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Xu Y.-H.,Macau University of Science and Technology | Xiong J.,Macau University of Science and Technology | Wang S.-S.,Macau University of Science and Technology | Tang D.,Macau University of Science and Technology | And 2 more authors.
Journal of Natural Medicines | Year: 2014

Endothelial cell (EC) apoptosis plays a pivotal role in the progression of diabetic complications. Abundant studies have demonstrated the pivotal role of advanced glycation end products (AGEs) on the development of diabetes. The aim of the present study was to investigate the effect of calycosin, a phytoestrogen, on AGEs-induced human umbilical vein endothelial cell (HUVEC) apoptosis. Fluorescence polarization and fluorescence absorption assays indicated that calycosin interacted with AGEs in a time-dependent manner. Further studies found that calycosin entered the cells as detected by HPLC. The MTT method demonstrated that calycosin ameliorated AGEs-induced HUVEC apoptosis in a dose-dependent manner, and statistical significance was observed at 1 × 10-8 Mof calycosin; this behavior was further demonstrated by acridine orange/ethidium bromide staining in that the presence of calycosin dramatically reduced AGEs-induced red staining in HUVECs. Further studies found that pre-incubation with calycosin at 1 × 10-8 Mdramatically increased anti-apoptotic Bcl-2 while decreased pro-apoptotic Bax and Bad expressions as detected by immunocytochemistry, and the effect of calycosin on rebalancing the ratio of Bcl-2/Bax was more significant than that of its glycoside, calycosin-7-O-β-Dglucopyranoside (CG). Furthermore, calycosin slightly reversedAGEs-induced cell oxidative stress at 1 × 10-8 M, but its antioxidative stress effect was less significant than that of CG. The present study strongly indicates that calycosin can enter the cell and modulate endothelial cell dysfunction by ameliorating AGEs-induced cell apoptosis. © The Japanese Society of Pharmacognosy and Springer Japan 2013.

Xu Y.,Macau University of Science and Technology | Wang S.,Macau University of Science and Technology | Feng L.,Macau University of Science and Technology | Zhu Q.,Macau University of Science and Technology | And 3 more authors.
International Immunopharmacology | Year: 2010

Advanced glycation end products (AGEs) have been recognized as a pivotal inducer in diabetes and kinds of aging-related vasculopathy. Endothelial dysfunction and inflammatory cells adhesion to endothelium have been regarded as important and early factors in the pathogenesis of vascular complications in diabetic patients. Owing to the key role of PKC-beta in AGEs-induced vascular dysfunction, we investigated effects of blocking PKC-beta by LY333531 on macrophage adhesion to HUVEC and the related mechanism. Transwell HUVEC-macrophage co-culture system was established to evaluate macrophage migration and adhesion ability. Immunocytochemistry was applied to examine TGF-beta1, ICAM-1 and RAGE protein expressions by SABC or SABC-AP method; mRNA expression of TGF-beta1, ICAM-1 and RAGE was determined by real-time RT-PCR. SOD and MDA levels in culture supernatant were detected. We found that LY333531 significantly reduced AGEs-induced macrophage adhesion to HUVEC. Blockade of PKC-beta strikingly decreased HUVEC TGF-beta1 and ICAM-1 expression in both protein and mRNA levels, RAGE protein level was also down-regulated. Furthermore, the anti-oxidative stress index, SOD/MDA was dramatically elevated on LY333531 application. Therefore we conclude that LY333531 can reduce AGEs-induced macrophage adhesion to endothelial cells and relieve the local inflammation, this was realized by its effect on decreasing inflammatory cytokines' expression and increasing cell anti-oxidative ability.

Xu Y.,Macau University of Science and Technology | Xiong J.,Macau University of Science and Technology | Zhao Y.,Macau University of Science and Technology | He B.,Institute of Consun Co. for Chinese Medicine in Kidney Diseases | And 3 more authors.
American Journal of Chinese Medicine | Year: 2015

Diabetes mellitus (DM) often accompanies liver dysfunction. Astragali Radix is a traditional Chinese herbal medicine that is widely administrated to ameliorate the symptoms of diabetes as well as liver dysfunction, but its acting mechanism is still not yet fully recognized. Advanced glycation end products (AGEs) play a key role in promoting diabetic organ dysfunction. Both hyperglycemia and AGEs can induce insulin resistance, hepatocyte damage and liver dysfunction. We designed this study to explore the effects of the phytoestrogen Calycosin, a major active component of Astragali Radix, on AGEs-induced glucose uptake dysfunction in the hepatocyte cell line and relevant mechanisms. MTT and BrdU methods were applied to evaluate cell viability. 2-NBDG was used to observe glucose uptake by a live cell imaging system. Immunofluorescence method was carried out to investigate GLUT1, GLUT4, and RAGE protein expressions on cell membrane. cAMP content was determined by an EIA method. We found Calycosin concentration-dependently ameliorated AGEs-induced hepatocyte viability damage. AGEs dramatically reduced basal glucose uptake in hepatocytes, and this reduction could be reversed by Calycosin administration. By immunofluorescence detection, we observed that Calycosin could inhibit AGEs-induced GLUT1 expression down-regulation via estrogen receptor (ER). Furthermore, Calycosin decreased AGEs-promoted RAGE and cAMP elevation in hepatocytes. These findings strongly suggest that Calycosin can ameliorate AGEs-promoted glucose uptake dysfunction in hepatocytes; the protection of cell viability and ER-RAGE and GLUT1 pathways play a significant role in this modulation. © 2015 World Scientific Publishing Company & Institute for Advanced Research in Asian Science and Medicine.

Wu A.,Macau University of Science and Technology | Wu A.,Institute of Consun Co. for Chinese Medicine in Kidney Diseases | Duan T.,Macau University of Science and Technology | Duan T.,Institute of Consun Co. for Chinese Medicine in Kidney Diseases | And 12 more authors.
Current Analytical Chemistry | Year: 2014

Nitric oxide (NO), a reactive free radical, is an important messenger molecule in biological cells. It plays a critical role in regulating vascular tone, host immunity, neurotransmission, nutrient metabolism and the whole-body homeostasis. NO is metabolized into various products, including plentiful nitrite and nitrate in the circulatory and excretory in vivo, others such as peroxynitrite, nitronium ions, nitroxide ions, and S-nitroso/nitro compounds are unstable and degraded to nitrite and nitrate. The direct measurement of NO is difficult for its reactive chemical properties and short half life. The determinations of nitrite and nitrate could be used to evaluate NO level. Numerous methods are available for measurement of nitrite and nitrate, such as high performance liquid chromatography (HPLC), gas chromatography (GC), ion chromatography (IC), Griess, spectrophotometry, chemiluminescence, fluorescence, catalytic kinetic, infrared spectrophotometry and electron paramagnetic resonance. Of note, chromatography analysis is a common and sensitive method. Based on the reaction of nitrite with 2, 3-diaminonaphthalene (DAN) to form N-naphthotriazole (NAT), HPLC coupled with fluorescence detector could lower the limit of detection of nitrite and nitrate, and has no interference of impurities in biological samples. Furthermore, the method involves the GC/LC-MS with the use of 15N as a stable-isotope label. GC-MS can determine the PFB-NO2 and PFB-NO3 derived from nitrite and nitrate with PFB-Br, while LC-MS can determine NAT derived from nitrite with DAN. GC/LC-MS could not only enable specific assessment for low nitrite and nitrate, but also enable reliable investigations on the metabolic fate and reactions of NO and its metabolites S-nitro compounds in vitro and in vivo. Here, we reviewed the applications of HPLC, GC and IC on the analysis of nitrite and nitrate in various biological fluids. It will provide some suggestions for the study of NO in biochemistry, physiology, and pharmacology researches in the future. © 2014 Bentham Science Publishers.

Tang D.,Macau University of Science and Technology | Tang D.,Institute of Consun Co. for Chinese Medicine in Kidney Diseases | Zhu J.-X.,Macau University of Science and Technology | Zhu J.-X.,Institute of Consun Co. for Chinese Medicine in Kidney Diseases | And 9 more authors.
Journal of Chromatography A | Year: 2013

Methylglyoxal (MGO), a very reactive metabolite of glucose, plays a pivotal role in the pathogenesis of several chronic diseases associated with diabetes, and it has been validated as an attractive target for them. In the present study, a simple and effective method, namely pre-column incubation followed by fast high performance liquid chromatography based on superficially porous particles (shell), coupled with diode array detection and tandem mass spectrometry (UHPLC-DAD-MSn), was proposed for rapid and high-throughput screening of natural MGO scavengers directly from the crude extract of Polygonum cuspidatum Sieb. et Zucc, a well-known traditional Chinese medicine which was used for treatment of diabetic complications. The hypothesis is that upon reaction with MGO, the peak areas of components with MGO scavenging potency in the chromatogram will be significantly reduced or disappear, and the structural characterization could be achieved by UHPLC-DAD-MSn hyphenated technique. First of all, 12 compounds in P. cuspidatum were well separated within shorter time (~12min) than previous methods and identified, and two of them, i.e. 3,5,4'-trihydroxystilbene-3-O-(6'-galloyl)-glucoside (3) and emodin-8-O-(6'-malonyl)-glucoside (8) were firstly reported ingredients. After incubation with MGO, four stilbene derivatives were demonstrated to possess potential MGO trapping activities. Furthermore, it was proved that both polydatin (piceid) and resveratrol exhibited effective MGO-trapping capacity by UHPLC analysis, and they could significantly inhibit the formation of advanced glycation end products (AGEs) in the human serum albumin (HSA)-MGO assay, indicating that they were potential candidate agents for delaying and preventing diabetic complications. Additionally, MGO trapping mechanism exploration by UHPLC-MSn showed that the positions 2 and 4 of the A ring of stilbene were major active sites for trapping MGO to form both mono- and di-MGO adducts, however, the glucosylation of the hydroxyl group would significantly decrease the trapping efficiency. Collectively, the current work provides a very promising method for rapid discovery of natural MGO scavengers directly from complex matrices such as herbal medicines with huge resources. © 2013 Elsevier B.V.

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