海参蛋白肽对H_2O_2诱导RAW264.7细胞氧化损伤的保护作用

Protective Effects of Sea Cucumber Peptides on H_2O_2-Induced Oxidative Injury in RAW 264.7 Macrophages

以海参为原料,研究海参蛋白肽的抗氧化性以及对H2O2诱导RAW264.7巨噬细胞氧化损伤的保护效果和作用机制。以清除羟自由基(·OH)能力、Fe2+螯合能力和Fe3+还原能力为指标,评价海参蛋白肽的体外抗氧化活性。以H2O2刺激RAW264.7巨噬细胞建立氧化损伤细胞模型,采用2′,7′-二氯荧光黄双乙酸盐(DCFH-DA)荧光探针法测定细胞活性氧(ROS)水平,四甲基偶氮唑盐(MTT)法测定细胞活力,实时荧光定量PCR测定细胞血红素氧合酶-1(HO-1)m RNA表达水平。结果表明,海参蛋白肽能够清除·OH,具有Fe2+螯合能力和Fe3+还原能力,海参蛋白肽的这种体外抗氧化能力呈现浓度依赖效应。海参蛋白肽显著降低氧化损伤RAW264.7巨噬细胞ROS水平和提高氧化损伤细胞活力(P<0.05)。而且,海参蛋白肽显著提高巨噬细胞内HO-1 m RNA表达(P<0.05),HO-1抑制剂锌原卟啉IX(Zn PPIX)部分逆转海参蛋白肽对氧化损伤巨噬细胞活力的促进作用。这些结果说明,海参蛋白肽通过上调RAW264.7巨噬细胞HO-1 m RNA表达水平,发挥对巨噬细胞氧化损伤的保护作用。

The antioxidant activities of sea cucumber peptides, and the protective effects and underlying mechanisms of sea cucumber peptides on H202-induced oxidative injury in RAW 264.7 macrophages were investigated in current study. Hydroxyl radical-scavenging capacity, ferrous ion （Fe2＋）-ehelating ability and ferric reducing activity were deter- mined to assess the antioxidant activities of sea cucumber peptides in vitro. The model of oxidative injury in RAW 264.7 macrophages was established by hydrogen peroxide treatment. The levels of intracellular reactive oxygen species （ROS） and cell viability were assayed using 2＇ ,7＇-dichlorodihydrofluoreseein diacetate （DCFH-DA） and 3-（4,5-dimethylthiazol- 2-yl）-2,5-diphenyhetrazolium bromide （MTF）, respectively. The mRNA expression levels of heme oxygenase-1 （HO-1） were evaluated by real time quantitative polymerase chain reaction. Results showed that sea cucumber peptides possessed hydroxyl radical-scavenging capacity, Fe2＋-chelating ability and ferric reducing activity in a dose-dependent manner. Sea cucumber peptides significantly reducedthe levels of intracellular ROS and increased cell viabilityin RAW 264.7 macrophages under oxidative injury （P〈0.05）. Moreover, the mRNA expression levels of HO-1 were significantly increased by sea cucumber peptides in RAW 264.7 macrophages （P〈0.05）. The improvement effects of sea cucumber peptides on the cell viability of RAW 264.7 macrophages subjected to H2O2-induced oxidative injury were partially reversed by co- treatment with zinc（Ⅱ）-protoporphyrin IX （ZnPPIX）, a specific inhibitor of HO-1. These results suggested that sea cu- cumber peptides protected against H2O2-induced oxidative injury in RAW 264.7 macrophages by increasing HO-1 mRNA expression