AIM:To establish a liquid chromatography method for determining the monosaccharide composition of human lipoproteins, and to investigate the differences between diabetic patients and healthy participants.METHODS:Liquid chromatography with pre-column derivatization was used to determine the neutral and basic monosaccharides, and liquid chromatography tandem mass spectrometry was applied to quantify N-acetylneuraminic acid content.RESULTS:The contents of mannose, glucosamine, N-acetylglucosamine, glucose, galactose and N-acetylneuraminic acid in high-density lipoprotein from healthy participants and diabetic patients were (5.88 ±0.94),(16.49 ±4.11),(1.31 ±0.33), (0.87 ±0.16), (7.18 ±1.64), (2.14 ±0.12) mmol/(g protein) and (8.68 ±0.39), (24.73 ±5.50), (1.91 ±0.54), (1.23 ±0.35), (9.73 ±2.85), (3.53 ±0.27) mmol/(g protein), respectively.The contents of mannose, glu-cosamine,N-acetylglucosamine, glucose, galactose and N-acetylneuraminic acid in low-density lipoprotein from healthy par-ticipants and diabetic patients were ( 29.20 ±3.57 ) , ( 50.77 ±4.72 ) , ( 5.28 ±0.64 ) , ( 10.06 ±1.37 ) , ( 28.44 ± 3.96),(6.86 ±0.11) mmol/(g protein) and (30.08 ±3.78), (38.52 ±6.38), (3.79 ±0.78), (7.63 ±1.50), (20.05 ±2.63), (6.45 ±0.18) mmol/(g protein), respectively.The contents of mannose, glucosamine, glucose, ga-lactose and N-acetylneuraminic acid in very-low-density lipoprotein from healthy participants and diabetic patients were (91.21 ±4.12), (27.05 ±2.34), (4 230.95 ±15.83), (43.40 ±3.75), (2.95 ±0.24) mmol/(g protein) and (82.40 ±0.51), (30.16 ±0.32), (4 722.73 ±93.27), (34.05 ±2.81), (4.42 ±0.15) mmol/(g protein), respec-tively.CONCLUSION:Liquid chromatography with pre-column derivatization is suitable for the neutral and basic mono-saccharide analysis in human lipoproteins, and the glycosylation of lipoproteins in diabetic patients are significantly changed compared with the healthy controls.

Mononuclear cells(MNCs) isolatedfrommammal'sbonemarrowandperipheral bloodbymeansof Ficoll density grandient centrifugation,which are composed of monocytes and lymphocytes,can differentiate into different progenitor cells with different functions in the development of atherosclerosis under different inducing conditions. This article described the induction conditions of MNCs,the identification methods of different progenitor cells,and the relationship between these progenitor cells and the development of atherosclerosis,thus provide a new idea for the prevention of the atherosclerosis.

Atherosclerosis is a multifactorial process associated with endothelial cell injury and dysfunction , inflammation, oxidative stress, cell proliferation, angiogenesis and so on , all of which play a crucial role in atherosclerosis . Wnt/β-catenin signaling pathway is highly conservative in the development of body , abnormal activation of which is related to types of diseases including cancer .Accumulating studies have shown that Wnt/β-catenin signaling pathway is involved in inflammation, oxidative stress and so on .This article would make a review about the role of Wnt/β-catenin signaling path-way in atherosclerosis based on the pathogenic mechanisms underlying atherosclerosis as mentioned above .

[ ABSTRACT] AIM:To investigate the effect of oxidized low-density lipoprotein ( ox-LDL) on autophagy in mac-rophages and the underlying molecular mechanisms.METHODS:RAW264.7 macrophages were pretreated with 2 mg/L anti-CD36 monoclonal antibody (anti-CD36 mAb), 5 μmol/L diphenyleneiodonium (DPI), 3 mmol/L 3-methyladenine (3-MA) or 1μmol/L rapamycin for 1 h and then treated with ox-LDL (100 mg/L) for 12 h.The viability of the cells was measured by MTT assay.The activities of lactic dehydrogenase ( LDH) in the medium and nicotinamide adenine dinucleoti-de phosphate ( NADPH) oxidase, superoxide dismutase ( SOD) in the cells as well as the levels of intracellular reactive ox-ygen species ( ROS) and malondialdehyde ( MDA) were determined to characterize the membrane integrity and the oxida-tive stress, respectively.The protein levels of beclin-1 and microtubule-associated protein 1 light chain 3-II ( LC3-II) , 2 important molecular markers of autophagy, were examined by Western blotting.RESULTS:ox-LDL induced autophagy in
RAW264.7 macrophages as assessed by upregulation of beclin-1 and LC3-II.Similar to 3-MA, an autophagy inhibitor, an-ti-CD36 mAb significantly inhibited the ox-LDL-induced upregulation of beclin-1 and LC3-II.Anti-CD36 mAb suppressed the ox-LDL-induced oxidative stress as revealed by decreased NADPH oxidase activation, ROS and MDA generation as well as increased SOD activity.Similar results were observed in the cells pretreated with DPI, a NADPH oxidase inhibitor.Mo-reover, DPI significantly inhibited the ox-LDL-induced upregulation of beclin-1 and LC3-II.Inaddition, the decrease in the cell viability and increase in LDH release induced by ox-LDL were promoted by 3-MA and blocked by rapamycin ( an auto-phagy inducer).CONCLUSION: ox-LDL induces autophagy in RAW264.7 macrophages, which may be involved in CD36-mediated ox-LDL uptake and subsequent activation of oxidative stress, and moderate activation of autophagy may pro-tect macrophages from ox-LDL-induced injury.

[ ABSTRACT] AIM:To observe the influences of different concentrations of MG132 on apoptosis and beta-amyloid protein ( Aβ) generation in SH-SY5Y cells, and to explore the underlying mechanism.METHODS:SHSY-5Y cells were incubated with MG132 for 24 h.The final concentrations of MG132 were 2.5, 5 and 10μmol/L.The cell viability was de-termined by MTT assay.The cell apoptosis was assessed by flow cytometry.The levels of Aβwere measured by ELISA. The relative protein levels were detected by Western blot.RESULTS:In the SH-SY5Y cells, MG132 reduced the cell via-bility, induced the cell apoptosis, increased the level of Aβ, and increased the expression of the related proteins for Aβgeneration in a concentration-dependent manner.CONCLUSION: MG132 induces apoptosis and increases the levels of Aβ1-42 and Aβ1-40 by regulating the proteins related to Aβgeneration in the SH-SY5Y cells.

Objective To investigate the effects of age and gender on anti-oxidative,antiinflammatory and anti-apoptosis functions of high-density lipoprotein (HDL).Methods Totally 120 healthy subjects aged from 20 to 60 years were randomly divided into young and middle-aged male (n=60) group and female (n =60) group,and the 120 healthy elderly aged from 60 to 78 years divided into elderly male (n =60) and elderly female (n =60) groups.Serum levels of high-and low-density lipoprotein cholesterol (HDL-C,LDL-C),total cholesterol (TC),and triacylglycerol (TG) were detected.Content of malonaldehyde (MDA) was detected to determine anti-oxidative function of HDL.Adhesion assay of endothelial cells and monocytes (THP1) was adopted to test the protective effects of HDL on endothelial cells.The expressions of endothelial cell adhesion molecules,VCAM-1 and ICAM-1,were analyzed by Western blot.MTT and flow cytometry assays were used to detect the viability and apoptosis of the cells to test anti-apoptosis function of HDL.Results The levels of low-density lipoprotein,triglycerides and total cholesterol were higher in elder female group than in other three groups (all P＜0.05).The level of HDL-C was higher in young and middle-aged females than in other three groups(all P＜0.05).The level of MDA was higher in elder female group than in other three groups(all P＜0.05).The level of MDA was higher in elder male group than in the young and middle-aged male and female groups(all P＜0.05).After protection of HDL,the number of monocytes adhesion and expression levels of VCAM-1 and ICAM-1 were higher in elder groups than in young and middle-aged male and female groups(all P＜ 0.05).Relative survival and viability rates of endothelial cells were higher in young and middle-aged groups than in elder groups (all P＜0.05).Conclusions Ageing in both male and female induces impairments of anti-oxidative,anti-inflammatory and anti-apoptosis functions of HDL,with more evident decrease in anti-oxidative function in females.

AIM:To investigate the effect of quercetin on the biological functions of rat bone marrow-derived endothelial progenitor cells (EPCs) and its potential mechanisms.METHODS:The bone marrow-derived mononuclear cells of Sprague-Dawley rats were isolated by density gradient centrifugation.The differentiated EPCs were cultured specially and stained with DiI-Ac-LDL and FITC-UEA-1.CD133+ and FLK-1+ were detected on the cell surfaces.After 14 d, the EPCs were incubated with a PI3K inhibitor BYL719 (3 μmol/L) and an ERK inhibitor FR180204 (15 μmol/L).After incubation of the inhibitors for 2 h, the cells were treated with quercetin at different concentrations (0, 10, 20, 40, 80 and 100 μmol/L).MTT assay and Transwell assay were used to detect cell viability and the number of migratory cells.The protein levels of AKT, eNOS, ERK and their phosphorylated status were determined by Western blot.RESULTS:Quercetin enhanced the viability and migration of the EPCs at a dose-dependent manner.However, the PI3K inhibitor BYL719 suppressed the QUE-induced cell viability and migration.Moreover, ERK inhibitor FR180204 exerted the similar inhibitory effect on the cell viability but had no effect on cell migration.Quercetin activated the phosphorylation of AKT, eNOS and ERK.On the other hand, BYL719 was observed to inhibit the phosphorylation of AKT and ERK.FR180204, however, was showed to inhibit the phosphorylation of ERK only.On the contrast, the stimulatory effects that quercetin exerted on the expression of eNOS and its phosphorylation were suppressed by BYL719 and FR180204.CONCLUSION:Quercetin stimulates the viability and migration of EPCs via PI3K/AKT/eNOS and ERK/eNOS signaling pathway, which would be beneficial for cardiovascular health.

AIM:To investigate the inhibitory effect of ethanol extract of propolis (EEP) on oxidized low-den-sity lipoprotein ( ox-LDL )-induced macrophage apoptosis and the underlying molecular mechanisms . METHODS:RAW264.7 macrophages were pretreated with EEP (7.5, 15 and 30 mg/L), 4-phenylbutyric acid (PBA, 5 mmol/L) or diphenyleneiodonium ( DPI, 5μmol/L) for 1 h and then treated with ox-LDL (100 mg/L) or tunicamycin ( TM, 4 mg/L) for 24 h.The cell viability and apoptosis were determined by MTT assay and Annexin V-FITC apoptosis detection kit , re-spectively.The activity of superoxide dismutase (SOD), and the levels of reactive oxygen species (ROS) and malondial-dehyde (MDA) in the cells were measured.The protein levels of caspase-12, a proapoptotic molecule under endoplasmic reticulum stress ( ERS) , were examined by Western blot analysis .RESULTS:Like PBA ( an ERS inhibitor ) , EEP pro-tected RAW264.7 macrophages from ox-LDL-induced injury in a dose-dependent manner , as assessed by the increased cell viability and the decreased apoptotic rate .The decrease in cell viability and increase in apoptotic rate induced by TM , an ERS inducer, were also attenuated by EEP .Moreover, EEP suppressed ox-LDL-induced oxidative stress as revealed by the decreased generation of ROS and MDA as well as elevated SOD activity , which were similar to DPI , an oxidative stress in-hibitor.Furthermore, EEP significantly suppressed ox-LDL-or TM-induced activation of caspase-12.Similar results were observed in the cells pretreated with PBA or DPI and then treated with ox-LDL.CONCLUSION: EEP may protect RAW264.7 macrophages from ox-LDL-induced apoptosis and the mechanism is at least partially involved in the ability of EEP to suppress oxidative stress and subsequent activation of caspase -12.

[ ABSTRACT] AIM: To investigate the protective effect of autophagy on oxidized low density lipoprotein ( ox-LDL)-induced macrophage apoptosis and the underlying molecular mechanisms .METHODS:The RAW264.7 macropha-ges were pretreated with 3 mmol/L 3-methyladenine (3-MA), 1 μmol/L rapamycin (Rap) or 4 mmol/L 4-phenylbutyric acid ( PBA) respectively for 1 h and then treated with ox-LDL (100 mg/L) for 12 h.The cell viability and apoptosis were determined by MTT assay and flow cytometry with Annexin V-FITC/PI staining, respectively.The activities of lactate de-hydrogenase ( LDH) in the medium and caspase-3 in the cells were determined by detection kits .The protein levels of bec-lin-1 (a molecular marker of autophagy ), glucose-regulated protein 78 (GRP78, an endoplasmic reticulum stress marker) and C/EBP homologous protein ( CHOP, a key-signaling component of endoplasmic reticulum stress-induced apoptosis ) were examined by Western blot .Microtubule-associated protein 1 light chain 3 (LC3, another molecular marker of autoph-agy) was observed under laser scanning confocal microscope .RESULTS: Treatment of the RAW264.7 macrophages with ox-LDL at 100 mg/L for 12 h resulted in significant decrease in cell viability , and dramatic elevation in LDH leakage , cell apoptosis and caspase-3 activity, which were promoted by 3-MA (an autophagy inhibitor) and inhibited by Rap (an autoph-agy inducer ) .ox-LDL induced autophagy in the macrophages as assessed by beclin-1 upregulation and frequent granulation of LC3, which were inhibited by 3-MA and promoted by Rap.Interestingly, 3-MA enhanced, while Rap blocked, the CHOP upregulation induced by ox-LDL.Moreover , PBA ( endoplasmic reticulum stress inhibitor ) significantly inhibited ox-LDL-induced GRP78 upregulation and autophagy as determined by the attenuation of beclin-1 upregulation and frequent granula-tion of LC3.CONCLUSION: Endoplasmic reticulum stress mediates ox-LDL-induced autophagy in macrophages , and moderates activation of autophagy may protect macrophages from ox-LDL-induced apoptosis by inhibiting CHOP expression .

[ABSTRACT]AIM:ToinvestigatetheeffectofD4F,anapolipoproteinA-Imimeticpeptide,onoxidizedlow-density lipoprotein ( ox-LDL)-induced macrophage apoptosis and activation of caspase-12, a key molecule in endoplasmic reticulum stress ( ERS )-associated apoptotic pathway, and to elucidate the underlying molecular mechanisms. METHODS:RAW264.7 macrophages were pretreated with D4F (12.5, 25 and 50 mg/L), 4-phenylbutyric acid (PBA, 5 mmol/L) or diphenyleneiodonium ( DPI, 5 μmol/L) for 1 h and then treated with ox-LDL (100 mg/L) or tunicamycin ( TM, 4 mg/L) for 24 h.The cell viability and apoptosis were determined by MTT assay and TUNEL detection, respective-ly.The levels of malondialdehyde ( MDA) and reactive oxygen species ( ROS) in the cells and the activities of superoxide dismutase ( SOD) and nicotinamide adenine dinucleotide phosphate ( NADPH) oxidase were determined.The protein level of caspase-12 was examined by Western blot analysis.RESULTS: Similar to the ERS inhibitor PBA, D4F protected RAW264.7 macrophages from ox-LDL or TM ( an ERS inducer)-induced decrease in the viability and increase in apoptotic rate in a dose-dependent manner.Like DPI (an oxidative stress inhibitor), D4F significantly inhibited ox-LDL-induced ox-idative stress, as expressed by the decreased generation of ROS and MDA ( P<0.01) , the increased activity of SOD and the decreased activity of NADPH oxidase (P<0.05).Moreover, similar to PBA and DPI, D4F significantly suppressed ox-LDL-induced activation of caspase-12 in a concentration-dependent manner ( P<0.05) .Furthermore, D4F also inhibi-ted the caspase-12 activation induced by TM (P<0.05).CONCLUSION: D4F inhibits macrophage apoptosis induced by ox-LDL, and the mechanism is at least partially by reducing oxidative stress and inhibiting the activation of caspase-12.