Allium Macrostemon Bge. (AMB) is a well-known homology of herbal medicine and food that has been extensively used for thousands of years to alleviate cardiovascular diseases. It contains a significant amount of polysaccharides, yet limited research exists on whether these polysaccharides are responsible for its cardiovascular protective effects. In this study, the anti-atherosclerosis effect of the crude polysaccharides of AMB (AMBP) was evaluated using ApoE-/- mice fed a high-fat diet, along with ox-LDL-induced Thp-1 foam cells. Subsequently, guided by the inhibitory activity of foam cells formation, a major homogeneous polysaccharide named AMBP80-1a was isolated and purified, yielding 11.1 % from AMB. The molecular weight of AMBP80-1a was determined to be 10.01 kDa. AMBP80-1a was firstly characterized as an agavin-type fructan with main chains consisting of →1)-β-d-Fruf-(2→ and →1,6)-β-d-Fruf-(2→ linked to an internal glucose moiety, with →6)-β-d-Fruf-(2→ and β-d-Fruf-(2→ serving as side chains. Furthermore, the bio-activity results indicated that AMBP80-1a reduced lipid accumulation and cholesterol contents in ox-LDL-induced Thp-1 foam cell. These findings supported the role of AMBP in alleviating atherosclerosis in vivo/vitro. AMBP80-1a, as the predominant homogeneous polysaccharide in AMB, was expected to be developed as a functional agent to prevent atherosclerosis.

OBJECTIVE: Tripartite motif (TRIM65) is an important member of the TRIM protein family, which is a newly discovered E3 ligase that interacts with and ubiquitinates various substrates and is involved in diverse pathological processes. However, the function of TRIM65 in atherosclerosis remains unarticulated. In this study, we investigated the role of TRIM65 in the pathogenesis of atherosclerosis, specifically in vascular smooth muscle cells (VSMCs) phenotype transformation, which plays a crucial role in formation of atherosclerotic lesions.
RESULTS: Both non-atherosclerotic and atherosclerotic lesions during autopsy were collected singly or pairwise from each individual (n = 16) to investigate the relationship between TRIM65 and the development of atherosclerosis. In vivo, Western diet-fed ApoE-/- mice overexpressing or lacking TRIM65 were used to assess the physiological function of TRIM65 on VSMCs phenotype, proliferation and atherosclerotic lesion formation. In vitro, VSMCs phenotypic transformation was induced by platelet-derived growth factor-BB (PDGF-BB). TRIM65-overexpressing or TRIM65-abrogated primary mouse aortic smooth muscle cells (MOASMCs) and human aortic smooth muscle cells (HASMCs) were used to investigate the mechanisms underlying the progression of VSMCs phenotypic transformation, proliferation and migration. Increased TRIM65 expression was detected in α-SMA-positive cells in the medial and atherosclerotic lesions of autopsy specimens. TRIM65 overexpression increased, whereas genetic knockdown of TRIM65 remarkably inhibited, atherosclerotic plaque development. Mechanistically, TRIM65 overexpression activated PI3K/Akt/mTOR signaling, resulting in the loss of the VSMCs contractile phenotype, including calponin, α-SMA, and SM22α, as well as cell proliferation and migration. However, opposite phenomena were observed when TRIM65 was deficient in vivo or in vitro. Moreover, in cultured PDGF-BB-induced TRIM65-overexpressing VSMCs, inhibition of PI3K by treatment with the inhibitor LY-294002 for 24 h markedly attenuated PI3K/Akt/mTOR activation, regained the VSMCs contractile phenotype, and blocked the progression of cell proliferation and migration.
CONCLUSIONS: TRIM65 overexpression enhances atherosclerosis development by promoting phenotypic transformation of VSMCs from contractile to synthetic state through activation of the PI3K/Akt/mTOR signal pathway.

Caffeic acid has been indicated to benefit cholesterol balance, but the effect of pure caffeic acid on atherosclerosis in vivo has not been tested. Given that atherosclerosis and Alzheimer\'s disease share common features including distracted lipid balance and chronic inflammation, the concurrent effects of caffeic acid on atherosclerotic lesions and cognitive decline were explored here by using the ApoE-/- mice model. A two months\' administration of 20 mg/kg caffeic acid or saline was given once two days intraperitoneally to 5-month-old female ApoE-/- mice. We found that the caffeic acid treatment reduced the atherosclerotic lesions in the whole aorta and aortic sinus of the resulting 7-month-old ApoE-/- mice by roughly 50%, compared with the saline control. Meanwhile, the cognitive decline of treated mice were significantly alleviated, as measured by Y-maze and Morris water maze tasks. A reduced accumulation of β-amyloid in the hippocampus was also observed. These effects were associated with elevated serum HDL-c concentration, upregulated ABCA1 and ABCG1 mRNA levels, as well as decrease local inflammation and reduced levels of serum pro-inflammatory cytokines including TNF-α, IL-6 and MCP-1. These obtained results suggested the preventive and therapeutic potential of caffeic acid against atherosclerosis and Alzheimer\'s disease during aging.

Hyperlipidemia is one major cause of atherosclerosis, which is a basic pathological change of cardiovascular diseases. Polysaccharide is a water-soluble component with lipid-lowering effects. In this study, alkaline-extracted polysaccharides were obtained from the fruiting body of C. militaris. Polysaccharides were purified via anion exchange and size exclusion chromatography. Their structural characteristics were investigated via chemical and spectroscopic methods. CM3I was mainly composed of →4)α-D-Glcp(1 → glycosyls and differed from starch due to the presence of →4,6)β-D-Glcp(1 → glycosyls. CM3II was characterized by its backbone, which was composed of →4)-β-D-Manp(1 → 6)-α-D-Manp(1 → 6)-β-D-Manp(1 → linked glycosyls, and especially the presence of O-methyl. Moreover, CM3II exhibited powerful anti-atherosclerotic effects via lowering plasma lipid levels in apolipoprotein E-deficient mice. The underlying mechanisms were attributed to its promoting effect on LXRα and inhibitory effect on SREBP-2. Collectively, CM3I and CM3II are different from the previously reported polysaccharides from C. militaris, and CM3II has a potential application in hypolipidemia and anti-atherosclerosis.

BACKGROUND: Naringenin is naturally isolated from citrus fruits possessing many pharmacological activities. However, little is known about the effect of naringenin on nonalcoholic steatohepatitis (NASH) in the model of metabolic syndrome.
OBJECTIVE: The present study is aimed to investigate the effect of naringenin on NASH in 12-mo-old male ApoE-/- mice and its possible underlying mechanism.
METHODS: In vivo, 12-mo-old male ApoE-/- mice were administrated with naringenin by intragastric gavage for 12 weeks. At the end of experiment, the blood samples and liver tissues were collected. Metabolic parameters in serum, levels of triglyceride, cholesterol and hydroxyproline, activities of antioxidant enzymes, and content of inflammatory cytokines (TNF-α and IL-6) in liver were examined by corresponding assay kits. Pathological changes in liver were observed by hematoxylin-eosin, oil red O, masson\'s trichrome, picro-sirius red and senescence β-galactosidase staining. Dihydroethidium was used for detection of reactive oxygen species (ROS). In vitro, AML-12 cells were treated with oleic acid in the presence or absence of naringenin for 24 h. Transfection of SIRT1 siRNA was also conducted in vitro. Lipid accumulation, cellular ROS generation, malondialdehyde content, antioxidant enzyme activities and secretion levels of TNF-α and IL-6 were examined. Both in vivo and in vitro, gene expressions were detected by real-time PCR or western blot.
RESULTS: Naringenin administration improved metabolic parameters, suppressed hepatic steatosis, regulated expression of genes involved in lipid metabolism (FASN, SCD1, PPARα and CPT1α), reduced hepatic fibrosis and cell senescence, inhibited hepatic inflammation as evidenced by the decreased macrophage recruitment and content of TNF-α and IL-6, and reduced hepatic oxidative stress by suppressing ROS generation and normalizing activities of antioxidant enzymes. Notably, naringenin administration increased hepatic SIRT1 protein expression and activity along with the increased deacetylation of liver kinase B1 (LKB1), PGC1α and NF-κB. In vitro study, the benefits of naringenin on lipid accumulation, oxidative stress and inflammation were diminished by SIRT1 siRNA transfection.
CONCLUSIONS: These results indicate that naringenin administration may be a potential curative therapy for NASH treatment and the activation of hepatic SIRT1-mediated signaling cascades is involved in its beneficial effects.

Inflammation plays an important role in the process of atherosclerosis (AS). Inhibition of inflammation is an effective way to prevent AS. Imperatorin (IMP) is a kind of furan coumarin with various activities. In this study, the anti-inflammatory effect of IMP was explored in oxidized low-density lipoprotein (ox-LDL)-induced VSMCs and high fat diet (HFD)-induced ApoE-/- mice. The results showed that IMP attenuated the elevation of TNF-α, IL-6, MCP-1 and NO induced by ox-LDL in supernatant of VSMCs. IMP has normalized the levels of serum lipids (TC, TG, LDL-C and HDL-C) and attenuated inflammatory cytokines in serum. IMP also improved pathological changes and lipid accumulation in aorta. Matrix metalloproteinase-2 (MMP-2) expression in aorta was down-regulated by IMP. IMP could inhibit the phosphorylation of MAPKs pathway in the aorta and VSMCs, resulting in a significant decrease in the contents of p-ERK 1/2, p-JNK and p-P38. Overall, IMP could exert anti-inflammatory effects in vivo and in vitro to interfere with AS.

The aim of this study was to investigate the beneficial effect of swimming exercise on autophagy and atherosclerosis in mice aorta, so as to clarify the possible causal relationship between autophagy activation and atherosclerosis.
The body weight was monitored regularly. Hematoxylin-eosin staining and Oil Red O staining was conducted to observe vascular morphology and plaque burden respectively. The levels of serum total cholesterol (TC), triglyceride (TG), soluble intercellular adhesion molecule-1 (sICAM-1), matrix metalloproteinase-9 (MMP-9) and interleukin-6 (IL-6) was examined via Enzyme-linked immu-nosorbent assays (ELISA). The mRNA expression level of autophagy markers, including LC3 and Beclin-1, was examined by real-time quantitative polymerase chain reaction (RT-PCR). The expressions of LC3-II/LC3-I and Beclin-1 are detected by Western blotting and immunohistochemistry.
Compared with the model group, long-term swimming exercise decreased the weight gain of ApoE-/- mice, improved the structural disorder of artery, reduced the load of atherosclerotic lesion, and attenuated the concentrations of serum TC, TG, sICAM-1, MMP-9, and IL-6. In addition, the expression of autophagy markers LC3 and Beclin-1 increased significantly at the mRNA and protein levels.
Long-term swimming exercise could activate the autophagy and reduce atherosclerotic lesion in the aorta of ApoE-/- mice. Autophagy activation may be one of the mechanisms by which atherosclerosis is improved through exercise.

Atherosclerosis (AS), a severe disease characterized by an accumulation of lipids and fibers in the large arteries, is the most important contributor to ischemic stroke (IS). Although microRNAs (miRNAs) have been found in circulating blood, the role of miRNAs in the progression of AS remains unknown. In a previous study, we demonstrated that the spleen tyrosine kinase (Syk) gene plays a vital role in the process of IS. In the present study, we aimed to clarify whether the miRNAs targeting the Syk gene might slow the development of AS. Candidate miRNAs were screened in U937 and THP-1 cells via Bioinformatics analyses, RT-qPCR and dual-luciferase reporter assay. ApoE-/- mice were used as an AS animal model. RAAV transfection was performed to identify the roles of Syk gene and miRNAs in the development of AS in ApoE-/- mice. HE staining, Oil red O staining and immunohistochemistry were used to determine the mechanism of AS. RT-qPCR and western blotting were performed to determine the expressions of miRNAs and proteins, respectively. Over-expression of the Syk gene accelerated the development of AS. miR-377 effectively mediated the expression of the Syk gene in vitro and in vivo experiments. Further analysis indicated that over-expression of miR-377 partly alleviated the development of AS by down-regulating the expression of the Syk gene. This study identifies a novel role of miR-377 in AS via targeting Syk.

The F11 Receptor (F11R), AKA Junctional Adhesion Molecule-A (JAM-A) (F11R/JAM-A), is an adhesion protein constitutively expressed on the membrane surface of circulating platelets and the luminal surface of inflamed endothelial cells (EC). Platelet adhesion to an inflamed endothelium is one of the early steps of atherosclerotic plaque formation. Our previous studies, conducted with cultured EC in vitro, have demonstrated the expression of F11R/JAM-A on the luminal surface of inflamed EC, platelet adhesion to inflamed EC through F11R/JAM-A interactions, and inhibition of this interaction by the presence of F11R/JAM-A antagonistic peptide (F11Rpeptide 4D). In the present study, we examined in vivo the overall health-benefits and cardiovascular effects of long-term treatment of animals prone to atherosclerosis, ApoE-/- mice, with F11R-peptide 4D.
Twenty ApoE-/- mice were assigned to daily treatment with peptide 4D and compared to their counterparts control untreated mice. Mice were observed for wellness and survival. Plaque size in the aorta and heart was measured using histological analysis. Effects of peptide 4D (or scramble control) on platelet adhesion to inflamed endothelium were measured using intravital microscopy.
Significant reductions in atherosclerotic plaques number and size, an overall robust health with longer survival were found in the peptide 4D treated group of ApoE-/- mice. Intravital microscopic studies conducted in exposed vessels of ApoE-/- mice demonstrated significant inhibition by peptide 4D of platelet adhesion to the cytokine-inflamed endothelium.
Our results demonstrate that peptide 4D significantly reduces atherosclerotic plaque formation in ApoE-/- mice and inhibits platelet adhesion to the inflamed arterial endothelium.

COX-2-selective inhibitors have been associated with an increased risk of cardiovascular complications, and their impact on atherosclerosis (AS) remains controversial. The proinflammatory COX-2 and 5-LO pathways both play essential roles in AS and related cardiovascular diseases. Previous clinical studies have provided evidence of the ability of COX-2-selective inhibitors to shunt AA metabolism from the COX-2 pathway to the 5-LO pathway. In this study, the effects of celecoxib, a selective COX-2 inhibitor, on AS and the COX-2 and 5-LO pathways were investigated in vivo and in vitro.
Male ApoE-/- mice fed a western-type diet for 18 weeks and cultured mouse RAW264.7 macrophages stimulated with 1 μg/mL LPS for 24 h were used in this study.
In ApoE-/- mice, intragastric administration of celecoxib (80 mg/kg/d) for 18 weeks significantly increased aortic atherosclerotic lesion area but had no effect on hyperlipidemia. In addition, celecoxib significantly lowered TNF-α and PGE2 levels but increased both LTB4 and CysLTs levels in aortic tissues. In LPS-stimulated RAW264.7 macrophages, pretreatment with 8 μmol/L celecoxib for 1 h significantly lowered the TNF-α, NO, and PGE2 levels but increased the LTB4 and CysLTs levels. Celecoxib also decreased the protein and mRNA expression of COX-2 but increased the expression of 5-LO and LTC4S in both ApoE-/- mouse aortic tissues and LPS-stimulated RAW264.7 macrophages.
The COX-2-selective inhibitor celecoxib can aggravate atherogenesis, an effect that may be related to upregulation of LTs via a 5-LO pathway shunt.