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目的:探讨吡哆胺对血管紧张素Ⅱ(AngⅡ)诱导的自发性高血压大鼠血管平滑肌细胞(VSMCs)增殖的影响及其作用机制。方法:原代培养自发性高血压大鼠胸主动脉VSMCs,选3～4代处于对数生长期的细胞进行药物干预。以未加任何干预的自发性高血压大鼠VSMCs为对照组,以10-7 mol/L AngⅡ刺激作为AngⅡ组,以不同浓度(0.1mmol/L、1.0mmol/L、10.0mmol/L)吡哆胺预处理作为吡哆胺组。采用四唑盐比色法检测吡哆胺对VSMCs增殖的影响,酶联免疫吸附法检测细胞上清液晚期糖基化终末产物(AGEs)水平,流式细胞仪分析细胞内活性氧簇(ROS)水平,实时荧光半定量PCR检测晚期糖基化终末产物受体(RAGE)、核因子κB(NF-κB)P65、还原型烟酰胺腺嘌呤二核苷磷酸(NADPH)氧化酶P47phox的mRNA水平。结果:与对照组相比,AngⅡ组促进细胞增殖(P<0.01),升高细胞上清液中AGEs浓度(P<0.01),使细胞内ROS生成增多(P<0.01),胞内RAGE、NF-κB P65、NADPH氧化酶P47phox mRNA相对量的表达均较对照组显著升高(P<0.01);1.0mmol/L和10.0mmol/L吡哆胺预处理可以逆转AngⅡ作用下的细胞增殖(P<0.01),降低细胞上清液中AGEs浓度(P<0.01),减少ROS生成(P<0.01),使RAGE、NF-κB P65、NADPH氧化酶P47phox mRNA表达下降(P<0.01),且吡哆胺10mmol/L作用比1mmol/L更显著(P<0.01)。结论:吡哆胺可能通过抑制AGEs的形成、降低胞内ROS水平,减少RAGE、NF-κB P65、NADPH氧化酶P47phox表达,从而有效抑制AngⅡ诱导的VSMCs增殖作用。
Objective: To investigate the effect of pyridoxamine on the proliferation of vascular smooth muscle cells (VSMCs) induced by angiotensin Ⅱ (Ang Ⅱ) in spontaneously hypertensive rats and its mechanism. Methods: Thoracic aorta VSMCs of spontaneously hypertensive rats were cultured in primary culture, and the cells in logarithmic growth phase were selected for 3 ~ 4 generations for drug intervention. VSMCs of spontaneously hypertensive rats without any intervention were used as the control group. Angiotensin Ⅱ (AngⅡ) was stimulated with 10-7 mol / L Ang Ⅱ and the cells were treated with 0.1 mmol / L, 1.0 mmol / L, 10.0 mmol / L Dopamine pretreatment as pyridoxamine group. The effects of pyridoxamine on the proliferation of VSMCs were detected by tetrazolium salt colorimetric assay. The levels of advanced glycation end products (AGEs) in supernatants of cells were detected by enzyme-linked immunosorbent assay (ELISA). The intracellular reactive oxygen species (ROS), and the levels of RAGE, NF-κB P65, reduced nicotinamide adenine dinucleoside phosphate (NADPH) oxidase P47phox mRNA level. Results: Compared with the control group, the AngⅡ group increased the cell proliferation (P <0.01), increased the concentration of AGEs in the cell supernatant (P <0.01), increased the intracellular ROS production (P <0.01) The expressions of NF-κB P65 and NADPH oxidase P47phox mRNA were significantly increased compared with the control group (P <0.01). Pretreatment with 1.0 mmol / L and 10.0 mmol / L pyridoxamine could reverse the cell proliferation induced by AngⅡ (P <0.01), decreased the concentration of AGEs in the cell supernatant (P <0.01), reduced ROS production (P <0.01), and decreased the expression of RAGE, NF-κB P65 and NADPH oxidase P47phox mRNA The effect of 10mmol / L pyridoxamine was more significant than that of 1mmol / L (P <0.01). CONCLUSION: Pyridoxamine can inhibit the proliferation of VSMCs induced by AngⅡ by inhibiting the formation of AGEs, decreasing the level of intracellular ROS, decreasing the expression of RAGE, NF-κB P65 and NADPH oxidase P47phox.