This study explores the regulatory effect of astragaloside Ⅳ on miR-17-5 p and its downstream proprotein convertase subtillisin/kexin type 9(PCSK9)/very low density lipoprotein receptor(VLDLR) signal pathway, aiming at elucidating the mechanism of astragaloside Ⅳ against atherosclerosis(AS). In cell experiment, oxidized low-density lipoprotein(ox-LDL) was used for endothelial cell injury modeling with vascular smooth muscle cells(VSMCs). Then cells were classified into the model group, miR-17-5 p inhibitor group, blank serum group, and astragaloside Ⅳ-containing serum group based on the invention. Afterward, cell viability and the expression of miR-17-5 p, VLDLR, and PCSK9 mRNA and protein in cells in each group were detected. In animal experiment, 15 C57 BL/6 mice were used as the control group, and 45 ApoE~(-/-) mice were classified into the model group, miR-17-5 p inhibitor group, and astragaloside Ⅳ group, with 15 mice in each group. After 8 weeks of intervention, the peripheral serum levels of interleukin-6(IL-6), interleukin-10(IL-10), and tumor necrosis factor-α(TNF-α), and the expression of miR-17-5 p, VLDLR, and PCSK9 mRNA in the aorta of mice were detected. The pathological changes of mice in each group were observed. According to the cell experiment, VSMC viability in the miR-17-5 p inhibitor group and the astragaloside Ⅳ-containing serum group was higher than that in the model group(P<0.05). The mRNA and protein expression of miR-17-5 p and VLDLR in VSMCs in the miR-17-5 p inhibitor group and the astragaloside Ⅳ-containing serum group was lower than that in the model group(P<0.05), but the mRNA and protein expression of PCSK9 was higher than that in the model group(P<0.05). As for the animal experiment, the levels of IL-6 and TNF-α in the peripheral serum of the miR-17-5 p inhibitor group and the astragaloside Ⅳ group were lower(P<0.05) and the serum level of IL-10 was higher(P<0.05) than that of the model group. The mRNA expression of miR-17-5 p and VLDLR in the aorta in the miR-17-5 p inhibitor group and the astragaloside Ⅳ group was lower(P<0.05), and PCSK9 mRNA expression was higher(P<0.05) than that in the model group. Pathological observation showed mild AS in the miR-17-5 p inhibitor group and the astragaloside Ⅳ group. In summary, astragaloside Ⅳ can prevent the occurrence and development of AS. The mechanism is that it performs targeted regulation of miR-17-5 p, further affecting the PCSK9/VLDLR signal pathway, inhibiting vascular inflammation, and thus alleviating endothelial cell injury.
Animals ; Atherosclerosis/genetics* ; Lipoproteins, LDL/metabolism* ; Mice ; MicroRNAs/metabolism* ; Proprotein Convertase 9/metabolism* ; Receptors, LDL/metabolism* ; Saponins ; Signal Transduction ; Triterpenes

OBJECTIVETo investigate the short- and long-term effects of Xuezhikang (XZK), an extract of cholestin, on proprotein convertase subtilisin/kexin type 9 (PCSK9) level.

METHODSThirty rats were randomly divided into three groups and were given saline, XZK 1,200 mg/kg or lovastatin 10 mg/kg respectively by daily gavage for 3 days (n=10 for each). Sixteen patients without previous lipid-lowering drug treatment for dyslipidemia received XZK 1,200 mg daily for 8 weeks. Fasting blood samples and liver tissue were collected at day 3 for rats, while the blood samples were obtained at baseline and week 8 from patients. The serum PCSK9 and lipid profile were measured. The expression of hepatic low density lipoprotein (LDL) receptor and sterol regulatory element binding protein 2 (SREBP-2) were measured by real time-PCR.

RESULTSPCSK9 levels in rats were significantly increased in the XZK and lovastatin groups (P=0.002, P=0.003 vs. control) at day 3, while no significant differences were found in the levels of lipid parameters. PCSK9 levels in patients increased by 34% (P=0.006 vs. baseline) accompanied by total cholesterol and LDL-cholesterol decreased by 22% and 28% P=0.001, P=0.002 vs. baseline). The hepatic mRNA levels of LDL-receptor and SREBP-2 were significantly increased in the XZK and lovastatin groups.

CONCLUSIONXZK has significant impact on PCSK9 in a short- and long-term manner in both rats and humans. Moreover, the data indicated that as lovastatin, XZK increased PCSK9 levels through SREBP-2 pathway.

Animals ; Biological Products ; chemistry ; Drugs, Chinese Herbal ; pharmacology ; Female ; Humans ; Lipids ; blood ; Male ; Middle Aged ; Proprotein Convertase 9 ; blood ; Rats, Sprague-Dawley ; Receptors, LDL ; genetics ; metabolism ; Sterol Regulatory Element Binding Protein 2 ; genetics ; metabolism ; Time Factors

To study the effect of cholesterol and 25-OH-cholesterol on cholesterol metabolism in HepG2 cells and the effect of coptisine (Cop) extracted from Coptidis Rhizoma (CR) in reducing and regulating cholesterol. In this study, TC, TG, LDL-c and HDL-c were measured by biochemical analysis; mRNA and protein expressions of LDLR, HMGCR and CYP7A1 were detected by qRT-PCR and Western blot. According to the results, cholesterol and 25-OH-cholesterol inducing could decrease in mRNA and protein expressions of LDLR and CYP7A1, so as to increase TC and LDL-c contents. However, Cop could up-regulate mRNA and protein expressions of LDLR and CYP7A1 and down-regulate that of HMGCR, so as to reduce TC and LDL-c levels. These findings suggested that Cop has potential pharmacological activity for reducing cholesterol, and may reduce cholesterol by regulating mRNA and protein expressions of key genes involved in cholesterol metabolism, such as LDLR, CYP7A1 and HMGCR. This study laid a firm theoretical foundation for developing new natural drugs with the cholesterol-lowering activity.
Berberine ; analogs & derivatives ; pharmacology ; Cholesterol ; metabolism ; Cholesterol 7-alpha-Hydroxylase ; genetics ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Gene Expression Regulation, Enzymologic ; drug effects ; Hep G2 Cells ; Humans ; Hydroxymethylglutaryl CoA Reductases ; genetics ; metabolism ; Receptors, LDL ; genetics ; metabolism ; Triglycerides ; metabolism

The effect of thymic stromal lymphopoietin (TSLP) on macrophage-derived foam cell formation and the underlying mechanism were studied. Macrophages isolated from C57BL/6 mice were co-cultured in vitro with different concentrations of TSLP or TSLPR-antibody in the presence of oxidized low density lipoprotein (ox-LDL). The effects of TSLP on macrophage-derived foam cell formation were observed by using oil red O staining and intracellular lipid determination. The expression levels of foam cell scavenger receptors (CD36 and SRA) as well as ABCA1 and TSLPR were detected by using RT-PCR and Western blotting. As compared with the control group, TSLP treatment significantly promoted lipid accumulation in macrophages, significantly increased protein expression of CD36 and TSLPR in a dose-dependent manner, and significantly reduced the expression of ABCA1 protein in a dose-dependent manner. No significant differences were noted between the TSLPR-antibody group and the control group. TSLP may down-regulate the expression of cholesterol efflux receptor ABCA1 and up-regulate scavenger receptor expression via the TSLPR signaling pathway, thereby promoting macrophage-derived foam cell formation.
ATP Binding Cassette Transporter 1 ; genetics ; metabolism ; Animals ; Antibodies ; immunology ; pharmacology ; Blotting, Western ; CD36 Antigens ; genetics ; metabolism ; Cells, Cultured ; Cholesterol ; metabolism ; Cholesterol Esters ; metabolism ; Cytokines ; pharmacology ; Dose-Response Relationship, Drug ; Foam Cells ; cytology ; drug effects ; metabolism ; Gene Expression ; drug effects ; Immunoglobulins ; immunology ; metabolism ; Lipoproteins, LDL ; pharmacology ; Macrophages ; cytology ; drug effects ; metabolism ; Mice ; Mice, Inbred C57BL ; Receptors, Cytokine ; immunology ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Scavenger Receptors, Class A ; genetics ; metabolism

In the previous study, a high-throughput screening method was established to find the antagonists of CD36. In the present study, a new compound named IMB-1680 was found using this method. The anti-atherosclerotic activities of IMB-1680 were then evaluated. Dose-dependent activities of IMB-1680 were detected by using Sf9 [hCD36] and CHO [hCD36] models. Fluorescence microscopic photography and flow cytometry were used to analyze uptake of mLDL. Foam cell test with RAW264.7 macrophages was used to examine lipid accumulation. The results showed that IMB-1680 inhibited CD36 activity with IC50 of 2.80 and 8.79 micromol x L(-1) in Sf9[hCD36] and CHO [hCD36] cells, respectively. Fluorescence microscopic photography and flow cytometry revealed that IMB-1680 could significantly reduce DiI-AcLDL uptake. Meanwhile, IMB-1680 also could reduce lipids accumulation in RAW264.7 macrophages. In all, the data indicated that IMB-1680 might be a potent effective anti-atherosclerotic leading compound.
Animals ; CD36 Antigens ; antagonists & inhibitors ; genetics ; metabolism ; CHO Cells ; Cells, Cultured ; Cricetulus ; Dose-Response Relationship, Drug ; Foam Cells ; cytology ; High-Throughput Screening Assays ; Humans ; Lipoproteins, LDL ; metabolism ; Macrophages ; cytology ; metabolism ; Mice ; Molecular Structure ; Plasmids ; Receptors, Scavenger ; antagonists & inhibitors ; Sf9 Cells ; Spodoptera ; Transfection

To study the effects of alkaloids from Coptidis Rhizoma on low-density lipoprotein receptor (LDLR) mRNA expression and antihyperlipedemic levels. The LDLR mRNA expression were detected by real time fluorescence quantitative PCR, and the levels of total cholesterol (TC), triglyceride (TG), low density lipoprotein (LDL-c) and high-density lipoprotein cholesterol (HDL-c) in serum were measured at the first and last examination. The results show that, after the drug treatment, compared with the model group, each drug group showed a lipid-lowering effect. Especially, coptisine, palmatine, jatrorrhinze were significantly reduced TC, TG, LDL-c (P < 0.05, P < 0.01), and increased HDL-c (P < 0.01). In addition, they also increased mRNA expression of the LDLR in liver and HepG2 cells. The results showed that alkaloids from Coptidis Rhizoma can regulate lipid metabolism disorder, and coptisine have the best lipid-lowering effect.
Alkaloids ; administration & dosage ; Animals ; Cholesterol ; metabolism ; Cricetinae ; Drugs, Chinese Herbal ; administration & dosage ; Humans ; Hyperlipidemias ; drug therapy ; genetics ; metabolism ; Hypoglycemic Agents ; administration & dosage ; Lipid Metabolism ; drug effects ; Lipids ; blood ; Lipoproteins, LDL ; metabolism ; Mesocricetus ; Receptors, Lipoprotein ; genetics ; metabolism ; Triglycerides ; metabolism

OBJECTIVETo investigate the effect of Antrodia cinnamomea on gene expression related to aortal endothelial injury of rats with hyperlipidemia.

METHODFifty SD rats were randomly divided into five groups: the normal control group (NG), the model group (MG), the antrodia cinnamomea groups of low, middle and high doses (AC-LG, AC-MG, AC-HG, 250, 500, 1 000 mg x kg(-1)). The rats were fed with high-fat diets to establish the hyperlipidemia model. After the drug administration for 10 weeks, their serum lipid, SOD, MDA and ox-LDL, LOX-1, P38 MAPK and NF-kappaB mRNA and protein expression were respectively determined, and the aortal endothelial injury was observed under electron microscope.

RESULTIn the model group, the contents of TC, TG and LDL-C significant increased (P < 0.01), whereas the content of HDL-C significant decreased (P < 0.01). Compared with the model group, both the AC-M group and the AC-H group showed reduction in endothelial injury and significant decrease in the content of TC, TG and LDL-C (P < 0.05 or P < 0.01). The content of HDL-C increased, but with no significant difference. SOD activity in serum remarkably increased (P < 0.05 or P < 0.01), MDA and ox-LDL levels dramatically decreased (P < 0.05 or P < 0.01).

CONCLUSIONA. cinnamomea can alleviate endothelial lipid injury by inhibiting the expressions of LOX-1, P38MAPK and NF-kappaB in aorta and better protect aortal endothelial cells from oxidative lipid injury.

Animals ; Antrodia ; chemistry ; Aorta ; drug effects ; metabolism ; ultrastructure ; Atherosclerosis ; blood ; genetics ; prevention & control ; Biological Products ; pharmacology ; Cholesterol ; blood ; Cholesterol, HDL ; blood ; Cholesterol, LDL ; blood ; Endothelium, Vascular ; drug effects ; metabolism ; pathology ; Enzyme-Linked Immunosorbent Assay ; Gene Expression ; drug effects ; Hyperlipidemias ; blood ; genetics ; prevention & control ; Lipoproteins, LDL ; blood ; Male ; Malondialdehyde ; blood ; Microscopy, Electron ; NF-kappa B ; blood ; genetics ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; Scavenger Receptors, Class E ; blood ; genetics ; metabolism ; Superoxide Dismutase ; blood ; Triglycerides ; blood ; p38 Mitogen-Activated Protein Kinases ; blood ; genetics ; metabolism

OBJECTIVETo explore action mechanism of electroacupuncture for coronary atherosclerotic heart disease (CHD) in order to provide experimental support for clinical acupoint selection.

METHODSAmong sixty clean-grade healthy male Wistar rats, twenty-four cases were randomly selected as a normal control group and an electroacupuncture (EA) preconditioning group, 12 cases in each one. Then rats in the EA preconditioning group and the rest 36 rats were fed with high fat diet for 12 weeks to duplicate the CHD model. When the models were successfully established, the rats were randomly divided into a model control group, an EA group and a medication group, 12 cases in each one. EA was applied with Hwa-to SDZ-IV apparatus in the EA preconditioning group at "Neiguan" (PC 6) and "Xinshu" (BL 15), 1 mA in current intensity, 2 Hz in frequency, 30 min per times, once every other day for 14 weeks. When model was established, the same acupoint and method was used in the EA group for 2 weeks while intragastric administration of atorvastatin mixed suspension, 0.25 mg/kg, once a day, was applied in the medication group for 2 weeks. The content of oxidized low-density lipoprotein (oxLDL) in the serum was tested by double antibody enzyme-linked immunosorbent assay (ELISA) while content of lectin-like oxidized low density lipoprotein receptor-1 (LOX-1) in coronary arterial tissue was test by western blot method. Expression of LOX-1 mRNA was tested by fluorogenic quantitative polymerase chain reaction (PCR).

RESULTSAfter model was duplicated successfully, the content of oxLDL in the serum and the expression of LOX-1 and its mRNA in coronary arterial tissue in the model control group were increased significantly compared with those in the normal control group (all P < 0.01). Compared with the model control group, the content of oxLDL in the serum and the expression of LOX-1 and its mRNA in coronary arterial tissue in the EA preconditioning group, EA group and medication group were significantly reduced (all P < 0.01).

CONCLUSIONThe electroacupuncture at "Neiguan" (PC 6) and "Xinshu" (BL 15) could effectively reduce the content of oxLDL in the serum and expression of LOX-1 and its mRAN in coronary arterial tissue in CHD rats. The oxidative modificatory low-density lipoprotein and its specific receptor system could be one of the ways to prevention and treatment of acupuncture for CHD.

Animals ; Coronary Disease ; genetics ; metabolism ; therapy ; Disease Models, Animal ; Electroacupuncture ; Humans ; Lipoproteins, LDL ; genetics ; metabolism ; Male ; Rats ; Rats, Wistar ; Scavenger Receptors, Class E ; genetics ; metabolism

KR-31543, (2S, 3R, 4S)-6-amino-4-[N-(4-chlorophenyl)-N-(2-methyl-2H-tetrazol-5-ylmethyl) amino]-3,4-dihydro-2-dimethyoxymethyl-3-hydroxy-2-methyl-2H-1-benz opyran is a new neuroprotective agent for ischemia-reperfusion damage. It has also been reported that KR-31543 has protective effects on lipid peroxidation and H2O2-induced reactive oxygen species production. In this study, we investigated the anti-inflammatory and anti-atherogenic properties of KR-31543. We observed that KR-31543 treatment reduced the production of MCP-1, IL-8, and VCAM-1 in HUVECs, and of MCP-1 and IL-6 in THP-1 human monocytes. We also examined the effect of KR-31543 on monocytes migration in vitro. KR-31543 treatment effectively reduced the migration of THP-1 human monocytes to the HUVEC monolayer in a dose-dependent manner. We next examined the effects of this compound on atherogenesis in LDL receptor deficient (Ldlr-/-) mice. After 10 weeks of western diet, the formation of atherosclerotic lesion in aorta was reduced in the KR-31543-treated group compared to the control group. The accumulation of macrophages in lesion was also reduced in KR-31543 treated group. However, the plasma levels of total cholesterol, HDL, LDL, and triglyceride were not affected by KR-31543 treatment. Taken together, these results show that KR-31543 has anti-inflammatory properties on human monocytes and endothelial cells, and inhibits fatty streak lesion formation in mouse model of atherosclerosis, suggesting the potential of KR-31543 for the treatment for atherosclerosis.
Animals ; Aorta/pathology ; Atherosclerosis/blood/*drug therapy/pathology ; Benzopyrans/*pharmacology/therapeutic use ; Cholesterol, HDL/blood ; Cholesterol, LDL/blood ; Diet ; Disease Models, Animal ; Human Umbilical Vein Endothelial Cells/drug effects/metabolism ; Inflammation Mediators/*metabolism ; Interleukin-6/metabolism ; Interleukin-8/metabolism ; Macrophages/metabolism ; Mice ; Mice, Transgenic ; Monocytes/drug effects/*metabolism ; Neuroprotective Agents/*pharmacology/therapeutic use ; Receptors, CCR2/metabolism ; Receptors, LDL/genetics ; Tetrazoles/*pharmacology/therapeutic use ; Transendothelial and Transepithelial Migration/drug effects ; Triglycerides/blood ; Vascular Cell Adhesion Molecule-1/metabolism

Low density lipoprotein receptor (LDLR) plays an important role in the cholesterol homeostasis. We examined the possible circadian regulation of LDLR and mechanism(s) underlying it. In mice, blood glucose and plasma triglyceride, total and high density lipoprotein cholesterol varied distinctively throughout a day. In addition, LDLR mRNA oscillated in the liver in a functional clock-dependent manner. Accordingly, analysis of human LDLR promoter sequence revealed three putative E-boxes, raising the possible regulation of LDLR expression by E-box-binding transcription factors. To test this possibility, human LDLR promoter reporter constructs were transfected into HepG2 cells and the effects of CLOCK/BMAL1, Hes1, and Hes6 expression were analyzed. It was found that positive circadian transcription factor complex CLOCK/BMAL1 upregulated human LDLR promoter activity in a serum-independent manner, while Hes family members Hes1 and Hes6 downregulated it only under serum-depleted conditions. Both effects were mapped to proximal promoter region of human LDLR, where mutation or deletion of well-known sterol regulatory element (SRE) abolished only the repressive effect of Hes1. Interestingly, hes6 and hes1 mRNA oscillated in an anti-phasic manner in the wild-type but not in the per1-/-per2-/- mouse. Comparative analysis of mouse, rat and human hes6 genes revealed that three E-boxes are conserved among three species. Transfection and site-directed mutagenesis studies with hes6 reporter constructs confirmed that the third E-box in the exon IV is functionally induced by CLOCK/BMAL1. Taken together, these results suggest that LDLR expression is under circadian control involving CLOCK/BMAL1 and Hes family members Hes1 and Hes6.
ARNTL Transcription Factors/physiology ; Animals ; Base Sequence ; Basic Helix-Loop-Helix Transcription Factors/*genetics/metabolism/physiology ; CLOCK Proteins/physiology ; Cholesterol/blood ; *Circadian Rhythm ; E-Box Elements ; Exons ; *Gene Expression Regulation ; Hep G2 Cells ; Homeodomain Proteins/*genetics/metabolism/physiology ; Homeostasis ; Humans ; Liver/metabolism ; Male ; Mice ; Mice, Inbred C57BL ; *Promoter Regions, Genetic ; Receptors, LDL/*genetics/metabolism ; Repressor Proteins/*genetics/metabolism/physiology ; Transcription, Genetic