（+）-儿茶素及（-）-表儿茶素对HEK293T细胞抗氧化的影响及差异

Effects and Differences of (+)-Catechin and (-)-Epicatechin on Antioxidant Activity of HEK293T Cells

摘要:

【目的】探究天然抗氧化剂（+）-儿茶素及其同分异构体（-）-表儿茶素基于Nrf2-ARE通路对HEK293T细胞抗氧化的影响及差异。【方法】以（+）-儿茶素、（-）-表儿茶素为试验对象，利用MTT法探究对HEK293T细胞活力的影响，应用报告基因探究对ARE基因转录水平的调控作用，利用RT-PCR和Western blot方法探究对Nrf2-ARE信号通路下游基因（GCLM、NQO1和HO-1）和蛋白（GCLM、NQO1和HO-1）表达的影响。【结果】报告基因显示，（+）-儿茶素及（-）-表儿茶素分别在20、10 μmol/L显著诱导了ARE介导的转录激活。RT-PCR及Western blot结果表明，当浓度为40 μmol/L时，（+）-儿茶素可以显著上调GCLM、NQO1和HO-1基因表达，（-）-表儿茶素可以显著上调NQO1和HO-1基因表达；当浓度为10 μmol/L时，（+）-儿茶素可以上调GCLM、NQO1和HO-1蛋白表达，（-）-表儿茶素可以上调GCLM、HO-1蛋白表达。【结论】（+）-儿茶素、（-）-表儿茶素对细胞的最低影响浓度不同。二者均有激活Nrf2-ARE信号通路的能力，（-）-表儿茶素激活通路所需浓度低于（+）-儿茶素，对于Nrf2-ARE信号通路下游基因及蛋白的表达也有差异，相同浓度时（+）-儿茶素抗氧化基因和蛋白表达效果更好。

Abstract:

[Objective]This study aims to investigate the effects and differences of the natural antioxidant (+)-catechin and its isomer (-)-epicatechin on the antioxidant activity of HEK293T cells based on the nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway.[Method]The effects of (+)-catechin and (-)-epicatechin on the viability of HEK293T cells were investigated via the methyl thiazolyl tetrazolium (MTT) assay. Reporter genes were employed to investigate the regulatory effects on the activation of ARE. The effects of (+)-catechin and (-)-epicatechin on the mRNA and proteins levels of the downstream factors [glutamate-cysteine ligase modifier subunit (GCLM), NAD(P)H quinone dehydrogenase 1 (NQO1), and heme oxygenase-1 (HO-1)] of the Nrf2-ARE signaling pathway were explored by RT-PCR and Western blot, respectively.[Result]The expression of reporter genes showed that ARE-mediated transcriptional activation was significantly induced by (+)-catechin and (-)-epicatechin at 20 μmol/L and 10 μmol/L, respectively. RT-PCR and Western blot results showed that (+)-catechin at 40 μmol/L significantly up-regulated the mRNA levels of GCLM, NQO1, and HO-1, and (-)-epicatechin at the same concentration significantly up-regulated the mRNA levels of NQO1 and HO-1. At 10 μmol/L, (+)-catechin up-regulated the protein levels of GCLM, NQO1, and HO-1, and (-)-epicatechin up-regulated the protein levels of GCLM and HO-1.[Conclusion](+)-Catechin and (-)-epicatechin have different minimum effect concentrations on cells. Both of them have the ability to activate the Nrf2-ARE signaling pathway, and the required concentration of (-)-epicatechin to activate the pathway is lower than that of (+)-catechin. In addition, the two compounds have different effects on the expression of genes and proteins downstream of the Nrf2-ARE signaling pathway, with (+)-catechin outperforming (-)-epicatechin at the same concentration.