CONTEXT The potential hepatotoxicity of Polygoni Multiflori Radix (PMR) has attracted much attention, but the specific mechanism of inducing hepatotoxicity is still unclear due to the complexity of its components. OBJECTIVE This study investigated the specific mechanism by which 2,3,5,4'-tetrahydroxy-stilbene-2-O-β-d-glucoside (TSG) regulates hepatotoxicity. MATERIALS AND METHODS 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. RESULTS 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. DISCUSSION AND CONCLUSIONS 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 in vivo research is necessary to verify our conclusion.

中文翻译：

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2,3,5,4'-四羟基-二苯乙烯-2-O-β-d-葡萄糖苷通过上调 miR-122 和抑制 PI3K/Akt/mTOR 通路诱导自噬介导的肝细胞凋亡：对其肝毒性的影响

背景 何首乌（PMR）潜在的肝毒性引起了广泛关注，但由于其成分复杂，导致肝毒性的具体机制尚不清楚。目的 本研究调查了 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。材料与方法 孵育 24 小时后，使用 MTT、流式细胞术和 LDH 测定法检查 TSG（10、100、1000 μg/mL）对 WRL-68 细胞的毒性作用。未处理的细胞用作对照。基因和蛋白质表达水平分别通过定量实时 PCR 和蛋白质印迹确定。进行免疫荧光分析以研究轻链 3 (LC3) 的表达。荧光素酶活性测定用于评估 miR-122 对 RUNX1 的靶向调节。结果 TSG 在 WRL-68 细胞中的半数抑制浓度 (IC50) 计算为 1198.62 μg/mL。TSG (1000 μg/mL) 通过诱导自噬抑制细胞活力和 LDH 活性并促进 WRL-68 细胞凋亡。随后的研究结果表明，TSG 通过下调 p-PI3K、p-Akt 和 p-mTOR 蛋白的水平，在 WRL-68 细胞中诱导自噬并促进细胞凋亡，而 RUNX1 过表达则挽救了这种抑制。此外，抑制 miR-122 可以逆转 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。讨论和结论我们的数据首次证明 TSG 调节肝毒性，可能通过上调 miR-122 和抑制 RUNX1 介导的 PI3K/Akt/mTOR 通路来促进自噬和诱导肝细胞凋亡。需要进一步的体内研究来验证我们的结论。