Research on the antioxidant capacity of Parabarium micranthum extracted by different ethanol concentrations and its antioxidant mechanism on HaCaT cells
- 医学院－已发表论文 
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 H_2O_2-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,H_2O_2 and PEE + H_2O_2 groups. The cultured Ha Ca T cells were treated with different concentrations of H_2O_2 and 200μmol /L H_2O_2 was selected to establish H_2O_2-induced oxidative stress model. For the PEE + H_2O_2 group,the cells were treated with PEE extracts of various ethanol concentrations before exposure to H_2O_2. 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). H_2O_2 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 H_2O_2-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.