OBJECTIVETo investigate neuroprotection of resveratrol against cytotoxicity of oxidized low density lipoprotein (oxLDL) in PC12 cells.

METHODSPC12 cells were pretreated with resveratrol for one hour as a neuron model and then exposed to oxLDL at varied concentrations. Effects of resveratrol on cell viability, permeability of cell membrane, cell nucleus and activity of caspase-3 were evaluated with MTT assay, lactic acid dehydrogenase (LDH) release assay, DNA fragmentation (TUNEL) assay and assay for caspase-3 activity (caspase assay).

RESULTSCell viability, LDH release rate, percentage of cells with positive TUNEL and activity of caspase-3 were (62 +/- 3)%, (23 +/- 3)%, (26 +/- 5)% and (0.811 +/- 0.049) mol.min(-1).microg(-1), vs (84 +/- 7)%, (13 +/- 4)%, (12 +/- 4)% and (0.553 +/- 0.048) mol.min(-1).microg(-1) in PC12 cells treated with 10 mg/L oxLDL vs in those treated with 10 mg/L oxLDL plus 50 micromol/L resveratrol, respectively, with statistically significant difference.

CONCLUSIONResveratrol could attenuate cytotoxicity induced by oxLDL in PC12 cells with neuroprotection.

Animals ; Cell Survival ; drug effects ; Lipoproteins, LDL ; toxicity ; Neuroprotective Agents ; pharmacology ; PC12 Cells ; Rats ; Stilbenes ; pharmacology

Lipoprotein lipase (LPL) is known to be attached to the luminal surface of vascular endothelial cells in a complex with membrane-bound heparan sulfate, and released into blood stream by heparin. LPL that catalyzes hydrolysis of triglyceride (TGL) on chylomicron and VLDL into two fatty acids and monoacylglycerol, is also implicated to participate in an enhancement of cholesterol uptake by arterial endothelial cells in vitro. But little is known about the LPL-mediated cholesterol uptake in physiological state. In this study, changes in blood lipid composition and levels of lipoproteins were determined after the injection of heparin in human. The level of LPL in plasma was increased from 0 to 11 mU/ml within 30-40 min post-heparin administration and decreased to the basal level within 2 h. The level of TGL in plasma decreased from 70 mg/dl to 20 mg/dl within 1 h and gradually increased to 80 mg/dl within 4 h. However the level of total cholesterol in plasma remained at 140 mg/dl during an experimental period of 4 h. Analysis of Lipoproteins in plasma by NaBr density gradient ultracentrifugation showed that the level of VLDL decreased from 50 mg/dl to 10 mg/dl within 1-2 h and returned to normal plasm level at 4 h. However there were no significant changes in the level of LDL and HDL. These results suggest that, at least, in normo-lipidemic subjects, increased free plasm LPL acts primarily on VLDL and failed to show any significant uptake of cholesterol-rich lipoproteins in human.
Adult ; Cholesterol/blood ; Heparin/pharmacology* ; Heparin/administration & dosage ; Human ; Immunoblotting ; Lipoprotein Lipase/blood* ; Lipoproteins/blood* ; Lipoproteins, HDL/blood ; Lipoproteins, LDL/blood ; Lipoproteins, VLDL/blood ; Triglycerides/blood

To observe the effect of oxidized low density lipoprotein (OxLDL) on arterial endothelial cells apoptosis in vivo, we established a model in which Sprague-Dawley rats were given intraperitoneal and intravenous injection of unmodified LDL (8 mg/kg every day) via the tail vein. Seven days after the injection, the aortic endothelial cells specimens were prepared by an en face preparation of rat aorta. The apoptotic cells were identified and counted by in situ nick and labelling (TUNEL) method and light microscopy. The numbers of the apoptotic cells were 12.52 +/- 4.71/field in the intraperitoneal injection control group, 11.41 +/- 2.94/field in the intravenous injection control group, 22.98 +/- 8.01/field in the intraperitoneal injection LDL group and 103.8 +/- 11.5/field in the intravenous injection LDL group, respectively. The difference was significant between injection LDL group and control (P < 0.01), and the difference was also significant between two LDL injection groups (P < 0.01). These findings suggest that injection of LDL can induce apoptosis in arterial endothelial cells and the effect is especially significant with intravenous injection LDL. After injection, oxidative modification of LDL may occur in local arteries and causes injury to the endothelial cells.
Aorta ; Apoptosis/*drug effects ; Endothelium, Vascular/*pathology ; In Situ Nick-End Labeling ; Injections, Intraperitoneal ; Injections, Intravenous ; Lipoproteins, LDL/*metabolism ; Lipoproteins, LDL/*pharmacology ; Oxidation-Reduction ; Random Allocation ; Rats, Sprague-Dawley

Animals ; Arteriosclerosis ; drug therapy ; Cholesterol, LDL ; blood ; Drugs, Chinese Herbal ; pharmacology ; Hyperlipidemias ; drug therapy ; Lipids ; blood ; Lipoproteins ; blood ; Lipoproteins, LDL ; blood ; Male ; Rabbits ; Tablets ; Triglycerides ; blood

The preparation of a novel amphiphilic dietary fiber LDL adsorbent with sulfonic group and laurylamine group was studied. The effects of reaction time and reaction temperature on the adsorption rate were studied. The results show that the adsorption rates for the removal of Total cholesterol (TC), Low-density lipoprotein cholesterol (LDL-C) and High-density lipoprotein cholesterol (HDL-C) are 40.8%, 50.8% and 23.6%, respectively. The amphiphilic adsorbent has better selectivity in removing LDL-C.
Adsorption ; Binding, Competitive ; Cholesterol ; blood ; isolation & purification ; Dietary Fiber ; pharmacology ; Lipoproteins, LDL ; blood ; isolation & purification

OBJECTIVE: To investigate the effect of reinioside C (RC) on the expression of lectin-like oxidized low density lipoprotein receptor (LOX)-1 mRNA and LOX-1 protein induced by oxidized low density lipoprotein (ox-LDL) in cultured human umbilical vein endothelial cells (HUVEC). METHODS: HUVECs were cultured with ox-LDL (50 mg/L) for 24 h in the absence or presence of RC (1, 3, and 10 micromol/L). The expressions of LOX-1 mRNA and LOX-1 protein were examined by RT-PCR and Western-blot. RESULTS: Incubation with ox-LDL (50 mg/L) significantly raised the expression of LOX-1 mRNA and LOX-1 protein,which was concentration-dependent. CONCLUSION RC can inhibit the increased expression of LOX-1 mRNA and LOX-1 protein induced by ox-LDL in HUVECs.
Cells, Cultured ; Drugs, Chinese Herbal ; pharmacology ; Endothelium, Vascular ; Humans ; Lipoproteins, LDL ; pharmacology ; Polygala ; chemistry ; RNA, Messenger ; biosynthesis ; genetics ; Receptors, LDL ; biosynthesis ; genetics ; Saponins ; pharmacology ; Umbilical Veins ; cytology ; metabolism

The role of very low density lipoprotein receptor (LVLDR) in the process of foam cell formation was investigated. After the primary cultured mouse peritoneal macrophages were incubated with VLDL, beta-VLDL or low density lipoprotein (LDL), respectively for 24 h and 48 h, foam cells formation was identified by oil red O staining and cellular contents of triglyceride (TG) and total cholesterol (TC) were determined. The mRNA levels of LDLR, LDLR related protein (ILRP) and VLDLR were detected by semi-quantitative RT-PCR. The results demonstrated that VLDL, beta-VLDL and LDL could increase the contents of TG and TC in macrophages. Cells treated with VLDL or beta-VLDL showed markedly increased expression of VLDLR and decreased expression of LDLR, whereas LRP was up-regulated slightly. For identifying the effect of VLDL receptor on cellular lipid accumulation, ldl-A7-VR cells, which expresses VLDLR and trace amount of LRP without functional LDLR, was used to incubate with lipoproteins for further examination. The results elucidated that the uptake of triglyceride-rich lipoprotein mediated by VLDLR plays an important role in accumulation of lipid and the formation of foam cells.
Animals ; Arteriosclerosis ; metabolism ; pathology ; Cells, Cultured ; Cholesterol, LDL ; metabolism ; pharmacology ; Female ; Foam Cells ; cytology ; metabolism ; Lipoproteins, VLDL ; pharmacology ; Macrophages, Peritoneal ; cytology ; metabolism ; Mice ; Receptors, LDL ; metabolism ; Triglycerides ; metabolism

OBJECTIVE: : To investigate the effect of chlorogenic acid (CGA) on non-enzymatic glycation and oxidation of low density lipoprotein (LDL). METHODS: : The non-enzymatic glycation incubation system of LDL-glucose was established. The contents of early glycation products (Amodori product) and intermediate products (dicarbonyl compound) were determined by ultraviolet-visible spectrophotometry, and the content of advanced glycation end products (AGEs) was determined by fluorescence spectrophotometry. The LDL oxidation incubation system was established. The contents of thiobarbituric acid reactive substances(TBARS) and conjugated diene were determined by ultraviolet-visible spectrophotometry. The tryptophan fluorescence quenching, and the content of lipofuscin, total fluorescence products, active aldehydes and malondialdehyde were determined by fluorescence spectrophotometry, and further verified by three-dimensional fluorescence spectroscopy. RESULTS: : In the LDL glycation experiment, 150 μg/mL and 300 μg/mL CGA inhibited the formation of Amadori product, dicarbonyl compounds and AGEs. In the LDL oxidation experiment, 15 μg/mL and 25 μg/mL CGA inhibited the formation of TBARS effectively; 5 μg/mL and 10 μg/mL CGA inhibited tryptophan fluorescence quenching, and the formation of active aldehydes, malondialdehyde, total fluorescence products, lipofuscin and conjugated diolefine. And the three-dimensional fluorescence spectroscopy showed the same results. CONCLUSIONS : CGA can inhibit non-enzymatic glycation and oxidation of LDL.
Chlorogenic Acid ; pharmacology ; Glycosylation ; drug effects ; Lipoproteins, LDL ; metabolism ; Oxidation-Reduction ; drug effects ; Thiobarbituric Acid Reactive Substances ; analysis

The purpose of the present study was to investigate the effect of pravastatin on cholesteryl esters in foam cells of murine macrophages and the relation with caveolin-1. RAW 264.7 murine macrophages were coincubated with 80 mg/L oxidized low density lipoprotein (ox-LDL) and pravastatin (0~100 mumol/L) respectively for 24 h. When the best control concentration of pravastatin was confirmed, RAW 264.7 murine macrophages were coincubated with 80 mg/L ox-LDL and pravastatin of the best concentration respectively for 0, 6, 12, 24 h. Oil red O dyeing experiment was used to show the lipid droplets in foam cells. High performance liquid chromatography (HPLC) analysis was performed to determine the content of cellular cholesterol. The level of caveolin-1 was determined by Western blot analysis. The result showed that when macrophages were incubated with 80 mg/L ox-LDL, the ratio of cellular cholesteryl ester to total cholesterol (CE/TC) was beyond 50% through HPLC analysis, and a great deal of lipid droplets displayed in cells through Oil red O dyeing experiment, which manifested the formation of the foam cells. Pravastatin could decrease CE in foam cells in a concentration-dependent manner (1~100 mumol/L). At the concentration of 100 mumol/L, pravastatin decreased cellular CE more than 50%. The effects of pravastatin on the decrease of CE in murine macrophages also displayed a time-dependent manner (incubated with 100 mumol/L pravastatin from 6 to 24 h). Moreover, the expression of caveolin-1 was decreased when the macrophages were incubated with ox-LDL (80 mg/L), while treatment with pravastatin increased the level of caveolin-1 and displayed a concentration- and time-dependent manner. These results suggest that pravastatin could inhibit the development of foam cells through the decrease of cellular CE, which may be related to the upregulation of caveolin-1.
Animals ; Anticholesteremic Agents ; pharmacology ; Caveolin 1 ; metabolism ; Cell Line ; Cells, Cultured ; Cholesterol Esters ; metabolism ; Foam Cells ; metabolism ; Lipoproteins, LDL ; pharmacology ; Macrophages ; cytology ; Pravastatin ; pharmacology ; Rats

BackgroundOxidized low-density lipoprotein (ox-LDL)-induced oxidative stress and endothelial apoptosis are essential for atherosclerosis. Our previous study has shown that ox-LDL-induced apoptosis is mediated by the protein kinase RNA-like endoplasmic reticulum kinase (PERK)/eukaryotic translation initiation factor 2α-subunit (eIF2α)/CCAAT/enhancer-binding protein homologous protein (CHOP) endoplasmic reticulum (ER) stress pathway in endothelial cells. Statins are cholesterol-lowering drugs that exert pleiotropic effects including suppression of oxidative stress. This study aimed to explore the roles of simvastatin on ox-LDL-induced ER stress and apoptosis in endothelial cells.

MethodsHuman umbilical vein endothelial cells (HUVECs) were treated with simvastatin (0.1, 0.5, or 2.5 μmol/L) or DEVD-CHO (selective inhibitor of caspase-3, 100 μmol/L) for 1 h before the addition of ox-LDL (100 μg/ml) and then incubated for 24 h, and untreated cells were used as a control group. Apoptosis, expression of PERK, phosphorylation of eIF2α, CHOP mRNA level, and caspase-3 activity were measured. Comparisons among multiple groups were performed with one-way analysis of variance (ANOVA) followed by post hoc pairwise comparisons using Tukey's tests. A value of P < 0.05 was considered statistically significant.

ResultsExposure of HUVECs to ox-LDL resulted in a significant increase in apoptosis (31.9% vs. 4.9%, P < 0.05). Simvastatin (0.1, 0.5, and 2.5 μmol/L) led to a suppression of ox-LDL-induced apoptosis (28.0%, 24.7%, and 13.8%, F = 15.039, all P < 0.05, compared with control group). Ox-LDL significantly increased the expression of PERK (499.5%, P < 0.05) and phosphorylation of eIF2α (451.6%, P < 0.05), if both of which in the control groups were considered as 100%. Simvastatin treatment (0.1, 0.5, and 2.5 μmol/L) blunted ox-LDL-induced expression of PERK (407.8%, 339.1%, and 187.5%, F = 10.121, all P < 0.05, compared with control group) and phosphorylation of eIF2α (407.8%, 339.1%, 187.5%, F = 11.430, all P < 0.05, compared with control group). In contrast, DEVD-CHO treatment had no significant effect on ox-LDL-induced expression of PERK (486.4%) and phosphorylation of eIF2α (418.8%). Exposure of HUVECs to ox-LDL also markedly induced caspase-3 activity together with increased CHOP mRNA level; these effects were inhibited by simvastatin treatment.

ConclusionsThis study suggested that simvastatin could inhibit ox-LDL-induced ER stress and apoptosis in vascular endothelial cells.

Apoptosis ; drug effects ; Cells, Cultured ; Endoplasmic Reticulum Stress ; drug effects ; Human Umbilical Vein Endothelial Cells ; drug effects ; metabolism ; Humans ; Lipoproteins, LDL ; pharmacology ; Oligopeptides ; pharmacology ; Simvastatin ; pharmacology