马齿苋总黄酮对H2O2致PC12细胞氧化应激损伤的保护作用

Protective effect of Portulaca oleracea total flavones on H2O2 induced PC12 cells oxidative damage

目的:探讨马齿苋总黄酮(portulaca oleracea total flavones, POTF)对过氧化氢(H2O2)所诱导的PC12细胞氧化应激损伤的保护作用及其机制。方法:通过MTT比色法检测马齿苋总黄酮对PC12细胞增殖的影响;并用H2O2诱导PC12细胞建立氧化损伤模型,通过四唑盐(MTT)和乳酸脱氢酶(LDH)比色法检测马齿苋总黄酮保护PC12细胞免受H2O2损伤的效果;利用试剂盒检测细胞内活性氧(ROS)含量和培养液中丙二醛(MDA)、谷胱甘肽(GSH)和一氧化氮(NO)含量观察马齿苋总黄酮的抗氧化效果;通过试剂盒检测超氧化物歧化酶(SOD)、过氧化氢酶(catalase,CAT)和谷胱甘肽过氧化物酶(GSH-Px)活性以及实时荧光定量PCR(q-PCR)和Western blot检测硫氧还蛋白(Trx)和诱导型一氧化氮合酶(iNOS)的转录和蛋白水平,初步探讨马齿苋总黄酮的抗氧化机制。结果:马齿苋总黄酮在320μg/mL浓度范围内对PC12细胞增殖无影响,而马齿苋总黄酮浓度达到640μg/mL时对PC12细胞有一定的毒性作用;马齿苋总黄酮随剂量依赖地提高细胞内SOD、CAT和GSH-Px活性和调节Trx和iNOS的转录水平,减少ROS和NO的产生和增加GSH的含量,从而保护H2O2对PC12细胞的氧化损伤,尤以160 mg/kg最显著。结论:马齿苋总黄酮对H2O2致PC12细胞损伤具有保护作用,其作用机制可能与提高细胞的抗氧化能力有关。

Objective: To explore the protective effect of Portulaca oleracea total flavones(POTF) on the H2O2 induced oxidative damage of PC12 cells. Methods: The effect of POTF on the proliferation of PC12 cells was determined by MTT assay. PC12 cells oxidative damage model was established by H2O2, then the protective effect of POEF on the model was determined by MTT assay and LDH assay. The intracellular levels of reactive oxygen species(ROS)and the supernatant level of malondialdehyde(MDA), glutathione(GSH) and nitric oxide(NO) were measured by commercial kits to observe the antioxidant effect of POTF. Then the changes in superoxide dismutase(SOD), catalase(CAT), glutathione peroxidase(GSH-Px) activity, the mRNA and protein levels of thioredoxin(Trx) and inducible Nitric Oxide Synthase(iNOS) were determined to explore the antioxidant mechanism of POTF. Results: POTF(10~320 mg/L) showed no effect on the proliferation of PC12. 640 mg/L POTF had a significant cytotoxic effect on PC12 cells. POTF protected the oxidative damage in PC12 cells by reducing the production of ROS and NO, and increasing the content of GSH through regulating the activities of SOD, CAT, GSH-Px, and the expressions(mRNA and protein) of Trx and iNOS, the dose of 160 mg/kg showed the most significant difference.Conclusion: POTF has the protective effect on oxidative damage induced by H2O2 in PC12 cells, which may be related to the improvement of the antioxidant capacity of PC12 cells.