Protective Effects Of A Prescribed Traditional Chinese Medicine Preparation On Oxidative Stress Induced By High Glucose In BEnd.3 Cells

Aims:vascular diseases are the principal causes of morbidity and mortality in patients with diabetes mellitus.It has been suggested that endothelial dysfunction may be a critical and initiating factor in the pathogenesis of diabetic vascular complications.An underlying mechanism for endothelial dysfunction is the enhanced generation of endothelial mitochondrial reactive oxygen species(ROS).We previously observed that a prescribed traditional Chinese medicine preparation Qinghuoyihao(QHYH) can decrease urinary microalbumin excretion in type 2 diabetic patients and improve microvascular endothelial cells growth in high glucose medium.This study was designed to gain the insight into the antioxidant mechanism of QHYH against oxidative stress induced by high glucose in endothelial cells.We studied the effect of QHYH on ROS production,NO generation and the phosphorylation of Akt and eNOS. Since a recent study indicates that genetic ablation of UCP2 produces more ROS in endothelial cells,we therefore investigated the effect of QHYH on UCP2 expression. Finally,using siRNA technique,we studied whether UCP2 is involved in the increase of ROS in mice brain microvascular cells(bEnd.3 cells) induced by high glucose,and whether it is accounted for the antioxidant mechanism of QHYH in endothelial cells.Materials and Methods:bEnd.3 cells were cultured in normal glucose(NG group, 5.6 mM),high glucose(HG group),high glucose in the presence of QHYH(1:100 dilution).35 mM glucose was used as high glucose working concentration except 25 mM in ROS detection experiments.ROS and nitric oxide(NO) production was measured by the fluorescent marker H₂DCF-DA and DAF-FM DA,respectively. Endothelial nitric oxide synthase(eNOS),and uncoupling protein 2(UCP2) mRNA were detected by quantitative real-time reverse transcription- polymerase chain reaction.Phosphorylation of eNOS,protein kinase B(Akt) and UCP2 protein expression were measured by western blotting.UCP2 siRNA was used to evaluate the involvement of UCP2 in the antioxidant effect of QHYH.Results:High glucose(25 mM) increased ROS production(142.27±11.44%vs. 100.00±1.92%in NG group,P＜0.01) in bEnd.3 cells.UCP2 mRNA and protein expression were decreased at the same time,glucose reduction of UCP2 mRNA in a dose-dependent manner,with the maximum effect at 35 mM glucose(69.90±4.84%, P＜0.01 vs.NG group),and was time-dependent,with maximal effect occurring after 48 hours of incubation(66.93±4.73%,P＜0.001 vs.0h group).High glucose(35 mM) downregulated phosphorylation of eNOS at Ser1177 and Akt at Ser473(45.49±2.51%and 76.71±4.00%,respectively,P＜0.01 vs.NG group) and decreased NO generation(53.83±3.98%,P＜0.01 vs.NG group) in bEnd.3 cells compared to NG group.Addition of QHYH reversed high glucose's effects on ROS production(53.65±8.11%vs.142.27±11.44%in HG group,P＜0.001),eNOS and Akt phosphorylation (72.81±7.02%vs.45.49±2.51%in HG group,P＜0.05 and 76.71±4.00%vs.120.98±1.20%in HG group,P＜0.001,respectively),NO generation(118.82±2.95%vs. 53.83±3.98%in HG group,P＜0.001),and UCP2 protein expression(94.09±6.30% vs.53.83±3.98%in HG group,P＜0.001).When UCP2 expression was blocked by UCP2 siRNA,the anti-oxidant effects of QHYH against high glucose were attenuated; QHYH could not reverse the high glucose's effects on ROS production and NO generationConclusions:QHYH could protect endothelial cells from high glucose induced ROS production,downregulation of Akt and eNOS phosphorylation and reduction of NO generation.The protective effects of QHYH were partially related to UCP2 protein expression,since deletion of UCP2 by siRNA lead to the loss of the effects.These results suggest the therapeutic potential of QHYH in treatment of diabetic vascular complications. Background:We have confirmed that QHYH has antioxidative effects.The purpose of this experiment is to investigate the chemical constituents of QHYH,determine their antioxidative effects on high glucose induced ROS production,and study the underlying mechanism of their antioxidative effects.Genetic ablation of UCP2 produces more ROS in endothelial cells,and our study has confirmed that QHYH could improve glucose induced inhibition of UCP2 mRNA,deletion of UCP2 by siRNA lead to the loss of protective effects of QHYH,QHYH's protective effects were partially related to UCP2 protein expression,so we detected effects of the chemical compounds extracted from QHYH on UCP2 mRNA level in bEnd.3 cells. NADPH oxidase is one of the most important sources of ROS.It is composed of gp91^phox,p22^phox,p67^phox and p47^phox,so we also detected the effects of chemical compounds extracted from QHYH on NADPH submits mRNA levels.Materials and methods:We got 3 fractions from QHYH by solvent extraction and 5 fractions by using of macroporous resin,bEnd.3 cells were cultured in normal glucose (NG group,5.6 mM),high glucose(HG group),high glucose in the presence of QHYH(1:100 dilution) or the chemical compounds extracted from QHYH.35 mM glucose was used as high glucose working concentration except 25 mM in ROS detection experiments.ROS production was measured by the fluorescent marker H₂DCF-DA.Column chromatogram was used for the furhther separation and extraction.The effects of chemical compounds on endothelial cell viatality were determined by MTT chromatometry.The mRNA levels of UCP2,p91^phox,p22^phox, p67^phox,p47^phox were determined by real-time quantitive PCR.Results:28 fractions were isolated from QHYH,and by detecting their effects on high glucose induced ROS production in bEnd.3 cells and further separation and extraction,12 chemical compounds were extracted from QHYH,9 of which are known,including geniposidic acid,geniposide,astragaloside,puerarin,hesperidin, crocin,tetramethylpyrazine(TMP),ferulic acid(FA) and chlorogenic acid(CHA), and all of them have been reported to have antioxidative effects.The structure of the other 3 compounds:E3-M15-1,E3-M15-2 and E1-H1-1 have not been determined yet. crocin,TMP,FA,CHA,E3-M15-1,E3-M15-2 and E1-H1-1 could inhibit high glucose induced ROS production in both bEnd.3 and HUVEC cell,and have no significant cytotoxicity effects on both cell lines(except E3-M15-2 in high concentration).High glucose could decrease UCP2 mRNA level in bEnd.3 cells.Addition of QHYH and extractions(crocin,TMP,FA,CHA,E3-M15-1,E3-M15-2 and E1-H1-1) could inhibit the effect of high glucose.High glucose increased the mRNA level of gp91^phox and p67^phox.QHYH and TMP could inhibit high glucose induced increase of gp91^phox, p22^phoxand p67^phox mRNA.E3-M15-1 could inhibit high glucose induced increase of gp91^phox and p22^phox mRNA.Crocin could inhibit high glucose induced increase of gp91^phox and p67^phox mRNA level.FA,CHA could only inhibit high glucose induced increase of p22^phox mRNA.Conclusions:The chemical compounds extracted from QHYH including crocin,TMP, FA,CHA,E3-M15-1,E3-M15-2 and E1-H1-1 could inhibit high glucose induced ROS production in bEnd.3 and HUVEC cells,they are among the important constituents of QHYH for its antioxidative effects.The protective effects of QHYH and its extraction(crocin,TMP,FA,CHA,E3-M15-1) were partially related to their improvement of UCP2 mRNA suppressed by high glucose,and their inhibition of NADPH oxidase mRNA expression induced by high glucose.