人参-三七-川芎提取物对内皮微粒介导HUVECs衰老的干预作用

目的:观察人参-三七-川芎提取物对延缓内皮微粒(EMPs)诱导的血管内皮细胞衰老的影响,并探索其作用机制。方法:以人脐静脉内皮细胞(HUVECs)为研究对象,复制性衰老10~12代细胞作为衰老模型,实验分为年轻组(2~4代细胞)、衰老组(10~12代细胞)、单纯EMPs干预组(提取衰老细胞产生的EMPs来干预年轻细胞)以及中药低、中、高剂量组(200,300,400 mg·L-1)。结合衰老相关β半乳糖苷酶(SA-β-gal)染色法和碘化丙啶(PI)单染法检测细胞周期来判断细胞衰老程度。采用细胞活性和细胞增殖检测(CCK-8)法筛选给药浓度,两步离心法分离出EMPs,藻红蛋白(PE)CD31抗体或异硫氰酸荧光素(FITC)Annexin V标记分离得到的EMPs,并用流式细胞术进行定量,2’,7’-二氯荧光素二乙酸酯(DCFDA)染色检测细胞内活性氧(ROS)水平。结果:与衰老组比较,中药给药组可显著降低衰老细胞SA-β-gal活性(P<0.01),使细胞周期S期阻滞得到恢复(P<0.01),并降低衰老细胞分泌CD31+EMPs及Annexin V+EMPs的数量(P<0.05)。与年轻组比较,单纯EMPs干预组可诱导年轻细胞SA-β-gal活性增强(P<0.01),细胞周期阻滞于S期(P<0.05),与衰老组比较,但在EMPs干预的同时给予中药干预后,可明显抑制EMPs介导增强的SA-β-gal活性(P<0.05),并使S期阻滞得到恢复。中药组还可明显抑制EMPs诱导的细胞内ROS增高(P<0.05,P<0.01)。结论:人参-三七-川芎提取物可通过影响EMPs延缓复制性血管内皮细胞衰老,其机制可能与抑制EMPs诱导的细胞内ROS水平增高有关。

Effect of Ginseng Radix et Rhizoma-Notoginseng Radix et Rhizoma-Chuanxiong Rhizoma Extract on HUVECs Senescence Mediated by Endothelial Microparticles

Objective: To investigate the effect of Ginseng Radix et Rhizoma-Notoginseng Radix et Rhizoma-Chuanxiong Rhizoma extract on endothelial microparticles(EMPs)-induced vascular endothelial cell senescence,and explore the possible mechanism. Method: Human umbilical vein endothelial cells(HUVECs)were used as the research objects,and the aged model was established with 10-12 passages of replicative senescence cells. The experimental cells were divided into young group(2-4 passage cells),aged group(10-12 passage cells),only EMPs intervention group(extract EMPs produced by aged cells to intervene young cells)and low dose,middle dose and high dose drug intervention groups(200,300,400 mg·L-1). Senescence relatedβ-galactosidase(SA-β-gal)staining and cell cycle propidium iodide(PI)staining were used to determine cell senescence. Cell counting kit-8(CCK-8)assay was used to screen the drug concentration. EMPs were extracted by two-step centrifugation,EMPs labeled with phycoerythrin(PE)anti-human CD31 antibody or fluorescein isothiocyanate(FITC)annexin V were detected by flow cytometry,intracellular reactive oxygen species(ROS)were detected by 2’,7’-dichlorofluorescein diacetate(DCFDA)staining. Result:After treatment with the drug,SA-β-gal activity of the aged cells significantly decreased(P<0.01),the S phase arrest was restored(P<0.01),and the number of CD31+EMPs and annexin V+EMPs secreted by aged cells decreased(P<0.05). Compared with the young group,only EMPs intervention group could induce increased SA-β-gal activity and S phase arrest in young cells(P<0.05,P<0.01). However,after intervention of EMPs and the drug,EMPs-mediated increase of SA-β-gal activity was significantly inhibited and S phase arrest was restored(P<0.05). The increase of intracellular ROS induced by EMPs was also significantly inhibited by the drug(P<0.05,P<0.01). Conclusion:Ginseng Radix et Rhizoma-Notoginseng Radix et Rhizoma-Chuanxiong Rhizoma extract can delay the senescence of vascular endothelial cells by influencing EMPs, and the mechanism may be related to the inhibition of increased intracellular ROS induced by EMPs.