人参三七川芎提取物对复制性衰老血管平滑肌细胞SM22α蛋白的影响

Effects of Extract from Ginseng Radix et Rhizoma,Notoginseng Radix et Rhizoma and Chuanxiong Rhizoma on SM22α Protein Expressions of Replicative Senescence Vascular Smooth Muscle Cells

目的:观察人参三七川芎提取物对复制性衰老血管平滑肌细胞及其骨架蛋白相关蛋白平滑肌22α(SM22α)的影响。方法:以人主动脉平滑肌细胞作为研究对象,复制性衰老9代细胞作为衰老模型,实验分为青年组(5代细胞),模型组(9代细胞),人参三七川芎提取物低、中、高(100,200,400 mg·L-1)剂量组及白藜芦醇组(10μmol·L-1),药物干预时间为48 h。采用β-半乳糖苷酶特异染色法计算蓝染细胞比率,流式细胞技术分析细胞周期,免疫荧光染色观察细胞SM22α形态的改变,实时荧光定量-聚合酶链式反应(q PCR)和蛋白质免疫印迹(Western blot)检测SM22α的mRNA及蛋白表达情况。结果:与青年组比较,模型组细胞体积增大,形态不规则,G0/G1期数量增多,S期数量减少,且β-半乳糖苷酶染色蓝染数量减少(P<0.01),符合衰老模型特点,且SM22α染色模糊暗淡,边缘不显,荧光强度明显减弱,SM22αmRNA及蛋白表达均下降(P<0.01)。与模型组比较,药物干预后,可以有效的增加β-半乳糖苷酶染色蓝染细胞数量,减少G0/G1期的细胞数量,同时增加S期的细胞数量,增强SM22α荧光染色强度,使得SM22αmRNA及蛋白表达均增加,并具有统计学意义(P<0.05,P<0.01)。结论:复制性衰老的血管平滑肌细胞可以作为衰老研究的模型;骨架蛋白相关蛋白SM22α的形态、基因表达和蛋白表达在细胞衰老过程中改变明显,其可能与骨架蛋白一同参与了平滑肌细胞衰老的进程;人参三七川芎提取物一定程度上延缓了血管平滑肌细胞的老化,且对于SM22α有明显干预作用,并可能通过此作用及对骨架蛋白的作用一同延缓血管老化的进程。

Objective： To observe the effect of extract from Ginseng Radix et Rhizoma,Notoginseng Radix et Rhizoma and Chuanxiong Rhizoma（ EGNC） on the smooth muscle 22α（ Sm22α） expressions of replicative senescence vascular smooth muscle cells and cytoskeleton-associated protein. Method： With human aortic smooth muscle cells as the research objects and the replicative senescence 9thgeneration cells as the aging model,this experiment was divided into youth group（ 5thgeneration cells）,model group（ 9thgeneration cell）,EGNC low dose group（ 100 mg·L^-1）,middle dose group（ 200 mg·L^-1）,and high dose group（ 400 mg·L^-1）,resveratrol group（ 10 μmol·L^-1）. The drug intervention time was 48 h. β-galactosidase staining method was used to calculate blue dye cell ratio. The cell cycle was analyzed by the flow cytometry. Immunofluorescent staining method was used to observe the morphological changes of Sm22α; while the mRNA and protein expressions of Sm22α were detected by q PCR and Western blot respectively. Result： Compared with the youth group,the cell size was increased and shape was irregular in model; number of cells in G0/ G1 stage was increased and number of cells in S stage was reduced; number of blue-dyed cells in β-galactosidase staining method was reduced（ P〈0. 01）,conforming to the characteristics of aging model; in addition,Sm22α staining was fuzzy and bleak,and the fluorescence intensity was weakened significantly,with reduced mRNA and protein expressions of Sm22α（ P〈0. 01）. As compared with the model group,drug intervention effectively reduced the number of blue-dyed cells,reduced the number of cells in G0/ G1,increased the number of cells in S stage,enhanced the fluorescence staining intensity of Sm22α,and increased mRNA and protein expressions of Sm22α,with statistically significant difference（ P〈0. 05,P〈0. 01）. Conclusion： The replicative senescence vascular smooth muscle cells can be used as aging research models. The morphology,gene and protein expressions of Sm22α were changed obviously in the process of cell aging,and it may involve in the process of cell aging together with cytoskeleton. EGNC delayed the aging of vascular smooth muscle cells to a certain extent and had obvious intervention effect on Sm22α,so it might delay the aging of vessels with the cytoskeleton indirectly.