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目的:研究葛根素对外周血内皮祖细胞(EPCs)缺氧/复氧损伤的保护作用。方法:采用密度梯度离心法和贴壁培养法相结合,使用专用培养基EGM-2,成功分离纯化EPCs,通过形态学、细胞表面分子标志物检测、内皮祖细胞吞噬功能对其鉴定;采用Krebs液建立缺氧/复氧损伤模型,用葛根素干预后,采用MTT法测定细胞活力、2,4-二硝基苯肼显色法测定细胞外乳酸脱氢酶(LDH)释放量、黄嘌呤氧化酶法测定细胞内超氧化物歧化酶(SOD)活性、硫代巴比妥酸法测定丙二醛(MDA)含量、硝酸还原酶法测定一氧化氮(NO)含量;Western blot法考察胞核、胞质Nrf2和全细胞HO-1蛋白表达。结果:与模型组相比,葛根素20.8 mg·L~(-1)及41.6 mg·L~(-1)组显著提高细胞活力(P<0.05,P<0.01);葛根素20.8 mg·L~(-1)及41.6 mg·L~(-1)能显著降低NO、MDA含量和LDH释放量(P<0.01);葛根素20.8 mg·L~(-1),41.6 mg·L~(-1),83.2 mg·L~(-1)均显著提高缺氧/复氧后细胞SOD活性(P<0.01),并上调细胞HO-1、胞核Nrf2蛋白表达(P<0.01),显著降低胞浆Nrf2蛋白表达(P<0.01)。结论:葛根素可显著拮抗缺氧/复氧对EPCs的损伤,可能与其抑制细胞过氧化损伤,增强细胞抗氧化能力有关。
Objective: To study the protective effect of puerarin on hypoxia / reoxygenation injury in peripheral blood endothelial progenitor cells (EPCs). Methods: EPCs were successfully isolated and purified by using special medium EGM-2 by density gradient centrifugation and adherent culture. EPCs were identified by morphology, cell surface markers and phagocytosis of endothelial progenitor cells. Hypoxia / reoxygenation injury model was established. After intervention with puerarin, cell viability was determined by MTT assay. The release of extracellular lactate dehydrogenase (LDH) and xanthine oxidation were determined by 2,4-dinitrophenylhydrazine The activity of superoxide dismutase (SOD) was measured by enzymatic method, the content of malondialdehyde (MDA) was measured by thiobarbituric acid method and the content of nitric oxide (NO) by nitrate reductase method. , Cytoplasmic Nrf2 and whole cell HO-1 protein expression. Results: Compared with the model group, puerarin significantly increased cell viability (P <0.05, P <0.01) at 20.8 mg · L -1 and 41.6 mg · L -1, puerarin 20.8 mg · L -1 (-1) and 41.6 mg · L -1 could significantly decrease NO, MDA content and LDH release (P <0.01), puerarin 20.8 mg · L -1, 41.6 mg · L -1 -1) and 83.2 mg · L -1 significantly increased the activity of SOD (P <0.01) and increased the expression of HO-1 and Nrf2 protein in hypoxia / reoxygenation group (P <0.01) Reduce the expression of Nrf2 protein in cytoplasm (P <0.01). CONCLUSION: Puerarin can significantly antagonize the hypoxic / reoxygenation injury of EPCs, which may be related to its inhibition of cell peroxidation injury and enhancement of cell antioxidant capacity.