Institute of Translational Medicine, The First Hospital of Jilin University, Changchun, China.
Department of Ophthalmology, The Second Hospital of Jilin University, 218 Ziqiang Street, Changchun, China.
Phytomedicine. 2020 Jun;71:153241. doi: 10.1016/j.phymed.2020.153241. Epub 2020 May 17.
Oxidative stress-triggered fatal hepatotoxicity is an essential pathogenic factor in acute liver failure (ALF).
To investigate the protective effect of daphnetin (Daph) on tert-butyl hydroperoxide (t-BHP) and acetaminophen (APAP)-induced hepatotoxicity through altering Nrf2/Trx-1 pathway activation.
In vivo, male C57BL/6 mice with Wild-type (WT) and Nrf2 were divided into five groups and acute liver injury model were established by APAP or LPS/GalN after injection with Daph (20, 40, or 80 mg/kg), seperately. Then, liver tissue and serum were collected for biochemical determination, TUNEL and H & E staining, and western blot analysis. In vitro, HepG2 cells were used to investigate the protective effect and mechanism of daphnetin against ROS and apoptosis induced by t-BHP via apoptosis detection, western blot, immunofluorescence analysis, and sgRNA transfection.
Our results indicated that Daph efficiently inhibited t-BHP-stimulated hepatotoxicity, and modulated Trx-1 expression and Nrf2 activation which decreased Keap1-overexpression in HepG2 cells. Moreover, Daph inhibited t-BHP-excited hepatotoxicity and enhanced Trx-1 expression, which was reversed in Nrf2 HepG2 cells. In vivo, a survival rate analysis first suggested that Daph significantly reduced the lethality induced by APAP or GalN/LPS in a Nrf2-dependent or -independent manner by using Nrf2 mice, respectively. Next, further results implicated that Daph not only effectively alleviated APAP-induced an increase of ALT and AST levels, histopathological changes, ROS overproduction, malondialdehyde (MDA) formation and GSH/GSSG reduction, but it also relieved hepatic apoptosis by strengthening the suppression of cleaved-caspase-3 and expression of P53 protein. Additionally, Daph attenuated mitochondrial dysfunction by suppressing ASK1/JNK activation and decreasing apoptosis-inducing factor (AIF) and Cytochrome c release and Bax mitochondrial translocation. Daph inhibited inflammatory responses by inactivating the thioredoxin-interacting protein (Txnip)/NLRP3 inflammasome. Furthermore, Daph efficiently enhanced Nrf2 nuclear translocation and Trx-1 expression. However, these effects in WT mice were eliminated in Nrf2 mice.
These investigations demonstrated that Daph treatment has protective potential against oxidative stress-driven hepatotoxicity by inhibition of ASK1/JNK and Txnip/NLRP3 activation, which may be strongly related to the Nrf2/Trx-1 upregulation.
氧化应激引发的致命肝毒性是急性肝衰竭（ALF）的一个重要发病因素。
通过改变 Nrf2/Trx-1 通路的激活，研究瑞香素（Daph）对叔丁基过氧化氢（t-BHP）和对乙酰氨基酚（APAP）诱导的肝毒性的保护作用。
体内实验中，雄性 C57BL/6 小鼠（WT 型和 Nrf2 型）分为五组，在注射 Daph（20、40 或 80mg/kg）后，通过 APAP 或 LPS/GalN 分别建立急性肝损伤模型。然后，收集肝组织和血清进行生化测定、TUNEL 和 H&E 染色以及 Western blot 分析。体外实验中，用瑞香素处理 HepG2 细胞，通过检测细胞凋亡、Western blot、免疫荧光分析和 sgRNA 转染，研究瑞香素对 t-BHP 诱导的 ROS 和细胞凋亡的保护作用及其机制。
研究结果表明，Daph 能有效抑制 t-BHP 刺激引起的肝毒性，调节 Trx-1 表达和 Nrf2 激活，降低 HepG2 细胞中 Keap1 的过表达。此外，Daph 抑制 t-BHP 引起的肝毒性，增强 Trx-1 表达，但在 Nrf2 HepG2 细胞中被逆转。体内实验中，生存分析首先表明，Daph 通过分别利用 Nrf2 型和非依赖型小鼠，显著降低了由 APAP 或 GalN/LPS 诱导的致死率。接下来，进一步的结果表明，Daph 不仅有效缓解了 APAP 引起的 ALT 和 AST 水平升高、组织病理学变化、ROS 过度产生、丙二醛（MDA）形成和 GSH/GSSG 减少，还通过增强对 cleaved-caspase-3 和 P53 蛋白表达的抑制，缓解了肝凋亡。此外，Daph 通过抑制 ASK1/JNK 激活、减少凋亡诱导因子（AIF）和细胞色素 c 释放以及 Bax 线粒体易位来减轻线粒体功能障碍。Daph 通过失活硫氧还蛋白相互作用蛋白（Txnip）/NLRP3 炎性小体来抑制炎症反应。此外，Daph 有效地促进了 Nrf2 的核易位和 Trx-1 的表达。然而，在 Nrf2 型小鼠中，这些在 WT 型小鼠中的作用被消除。
这些研究表明，Daph 治疗具有通过抑制 ASK1/JNK 和 Txnip/NLRP3 激活来抵抗氧化应激驱动的肝毒性的潜力，这可能与 Nrf2/Trx-1 的上调密切相关。
