School of Pharmacy, The First Clinical Medical College, Nanjing University of Chinese Medicine, Nanjing, 210023, China.
Department of Pharmacy, Wuxi No.9 People's Hospital, Soochow University, Wuxi Hand Surgery Hospital, Wuxi, Jiangsu Province, 214062, China.
Chin J Integr Med. 2017 Dec;23(12):929-936. doi: 10.1007/s11655-017-2546-6. Epub 2017 May 18.
To find the signaling pathway of triptolide (TP)-induced liver injury and to reveal whether NF-E2-related factor 2 (Nrf2) plays an important role in cellular self-protection.
The L-02 and HepG2 cells were cultured and treated with various concentrations of TP. The cell viability was observed, and the cell medium was collected for detecting the aspartate aminotransferase (ALT), alanine aminotransferase (AST), lactate dehydrogenase (LDH), superoxide dismutase (SOD) and L-glutathione production (GSH) levels. Nrf2 and its downstream target NAD(P)H: quinine oxidoreductase 1 (NQO1) and heme oxygenase-1 (HO-1) expression, the nuclear translocation of Nrf2, and the binding ability of Nrf2 and antioxidant response element (ARE) were also identified. Meanwhile, shRNA was used to silence Nrf2 in L-02 cells to find out whether Nrf2 plays a protective role.
The viability of the L-02 and HepG2 cells treated with TP decreased in a doseand time-dependent manner, and TP (20-80 μg/mL) markedly induced the release of ALT, AST and LDH (P<0.05 or P<0.01), reduced the levels of SOD and GSH (P<0.01), and increased the intracellular reactive oxygen species. Meanwhile, TP augmented the Nrf2 expression in L-02 and HepG2 cells (P<0.05 or P<0.01), induced Nrf2 nuclear translocation, increased the Nrf2 ARE binding activity, and increased HO-1 and NQO1 expressions. Nrf2 knockdown revealed a more severe toxic effect of TP (P<0.05 or P<0.01).
Human hepatic cells treated with TP induced oxidative stress, and led to cytotoxicity. Self-protection against TP-induced toxicity in human hepatic cells might be via Nrf2-ARE-NQO1 transcriptional pathway.
寻找雷公藤红素（TP）诱导肝损伤的信号通路，并揭示核因子 E2 相关因子 2（Nrf2）是否在细胞自我保护中发挥重要作用。
培养 L-02 和 HepG2 细胞，并使用不同浓度的 TP 处理细胞。观察细胞活力，收集细胞培养液，检测天冬氨酸转氨酶（ALT）、丙氨酸转氨酶（AST）、乳酸脱氢酶（LDH）、超氧化物歧化酶（SOD）和 L-谷胱甘肽生成（GSH）水平。鉴定 Nrf2 及其下游靶标 NAD(P)H：醌氧化还原酶 1（NQO1）和血红素加氧酶-1（HO-1）表达、Nrf2 的核易位以及 Nrf2 与抗氧化反应元件（ARE）的结合能力。同时，使用 shRNA 沉默 L-02 细胞中的 Nrf2，以确定 Nrf2 是否具有保护作用。
TP 处理的 L-02 和 HepG2 细胞活力呈剂量和时间依赖性下降，TP（20-80μg/mL）显著诱导 ALT、AST 和 LDH 的释放（P<0.05 或 P<0.01），降低 SOD 和 GSH 水平（P<0.01），并增加细胞内活性氧。同时，TP 增强了 L-02 和 HepG2 细胞中的 Nrf2 表达（P<0.05 或 P<0.01），诱导 Nrf2 核易位，增加 Nrf2 ARE 结合活性，增加 HO-1 和 NQO1 的表达。Nrf2 敲低显示出 TP 更严重的毒性作用（P<0.05 或 P<0.01）。
TP 处理的人肝细胞诱导氧化应激，导致细胞毒性。人肝细胞对 TP 诱导的毒性的自我保护可能是通过 Nrf2-ARE-NQO1 转录途径。
