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目的:研究中药单体组方梓葛冻干粉对人脐静脉内皮细胞(HUVECs)缺氧/复氧损伤的保护作用。方法:体外培养HUVECs,采用Krebs液建立缺氧/复氧损伤模型。用梓葛冻干粉干预后,采用MTT比色法测定细胞活力、黄嘌呤氧化酶法测定细胞内超氧化物歧化酶(SOD)活性、硫代巴比妥酸显色法测定丙二醛(MDA)含量、硝酸还原酶法测定一氧化氮(NO)含量、2,4-二硝基苯肼显色法测定细胞外乳酸脱氢酶(LDH)释放量;Western blot法分析细胞凋亡相关蛋白Bcl-2,Bax,Caspase-3的表达。结果:与模型组相比,梓葛冻干粉12.5 mg.L-1显著提高细胞活力(P<0.05);梓葛冻干粉49.0,24.5,12.5 mg.L-1均显著提高缺氧/复氧后细胞SOD活性(P<0.05);梓葛冻干粉24.5,12.5 mg.L-1能显著减少LDH的释放量及降低MDA和NO的含量(P<0.05),并上调细胞Bcl-2蛋白表达(P<0.05,P<0.01);梓葛冻干粉49.0,24.5,12.5 mg.L-1显著降低Bax的表达并上调Bcl-2/Bax(P<0.05,P<0.01),且梓葛冻干粉49.0,24.5 mg.L-1显著降低Caspase-3的表达(P<0.01)。结论:梓葛冻干粉可显著拮抗缺氧/复氧对HUVECs的损伤,可能与其抑制细胞过氧化损伤,增强细胞抗氧化和抗凋亡能力有关。
OBJECTIVE: To study the protective effect of Zijuge freeze-dried powder of Chinese traditional medicine on hypoxic / reoxygenation injury of human umbilical vein endothelial cells (HUVECs). Methods: HUVECs were cultured in vitro. Krebs fluid was used to establish hypoxia / reoxygenation injury model. After the intervention of Zijuge freeze-dried powder, MTT colorimetric assay was used to determine the cell viability, the activity of superoxide dismutase (SOD) was measured by xanthine oxidase method, the content of malondialdehyde MDA), nitrate reductase (NO) content and 2,4-dinitrophenylhydrazine colorimetric method were used to determine the release of extracellular lactate dehydrogenase (LDH). Western blot was used to analyze the correlation between cell apoptosis Protein Bcl-2, Bax, Caspase-3 expression. Results: Compared with the model group, the freeze-dried Zi Zi freeze-dried powder 12.5 mg.L-1 significantly increased the cell viability (P <0.05). The freeze-dried Zi Zi freeze-dried powder 49.0,24.5,12.5 mg.L- The activities of SOD in the cells after reoxygenation were significantly decreased (P <0.05). The plasma levels of LDH and the contents of MDA and NO were significantly decreased (P <0.05) and 24.5,12.5 mg.L-1 (P <0.05, P <0.01). Zi Zi freeze-dried powder 49.0,24.5,12.5 mg.L-1 significantly decreased Bax expression and up-regulated Bcl-2 / Bax Moreover, 49.0 and 24.5 mg.L-1 of Zi Ge freeze-dried powder significantly decreased Caspase-3 expression (P <0.01). Conclusion: Zijuge freeze-dried powder can significantly antagonize hypoxia / reoxygenation injury of HUVECs, which may be related to its ability of inhibiting cell peroxidation injury, enhancing cell anti-oxidation and anti-apoptosis.