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Hyperlipidemia is induced by abnormal lipid metabolism, which can cause the occurrence of cardiovascular diseases and lead to grievous injury to health. Studies showed that AEE had a significant therapeutic effect on hyperlipidemia and is likely to be associated with the up-regulation of cholesterol 7-alpha hydroxylase (CYP7A1), the key enzyme for cholesterol conversion to bile acids, but no research confirmed whether the effect of AEE on hyperlipidemia was related to the gut microbiota and liver lipids. At the same time, more and more studies have shown that gut microbiota and lipids are closely related to hyperlipidemia. Hence, in this study, we investigated the effects of AEE on liver lipids through LC-MS-based untargeted lipidomics and the effects of AEE on gut microbiota based on cecal contents metagenomics by Illumina sequencing in HFD-induced hyperlipidemia ApoE−/− mice at the overall level. The results of lipidomics showed that AEE relieved hyperlipidemia by decreasing the concentration of 10 PEs and 12 SMs in the liver and regulating the pathways of glycerophospholipid metabolic pathway, sphingolipid signaling pathway, and NF-kB signaling pathway. The results of metagenomics concluded that AEE treatment changed the composition of gut microbiota and regulated the functions of lipid transport and metabolism, as well as the metabolism of bile acids and secondary bile acids. The results of the joint analysis between lipidomics and metagenomics showed that the abundance of Verrucomicrobia, Verrucomicrobiales, Candidatus_Gastranaerophilales, and Candidatus_Melainabacteria was significantly positively correlated with the concentration of SM (d18:1/18:0) and PE (16:0/18:1) in the process of AEE alleviating hyperlipidemia in mice. In conclusion, these results suggested that the effect of AEE on hyperlipidemia was closely related to the gut microbiota by the change of bile acids and liver lipids.

ObjectiveThis study aimed to investigate and compare the morphological and biochemical characteristics of the hippocampus and the spatial memory of young adult ApoE–/– mice on a standard chow diet, a low-fat diet (LFD), a high-fat diet (HFD), and an HFD supplemented with lingonberries.MethodsEight-week-old ApoE–/– males were divided into five groups fed standard chow (Control), an LFD (LF), an HFD (HF), and an HFD supplemented with whole lingonberries (HF+WhLB) or the insoluble fraction of lingonberries (HF+InsLB) for 8 weeks. The hippocampal cellular structure was evaluated using light microscopy and immunohistochemistry; biochemical analysis and T-maze test were also performed. Structural synaptic plasticity was assessed using electron microscopy.ResultsApoE–/– mice fed an LFD expressed a reduction in the number of intact CA1 pyramidal neurons compared with HF+InsLB animals and the 1.6–3.8-fold higher density of hyperchromic (damaged) hippocampal neurons relative to other groups. The LF group had also morphological and biochemical indications of astrogliosis. Meanwhile, both LFD- and HFD-fed mice demonstrated moderate microglial activation and a decline in synaptic density. The consumption of lingonberry supplements significantly reduced the microglia cell area, elevated the total number of synapses and multiple synapses, and increased postsynaptic density length in the hippocampus of ApoE–/– mice, as compared to an LFD and an HFD without lingonberries.ConclusionOur results suggest that, in contrast to the inclusion of fats in a diet, increased starch amount (an LFD) and reduction of dietary fiber (an LFD/HFD) might be unfavorable for the hippocampal structure of young adult (16-week-old) male ApoE–/– mice. Lingonberries and their insoluble fraction seem to provide a neuroprotective effect on altered synaptic plasticity in ApoE–/– animals. Observed morphological changes in the hippocampus did not result in notable spatial memory decline.

The cleavage of osteopontin (OPN) by thrombin results in an N-terminal fragment (OPN-N), which exposes a cryptic integrin-binding motif that promotes the adherence of cells, and plays a proinflammatory role. However, the effect of OPN-N on abdominal aortic aneurysm (AAA) remains unknown. The aim of this study was to investigate the expression of OPN-N in aortic tissue samples obtained from patients, who underwent acute aortic dissection (AD), and normal aorta, effect of OPN-N on angiotensin (Ang) II-induced AAA in mice, and relationship between OPN-N and pyroptosis-related inflammatory factors in vitro. Hematoxylin and eosin staining was conducted to detect histological changes. Next, we detected the expression of the OPN-N protein. Additionally, ApoE−/− mice were divided into four groups: control, control + M5Ab (to block the OPN-N function in mice), Ang II, and Ang II + M5Ab. All mice were euthanized after a 28-day infusion and whole aortas, including thoracic and abdominal aortas, were collected for morphological and histological analysis of the AAA. The OPN-N protein expression was higher in patients with AD than in normal individuals, while histological changes in the aortas of Ang II mice were suppressed in Ang II + M5Ab mice. The expression of OPN-N, NOD-, LRR-, and pyrin domain-containing protein 3, pro-Caspase-1, ASC, Gasdermin-d, interleukin (IL)-18, IL-1β, matrix metalloproteinase (MMP) 2, and MMP9 was lower in the Ang II + M5Ab group than in the Ang II group. The gene expression of monocyte chemoattractant protein-1, IL-6, and tumor necrosis factor-α was suppressed in the aortic tissues of the Ang II + M5Ab group compared with the Ang II group. Moreover, the expression of α-smooth muscle actin was lower in the Ang II group than in the Ang II + M5Ab group. In vitro results showed that the increase in the expression of pyroptosis-related inflammatory factors induced by OPN was mediated through the nuclear factor (NF)-κB pathway. In conclusion, OPN-N promotes AAA by increasing the expression of pyroptosis-related inflammatory factors through the NF-κB pathway, inflammation, and extracellular matrix degradation. These results highlight the potential of OPN-N as a new therapeutic target to prevent AAA expansion.

Objective: Multiple mechanisms including vascular endothelial cell damage have a critical role in the formation and development of atherosclerosis (AS), but the specific molecular mechanisms are not exactly clarified. This study aims to determine the possible roles of proline-rich tyrosine kinase 2 (Pyk2)/mitochondrial calcium uniporter (MCU) pathway in AS mouse model and H2O2-induced endothelial cell damage model and explore its possible mechanisms.Approach and Results: The AS mouse model was established using apolipoprotein E-knockout (ApoE–/–) mice that were fed with a high-fat diet. It was very interesting to find that Pyk2/MCU expression was significantly increased in the artery wall of atherosclerotic mice and human umbilical vein endothelial cells (HUVECs) attacked by hydrogen peroxide (H2O2). In addition, down-regulation of Pyk2 by short hairpin RNA (shRNA) protected HUVECs from H2O2 insult. Furthermore, treatment with rosuvastatin on AS mouse model and H2O2-induced HUVEC injury model showed a protective effect against AS by inhibiting the Pyk2/MCU pathway, which maintained calcium balance, prevented the mitochondrial damage and reactive oxygen species production, and eventually inhibited cell apoptosis.Conclusion: Our results provide important insight into the initiation of the Pyk2/MCU pathway involved in AS-related endothelial cell damage, which may be a new promising target for atherosclerosis intervention.

Objective: To observe the effect of Si-Miao-Yong-An (SMYA) on atherosclerosis (AS) vulnerable plaques, and to further explore the mechanism by vasa vasorum (VV) angiogenesis and maturation as an entry point.Methods: SPF-class healthy male ApoE−/− mice were randomized into model group, simvastatin group and SMYA group, and C57BL/6 mice were used as the control group. After 8 weeks of intervention, the pathological morphology of plaque was observed by HE staining; the VV density in plaque and aortic adventitia were observed by immunohistochemistry; VV maturation was measured by double-labelling immunofluorescence; the critical proteins of HIF-1α-Apelin/APJ and Ang-1/Tie signal pathways were detected by western blotting.Results: SMYA decreased the plaque area and the ratio of plaque to lumen area; increased the minimum thickness of fibrous cap and its effect was greater than simvastatin. SMYA suppressed the VV neovascularization; promoted smooth muscle cells recruitment and VV maturation, which maintained plaque stability; its effect was obviously superior to simvastatin. SMYA deceased the expression of HIF-1α, Apelin, APJ, Phospho-MEK1/2 (Ser217/221), Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204), Phospho-p70 S6 Kinase (Thr421/Ser424), Ang-2 and Tie-2; it also increased the expression of Ang-1, Phospho-Akt (Ser473), Phospho-FOXO1 (Ser256) and Survivin.Conclusions: SMYA can decrease the AS plaque area in ApoE−/− mice, suppress the VV neovascularization and promote the VV maturation, and stabilize AS vulnerable plaque. The mechanism could be regulating the HIF-1α-Apelin/APJ and Ang-1/Tie signal pathways.

Background and AimsEndothelial senescence is an important risk factor leading to atherosclerosis. The mechanism of quercetin against endothelial senescence is worth exploring.MethodsQuercetin (20 mg/kg/d) was administered to ApoE-/- mice intragastrically to evaluate the effectiveness of quercetin on atherosclerotic lesion in vivo. In vitro, human aortic endothelial cells (HAECs) were used to assess the effect of quercetin on cellular senescence induced by oxidized low-density lipoprotein (ox-LDL). Transcriptome microarray and quantitative RT-PCR was conducted to study the pharmacological targets of quercetin.ResultsApoE-/- mice demonstrated obvious lipid deposition in arterial lumina, high level of serum sIcam-1 and IL-6, and high density of Vcam-1 and lower density of Sirt1 in aorta. Quercetin administration decreased lipid deposition in arterial lumina, serum sIcam-1, and IL-6 and Vcam-1 in aorta, while increased the density of Sirt1 in aorta of ApoE-/- mice. In vitro, quercetin (0.3, 1, or 3 μmol/L) decreased the expression of senescence-associated β-galactosidase and improved cell morphology of HAECs. And quercetin decreased the cellular apoptosis and increased mitochondrial membrane potential (ΔΨm) in dose-dependent manner, and decreased ROS generation simultaneously. Transcriptome microarray suggested 254 differentially expressed (DE) mRNAs (110 mRNAs were upregulated and 144 mRNAs were downregulated) in HAECs after quercetin treatment (fold change > 1.5, P < 0 .05, Que vs Ox-LDL). GO and KEGG analysis indicated nitrogen metabolism, ECM-receptor interaction, complement, and coagulation cascades, p53 and mTOR signaling pathway were involved in the pharmacological mechanisms of quercetin against ox-LDL.ConclusionsQuercetin alleviated atherosclerotic lesion both in vivo and in vitro.

Angong Niuhuang Pill (ANP) is a well-known patented Chinese medicine which is used for hundreds of years for treating the central nervous system diseases. Atherosclerosis is a poly-aetiological chronic inflammatory vascular disease. Preventing inflammation is fundamental for treating atherosclerosis in early stages. In this study, we investigated the protective effects and possible mechanisms of ANP action on a high-fat diet induced early and mid-term atherosclerosis ApoE-/- mice. The effects of ANP were compared with accepted drug simvastatin. Twelve male C57BL/6J mice were used as the control group, and 60 male ApoE-/- mice were randomly divided into five groups: Model group, Simvastatin group, Low-, Medium-, and High-dose ANP group these groups received, respectively, saline, simvastatin (3.0mg/kg), low-dose ANP (0.25 g/kg), medium-dose ANP (0.50 g/kg), and high-dose ANP (1.0 g/kg), once every other day for 10 weeks. After administration, serum biochemical indices were detected by the automatic biochemical analyzer, the concentrations of IL-6 and IL-10 in the serum were assayed by ELISA, expression levels of IL-1β, TNF-α, MMP-2, MMP-9, CCL2, and its receptor CCR2 in the full-length aorta, and expression levels of transcription factors Foxp3, RORγt in the spleen were assayed via western blotting and RT-qPCR. Flow cytometry was used to analyze Th17 cells and Treg cells. Pathological and histological analysis was completed on aortic root. ANP decreased LDL/HDL ratio, concentrations of IL-6 while increased IL-10 in serum. Moreover, ANP down-regulated the expression levels of IL-1β, TNF-α, MMP-2, MMP-9, CCL2, and CCR2 receptor in the full-length aorta. In addition, ANP decreased Th17 cells and expression levels of transcription factor RORγt, increased Treg cells and expression levels of transcription factor Foxp3. ANP decreased content of collagen fibers and infiltration of inflammatory cells in the aortic root. In conclusion, we demonstrated that ANP has anti-atherosclerosis effects on a high-fat diet induced ApoE-/- mice early and mid-term AS model via regulating Th17/Treg balance, inhibiting chronic inflammation, reducing plaque collagen fibers, and reducing inflammatory cells infiltration, to exert its multi-channel multi-target anti-early and mid-term AS effects.

Background and Aims: Endothelial senescence is an important risk factor leading to atherosclerosis. The mechanism of quercetin against endothelial senescence is worth exploring. Methods: Quercetin (20 mg/kg/d) was administered to ApoE-/- mice intragastrically to evaluate the effectiveness of quercetin on atherosclerotic lesion in vivo. In vitro , human aortic endothelial cells (HAECs) were used to assess the effect of quercetin on cellular senescence induced by oxidized low-density lipoprotein (ox-LDL). Transcriptome microarray and quantitative RT-PCR was conducted to study the pharmacological targets of quercetin. Results: ApoE-/- mice demonstrated obvious lipid deposition in arterial lumina, high level of serum sIcam-1 and IL-6, and high density of Vcam-1 and lower density of Sirt1 in aorta. Quercetin administration decreased lipid deposition in arterial lumina, serum sIcam-1, and IL-6 and Vcam-1 in aorta, while increased the density of Sirt1 in aorta of ApoE-/- mice. In vitro , quercetin (0.3, 1, or 3 μmol/L) decreased the expression of senescence-associated β-galactosidase and improved cell morphology of HAECs. And quercetin decreased the cellular apoptosis and increased mitochondrial membrane potential (ΔΨm) in dose-dependent manner, and decreased ROS generation simultaneously. Transcriptome microarray suggested 254 differentially expressed (DE) mRNAs (110 mRNAs were upregulated and 144 mRNAs were downregulated) in HAECs after quercetin treatment (fold change > 1.5, P < 0.05, Que vs Ox-LDL). GO and KEGG analysis indicated nitrogen metabolism, ECM-receptor interaction, complement, and coagulation cascades, p53 and mTOR signaling pathway were involved in the pharmacological mechanisms of quercetin against ox-LDL. Conclusions: Quercetin alleviated atherosclerotic lesion both in vivo and in vitro. [ABSTRACT FROM AUTHOR]

Angong Niuhuang Pill (ANP) is a well-known patented Chinese medicine which is used for hundreds of years for treating the central nervous system diseases. Atherosclerosis is a poly-aetiological chronic inflammatory vascular disease. Preventing inflammation is fundamental for treating atherosclerosis in early stages. In this study, we investigated the protective effects and possible mechanisms of ANP action on a high-fat diet induced early and mid-term atherosclerosis ApoE-/- mice. The effects of ANP were compared with accepted drug simvastatin. Twelve male C57BL/6J mice were used as the control group, and 60 male ApoE-/- mice were randomly divided into five groups: Model group, Simvastatin group, Low-, Medium-, and High-dose ANP group these groups received, respectively, saline, simvastatin (3.0mg/kg), low-dose ANP (0.25 g/kg), medium-dose ANP (0.50 g/kg), and high-dose ANP (1.0 g/kg), once every other day for 10 weeks. After administration, serum biochemical indices were detected by the automatic biochemical analyzer, the concentrations of IL-6 and IL-10 in the serum were assayed by ELISA, expression levels of IL-1β, TNF-α, MMP-2, MMP-9, CCL2, and its receptor CCR2 in the full-length aorta, and expression levels of transcription factors Foxp3, RORγt in the spleen were assayed via western blotting and RT-qPCR. Flow cytometry was used to analyze Th17 cells and Treg cells. Pathological and histological analysis was completed on aortic root. ANP decreased LDL/HDL ratio, concentrations of IL-6 while increased IL-10 in serum. Moreover, ANP down-regulated the expression levels of IL-1β, TNF-α, MMP-2, MMP-9, CCL2, and CCR2 receptor in the full-length aorta. In addition, ANP decreased Th17 cells and expression levels of transcription factor RORγt, increased Treg cells and expression levels of transcription factor Foxp3. ANP decreased content of collagen fibers and infiltration of inflammatory cells in the aortic root. In conclusion, we demonstrated that ANP has anti-atherosclerosis effects on a high-fat diet induced ApoE-/- mice early and mid-term AS model via regulating Th17/Treg balance, inhibiting chronic inflammation, reducing plaque collagen fibers, and reducing inflammatory cells infiltration, to exert its multi-channel multi-target anti-early and mid-term AS effects. [ABSTRACT FROM AUTHOR]

Numerous risk factors are responsible for the development of atherosclerosis, for which an increased serum level of low-density lipoprotein cholesterol (LDL-C) is a driving force. By binding to the low-density lipoprotein cholesterol receptor (LDLR) and inducing LDLR degradation, proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a key role in cholesterol homeostasis regulation. The inducement of PCSK9 expression is also an important reason for statin intolerance. The Di’ao Xinxuekang (DXXK) capsule extracted from Dioscorea nipponica Makino is a well-known traditional Chinese herbal medicinal product used in atherosclerotic cardiovascular disease. Although DXXK has been widely used in atherosclerotic cardiovascular treatment for nearly 30 years, studies on the potential mechanisms of the lipid-lowering effect are very limited. The purpose of the present study was to demonstrate the possible involvement of the PCSK9/LDLR signaling pathway in the lipid-lowering and antiatherosclerotic effect of DXXK in high-fat diet-fed ApoE–/– mice. The results showed that DXXK treatment alleviated hyperlipidemia, fat accumulation, and atherosclerosis formation in ApoE–/– mice. Furthermore, changes in the expression of PCSK9 mRNA in liver tissue and the circulating PCSK9 level in ApoE–/– mice were both reversed after DXXK treatment, and upregulation of LDLR in the liver was also detected in the protein level in DXXK-treated mice. Our study is the first to show that DXXK could alleviate lipid disorder and ameliorate atherosclerosis with downregulation of the PCSK9 in high-fat diet-fed ApoE–/– mice, suggesting that DXXK may be a potential novel therapeutic treatment and may support statin action in the treatment of atherosclerosis.

Quercetin, endothelial cellular senescence, atherosclerosis, oxidized low-density lipoprotein, ApoE-/- mice. [Extracted from the article]

Objective: To observe the effect of Si-Miao-Yong-An (SMYA) on atherosclerosis (AS) vulnerable plaques, and to further explore the mechanism by vasa vasorum (VV) angiogenesis and maturation as an entry point. Methods: SPF-class healthy male ApoE−/− mice were randomized into model group, simvastatin group and SMYA group, and C57BL/6 mice were used as the control group. After 8 weeks of intervention, the pathological morphology of plaque was observed by HE staining; the VV density in plaque and aortic adventitia were observed by immunohistochemistry; VV maturation was measured by double-labelling immunofluorescence; the critical proteins of HIF-1α-Apelin/APJ and Ang-1/Tie signal pathways were detected by western blotting. Results: SMYA decreased the plaque area and the ratio of plaque to lumen area; increased the minimum thickness of fibrous cap and its effect was greater than simvastatin. SMYA suppressed the VV neovascularization; promoted smooth muscle cells recruitment and VV maturation, which maintained plaque stability; its effect was obviously superior to simvastatin. SMYA deceased the expression of HIF-1α, Apelin, APJ, Phospho-MEK1/2 (Ser217/221), Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204), Phospho-p70 S6 Kinase (Thr421/Ser424), Ang-2 and Tie-2; it also increased the expression of Ang-1, Phospho-Akt (Ser473), Phospho-FOXO1 (Ser256) and Survivin. Conclusions: SMYA can decrease the AS plaque area in ApoE−/− mice, suppress the VV neovascularization and promote the VV maturation, and stabilize AS vulnerable plaque. The mechanism could be regulating the HIF-1α-Apelin/APJ and Ang-1/Tie signal pathways. [ABSTRACT FROM AUTHOR]