Yen Jui-Hung, Wu Pei-Shan, Chen Shu-Fen, Wu Ming-Jiuan
Department of Molecular Biology and Human Genetics, Tzu Chi University, Hualien 970, Taiwan.
Department of Biotechnology, Chia Nan University of Pharmacy and Science, Tainan 717, Taiwan.
Int J Mol Sci. 2017 Apr 17;18(4):852. doi: 10.3390/ijms18040852.
Fisetin (3,7,3',4'-tetrahydroxyflavone) is a dietary flavonol and exhibits antioxidant, anti-inflammatory, and neuroprotective activities. However, high concentration of fisetin is reported to produce reactive oxygen species (ROS), induce endoplasmic reticulum (ER) stress and cause cytotoxicity in cancer cells. The aim of this study is to investigate the cytoprotective effects of low concentration of fisetin against tunicamycin (Tm)-mediated cytotoxicity in neuronal-like catecholaminergic PC12 cells.
Cell viability was assayed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and apoptotic and autophagic markers were analyzed by Western blot. Gene expression of unfolded protein response (UPR) and Phase II enzymes was further investigated using RT-Q-PCR or Western blotting. Intracellular ROS level was measured using 2',7'-dichlorodihydrofluorescein diacetate (H₂DCFDA) by a fluorometer. The effects of fisetin on mitogen activated protein kinases (MAPKs) and SIRT1 (Sirtuin 1) signaling pathways were examined using Western blotting and specific inhibitors.
Fisetin (<20 µM) restored cell viability and repressed apoptosis, autophagy and ROS production in Tm-treated cells. Fisetin attenuated Tm-mediated expression of ER stress genes, such as glucose-regulated proteins 78 (GRP78), C/EBP homologous protein (CHOP also known as GADD153) and Tribbles homolog 3 (TRB3), but induced the expression of nuclear E2 related factor (Nrf)2-targeted heme oxygenase (HO)-1, glutamate cysteine ligase (GCL) and cystine/glutamate transporter (xCT/SLC7A11), in both the presence and absence of Tm. Moreover, fisetin enhanced phosphorylation of ERK (extracellular signal-regulated kinase), JNK (c-JUN NH₂-terminal protein kinase), and p38 MAPK. Addition of JNK and p38 MAPK inhibitor significantly antagonized its cytoprotective activity and modulatory effects on UPR. Fisetin also restored Tm-inhibited SIRT1 expression and addition of sirtinol (SIRT1 activation inhibitor) significantly blocked fisetin-mediated cytoprotection. In conclusion, this result shows that fisetin activates Nrf2, MAPK and SIRT1, which may elicit adaptive cellular stress response pathways so as to protect cells from Tm-induced cytotoxicity.
漆黄素（3,7,3',4'-四羟基黄酮）是一种膳食黄酮醇，具有抗氧化、抗炎和神经保护活性。然而，据报道高浓度的漆黄素会产生活性氧（ROS），诱导内质网（ER）应激并导致癌细胞的细胞毒性。本研究的目的是探讨低浓度漆黄素对衣霉素（Tm）介导的神经样儿茶酚胺能PC12细胞毒性的细胞保护作用。
通过MTT（3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐）测定细胞活力，通过蛋白质免疫印迹分析凋亡和自噬标志物。使用RT-Q-PCR或蛋白质免疫印迹进一步研究未折叠蛋白反应（UPR）和II期酶的基因表达。使用荧光计通过2',7'-二氯二氢荧光素二乙酸酯（H₂DCFDA）测量细胞内ROS水平。使用蛋白质免疫印迹和特异性抑制剂研究漆黄素对丝裂原活化蛋白激酶（MAPK）和SIRT1（沉默调节蛋白1）信号通路的影响。
漆黄素（<20μM）可恢复Tm处理细胞的活力，并抑制其凋亡、自噬和ROS产生。漆黄素减弱了Tm介导的ER应激基因的表达，如葡萄糖调节蛋白78（GRP78）、C/EBP同源蛋白（CHOP，也称为GADD153）和 Tribbles同源物3（TRB3），但在有或没有Tm的情况下均诱导了核E2相关因子（Nrf）2靶向的血红素加氧酶（HO）-1、谷氨酸半胱氨酸连接酶（GCL）和胱氨酸/谷氨酸转运体（xCT/SLC7A11）的表达。此外，漆黄素增强了细胞外信号调节激酶（ERK）、c-JUN氨基末端蛋白激酶（JNK）和p38 MAPK的磷酸化。添加JNK和p38 MAPK抑制剂可显著拮抗其细胞保护活性和对UPR的调节作用。漆黄素还恢复了Tm抑制的SIRT1表达，添加sirtinol（SIRT1激活抑制剂）可显著阻断漆黄素介导的细胞保护作用。总之，该结果表明漆黄素激活Nrf2、MAPK和SIRT1，这可能引发适应性细胞应激反应途径，从而保护细胞免受Tm诱导的细胞毒性。
