Primary Culture of Rat Aortic Vascular Smooth Muscle Cells: A New Method.

BACKGROUND Developing a simple and efficient method of obtaining primary cultured VSMCs is necessary for basic cardiovascular research. MATERIAL AND METHODS The procedure of our new method mainly includes 6 steps: isolation of the aortic artery, removal of the fat tissue around the artery, separation of the media, cutting the media into small tissue blocks, transferring the tissue blocks to cell culture plates, and incubation until the cells reach confluence. The cells were identified as VSMCs by morphology and immunofluorescence. Then, VSMCs obtained by this new tissue explants method, the traditional tissue explants method, the enzyme digestion method, and A7r5 cell line were divided into 4 groups. The purity of cells was test by multiple fluorescent staining. Western blotting was used to investigate the phenotype of VSMCs obtained by different methods. RESULTS Cells began to grow out at about 8 days and became relatively confluent within 16 days. Compared with VSMCs from the traditional tissue explants method and enzyme digestion method or A7r5 cell line, VSMCs obtained by our method showed higher purity and manifested a more "contractile" phenotype characteristic. CONCLUSIONS We have conquered the disadvantages in the previous primary culture methods and established a simple and reliable way to isolate and culture rat aortic VSMCs with high purity and stability.

Polyphenols and Polypeptides in Chinese Rice Wine Inhibit Homocysteine-induced Proliferation and Migration of Vascular Smooth Muscle Cells.

The beneficial effect of Chinese rice wine on atherosclerosis has been proved, but the exact components that have the cardiovascular protective effect are still unknown. This study aimed to explore the exact ingredients in Chinese rice wine that could inhibit homocysteine (Hcy)-induced vascular smooth muscle cell (VSMC) proliferation and migration. VSMCs were divided into 7 groups: control, Hcy (1 mmol/L), Hcy + oligosaccharide, Hcy + polypeptides, Hcy + polyphenols, Hcy + alcohol, and Hcy + Chinese rice wine. methyl thiazolyl tetrazolium (MTT) assay, Transwell chambers, and wound-healing assay were used to test the proliferation and migratory ability of the VSMCs. Western blot and gelatin zymography were used to investigate the expressions and activities of metal matrix proteinase 2/9 (MMP-2/9) and tissue inhibitor of metalloproteinase 2 (TIMP-2) in VSMCs. Polypeptides and polyphenols in the Chinese rice wine reduced the proliferation and migration ability of the VSMCs. Furthermore, they also decreased the expression and activity of MMP-2/9 but had no obvious impact on the expression of TIMP-2 in each group. This study further confirms that polypeptides and polyphenols in the Chinese rice wine could inhibit Hcy-induced proliferation and migration of VSMCs and maintain the balance between matrix metalloproteinases (MMPs) and TIMPs.

Effects of Chinese yellow wine on nitric oxide synthase and intercellular adhesion molecule-1 expressions in rat vascular endothelial cells.

OBJECTIVE: The objective of this study was to determine similarities in the effect of yellow wine as compared to statin and the possibility that yellow wine inhibits tumour necrosis factor-α (TNF-α)-induced nitric oxide (NO) synthesis, endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), and intercellular adhesion molecule-1 (ICAM-1) in cultured rat vascular endothelial cells (VECs). METHODS: We isolated VECs, and cultivated and purified Sprague Dawley (SD) rat thoracic aortas in vitro. We selected the optimal wine concentration using clonogenic and MTT assays to measure cell survival. Next, we divided the cells into 9 groups: (1) control, (2) TNF-α, (3) TNF-α + rosuvastatin (10 µmol/L), (4) TNF-α + ethanol 0.5%, (5) TNF-α + yellow wine 0.5%, (6) TNF-α + ethanol 1.0%, (7) TNF-α + yellow wine 1.0%, (8) TNF-α + ethanol 1.5%, and (9) TNF-α + yellow wine 1.5% and they were given the corresponding treatment for 24 h. We determined NO production with nitrate reductase. We then measured eNOS activity, and detected eNOS, iNOS, and ICAM-1 protein levels by Western blotting. RESULTS: Compared with the TNF-α group, NO production, eNOS activity, and eNOS protein expression in the rosuvastatin, and yellow wine 1.0%, and 1.5% groups were significantly increased. Protein expression of iNOS and ICAM-1 in the rosuvastatin, yellow wine 1.0%, and 1.5% groups were significantly decreased. Compared with the rosuvastatin group, eNOS, iNOS, and ICAM-1 protein expression in the yellow wine (0.5% -1.5%) groups were significantly different. CONCLUSION: Treatment with yellow wine increased NO production, eNOS activity, and eNOS protein expression, which decreases iNOS and ICAM-1 protein expression. We conclude that yellow wine may have similar beneficial effects as rosuvastatin on the cardiovascular system. These effects may be attributed to their anti-atherosclerotic actions.

Yellow wine polyphenolic compounds inhibit matrix metalloproteinase-2, -9 expression and improve atherosclerotic plaque in LDL-receptor-knockout mice.

Many epidemiological studies have strongly suggested an inverse correlation between dietary polyphenol consumption and reduced risks of cardiovascular diseases. Yellow rice wine is a Chinese specialty and one of the three most ancient wines in the world (Shaoxing rice wine, beer, and grape wine). There is a large amount of polyphenol substances in yellow rice wine. This experiment was designed to study the potential beneficial effects of yellow wine polyphenolic compounds (YWPC) from yellow rice wine on progression of atherosclerosis in vivo and to further explore its underlying mechanisms. Six-week-old male LDL-receptor-knockout mice were treated with high-fat diet to establish the mouse model with atherosclerosis. Animals received 10, 30, or 50 mg/kg per day of YWPC or 10 mg/kg per day rosuvastatin or water (vehicle) for 14 weeks. The results indicated that YWPC and rosuvastatin significantly decreased circulating total cholesterol and low-density lipoprotein cholesterol. Compared to the control group, the atherosclerosis lesion area in the rosuvastatin-intervention group and YWPC at doses of 10, 30, and 50 mg/kg per day intervention groups decreased by 74.14%, 18.51%, 40.09%, and 38.42%, respectively. YWPC and rosuvastatin decreased the expression and activity of matrix metalloproteinases (MMP)-2, 9, whereas the expression of the endogenous inhibitors of these proteins, namely, tissue inhibitors of matrix metalloproteinases (TIMP)-1, 2, increased when compared to the control group. It can be concluded that the YWPC is similar to the benefic effects of rosuvastatin on cardiovascular system. These effects may be attributed to their anti-atherosclerotic actions by lowering lipid and modulating the activity and expression of MMP-2, 9 and TIMP-1, 2.

LncRNA GHET1 from bone mesenchymal stem cell-derived exosomes improves doxorubicin-induced pyroptosis of cardiomyocytes by mediating NLRP3.

Doxorubicin (DOX) is an important chemotherapeutic agent for the treatment of hematologic tumors and breast carcinoma. However, its clinical application is limited owing to severe cardiotoxicity. Pyroptosis is a form of programmed cell death linked to DOX-induced cardiotoxicity. Bone mesenchymal stem cell-derived exosomes (BMSC-Exos) and endothelial progenitor cells-derived exosomes (EPC-Exos) have a protective role in the myocardium. Here we found that BMSC-Exos could improve DOX-induced cardiotoxicity by inhibiting pyroptosis, but EPC-Exos couldn't. Compared with EPCs-Exo, BMSC-Exo-overexpressing lncRNA GHET1 more effectively suppressed pyroptosis, protecting against DOX-induced cardiotoxicity. Further studies showed that lncRNA GHET1 effectively decreased the expression of Nod-like receptor protein 3 (NLRP3), which plays a vital role in pyroptosis by binding to IGF2 mRNA-binding protein 1 (IGF2BP1), a non-catalytic posttranscriptional enhancer of NLRP3 mRNA. In summary, lncRNA GHET1 released by BMSC-Exo ameliorated DOX-induced pyroptosis by targeting IGF2BP1 to reduce posttranscriptional stabilization of NLRP3.

Combined effects of vitamin D and neferine on the progression and metastasis of colorectal cancer.

PURPOSE: To investigate the synergistic effect of vitamin D and neferine on the growth and metastasis of colorectal cancer (CRC). METHODS: The synergistic effect of biologically active form of vitamin D, VD3 and neferine on the treatment of CRC was investigated by bliss analysis. Colony formation and wound healing ability, migration and invasion ability, and epithelial mesenchymal transition of HCT-116 cells, as a response to the combination treatment with VD3 and neferine were evaluated. RESULTS: VD3 and neferine showed a synergistic effect on CRC cell growth at a relatively low dose. The wound healing and colony formation capacity, cell migration and invasion abilities were all decreased by combination use of VD3 and neferine, compared to the VD3 or neferine treated single group. Furthermore, VD3 and neferine significantly decreased the expressions of N-cadherin, vimentin, snail, and slug in HCT-116 cells. CONCLUSION: These data suggest that neferine enhances the anticancer capability of VD3 and reduces the dose dependency of VD3. The combination of vitamin D with neferine appears to be a potential therapeutic strategy for CRC.

Rosuvastatin may modulate insulin signaling and inhibit atherogenesis beyond its plasma cholesterol-lowering effect in insulin-resistant mice.

OBJECTIVES: To provide evidence that rosuvastatin may improve insulin-resistance and inhibit atherogenesis by modulating insulin signaling, and whether this effect occurs beyond its plasma cholesterol-lowering effect. METHODS: Thirty-two 6-week-old low-density lipoprotein receptor deficient mice were randomized into 4 groups (n = 8 in each group): Normal control group (NC); High fat and high fructose diet group (HFF); HFF plus rosuvastatin group (HFFR); HFFR plus mevalonic acid group (HFFRMA). After 12 weeks, we measured fasting blood sugar, fasting insulin and cholesterol levels; the morphological concentrations of the aorta and aortic sinus; the expression of insulin receptor substrate 2, phosphorylated insulin receptor substrate 2, protein kinase B, phosphorylated protein kinase B and the glucose transporter 4 in the liver. RESULTS: Compared with other groups, fasting blood sugar and fasting insulin increased significantly in HFF group. Furthermore, HFF group had an increase in the morphological concentrations of the aorta and aortic sinus, but there was a significant decrease in the HFFRMA group and the HFFR group. Moreover, there was a high expression of insulin receptor substrate 2, phosphorylated insulin receptor substrate 2, protein kinase B, phosphorylated protein kinase B and the glucose transporter 4 in the HFFRMA and HFFR groups, but a low expression in the HFF group. No significant differences regarding each afore-mentioned index was observed in the HFFR and HFFRMA groups. CONCLUSIONS: Our data show that rosuvastatin may improve insulin-resistance and inhibit atherogenesis in HFF-fed mice by partially reversing the decrease in the insulin stimulated insulin receptor substrate 2/Phosphatidylinositol 3-kinase/protein kinase B/glucose transporter 4 pathway in the liver, and that this effect is independent of its cholesterol-lowering effect.

Relationship Between Dietary Knowledge, Socioeconomic Status, and Stroke Among Adults Involved in the 2015 China Health and Nutrition Survey.

Stroke is a public health threat that requires urgent attention in China. Nutrients have individual significant impacts on the prevalence of stroke. However, little research has been conducted on the impact of dietary knowledge on stroke and whether the impact is potentially heterogeneous under the effect of socioeconomic status. This study used the 2015 Chinese Health and Nutrition Survey to explore the impact of dietary knowledge and socioeconomic factors on populations suffering from stroke. Results indicated that risk of stroke decreased significantly with increasing dietary knowledge score. Additionally, the impact of dietary knowledge scores on the prevalence of stroke has obvious heterogeneity. First, dietary knowledge scores significantly influenced low-income groups and individuals with low educational levels. Second, the risk of stroke in females is more affected by dietary knowledge. Third, for people living in different areas, dietary knowledge determines whether rural populations suffer from stroke.

The differential diagnostic value of serum homocysteine for white coat hypertension.

OBJECTIVE: To assess the value of serum homocysteine (Hcy) in differential diagnosis of white coat hypertension (WCH). RESULTS: In this retrospective study, serum Hcy levels were elevated in hypertensive patients (P < 0.001) compared to WCH patients. Serum Hcy levels were positively correlated with 24-h mean systolic blood pressure, r = 0.1378, P < 0.001. The results of the receiving operating characteristic (ROC) curve showed that the AUC value of Hcy was 0.80 (95% CI, 0.77-0.83), the cut-off value was 13.8 µmol/L, the sensitivity was 68.58% and the specificity 87.21%. In the prospective study, the AUC value of Hcy was 0.73 (95% CI: 0.67-0.78), higher than N - terminal pro - brain natriuretic peptide(NT-pro-BNP) (0.64, 95% CI:0.58-0.70) and cystatin C (Cys-C) (0.62, 95% CI:0.55-0.68). Hcy, NT-proBNP and Cys-C combined, provided a better indication of a differential diagnosis of WCH, than Hcy alone. MATERIALS AND METHODS: This investigation involved both a retrospective and a prospective study. Clinical data including blood pressure, age, sex, height, weight, BMI, smoking status, past history, and behavioral electrocardiogram of patients who had undergone 24-hour ambulatory blood pressure monitoring (ABPM) with elevated clinical blood pressure (BP) were recorded. Pearson correlation analysis was used to test the correlation between Hcy and BP. The ROC curve was used to analyze the value of measuring Hcy levels in differential diagnosis of WCH. CONCLUSIONS: Serum Hcy was decreased in WCH patients and therefore could be a biomarker for differential diagnosis of WCH.

[Exploring the active ingredient of Chinese yellow wine which could inhibit the Hcy induced proliferation and migration of vascular smooth muscle cells].

OBJECTIVE: To explore the active ingredients in the Chinese yellow wine could inhibit the proliferation and migration of rat vascular smooth muscle cells induced by homocysteine (Hcy). METHODS: The primary culture and identification of rat vascular smooth muscle cells (VSMCs) was conducted, and the VSMCs in passage 4-7 were used in the following experiments. The VSMCs were divided into 7 groups: control, Hcy (1 mmol/L), Hcy + oligosaccharide, Hcy + polypeptides, Hcy + polyphenols, Hcy + alcohol, Hcy + Chinese yellow wine and were given the corresponding treatment. The proliferation of VSMCs was determined by MTT. Transwell chambers and would healing were employed to test the migratory ability of VSMCs. Wester blot and gelatin zymography were used to investigate the expressions and activities of metal matrix proteinase 2/9 (MMP-2/9) and tissue inhibitor of metalloproteinase 2 (TIMP-2) in VSMCs of each group. RESULTS: Compared with control group, the proliferation, migration and the expression and activity of MMP-2/9 of VSMCs were significantly increased in the VSMCs of Hcy group (P < 0.01). Compared with Hcy group, the proliferation, migration and the expression and activity of MMP-2/9 of VSMCs were significantly decreases in the VSMCs of polypeptides group, polyphenols group and Chinese yellow wine group. However, the expression of TIMP-2 among each group had no significant difference. CONCLUSION: Polypeptides and polyphenols in the Chinese yellow wine could inhibit the proliferation and migration of VSMCs induced by Hcy.

Hyperhomocysteinemia independently causes and promotes atherosclerosis in LDL receptor-deficient mice.

BACKGROUND: Hyperhomocysteine is an independent risk factor of coronary heart disease (CHD). However, whether hyperhomocysteine affects the progression of atherosclerosis is unclear. In the present study, we examined the effect of hyperhomocysteine on the formation of atherosclerosis in low-density lipoprotein receptor-deficient (LDLr(-/-)) mice. METHODS: Forty-eight 7-week-old LDLr(-/-) mice were assigned to the following groups: mice fed a standard rodent diet (control group), mice fed a high-methionine diet (high-methionine group), mice fed a high-fat diet (high-fat group), and mice fed a diet high in both methionine and fat (high-methionine and high-fat group). At the age of 19, 23, and 27 weeks, four mice at each interval in every group were sacrificed. RESULTS: At the end of the study, mice did not show atherosclerotic lesions in the aortic sinus and aortic surface until 27 weeks old in the control group. However, atherosclerotic lesions developed in the other three groups at 19 weeks. The amount of atherosclerotic lesions on the aortic surface was lower in the high-methionine group than in the high-fat group (P < 0.001). Atherosclerotic lesions on the aortic surface in the high-methionine and high-fat group were the most severe. The mean area of atherosclerotic lesions in the aortic sinus compared with atherosclerotic lesions on the aortic surface was lower in the high-methionine group than in the high-fat group (P < 0.001). Atherosclerotic lesions in the aortic sinus in the high-methionine and high-fat group were the most severe. CONCLUSIONS: Homocysteinemia accelerates atherosclerotic lesions and induces early atherosclerosis independently in LDLr(-/-) mice. Reducing the level of homocysteinemia may be beneficial for prevention and treatment of CHD.