Park Ji Young, Kang Kyoung Ah, Kim Ki Cheon, Cha Ji Won, Kim Eun Hee, Hyun Jin Won
School of Medicine and Institute for Nuclear Science and Technology, Jeju National University, Jeju.
College of Pharmacy, CHA University, Pocheon, Korea.
J Cancer Prev. 2013 Sep;18(3):249-56. doi: 10.15430/jcp.2013.18.3.249.
Oxidative stress damages to cells or tissues, however, cellular defense systems including heme oxygenase-1 (HO-1) protects them against oxidative stress. Flavonoid compounds can activate cellular defense mechanisms against oxidative stress and it can reduce cell damages. In the present study, the cytoprotective effects of morin (3,5,7,2',4'-pentahydroxyflavone), in terms of HO-1 enzyme, against the oxidative stress and its involved mechanisms was elucidated.
RT-PCR and western blot analysis were assessed to detect the mRNA and protein expression, respectively. Cell viability was measured by using MTT test. The immunocytochemistry was performed to define location of target protein. Electrophoretic mobility shift assay performed to measure transcription factor-promoter site binding activity.
Morin elevated mRNA and protein levels of HO-1 in human lens epithelial cells (HLE-B3). HO-1 inhibitor ZnPP attenuated the protective effect of morin against H2O2-induced cytotoxicity. Morin increased the protein level of transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2), which up-regulates HO-1 expression by binding to the antioxidant response element (ARE) within the HO-1 gene promoter. Moreover, morin induced the translocation of Nrf2 from the cytosol into the nucleus. Morin activated extracellular-regulated kinase (ERK), while ERK inhibitor attenuated morin-enhanced Nrf2 and HO-1 expression.
Morin activates ERK-Nrf2 signaling cascades in HLE-B3 cells, leading to the up-regulation of HO-1 and cytoprotection against oxidative stress.
氧化应激会对细胞或组织造成损伤，然而，包括血红素加氧酶-1（HO-1）在内的细胞防御系统可保护它们免受氧化应激的影响。类黄酮化合物可激活细胞对抗氧化应激的防御机制，并能减少细胞损伤。在本研究中，就HO-1酶而言，阐明了桑色素（3,5,7,2',4'-五羟基黄酮）对氧化应激的细胞保护作用及其相关机制。
分别采用逆转录聚合酶链反应（RT-PCR）和蛋白质免疫印迹分析来检测mRNA和蛋白质表达。使用MTT试验测定细胞活力。进行免疫细胞化学以确定靶蛋白的位置。采用电泳迁移率变动分析来测量转录因子与启动子位点的结合活性。
桑色素提高了人晶状体上皮细胞（HLE-B3）中HO-1的mRNA和蛋白质水平。HO-1抑制剂ZnPP减弱了桑色素对H2O2诱导的细胞毒性的保护作用。桑色素增加了转录因子核因子红细胞2相关因子2（Nrf2）的蛋白质水平，Nrf通过与HO-1基因启动子内的抗氧化反应元件（ARE）结合来上调HO-1的表达。此外，桑色素诱导Nrf2从细胞质转位到细胞核。桑色素激活了细胞外调节激酶（ERK），而ERK抑制剂减弱了桑色素增强的Nrf2和HO-1表达。
桑色素激活HLE-B3细胞中的ERK-Nrf2信号级联反应，导致HO-1上调并对氧化应激产生细胞保护作用。
