吡哆胺对血管紧张素Ⅱ诱导的自发性高血压大鼠血管平滑肌细胞增殖的影响

Effects of pyridoxamine on the preliferation of spontaneously hypertensive rat vascular smooth muscle cells pretreated with angiotensin Ⅱ

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 influence of pyridoxamine on the preliferation of vascular smooth muscle cells（VSMCs） pretreated by angiotensin Ⅱ in vitro and to explore the mechanism of this process.Method：Primary VSMCs were isolated from the thoracic aortas of spontaneously hypertensive rat.The VSMCs from 3rd to 5th generation at exponential phase of growth were selected for the experiments.Cells were devided into four groups： control group with no intervention,angiotensin Ⅱ group with 10-7 mol/L angiotensinⅡtreated,three pyridoxamine groups pretreated with 0.1 mmol/L,10 mmol/L and 10.0 mmol/L pyridoxamine respectively two hours before Angiotensin Ⅱ intervention.The proliferation of VSMCs was determined by 3-（4,5-dimehyl-2-thiazolyl）-2,5-diphenyl-2H-tetrazolium bromide（MTT） colorimetry.Advanced glycation end-products（AGEs） in cellular supernatant were determined by enzyme-linked immunosorbent assay.Reactive oxygen species（ROS） level was detected by flow cytometry.The mRNA expressions of receptor for AGEs（RAGE）,NF-κB P65,NADPH oxidase P47phox were detected by semiquantitative fluorescence real-time polymerase chain reaction.Result：Compared with the control group,angiotensin Ⅱ group showed increased proliferation of cells（P0.01）,AGEs level in cellular supernatant（P0.01） and intracellular ROS generation（P0.01）.The mRNA expressions of RAGE,NF-κB P65 and NADPH oxidase P47phox were all up-regulated（P0.01）.The pyndoxamine significantly inhibited the VSMCs proliferation（P0.01）,reduced the AGEs level in supernatant（P0.01） and decreased the ROS generation that induced by angiotensinⅡ（P0.01）.The mRNA expressions of RAGE,NF-κB P65,NADPH oxidase P47phox were also down-regulated significantly（P0.01）.Conclusion：The pyridoxamine may inhibit angiotensin Ⅱ-induced VSMCs proliferation by inhibition of AGEs formation,reduction of intracellular ROS and down-regulation of the mRNA expressions of RAGE,NF-κB P65 and NADPH oxidase P47phox.