同型半胱氨酸对大鼠血管内皮细胞MMP-2表达的影响和黄酒、红葡萄酒的逆转效应及其机制

摘要

Objective To study whether rice wine can inhibit the expression of matrix metalloproteinase-2 (MMP-2) in cultured rat vascular endothelial cells(VECs) induced by homocysteine (Hcy) and its mechanisms. Methods We carried out isolation, cultivation, purification and identification of VECs in rat aorta in vitro. Cell passages 3 and 4 were used for all studies. Either cells were incubated with different doses of Hcy (0, 50, 100, 500 and 1 000 μmol/L) for 48 h, or with 100 μmol/L Hcy for 24, 48 and 72 h to observe the effect of Hcy on mRNA and protein expressions of MMP-2 using fluorescent quantitative PCR (FQ-PCR) and Western blotting. The optimal concentration and time of Hcy were selected. The VECs were incubated with one type of wine (10, 12, 14, 16, 18, 20 mL/L) and 100 μmol/L Hcy for 48 h; then the optimal concentration of each type of wine was selected. In another experiment, cells were divided into 5 groups: control, Hcy, Hcy+rice wine, Hcy + red wine, Hcy+ethanol groups and then incubated for 48 h. The FQ-PCR was used to test MMP-2 expression at the mRNA level and the Western blotting was applied to detect MMP-2 expression at the protein level. Results The results showed that within the concentration range of 50 and 1 000 μmol/L, Hcy could significantly increase the mRNA and protein expressions of MMP-2 compared with those in the control group (P<0. 05 and P<0. 01), and could reach the maximum at 500 μmol/L. At 24 h, 48 h and 72 h after stimulation by Hcy at the concentration of 100 μmol/L, MMP-2 expression at the mRNA and protein levels was significantly increased compared with that in the control group (P<0.01), and could reach the maximum at 48 h. Compared with those in Hcy group, MMP-2 expression at the mRNA and protein levels in rice wine and red wine groups was significantly decreased (both P<0. 01). No significant difference was found between ethanol group and Hcy group (P>0.05). Compared with ethanol group, use of rice wine and red wine also significantly reduced the mRNA and protein expressions of MMP-2, and there were significant differences (both P<0. 01). However, the rice wine and red wine groups did not differ significantly (P>0.05). Conclusion The results suggest that Hcy promotes the mRNA and protein expressions of MMP-2, which may play an important role in pathogenesis of atherosclerosis. Extracellularly added rice wine and red wine can decrease Hcy-induced MMP-2 production. The inhibitory effect of rice wine and red wine on mRNA and protein expressions of MMP-2 might contribute to their beneficial effects on the cardiovascular system.%目的 观察黄酒能否抑制同型半胱氨酸(Hcy)诱导的大鼠血管内皮细胞(vascular endothelial cells,VECs)中基质金属蛋白酶-2(matrix metalloproteinase-2,MMP-2)表达及探讨其可能机制.方法 大鼠原代主动脉VECs经分离培养及纯化鉴定后,取第3～4代细胞用于实验.将Hcy(0、50、100、500、1 000μmol/L)与VECs共同孵育48 h及100μmol/L Hcy与VECs共同培养24、48、72h,分别用荧光定量PCR(FQ-PCR)和免疫印迹法检测Hcy对VECs中MMP-2 mRNA和蛋白表达的影响,确定Hcy最佳的刺激浓度和时间.每种酒(10、12、14、1 6、18、20 mL/L)与100μmol/L Hcy共同孵育VECs 48 h,MTT法检测酒类对细胞活性的影响,确定最佳干预浓度后处理分为正常组、Hcy组、Hcy+黄酒组、Hcy+红葡萄酒组、Hcy+酒精组5组.培养48 h后收集样品,FQ-PCR检测VECs中MMP-2mRNA水平,免疫印迹法测定VECs中MMP-2的表达量.结果 在50～1 000 μmol/L浓度范围内与对照组相比Hcy能明显地增强MMP-2 mRNA和蛋白表达(P＜0.05或P＜0.01),至500 μm.l/L时开始达到最高.Hcy(100 μmol/L)刺激24、48、72h后MMP-2 mRNA和蛋白表达较对照组有明显的增强(均P＜0.01),48h后开始达到最大值.实验组与Hcy组相比,黄酒组和红酒组MMP-2 mRNA和蛋白表达均明显下降,差异有统计学意义(均P＜0.01).酒精组MMP-2 mRNA和蛋白表达下降,差异均无统计学意义(P＞0.05).与酒精组相比,黄酒组和红酒组VECs中MMP-2 mRNA和蛋白表达均明显下降,差异有统计学意义(均P＜0.01),黄酒组和红酒组之间差异无统计学意义(P＞0.05).结论 Hcy能增强VECs中MMP 2 mRNA和蛋白的表达,这可能在动脉粥样硬化的发病机制中发挥重要作用.黄酒和红葡萄酒能抑制Hcy对MMP-2 mRNA和蛋白表达的增强,这可能是它们抗动脉粥样硬化和心血管保护作用的机制之一.