Efficacy of Qingre Huayu Fang on atherosclerotic vulnerable plaque in apolipoprotein E knockout mice: proteasome pathway involvement.

OBJECTIVE: To investigate the efficacy and mechanism of the Qingre Huayu Fang () on atherosclerotic vulnerable plaque in apolipoprotein E (ApoE) knockout mice through the ubiquitin proteasome pathway. METHODS: Sixty 8-week-old C57BL/6J ApoE knockout mice were fed a high-fat for 12 weeks and randomly divided into four treatment groups (n = 15 each): high-fat control, bortezomib (a proteasome inhibitor), bortezomib combined with Qingre Huayu Fang, and Qingre Huayu Fang alone. Aortic sections were examined for plaque development, inflammatory cell infiltration, type Ⅰ/Ⅲ collagen expression and immunohistochemical staining of CD40L, nuclear factor-kappa B (NF-κB)/P65 and ubiquitin. RESULTS: Mice in the high-fat control group had obvious atherosclerosis, with increased aortic plaque area. The degree of atherosclerosis of the atherosclerotic plaque was reduced in all of the treatment groups that received bortezomib and/or Duzhong (Cortex Eucommiae) Qingre Huayu. The expression of NF-?B, CD40L and ubiquitin were all reduced in the group that received combination bortezomib + Qingre Huayu Fang. CONCLUSION: The Qingre Huayu Fang inhibited aortic atherosclerosis in mice through a mechanism that may involve inhibition of the ubiquitin proteasome pathway.

Jianpi Huazhuo Tiaozhi granules reduce oxidative stress injury in macrophages by inhibiting the nicotinamide adenine dinucleotide phosphate oxidase/reactive oxygen species-nuclear transcription factor kappa B pathway.

OBJECTIVE: To assess the effect of Jianpi Huazhuo Tiaozhi granules (JHTG) on oxidative stress damage to macrophages and explore the relationship between the levels of this damage and the nicotinamide adenine dinucleotide phosphate oxidase (NOX)/reactive oxygen species (ROS)- nuclear transcription factor kappa B (NF-κB) signaling pathway. METHODS: Macrophages cultured in vitro were divided into seven groups: control, model control, inhibitor, positive control, 2.5% JHTG, 5% JHTG, and 10% JHTG. An oxidative stress injury model was established by stimulating macrophages with oxidized low-density lipoprotein. Cell survival and apoptosis were detected by 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di- phenytetrazoliumromide and flow cytometry assays, respectively. Malondialdehyde and superoxide dismutase levels were detected by enzyme-linked immunosorbent assays, while ROS levels were detected using a fluorescence probe. Proteins and mRNAs associated with the NOX/ROS-NF-κB pathway, including NOX4, p22phox, inhibitor of NF-κB kinase-α (IKK-α), inhibitor of NF-κB kinase-ß (IKK-ß), and NF-κB were detected by Western blot and PCR, respectively. RESULTS: After JHTG treatment, there were fewer damaged and apoptotic macrophages, while superoxide dismutase levels were elevated. The JHTG-treated groups also showed reduced ROS levels. The molecular changes following JHTG treatment included decreased expression of NOX4 and p22phox at the protein level and decreased IKK-α, IKK-ß, and NF-κB expression at the mRNA level. All of these effects were correlated with the JHTG concentration. CONCLUSION: These results demonstrated that the molecular mechanism underlying the antioxidant activity of JHTG in macrophages is through inhibiting the NOX/ROS-NF-κB pathway.

Dingxin recipe alleviates atherosclerosis injury in ApoE-knockout mice via downregulation of visfatin expression and inhibition of the visfatin-induced inflammatory response.

OBJECTIVE: To further elucidate the mechanism underlying the anti-atherosclerotic effect of Dingxin recipe (DXR). METHODS: Fifty 6-week-old male ApoE-/- mice were randomly divided into the following groups: model, simvastatin (5 mg·kg－1·d－1), DXR low-dose (9.30 g·kg－1·d－1), DXR middle-dose (18.59 g·kg－1·d－1) and DXR high-dose (37.18 g·kg－1·d－1) (n = 10). Ten male C57BL/6J mice were used as the control group. All ApoE-/- mice were fed a high-fat diet (HFD) and the control mice received a common diet. After HFD for 12 weeks, the mice were treated with DXR or simvastatin for another 12 weeks. The expression of inflammatory cytokines and visfatin was determined in serum and atherosclerotic lesions by enzyme-linked immunosorbent assay. Visfatin expression was also assessed in aortic atherosclerotic plaques. Cultured vessel endothelial cells (VECs) were pretreated with DXR sera prior to visfatin. The effects of DXR were analyzed to elucidate its protective mechanism against visfatin-induced inflammation in VECs. RESULTS: DXR regulated blood lipids and reduced tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), intercellular adhesion molecules-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and visfatin expression in ApoE-/- mice, particularly at the higher doses. The areas of atherosclerotic lesions in the DXR groups were significantly smaller than those in the model group. DXR alleviated visfatin-induced VEC injury via downregulation of TNF-α, IL-6, ICAM-1 and VCAM-1 through mitogen-activated protein kinase pathways. CONCLUSION: DXR alleviated atherosclerosis injury via downregulation of visfatin expression and inhibition of the visfatin-induced inflammatory response in VECs.

Bushen Jiangzhi formula reduces atherosclerosis in apoE-/- mice through autophagy.

OBJECTIVE: To study the effect of Bushen Jiangzhi formula (BSJZF) on atherosclerosis (AS) in apolipoprotein E knockout (apoE-/-) mice and the underlying mechanism. METHODS: We used a high fat diet to induce AS in apoE-/- mice. The mice were randomly divided into four groups: model, BSJZF, atorvastatin, and 3-methyladenine groups. Syngeneic C57BL/6 mice of the same age were used for the control group. Autophagosomes in the aorta were examined by transmission electron microscopy. Morphology, lipid accumulation, and collagen deposition in the aorta were examined by hematoxylin and eosin, Oil Red O, and Masson's staining, respectively. Serum levels of tumor necrosis factor alpha (TNF-), interferon gamma (IFN-), and interleukin 10 (IL-10) were measured by enzyme-linked immunoassays. Protein expression of microtubule-associated protein light chain 3 (LC3), Beclin 1, and p62 in the aorta were examined by Western blot analyses. RESULTS: ApoE-/- mice fed a high fat diet exhibited AS symptoms including less autophagosomes in the aorta, higher serum levels of TNF-a, IFN-r, and p62, and lower serum levels of IL-10, LC3, and Beclin 1. Treatment with BSJZF significantly reduced the area of the aortic plaque, decreased expression of TNF-a, IFN-r, and p62, and increased expression of IL-10, LC3, and Beclin 1. CONCLUSION: Our findings suggest that BSJZF promotes autophagy and reduces inflammation by regulating the expression of autophagy-related proteins LC3, Beclin 1, and p62, thereby effectively treating AS.

Danggui Buxue decoction ameliorates lipid metabolic defects involved in the initiation of diabetic atherosclerosis; identification of active compounds.

OBJECTIVE: To determine the constituent compounds of Danggui buxue decoction (DBD) involved and the potential mechanisms mediating its effects, with specific reference to lipids playing a role in the initiation of diabetic atherosclerosis. METHODS: Liquid chromatography-tandem mass spectrometry was used to identify and quantify the absorbed bioactive compounds (ABCs) present in DBD. Goto-Kakizaki (GK) rats were randomly allocated to a diabetes atherosclerosis (DA) group, a DBD group, and an ABC group (10 per group), which were all high-fat diet-fed. The treated rats were administered DBD (4 g/kg) or ABCs (in amounts equal to those present in DBD) once daily for 28 d, and a control group of Wistar rats were administered vehicle. Body mass gain, fasting blood glucose, and homeostasis assessment of insulin resistance (HOMA- IR) were measured. Serum triglyceride (TG), cholesterol (CHOL), high density lipoprotein-cholesterol (HDL-C), low-density lipoprotein-cholesterol (LDL- C) and tumor necrosis factor-α (TNF-α) concentrations were determined. Hematoxylin and eosin staining and microscopy were used to characterize the abdominal aorta and the expression of lipogenic genes was quantified in this vessel. RESULTS: Seven ABCs were identified in rat serum: ferulic acid, formononetin, calycosin, astragaloside, caffeic acid, ligustilide, and butyphthalide. DBD significantly reduced HOMA-IR, the serum concentrations of TG, CHOL, and LDL-C, and the expression of the lipogenic genes monocyte chemotactic protein 1, Fas, intercellular adhesion molecule 1, and Cd36 in aorta; and significantly increased the mRNA expression of Scd1 in aorta. CONCLUSION: DBD affects lipid metabolism in the early stage of atherosclerosis in diabetic GK rats, with the mechanism likely involving the regulation of lipid metabolic genes in vessels. The contribution of ABCs to the effect of DBD on lipid metabolism was 24%-101%.