Department of Preparations, The First Hospital of Hunan University of Chinese Medicine, Changsha, P.R. China.
Department of Intensive Care Medicine, The Third Xiangya Hospital of Central South University, Changsha, P.R. China.
Pharm Biol. 2020 Dec;58(1):806-814. doi: 10.1080/13880209.2020.1803367.
The potential hepatotoxicity of (PMR) has attracted much attention, but the specific mechanism of inducing hepatotoxicity is still unclear due to the complexity of its components.
This study investigated the specific mechanism by which 2,3,5,4'-tetrahydroxy-stilbene-2--β-d-glucoside (TSG) regulates hepatotoxicity.
The toxic effects of TSG (10, 100, 1000 μg/mL) on WRL-68 cells were examined using MTT, flow cytometry, and LDH assay after 24 h of incubation. Untreated cells served as the control. Gene and protein expression levels were determined by quantitative real-time PCR and Western blot, respectively. Immunofluorescence analysis was conducted to investigate the expression of light chain 3 (LC3). Luciferase activity assay was used to assess the targeted regulation of RUNX1 by miR-122.
The half maximal inhibitory concentration (IC50) of TSG in WRL-68 cells was calculated as 1198.62 μg/mL. TSG (1000 μg/mL) inhibited cell viability and LDH activity and promoted WRL-68 cell apoptosis by inducing autophagy. Subsequent findings showed that TSG induced autophagy and promoted apoptosis in WRL-68 cells by downregulating the levels of p-PI3K, p-Akt, and p-mTOR proteins, while RUNX1 overexpression rescued this inhibition. Additionally, the effect of TSG on hepatocyte apoptosis was reversed by miR-122 knockdown. Furthermore, bioinformatics and dual luciferase reporter assay results indicated that miR-122 targeted RUNX1.
Our data demonstrate for the first time that TSG regulates hepatotoxicity, possibly by upregulating miR-122 and inhibiting the RUNX1-mediated PI3K/Akt/mTOR pathway to promote autophagy and induce hepatocyte apoptosis. Further research is necessary to verify our conclusion.
对 PMR 的潜在肝毒性引起了广泛关注，但由于其成分的复杂性，诱导肝毒性的确切机制仍不清楚。
本研究旨在探讨 2,3,5,4'-四羟基二苯乙烯-2-O-β-D-葡萄糖苷（TSG）调节肝毒性的具体机制。
采用 MTT、流式细胞术和 LDH 检测法，在 24 h 孵育后，观察 TSG（10、100、1000μg/mL）对 WRL-68 细胞的毒性作用。未处理的细胞作为对照。通过实时定量 PCR 和 Western blot 分别检测基因和蛋白表达水平。免疫荧光分析检测 LC3 的表达。采用荧光素酶活性测定法评估 miR-122 对 RUNX1 的靶向调节作用。
计算得出 TSG 在 WRL-68 细胞中的半数抑制浓度（IC50）为 1198.62μg/mL。TSG（1000μg/mL）通过诱导自噬抑制细胞活力和 LDH 活性，促进 WRL-68 细胞凋亡。进一步的研究结果表明，TSG 通过下调 p-PI3K、p-Akt 和 p-mTOR 蛋白水平诱导自噬并促进 WRL-68 细胞凋亡，而过表达 RUNX1 则可挽救这种抑制作用。此外，miR-122 敲低可逆转 TSG 对肝细胞凋亡的影响。此外，生物信息学和双荧光素酶报告基因检测结果表明，miR-122 靶向 RUNX1。
本研究首次证明 TSG 可能通过上调 miR-122 并抑制 RUNX1 介导的 PI3K/Akt/mTOR 通路来调节肝毒性，促进自噬并诱导肝细胞凋亡。需要进一步的研究来验证我们的结论。
