A dual in vitro approach for systematic analysis of mitochondrial toxicity in traditional Chinese medicine injections

Abstract

Abstract Background There are technical obstacles in the safety evaluation of traditional Chinese medicine (TCM) injections due to their complex chemical nature and the lack of rapid and accurate in vitro methods. Objectives To establish a dual in vitro mitochondrial toxicity assay approach for systematically evaluating mitochondrial toxicity of TCM injections. Methods Using this dual in vitro approach, the effect of TCM injections exposure on ATP depletion in HepG2 cells was examined by the conventional “glucose/galactose” assay. The cytotoxicity of TCM injections on HCT116 WT and HCT116 SCO2 KO cell lines was determined by sulforhodamine B (SRB) colorimetric assay. Subsequently, mitochondrial toxicity of ingredients in identified TCM injections was validated by combining molecular docking analysis with experimental validation. Results Four of the 35 TCM injections, including Xiyanping, Dengzhanhuasu, Shuanghuanglian, and Yinzhihuang, significantly reduced cellular ATP production in galactose medium in the first assay, and presented less cytotoxic in the respiration deficient cells in the second assay, indicating that they have mitochondrial toxicity. Furthermore, we identified scutellarin, rutin, phillyrin, and baicalin could be the potential mitochondrial toxic ingredients in 4 TCM injections by combining molecular docking analysis with experimental validation. Conclusions Collectively, the dual in vitro approach is worth applying to the safety evaluation of more TCM products, and mitochondrial toxic TCM injections and ingredients found in this study deserve more attention.