Abstract

1. The aim of this study was to compare the in vitro cytotoxic effect of tramadol and M1 metabolite in HepG2 cell line, the underlying mechanism, and PI3K/AKT/mTOR as potential target.

2. Concentrations representing therapeutic level for tramadol (2 µM) and M1 metabolite (0.5 µM) were used. In addition, other increasing concentrations representing higher toxic levels were used (6, 10 µM for tramadol and 1.5, 2.5 µM for M1 metabolites). Cytotoxicity was assessed at 24, 48 and 72 h.

3. Both tramadol and M1 metabolites were able to produce cytotoxicity in a dose and time dependent manner. Insignificant difference was detected between cells exposed to tramadol and M1 metabolite at therapeutic concentrations. Tramadol-induced apoptotic and autophagic cell death while M1 metabolite-induced apoptosis only. For PI3K/AKT/mTOR pathway, the therapeutic concentration of tramadol was only able to increase phosphorylation of AKT while higher toxic concentrations were able to increase phosphorylation of whole pathway; Meanwhile, M1 metabolite was able to increase the phosphorylation of the whole pathway significantly in therapeutic and toxic concentrations.

4. In conclusion, both tramadol and M1 are equally cytotoxic. Apoptosis and autophagy both mediate hepatic cell death. PI3K/AKT pathway is involved in apoptosis induction while autophagy is regulated through mTOR independent pathway.

摘要

1. 本研究的目的是比较曲马多和 M1 代谢物在 HepG2 细胞系中的体外细胞毒作用、潜在机制以及作为潜在靶点的 PI3K/AKT/mTOR。

2. 使用了代表曲马多 (2 µM) 和 M1 代谢物 (0.5 µM) 治疗水平的浓度。此外，还使用了代表更高毒性水平的其他递增浓度（曲马多为 6、10 µM，M1 代谢物为 1.5、2.5 µM）。在 24、48 和 72 小时评估细胞毒性。

3.曲马多和M1代谢物都能够以剂量和时间依赖性方式产生细胞毒性。在暴露于治疗浓度的曲马多和 M1 代谢物的细胞之间检测到无显着差异。曲马多诱导细胞凋亡和自噬细胞死亡，而 M1 代谢物仅诱导细胞凋亡。对于PI3K/AKT/mTOR通路，曲马多的治疗浓度只能增加AKT的磷酸化，而较高的毒性浓度能够增加整个通路的磷酸化；同时，M1 代谢物能够在治疗和毒性浓度下显着增加整个途径的磷酸化。

4. 总之，曲马多和 M1 具有同等的细胞毒性。细胞凋亡和自噬均介导肝细胞死亡。PI3K/AKT 通路参与细胞凋亡诱导，而自噬通过 mTOR 独立通路进行调节。