黄芪甲苷对肿瘤坏死因子-α诱导的大鼠血管平滑肌细胞基质金属蛋白酶表达的影响  

Effects of Astragaloside IV on tumor necrosis factor-α-induced expressions of matrix metalloproteinases in rat vascular smooth muscle cells

作　　者：牛珩[1] 尉希清[1] 张金国[1] 

机构地区：[1]济宁医学院附属医院心内科、山东省心脏疾病诊疗重点实验室,272129

出　　处：《中华老年医学杂志》2016年第7期762-767,共6页Chinese Journal of Geriatrics

基　　金：山东省自然科学基金项目（ZR2011HL006）

摘　　要：目的观察黄芪甲苷（AS-IV）在肿瘤坏死因子（TNF-α）的诱导下，对离体大鼠血管平滑肌细胞（VSMCs）基质金属蛋白酶（MMPs）表达的影响。方法（1）采用组织贴块法培养大鼠血管平滑肌细胞，倒置相差显微镜和平滑肌肌动蛋白（α—SM—actin）进行细胞形态学和免疫组织化学鉴定；（2）以重组大鼠TNF-α（100Fg／L）作为刺激因素，建立体外平滑肌细胞增殖模型，并分组如下：①对照组；②TNFα组；③TNF-a＋AS-IV0．5mg／L；④TNF—α＋AS—IV5mg／L；⑤TNF—α＋AS-IV25mg／L；⑥TNF—α＋AS-IV50mg／L；（3）应用CCK-8法检测AS—IV对TNF-α诱导的VSMCs增殖能力影响；（4）应用实时PCR和Western—blot实验分别检测AΛ-IV对VSMCs分泌的MMP-2及其抑制因子（TIMP-2）mRNA和蛋白表达水平的影响。结果（1）在TNF—α刺激下，VSMCs增殖活性、迁移距离、侵袭能力与对照组相比均明显提高（P〈0．01），提示TNF—α可促进VSMCs的增殖、迁移，成功建立了体外细胞实验模型。（2）CCK-8实验结果显示，细胞经TNF-α孵育至预定时间后，细胞增殖明显，吸光度（A）值上升（P〈0．01）。经药物预处理后，各浓度组细胞增殖均受到抑制，与同期TNFα组相比A值降低（P〈0．05），且随着药物浓度和作用时间的增加，细胞生长抑制更加明显，提示AS-IV以时间和浓度依赖方式抑制了TNFα诱导的VSMCs增殖。（3）实时荧光定量PCR和Western—blot结果显示，TNF-α能够通过诱导actMMP-2的合成，对proMMP-2及TIMP-2的表达无明显影响来改变MMPs与TIMPs的比例，进而促进ECM的降解。经药物干预后，AS-IV以浓度依赖方式通过下调TNF—α诱导的MMP-2过表达，上调TIMP-2的mRNA及蛋白表达，恢复MMPs与TIMPs的比例，使ECM降解减少，从而实现对VSMCs增殖、迁移的抑制作用。结论黄芪甲苷能够以时间和浓度依赖方式抑制TNF-α诱导的VSMCs增殖、迁�Objective To investigate the effect of Astragaloside IV （AS-IV） on tumor necrosis factor-α （TNF-α）-induced expressions of matrix metalloproteinases （MMPs） in a rat vascular smooth muscle cells （VSMCs） proliferation model and its mechanism. Methods VSMCs were prepared from the thoracoabdominal aorta of rats by using issue-sticking method. Morphology of cells was observed by inverted microscope, and identified by immunohistochemical methods with antibody against SM-α-aetin. The model of VSMCs proliferation and migration was established by TNF-α inducer in vitro, and randomly divided into the following groups： the control group, the TNF group, the TNF-α AS-IV （0.5 μg/ml） group, the TNF α＋ AS-IV （5 μg/ml） group, the TNF-α＋ AS-IV （25 μg/ml） group, and the TNF-α＋ AS-IV （50μg/ml） group. The effect of AS-IV on TNF-α-induced VSMCs proliferation activity was detected by the caerulein and cholecystokinin octapeptide （CCK-8） method. The quantitative real-time polymerase chain reaction （real-time PCR） and Western blotting were used to examine the effects of AS-IV on the VSMCs-secreted mRNA and protein expressions of matrix metalloproteinase-2 （ MMP-2 ）, respectively. Results The proliferative activity, migratory distance and invasive capacity of VSMCs were obviously increased in TNF-α stimulation group versus in control group （all P〈0.01）, which suggested that TNF-α can promote VSMCs proliferation and migration, and that the rat model of VSMCs proliferation in vitro was successfully established. The results of CCK-8 tests showed that VSMCs proliferation was obvious and the optical density （OD） value was elevated （P〈0.01） after a preset time incubation with TNF-α. VSMCs proliferation was inhibited in each AS-IV treatment group, and the OD value was decreased as compared with the TNF-α group. And the inhibitive effect was increased along with the increments of AS-IV concentration and the acting time, which indicated that AS-IV can inhibit TNF-�

关 键 词：黄芪 肌 平滑 血管 肿瘤坏死因子Α 基质金属蛋白酶类 

分 类 号：R543[医药卫生—心血管疾病]