Cardio-cerebral vascular disease induced by atherosclerosis is a serious cause of human health. The pathogenesis of AS is very complex,and the oxidized low-density lipoprotein( ox LDL) induced foam cells formation is considered to be the most important cytological change in AS. Based on the definition of " TCM chemical biology",we clarified the chemical composition of Ilex hainanensis,the effective substances of I. hainanensis on the activity of anti-AS were screened. Then we found that saponin BF523 had the good inhibitory effect on foam cell formation. In this research,we studied the BF523 as the research object to clarify the molecular target of the active compound of I. hainanensis by foam cell formation model. The results showed that BF523 significantly inhibited the oxidation of ox LDL-induced macrophage foaming and decreased the lipid content in macrophages. BF523 had inhibited the phagocytosis of ox LDL in macrophages by reducing the mRNA and protein levels of scavenger receptor CD36,thereby inhibiting the occurrence and development of AS. These findings not only clarified the mechanism of the inhibition of foam cell formation by saponin BF523,but also provided a useful exploration for the enrichment of the theory of " TCM chemical biology".
Atherosclerosis ; CD36 Antigens ; metabolism ; Cells, Cultured ; Foam Cells ; cytology ; drug effects ; Humans ; Ilex ; chemistry ; Lipoproteins, LDL ; adverse effects

This study was designed to investigate the correlation between autophagy and polarization of macrophages in atherosclerosis (AS) plaque in arteriosclerosis obliterans amputees. Femoral artery specimens from arteriosclerosis obliterans amputees were performed hematoxylin and eosin (HE) staining, oil red O and immunofluorescence staining to observe the morphology of atherosclerotic plaque, phenotype of macrophages and autophagy in plaque; using real-time quantitative RT-PCR technology to detect the mRNA level of M1 and M2 type markers in arterial tissue; to analyze polarized signal pathway and autophagy protein levels in macrophages by Western blotting. Arterial specimens staining showed obvious lipid deposition and obvious infiltration of amount of foam cells and inflammatory cells. Macrophages were mainly expression M1 type in percentage in fibrous plaque. Although both M1 and M2 macrophages were upregulated in atheromatous plaque, the increase was dominant in M2 type in percentage. The level of autophagy was significantly higher in the atheromatous plaque than that of fibrous plaque. The expression of tumor necrosis factor- α (TNF-α), monocyte chemotactic protein-1 (MCP-1), inducible nitric oxide synthase (iNOS), interleukin-6 (IL-6) and interleukin-12 (IL-12) mRNA was significantly higher in fibrous plaque than that of atheromatous plaque (P < 0.01 or 0.05), and arginase-1 (Arg-1), transforming growth factor-β (TGF-β), CD163 and interleukin-10 (IL-10) mRNA was significantly lower than that in atheromatous plaque (P < 0.01). The levels of p-STAT1 and NF-κB were significantly increased in fibrous plaque (P < 0.01), while p-STAT6 expression was significantly increased in atheromatous plaque (P < 0.01). The level of LC3-II was significantly higher in atheromatous plaque than that in fibrous plaque (P < 0.01). Macrophages in early atherosclerotic plaque were induced to M1 type through p-STAT1/NF-κB pathway and expressed moderate levels of autophagy; while macrophages in advanced plaques were induced to polarization of M2 type through p-STAT6 pathway. M2 macrophages expressed a higher level of autophagy than M1 macrophages.
Amputees ; Arginase ; metabolism ; Arteriosclerosis Obliterans ; pathology ; Atherosclerosis ; pathology ; Autophagy ; Cell Polarity ; Chemokine CCL2 ; metabolism ; Foam Cells ; cytology ; Humans ; Interleukin-10 ; metabolism ; Interleukin-12 ; metabolism ; Interleukin-6 ; metabolism ; Macrophages ; cytology ; NF-kappa B ; metabolism ; Nitric Oxide Synthase Type II ; metabolism ; Phenotype ; STAT6 Transcription Factor ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism ; Up-Regulation

To explore the anti-atherosclerotic mechanism of estrogen and especially observe the effect of estradiol on the content of cholesterol in J774a.1 mouse mononuclear/macrophage-derived foam cells which were incubated with oxidized low-density lipoproteins (ox-LDL). J774a.1 mouse mononuclear/macrophages were incubated with ox-LDL or with both ox-LDL and estradiol (1, 0.1 or 0.01 micromol x L(-1)). Oil red O staining was used to observe the formation of foam cells, and cholesterol oxidase fluorometric was used to determine the content of cellular cholesterol content. Western blotting and RTFQ-PCR were used to observe the expressions of scavenger receptor class B type I (SR-B I ) in J774a.1 foam cells. Compared with the control cells, J774a.1 mouse mononuclear/macrophage-derived foam cells showed significantly increased contents of total cholesterol and cholesterol ester (P < 0.001) and decreased SR-B I mRNA expression (P < 0.01). Estradiol treatment significantly lowered the contents of total cholesterol and cholesterol ester (P < 0.05), and increased SR-B I protein and mRNA expression (P < 0.01) in the foam cells in a dose-dependent manner. Estradiol can inhibit the formation of mononuclear/macrophage-derived foam cells by decreasing the contents of total cholesterol and cholesterol ester and up-regulating the expression of SR-B I in the foam cells.
Animals ; Cell Line ; Cholesterol ; metabolism ; Cholesterol Esters ; metabolism ; Estradiol ; pharmacology ; Foam Cells ; cytology ; metabolism ; Lipoproteins, LDL ; metabolism ; Macrophages ; drug effects ; metabolism ; Mice ; Scavenger Receptors, Class B ; 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

OBJECTIVETo observe the effect of Huanglian Jiedu Decoction (HLJDD) in in vivo regulating differentiation of monocytes in an apolipoprotein E knockout (ApoE(-/-)) mouse model, and to observe the effect of HLJDD-containing serum in in vitro regulating differentiation of macrophages and foam cells.

METHODSFifteen apoE(-/-) mice were randomly divided into the common diet group, the hyperlipidemia group, and the hyperlipidemia +HLJDD treatment group, 5 in each group. Mice in the common diet group were fed with a chow diet. Mice in the hyperlipidemia group were fed with high cholesterol wild diet (WD). Those in the hyperlipidemia +HLJDD treatment group were fed with high cholesterol WD supplemented with HLJDD. All mice were fed for 4 weeks. Five C57BL/6 wild types were recruited as the wild common diet control group. HLJDD was administered to mice in the hyperlipidemia + HLJDD treatment group by gastrogavage at the daily dose of 5 g/kg. Equal volume of purified water was given by gastrogavage to mice in the rest 3 groups. Four weeks later, subtypes of monocytes in the peripheral blood were detected by FACS. HLJDD administered to another 30 SD rats by gastrogavage at the daily dose of 5 g/kg, once for every 12 h for 5 times in total, thereby preparing 5% HLJDD containing serum to intervene the differentiation of in vitro primary bone marrow-derived macrophage (BMDM) and foam cells. The M2 subtype surface receptor CD206 of macrophages and foam cells were detected by FACS. The expression of Nos2 and Arg1 genes were assayed by Real-time PCR.

RESULTSThe ratio of inflammatory subset of monocytes (Ly6C(high)) increased in the peripheral blood after ApoE(-/-) mice were fed with high fat diet for 4 weeks. HLJDD significantly decreased the ratio of inflammatory subset of monocytes (P < 0.05). Compared with the vehicle serum, 5% HLJDD containing serum significantly increased differentiation of CD206 + M2 BMDM (P = 0.034). Results of real-time quantitative PCR showed that the expression level of Arg1 mRNA could be up-regulated by HLJDD containing serum (P < 0.05), and that of Nos2 mRNA down-regulated (P = 0.017). ox-LDL induced the differentiation of M2 subtype foam cells from BMDM, and HLJDD containing serum could further elevate the ratio of CD206 + M2 foam cells and increase the Arg1 mRNA expression level (both P < 0.01). HLJDD containing serum could inhibit the inversion of M2 subtype of foam cells to M1 subtype induced by Th1 factors, significantly elevate the Arg1 mRNA expression level, and decrease the Nos2 mRNA expression level (all P < 0.01).

CONCLUSIONSHLJDD could lower hyperlipidemia induced inflammatory monocyte subtype ratios in the peripheral blood of ApoE(-/-) mice. HLJDD containing serum promoted in vitro differentiation of M2 macrophages and foam cells. HLJDD attenuated and inhibited the occurrence and development of atherosclerosis induced by hyperlipidemia possibly through regulating the functional differentiation of monocytes, macrophages, and foam cells.

Animals ; Apolipoproteins E ; genetics ; Cell Differentiation ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Female ; Foam Cells ; cytology ; drug effects ; Macrophages ; cytology ; drug effects ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Monocytes ; cytology ; drug effects

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

OBJECTIVETo evaluate whether atorvastatin inhibits oxidized low-density lipoproteins (Ox-LDL)-stimulated foam cell formation from THP-1 macrophages by regulating the activation of peroxisome proliferator-activated receptor γ (PPARγ) and nuclear factor-κB (NF-κB). Methods THP-1 macrophages were pretreated with 10, 20, or 40 µmol/L atorvastatin for 2 h, and after washing with PBS twice, the cells were incubated with 60 µg/ml of Ox-LDL for 48 h. The quantity of intracellular lipid of the cells was detected with Oil red O staining and enzymatic fluorometric method. The expression of the scavenger receptors of CD36 and SRA were analyzed with Western blotting. We also examined the effect of atorvastatin on adenosine triphosphate (ATP)-binding cassette transporter A1 (ABCA1) expression and the activation of PPARγ and p-iκB, and further assessed the capacity of the macrophages to bind to Dil-oxLDL.

RESULTSAtorvastatin potently inhibited ox-LDL-induced macrophage-derived foam cell formation, down-regulated the expression of CD36 and SRA, and up-regulated the expression of ABCA1. Atorvastatin markedly suppressed the activation of PPARγ and p-iκB in ox-LDL-stimulated THP-1 macrophages (P<0.05) and significantly decreased the Dil-oxLDL-binding capacity of the macrophages (P<0.05).

CONCLUSIONAtorvastatin as an effective anti-atherosclerosis agent can suppress the activation of PPARγ and p-iκB to reduce lipid accumulation in macrophages.

ATP Binding Cassette Transporter 1 ; metabolism ; Atorvastatin Calcium ; Cell Line ; Foam Cells ; cytology ; drug effects ; Heptanoic Acids ; pharmacology ; Humans ; I-kappa B Proteins ; metabolism ; Lipoproteins, LDL ; metabolism ; Macrophages ; cytology ; drug effects ; NF-kappa B ; metabolism ; PPAR gamma ; metabolism ; Pyrroles ; pharmacology ; Signal Transduction ; drug effects ; Transcriptional Activation ; Up-Regulation

Mitofusin2 (Mfn2) plays a pivotal role in the proliferation and apoptosis of vascular smooth muscle cells (VSMCs). The purpose of this study was to investigate the effects of Mfn2 on the trafficking of intracellular cholesterol in the foam cells derived from rat VSMCs (rVSMCs) and also to investigate the effects of Mfn2 on the expression of adenosine triphosphate-binding cassette subfamily A member 1 (ABCA1), adenosine triphosphate-binding cassette subfamily G member 1 (ABCG1) and peroxisome proliferator-activated receptor gamma (PPARγ). The rVSMCs were co-cultured with oxidized low density lipoprotein (LDL, 80 μg/mL) to produce foam cells and cholesterol accumulation in cells. Before oxidized LDL treatment, different titers (20, 40 and 60 pfu/cell) of recombinant adenovirus containing Mfn2 gene (Adv-Mfn2) were added into the culture medium for 24 h to transfect the Mfn2 gene into the rVSMCs. Then the cells were harvested for analyses. The protein expression of Mfn2 was significantly higher in Adv-Mfn2-transfected group than in untransfected group (P<0.05), and the expression levels significantly increased when the titer of Adv-Mfn2 increased (P<0.05). At 24 or 48 h after oxidized LDL treatment, rVSMCs became irregular and their nuclei became larger, and their plasma abounded with red lipid droplets. However, the number of red lipid droplets was significantly decreased in Adv-Mfn2-transfected group as compared with untransfected group. At 48 h after oxidized LDL treatment, the intracellular cholesterol in rVSMCs was significantly increased (P<0.05), but it was significantly decreased in Adv-Mfn2-transfected group as compared with untransfected group (P<0.05), and it also significantly decreased when the titer of Adv-Mfn2 increased (P<0.05). The mRNA and protein expression levels of ABCA1 and ABCG1 were significantly increased in Adv-Mfn2-transfected group as compared with untransfected group (P<0.05). Though the mRNA and protein expression levels of PPARγ was not significantly increased (P>0.05), the phosporylation levels of PPARγ were significantly decreased in Adv-Mfn2-transfected group as compared with untransfected group (P<0.05). These results suggest that the transfection of Adv-Mfn2 can significantly reduce intracellular cholesterol in oxidized LDL-induced rVSMCs possibly by decreasing PPARγ phosporylation and then increasing protein expression levels of ABCA1 and ABCG1, which may be helpful to suppress the formation of foam cells.
ATP Binding Cassette Transporter 1 ; metabolism ; ATP Binding Cassette Transporter, Sub-Family G, Member 1 ; ATP-Binding Cassette Transporters ; metabolism ; Animals ; Cell Differentiation ; physiology ; Cells, Cultured ; Cholesterol ; metabolism ; Foam Cells ; cytology ; metabolism ; Intracellular Fluid ; metabolism ; Lipoproteins, LDL ; metabolism ; Membrane Proteins ; genetics ; metabolism ; Mitochondrial Proteins ; genetics ; metabolism ; Muscle, Smooth, Vascular ; cytology ; metabolism ; Oxidation-Reduction ; PPAR gamma ; metabolism ; Rats

OBJECTIVETo observe the effect of andrographolide on the activation of mitogen-activated protein kinases (MAPKs) and expression of nuclear factor-κB (NF-κB) in macrophage foam cells.

METHODSThe mouse peritoneal macrophages were cultured in the media in the presence of oxidized low-density lipoprotein (ox-LDL), ox-LDL+andrographolide, or neither (control). The phosphorylation of MAPK molecules (p38MAPK, JNK, ERK1/2) and the expressions of NK-κB p65 were examined by Western blot.

RESULTSAs compared with cells in the control group, the expressions of phospho-p38 and NF-κB p65 were increased in the cells cultured with either ox-LDL or ox-LDL+andrographolide (P<0.01), but attenuated significantly in the presence of ox-LDL+ andrographolide when compared with ox-LDL (P<0.05). The phospho-JNK increased in the presence of either ox-LDL or ox-LDL+andrographolide when compared with control cells (P<0.01), but no significant difference existed between ox-LDL and ox-LDL+andrographolide (P>0.05). The expression of phospho-ERK1/2 was increased in the presence of ox-LDL compared with the control cells (P<0.01), but no significant differences existed between the cells cultured in the presence of ox-LDL+andrographolide and the control medium (P>0.05).

CONCLUSIONSAndrographolide could inhibit the activation of ERK1/2, p38MAPK and NK-κB induced by ox-LDL in macrophage foam cells, which might be one of its mechanisms in preventing atherosclerosis.

Animals ; Anti-Inflammatory Agents ; pharmacology ; Atherosclerosis ; immunology ; metabolism ; prevention & control ; Cells, Cultured ; Diterpenes ; pharmacology ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Foam Cells ; cytology ; drug effects ; enzymology ; JNK Mitogen-Activated Protein Kinases ; metabolism ; Lipoproteins, LDL ; metabolism ; MAP Kinase Signaling System ; drug effects ; immunology ; Macrophages, Peritoneal ; cytology ; drug effects ; enzymology ; Mice ; Mice, Inbred Strains ; NF-kappa B ; metabolism ; Vasculitis ; drug therapy ; immunology ; metabolism ; p38 Mitogen-Activated Protein Kinases ; metabolism

OBJECTIVETo observe the effect of rosiglitazone on the content of cholesterol and expressions of Acy-coenzyme A: cholesterol acyltransferase 1 (ACAT-1) and scavenger receptor class B type I (SR-BI) in RAW264.7 macrophage-derived foam cells and explore the anti-atherosclerotic mechanism of rosiglitazone.

METHODSRAW264.7 macrophages were incubated with oxidized low-density lipoproteins (ox-LDL) or with both ox-LDL and rosiglitazone (5, 10, or 20 µmol/L). Oil red O staining was used to observe the formation of foam cells, and cholesterol oxidase was used to determine the content of cellular cholesterol contents. Western blotting was used observe the expressions of ACAT-1 and SR-BI in RAW264.7 foam cells.

RESULTSCompared with the control cells, RAW264.7 macrophage-derived foam cells showed significantly increased contents of total cholesterol and free cholesterol (P<0.01) and ACAT-1 expressions (P<0.05) with mildly increased SR-BI expression (P>0.05). Rosiglitazone treatments significantly lowered the contents of total cholesterol and free cholesterol (P<0.05), decreased the expression of ACAT-1 (P<0.05), and increased SR-BI expression (P<0.05) in the foam cells in a dose-dependent manner.

CONCLUSIONRosiglitazone can decrease the contents of total and free cholesterol, down-regulate ACAT-1 expression and up-regulate SR-BI expression in the foam cells produce the anti-atherosclerotic effect.

Acetyl-CoA C-Acetyltransferase ; metabolism ; Cell Line ; Cholesterol ; metabolism ; Foam Cells ; cytology ; drug effects ; metabolism ; Humans ; Scavenger Receptors, Class B ; metabolism ; Thiazolidinediones ; pharmacology