked by neutralizing antibodies to RAGE, while the expression of ROS and MCP-1 stood nearly unchanged after cultured with huamn IgG.

CONCLUSIONThe expression of ROS and MCP-1 in HRMCs is induced by AGE-BSA through RAGE, which may have potential effects in the pathgenic mechanism of diabetic nephropathy.

Cells, Cultured ; Chemokine CCL2 ; metabolism ; Glycation End Products, Advanced ; pharmacology ; Humans ; Mesangial Cells ; drug effects ; metabolism ; Oxidative Stress ; drug effects ; Reactive Oxygen Species ; metabolism ; Receptor for Advanced Glycation End Products ; Receptors, Immunologic ; metabolism ; Serum Albumin, Bovine ; pharmacology

Animals ; Cattle ; Cell Line ; Electrophoresis, Polyacrylamide Gel ; Fluorescent Dyes ; chemistry ; Malondialdehyde ; chemistry ; Mice ; Microscopy, Confocal ; Serum Albumin, Bovine ; analysis ; chemistry ; metabolism ; Ultraviolet Rays

OBJECTIVETo analyze and compare molecular mechanisms of active ingredients of honeysuckle (chlorogenic acid, CGA) with bovine lactoferrin (BLF) or bovine serum albumin (BSA).

METHODThe spectral experiment and the computer analog technology were combined to determine the binding parameters, energy transfer parameters and thermodynamic functions between CGA and proteins, study the molecular mechanism, and compare the differences in interactive mechanism between CGA and BLF or BSA.

RESULTThe interactive mechanism between CGA and BLF or BSA was a dynamic molecular mechanism, whereas the static quenching mechanism existed between the interaction of CGA and BSA, with differences in the bonding intensity due to difference temperature. The binding distance r between CGA and BLF/BSA was very short, indicating the phenomenon of energy transfer. The results of the molecular modeling showed that the main interaction force between CGA and BLF or BSA was hydrogen bonds, together with Van der Waals' forces and hydrophobic effect.

CONCLUSIONThe computer analog shows consistent results with spectral experiment.

Absorption ; Animals ; Cattle ; Drugs, Chinese Herbal ; chemistry ; metabolism ; Humans ; Hydrophobic and Hydrophilic Interactions ; Lactoferrin ; chemistry ; metabolism ; Lonicera ; chemistry ; Models, Molecular ; Protein Binding ; Protein Conformation ; Serum Albumin, Bovine ; chemistry ; metabolism ; Thermodynamics

Increased formation of advanced glycation end-products (AGEs) is occurred in hyperglyceamia and diabetes, leading to oxidative stress and progression of diabetic vascular diseases. Glucose-6-phosphate dehydrogenase (G6PD), the principal source of NADPH, serves as an antioxidant enzyme to modulate the redox milieu. Deficiency of G6PD activity is associated with increased endothelial cell oxidative stress. Current study is designed to investigate the effects of AGEs on G6PD activity and expression in human umbilical vein endothelial cells. Treatment of AGE-modified bovine serum albumin (AGE-BSA, 100 µg/mL, 24 h), but not native BSA, to human umbilical vein endothelial cells increased ROS generation by (48.89 ± 5.28)%. G6PD activity was decreased by AGE-BSA treatment by (61.25 ± 11.2)%. The expression of G6PD at mRNA and protein levels was also decreased by AGE-BSA treatment by (27.92 ± 6.73)% and (23.72 ± 2.44)%, respectively. These results suggest that AGEs could result in G6PD deficiency in human umbilical vein endothelial cells by inhibiting the expression of G6PD at mRNA and protein levels and G6PD activity.
Antioxidants ; metabolism ; Cells, Cultured ; Glucosephosphate Dehydrogenase ; antagonists & inhibitors ; metabolism ; Glycation End Products, Advanced ; pharmacology ; Human Umbilical Vein Endothelial Cells ; drug effects ; enzymology ; Humans ; Oxidative Stress ; Reactive Oxygen Species ; metabolism ; Serum Albumin, Bovine ; pharmacology

PURPOSE: To investigate the effect of advanced glycation end products (AGE) on oxidative stress and cellular senescence in cultured human trabecular meshwork cells (HTMC). METHODS: Primarily cultured HTMC were exposed to 0, 10, 50, 100, 200 microg/mL of glycated bovine serum albumin (G-BSA) for 5 days. Also co-exposed were L-arginine, sepiapterin, and antioxidant N-acetylcysteine (NAC). Cellular survival and production of nitric oxide (NO), superoxide, and reactive oxygen species were assessed by 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide assay, Griess assay, cytochrome c assay, and dichlorofluorescin diacetate assay, respectively. Senescence-associated beta-galactosidase staining was performed to quantify the degree of cellular senescence. RESULTS: G-BSA decreased cellular survival, NO production, and increased superoxide production significantly in a dose-dependent manner. The effects of G-BSA were abolished with co-exposure of L-arginine, sepiapterin, and NAC. G-BSA enhanced cellular senescence accompanied by increased production of reactive oxygen species. G-BSA-induced cellular senescence was suppressed by application of L-arginine, sepiapterin, and NAC. CONCLUSIONS: AGE enhances cellular senescence of HTMC accompanied with increased oxidative stress. AGE-induced oxidative stress and cellular senescence could be delayed by application of anti-oxidants.
Acetylcysteine/metabolism ; Apoptosis/drug effects/physiology ; Arginine/metabolism ; Cell Aging/drug effects/*physiology ; Cell Survival/drug effects/physiology ; Cells, Cultured ; Glycosylation End Products, Advanced/metabolism/*toxicity ; Humans ; Nitric Oxide/metabolism ; Oxidative Stress/*physiology ; Pterins/metabolism ; Reactive Oxygen Species/metabolism ; Serum Albumin, Bovine/metabolism/toxicity ; Trabecular Meshwork/drug effects/*metabolism/*pathology

A combination of spectral experiment and molecular modeling techniques has been used to characterize the binding mechanism between an active component 5-hydroxymethyl-furfural (5-HMF) of traditional Chinese medicine and human serum albumin (HSA) or bovine serum albumin (BSA). The interaction mechanism of 5-HMF binding with HSA/BSA is analyzed. Although the drug can bind with HSA/BSA to form stable complexes, there are some differences in the bond strength. The values of binding distances (r) are different and low, which indicated the occurrence of energy transfer. The drug had conformational effect on HSA/BSA, which resulted in different changes of hydrophobic environment of the binding domain in HSA/BSA. The 'phase diagram' of fluorescence revealed that the changes on the conformational pattern of proteins have been affected by drug conformed to the "all-or-none" pattern. The interactions between drug and protein influenced by Co(II) were also discussed. Its effects acting on 5-HMF-HSA/BSA interactions are different. The computational modeling method was used to study the interaction between 5-HMF and HSA/BSA. The results of molecular model studies revealed that the binding modes for drug-serum albumin systems are mainly hydrophobic interactions and hydrogen bonding. These results are in accordance with spectral results. The research results have given a better theoretical reference for the study of pharmacological mechanism of 5-hydroxymethyl-furfural.
Animals ; Drugs, Chinese Herbal ; chemistry ; Energy Transfer ; Furaldehyde ; analogs & derivatives ; chemistry ; isolation & purification ; metabolism ; Humans ; Hydrogen Bonding ; Hydrophobic and Hydrophilic Interactions ; Ions ; chemistry ; Models, Molecular ; Molecular Conformation ; Protein Binding ; Serum Albumin ; chemistry ; metabolism ; Serum Albumin, Bovine ; chemistry ; metabolism ; Spectrometry, Fluorescence ; Spectrophotometry, Ultraviolet

OBJECTIVE: To evaluate the effectiveness of IL-10 in suppressing 18F-FDG uptake in the inflammatory granuloma of SD rats. METHODS: Eight SD rats were killed, and their blood was collected sterily. After centrifugion, the white blood cells were incubated in PRMI 1640 for 3 days. Then each culture flask of white blood cells was divided into two equal parts. To one group was added 0.2 mL IL-10 solution (0.1 mg/mL); to the control was added with 0.2 mL of 0.9% sodium chloride solution. All cells were then incubated for 120 minutes at 37 degree, after which 18F-FDG (1.85 MBq) was added. Sixty minutes later, the cells were washed twice with PBS and the extent of uptake 18F-FDG determined. In vivo, an inflammatory granuloma was produced by hypodermic injection of rats with a mixture of Freund's complete adjuvant, bovine serum albumin and talcum powder. Each rat was maintained for 8 weeks. Imaging of the inflammatory granulomas was performed using the 18F-FDG signal. IL-10 was injected into SD rats at 10 μg/kg of body weight. Sixty minutes later, 7.4 MBq of 18F-FDG were injected, and, after a further 60 minutes, the rats underwent a PET-CT scan. The region of interest (ROI) of the inflammatory granuloma was delineated and the standard uptake value (SUV) calculated. A second PET-CT scan was done without IL-10 on the next day. The granulomatous tissue underwent pathological examination. RESULTS: In the intro test, the with blood cell uptaking ratio of 18F-FDG was (50.3±6.7)% without IL-10, and (34.6±3.5)% with IL-10(t=8.9, P<0.01). IL-10 suppressed the rat white blood cell uptaking 18F-FDG. In the PET-CT scan, the SUV of ROI on inflammatory granuloma was 1.7±0.4 with IL-10 and 2.1±0.3 without IL-10 (t=20.6, P<0.01). IL-10 suppressed the inflammatory granuloma uptaking 18F-FDG. CONCLUSION IL-10 can suppress the inflammatory granuloma of SD rats uptaking 18F-FDG.
Animals ; Female ; Fluorodeoxyglucose F18 ; pharmacokinetics ; Freund's Adjuvant ; Granuloma ; chemically induced ; metabolism ; Interleukin-10 ; pharmacology ; Lung Diseases ; chemically induced ; metabolism ; Male ; Radiopharmaceuticals ; pharmacokinetics ; Rats ; Serum Albumin, Bovine

OBJECTIVETo elucidate the effect of valsartan on human glomerular mesangial cells oxidative stress and the expression of the receptor for advanced glycation end products (RAGE) induced by the advanced glycation end-products (AGEs).

METHODSHuman glomerular mesangial cells were treated with advanced glycation end-product-bovine serum albumin (AGE-BSA) in the presence of valsartan. The reactive oxygen species (ROS) in cells were measured by Flow cytometry, and the mRNA of p47 phox, which was the primary subunits of NADPH oxidase, was detected by semi-quantitative reberse transcription polymerase chain reaction (RT-PCR). The mRNA of RAGE was detected by RT-PCR and the RAGE protein was assayed by immunocytochemistry.

RESULTSThe product of ROS, and the expression of p47 phox and RAGE in mesangial cells, which were treated with AGE-BSA in the presence of valsartan, were down-regulated compared with the groups treated with AGE-BSA (P < 0.05). Valsartan dose-dependently and time-dependently inhibited the AGE-elicited overexpression of RAGE, ROS and p47(phox) in mesangial cells.

CONCLUSIONValsartan could inhibit RAGE expression through downregulation of oxidative stress.

Angiotensin II Type 1 Receptor Blockers ; pharmacology ; Antioxidants ; pharmacology ; Glycation End Products, Advanced ; pharmacology ; Humans ; Mesangial Cells ; cytology ; metabolism ; Oxidative Stress ; drug effects ; RNA, Messenger ; genetics ; metabolism ; Receptor for Advanced Glycation End Products ; Receptors, Immunologic ; genetics ; metabolism ; Serum Albumin, Bovine ; pharmacology ; Tetrazoles ; pharmacology ; Valine ; analogs & derivatives ; pharmacology ; Valsartan

It is generally believed that liposomes modified with polyethylene glycol (PEG) have no or lower immunogenicity. However, based on many recent literatures, when the PEGylated liposomes were repeatedly applied to the same animal, the immune responses occurred. The first injection of PEGylated liposomes resulted in a reduction in the circulation time and an increase in hepatic and splenic accumulation of the second dose of PEGylated liposomes in a time-interval, which was called "accelerated blood clearance (ABC)" phenomenon. Such immunogenicity of PEGylated liposomes presents a barrier in the research of liposomal formulations and their use in the clinics. This review focused on the definition, the method of verification, the development of the reason for ABC phenomenon, influencing factors of ABC phenomenon, and discussed if other PEGylated nanocarriers also induce ABC phenomenon.
Animals ; Antibiotics, Antineoplastic ; administration & dosage ; pharmacokinetics ; Doxorubicin ; administration & dosage ; pharmacokinetics ; Drug Carriers ; Immunoglobulin M ; biosynthesis ; blood ; Liposomes ; administration & dosage ; blood ; pharmacokinetics ; Liver ; metabolism ; Metabolic Clearance Rate ; Particle Size ; Polyethylene Glycols ; administration & dosage ; metabolism ; pharmacokinetics ; Serum Albumin, Bovine ; pharmacokinetics ; Spleen ; immunology ; metabolism