Luteolin and Apigenin Attenuate 4-Hydroxy-2-Nonenal-Mediated Cell Death through Modulation of UPR, Nrf2-ARE and MAPK Pathways in PC12 Cells

Luteolin and apigenin are dietary flavones and exhibit a broad spectrum of biological activities including antioxidant, anti-inflammatory, anti-cancer and neuroprotective effects. The lipid peroxidation product 4-hydroxy-2-nonenal (4-HNE) has been implicated as a causative agent in the development of neurodegenerative disorders. This study investigates the cytoprotective effects of luteolin and apigenin against 4-HNE-mediated cytotoxicity in neuronal-like catecholaminergic PC12 cells. Both flavones restored cell viability and repressed caspase-3 and PARP-1 activation in 4-HNE-treated cells. Luteolin also mitigated 4-HNE-mediated LC3 conversion and reactive oxygen species (ROS) production. Luteolin and apigenin up-regulated 4-HNE-mediated unfolded protein response (UPR), leading to an increase in endoplasmic reticulum chaperone GRP78 and decrease in the expression of UPR-targeted pro-apoptotic genes. They also induced the expression of Nrf2-targeted HO-1 and xCT in the absence of 4-HNE, but counteracted their expression in the presence of 4-HNE. Moreover, we found that JNK and p38 MAPK inhibitors significantly antagonized the increase in cell viability induced by luteolin and apigenin. Consistently, enhanced phosphorylation of JNK and p38 MAPK was observed in luteolin- and apigenin-treated cells. In conclusion, this result shows that luteolin and apigenin activate MAPK and Nrf2 signaling, which elicit adaptive cellular stress response pathways, restore 4-HNE-induced ER homeostasis and inhibit cytotoxicity. Luteolin exerts a stronger cytoprotective effect than apigenin possibly due to its higher MAPK, Nrf2 and UPR activation, and ROS scavenging activity.

木犀草素（Luteolin）和芹菜素（apigenin）属于膳食类黄酮，具备抗氧化、抗炎、抗肿瘤及神经保护等多种生物学活性。脂质过氧化产物4-羟基-2-壬烯醛（4-hydroxy-2-nonenal, 4-HNE）被认为是神经退行性疾病发生的致病因子。本研究旨在探究木犀草素与芹菜素在儿茶酚胺能神经元样PC12细胞中，对抗4-HNE介导的细胞毒性的细胞保护作用。两种类黄酮均可恢复4-HNE处理细胞的存活率，并抑制半胱氨酸天冬氨酸蛋白酶-3（caspase-3）与多聚ADP核糖聚合酶-1（PARP-1）的活化。木犀草素还可缓解4-HNE介导的微管相关蛋白1轻链3（LC3）转化与活性氧（reactive oxygen species, ROS）生成。木犀草素与芹菜素可上调4-HNE介导的未折叠蛋白反应（unfolded protein response, UPR），使内质网（endoplasmic reticulum）分子伴侣葡萄糖调节蛋白78（GRP78）表达升高，同时降低UPR靶向促凋亡基因的表达水平。二者在无4-HNE存在时，可诱导核因子E2相关因子2（nuclear factor erythroid 2-related factor 2, Nrf2）靶向的血红素氧合酶-1（HO-1）与胱氨酸/谷氨酸反向转运体（xCT）的表达，但在4-HNE存在时则会拮抗二者的表达。此外，本研究发现c-Jun氨基末端激酶（JNK）与p38丝裂原活化蛋白激酶（p38 MAPK）抑制剂可显著拮抗木犀草素与芹菜素诱导的细胞存活率提升。与之一致的是，在木犀草素与芹菜素处理的细胞中，可观察到JNK与p38 MAPK的磷酸化水平增强。综上，本研究结果表明，木犀草素与芹菜素可激活MAPK与Nrf2信号通路，进而诱发适应性细胞应激反应通路，恢复4-HNE诱导的内质网稳态失衡，并抑制细胞毒性。相较于芹菜素，木犀草素的细胞保护作用更强，这可能与其更高的MAPK、Nrf2及UPR激活能力与ROS清除活性有关。