红杜仲不同乙醇浓度提取物对HaCaT细胞抗氧化作用的比较及其抗氧化机制的研究

Research on the antioxidant capacity of Parabarium micranthum extracted by different ethanol concentrations and its antioxidant mechanism on HaCaT cells

目的本研究对红杜仲不同乙醇浓度提取物抑制H_2O_2诱导的HaCaT细胞氧化应激损伤作用进行筛选。方法以不同浓度乙醇进行回流提取分别获得了30%、50%、70%及95%乙醇提取的四种红杜仲醇提取物(Parabarium micranthum ethanol extract,PEE),利用1,1-二苯基-2-三硝基苯肼(1,1-Diphenyl-2-picrylhydrazyl radical 2,2-Diphenyl-1-(2,4,6-trinitrophenyl)hydrazyl,DPPH)法测定其体外清除自由基的能力。以人永生化表皮细胞HaCaT为研究对象,通过不同浓度H_2O_2处理细胞并筛选以200μmol/L作为建立H_2O_2诱导的氧化应激损伤模型的最适浓度。设立对照组、H_2O_2损伤组和红杜仲-H_2O_2组。通过微板法和比色法测定内源性抗氧化酶活力及细胞存活率,2',7'-二氯荧光黄双乙酸盐(2,7-dichlorodihydrofluorescein diacetate,DCFH-DA)荧光探针检测细胞内氧自由基(reactive oxygen species,ROS)水平,逆转录PCR测定Nrf2 mRNA表达量。结果不同乙醇浓度提取的PEE在体外均具有清除DPPH自由基的作用,效果最强的70%乙醇提取的PEE的IC(50)为7.6 mg/L;在细胞水平上,H_2O_2诱导使得HaCaT细胞内抗氧化酶SOD及CAT活力降低,50%、70%及95%乙醇提取的PEE能够显著提高细胞内抗氧化酶活力。其中70%乙醇提取的PEE的作用最为显著,抗氧化酶活力的提高表现出剂量依赖性,并且能显著提高细胞存活率、降低细胞内ROS的水平(与氧化应激组比,P<0.05或P<0.01)。同时,70%乙醇提取的PEE能够显著诱导抗氧化相关信号通路基因Nrf2 mRNA的表达。结论 70%乙醇提取的PEE可能通过激活Nrf2抗氧化信号通路,提高抗氧化酶活性,降低ROS水平,从而具有保护HaCaT细胞免受H_2O_2诱导的氧化损伤作用。

Objective The aim of this research was to screen the antioxidant capacity of Parabarium micranthum extract of various ethanol concentrations against H2O2-induced oxidative stress in Ha Ca T cells. Methods Parabarium micranthum extracts reflux purified with 30%,50%,70% and 95% ethanol（ PEE） were objected to screening for their free radical scavenging capacities in vitro by DPPH. Cultured Ha Ca T human keratinocytes were randomly assigned to control,H2O2 and PEE ＋ H2O2 groups. The cultured Ha Ca T cells were treated with different concentrations of H2O2 and 200μmol /L H2O2 was selected to establish H2O2-induced oxidative stress model. For the PEE ＋ H2O2 group,the cells were treated with PEE extracts of various ethanol concentrations before exposure to H2O2. Endogenous antioxidant enzyme activity and cell viability were detected by microplate assay and colorimetric method. Intracellular reactive oxygen species（ ROS） were detected by DCFH-DA fluorescent probe. Nrf2 mRNA expression was evaluated by reverse transcription PCR. Results All PEE extracts of various ethanol concentrations showed effective DPPH free radical scavenging capacities of which the 70% ethanol PEE extract exhibited the strongest scavenging activity（ IC（50）= 7. 6 mg / L）. H2O2 treatments induced oxidative stress in Ha Ca T and thus caused a decrease in antioxidant enzyme activities of SOD and CAT. However,the PEE extracts of 50%,70% and 95% ethanol could observably increase the enzyme activity of SOD and CAT. Among these,the 70% ethanol extracted PEE showed significant effect on enzyme activities in a dosedependent manner. It could also obviously improve the cell viability and reduce the intracellular ROS level（ compared with oxidative stress group,P〈0. 05 or P〈0. 01）. In addition,70% ethanol extracted PEE could remarkably enhance the expression of Nrf2 mRNA,a gene that regulates the antioxidative signaling pathway. Conclusion The findings of the present study suggest that 70% ethanol extracted PEE can attenuate H2O2-induced oxidative damage,which functions via the regulation of the Nrf2-related anti-oxidative signaling pathway,enhancement of the antioxidant activity of SOD and CAT,and reduction of ROS level.