| Cardiovascular diseases are the leading cause of death and disability worldwide, and 80-90% cardiovascular patients had hypertension. One of important plans on "Dietary Approaches to Stop Hypertension (DASH)" is to increase fruit and vegetable intake. Tumor necrosis factor-alpha(TNF-a), a prototypic proinflammatory cytokine commonly found in atherosclerotic lesions, can have direct effects on vascular endothelial cells to induce protein and mRNA of monocyte chemoattractant protein-1 (MCP-1), intercellular adhesion molecule-1(ICAM-1), and vascular cell adhesion molecule-1(VCAM-1), etc., which are important in the onset and progress of inflammation. Blueberry could enhance heart function, improve blood circulation, relaxation, promote blood vessels, and prevent hypertension. Blueberry anthocyanins are the most effective antioxidants found in nature. Malvidin (Mv) is one of the most wide-spread anthocyanidins. Therefore, we guessed that antioxidant pigment Malvidin maybe have potential inflammation inhibitory activity. The objective of the study was to study the anti-inflammatory properties of Malvidin chloride (Mv-Cl), Malvidin-3-glucoside chloride (Mv-3-glc-Cl), Malvidin-3-galactoside chloride (Mv-3-gal-Cl) on TNF-a-induced inflammation in human vascular umbilical endothelial cells (HUVECs). In addition, we investigated the synergistic anti-inflammatory effect of Mv-3-glc-Cl and Mv-3-gal-Cl in human vascular umbilical endothelial cells.This provides a theoretical basis for the proinflammatory cytokines induced endothelial dysfunction and inflammation to result cardiovascular disease.Objective:1. The HUVECs are cultured in vitro. Expose endothelial cells to TNF-a to establish cell model of inflammation. To investigate Mv-Cl, Mv-3-glc-Cl, and Mv-3-gal-Cl respectively whether they can protect the injury of endothelial cells.2. To detect the MCP-1, ICAM-1 and VCAM-1 protein and mRNA content by ELISA, western Blot and PT-PCR. To explore the molecular mechanisms of their anti-inflammatory effect to endothelial cells.To detect the expression of IκBα and NF-κB nuclear transcription situation, and primarily analyze the protective mechanism of different Malvidin towards the TNF-a induced inflammatory response in HUVECs.Methods:1. The human umbilical vein endothelial cells were isolated by enzymatic digestionfrom the fresh baby umbilical cord. The endothelial cells were cultured with M199 complete medium, passaged and frozen. The second to sixth passage cells were used for experiments.2. The HUVECs were treated with different concentrations of tumor necrosis factor (TNF-a) for 4 h, and treated different time with optimal concentrations of TNF-a. MCP-1, ICAM-1 and VCAM-1 protein levels in cell supernatants were measured by ELISA. The ICAM-1 and VCAM-1 protein levels in cells were tested by Western Blot. According to the results of the analysis, the optimal agent efficiency and treatment time of TNF-a to induce inflammmatary response in HUVECs were obtained.-.3. The HUVECs were pretreated with different concentrations of Mv-Cl, Mv-3-glc-Cl and Mv-3-gal-Cl for 18 h, respectively, followed by TNF-α (10 μg/L) stimulation for 6 h. DMSO was used as control. Cell viability was measured by MTT assay. MCP-1, ICAM-1 and VCAM-1 protein levels in cell supernatants were measured by ELISA. The cell ICAM-1 and VCAM-1 protein levels were tested by Western Blot. The changes on mRNA expression level of MCP-1 and ICAM-1 in cells were detected by RT-PCR.4. The protein of κBα was assessed by Western-blot. The activity of NF-κB was evaluated by immunofluorescence. We analyzed the molecular basis of the anti-inflammatory effect mechanism.Results:1. The effect of TNF-a on the expression of MCP-1, ICAM-1 and VCAM-1protein in cell surface and cellsIn unstimulated cells, the surface expression of MCP-1, ICAM-1 and VCAM-1 protein was very low. The preoteins in cell supernatant and cells obtained were assessed by ELISA and Western Blot, respectively. Incubation with different concentrations of TNF-a, their expression increased in a dose-dependent manner. The protein expression change was not significant at low concentrations. The protein expression increased significantly as the concentration increases. Especially the protein expression level of MCP-1, ICAM-1, and VCAM-1 got the highest values when HUVECs were induced by 10μg/L of TNF-a (P< 0.01).2. The HUVECs were stimulated with 10 μg/L TNF-a for different time. We found that the expression of MCP-1, ICAM-1, and VCAM-1 proteins was less affected with short time. ELIS A results on the cell-free endothelial supernatants showed that MCP-1, ICAM-1, and VCAM-1 proteins released into medium all significantly (3.762 ± 0.041, 8.436 ± 0.367,8.554 ± 0.069 fold, respectively) increased after TNF-a stimulated 6 h (P< 0.01). The ICAM-1/β-Actin and VCAM-1/β-Actin relative expression to the control group were 2.268 ± 0.012 (P< 0.001),1.616 ± 0.022 (P< 0.01) folds in cells at 6 h. Thus, the protein expression level of MCP-1, ICAM-1 and VCAM-1 got the highest values when HUVECs were stimulated 6 h by 10 μg/L TNF-a. Effect of Mv-Cl on TNF-a-induced MCP-1, ICAM-1, and VCAM-1 protein expression in HUVECsMTT tells that Mv-Cl, Mv-3-glc-Cl and Mv-3-gal-Cl of 1,10,50,100 μmol/L could inhibit 92.6%,87.6%,70.1%,64.8%; 92.6%,87.6%,70.1%,64.8%and 86.3%, 85.1%,70.3%,61.5% cells compared with unstimulated cells(P< 0.01). It indicated that Mv-Cl has a protective effect to TNF-a-induced cells.The changes on protein and mRNA expression level of MCP-1, ICAM-1, and VCAM-1 in cells were detected by ELIS A, Western Blot and RT-PCR. In unstimulated cells, the protein and mRNA expression of MCP-1, ICAM-1, and VCAM-1 protein were very low. Incubation with TNF-a, their expression increased. Pretreatment with 1,10,50,100 μmol/L Mv-Cl, TNF-a-induced MCP-1, ICAM-1, and VCAM-1 expression in the cells were inhibited. Even,50 and 100 μmol/L of Mv-Cl could inhibit more than 90% increased three proteins, whose level was simial to the control, which indicated that high concentration of Mv-Cl might protect the inflammotary cells.3. Effect of Mv-3-glc-Cl and Mv-3-gal-Cl on TNF-a-induced MCP-1, ICAM-1, and VCAM-1 expression in HUVECsThe changes on protein and mRNA expression level of MCP-1, ICAM-1, and VCAM-1 in cells were detected by ELISA, Western Blot, and RT-PCR. Mv-3-glc-Cl and Mv-3-gal-Cl had different inhibitory effects on the protein and mRNA levels of endothelial MCP-1, ICAM-1, and VCAM-1 in a concentration-dependent manner. For most condition, Mv-3-glc-Cl showed stronger effect than Mv-3-gal-Cl, except that low concentration of Mv-3-glc-Cl (1 and 10 μmol/L) had no significant inhibition on TNF-a-induced ICAM-1 expression. Mv-3-glc-Cl had stronger effect than Mv-3-gal-Cl at high concentration.4. Synergistic effect of Mv-3-glc-Cl and Mv-3-gal-Cl and molecular mechanism of their anti-inflammationThe changes on protein and mRNA expression level of MCP-1, ICAM-1, and VCAM-1 in cells were detected by ELISA, Western Blot, and RT-PCR. The results showed that the synergistic effect existed between Mv-3-glc-Cl and Mv-3-gal-Cl. They could inhibited TNF-a-induced increases of MCP-1, ICAM-1, and VCAM-1 production both in the protein and mRNA levels. TNF-a induced IκB degradation, and subsequently increased the expression of MCP-1, ICAM-1, and VCAM-1. The inhibitory effect of Mv-3-glc-Cl, Mv-3-gal-Cl and (Mv-3-glc-Cl+Mv-3-gal-Cl) (10 μmol/1) on TNF-a-induced expression of IκB degradation was decreased. The activation of NF-κB requires the translocation of the p65 subunit of NF-κB from the cytoplasm to the nucleus. Immunocytochemistry was performed by using NFκB and fluorescein isothiocyanate (FITC)-conjugated antibody. In un-stimulated cells, the levels of p65 in the nucleus were very low. Upon simulation by TNF-a, the levels of p65 in the nucleus were increased. On the other hand, pretreated with Mv-3-glc-Cl, Mv-3-gal-Cl, and their mixture, the fluorescence intensity levels of p65 were decreased in the nucleus. The high concentration of Mv-3-glc-Cl, Mv-3-gal-Cl, and their mixture might completely inhibit the nuclear translocation of p65, with the p65 protein level in the cell nucleus similar to the control, which suggested the anti-inflammation mechanism of Malvidin by the NF-κB pathway. These results indicated the potential role of Mv in preventing chronic inflammation in many diseases. |
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