Screening of hepatotoxic components of Epimedium brevicornu Maxim. by liver microfluidic-organ-chip: Icariside Ⅱ induces hepatocyte apoptosis via ROS-mediated mitochondrial dysfunction and the p38 MAPK pathway.

BACKGROUND: Epimedium brevicornu Maxim., a traditional tonic Chinese herbal medicine traditionally regarded as non-toxic, is clinically widely used for the treatment of bone and reproductive system diseases. However, with the expanded clinical application of its preparations, the number of adverse reactions such as hepatobiliary toxicity has gradually increased, thereby raising concerns about its safety. Currently, the potential toxic components of Epimedium brevicornu Maxim. remain unclear, and there is an urgent need to identify these toxic components and clarify their mechanisms using multi-dimensional models.

METHODS: We developed a liver microfluidic organ chip model with a sinusoidal structure using HepaRG spheres and HUVEC. At the same time, using organ chip models and immortalized liver cell lines, we systematically evaluated the five main flavonoids in Epimedium brevicornu Maxim. to screen for potential toxic monomers and explore their mechanisms.

RESULTS: First, the established liver microfluidic organ chip exhibited selective permeability and maintained stable hepatic synthetic-secretory functions for 7 days, providing a reliable platform for hepatotoxicity screening. Second, using this validated chip and an immortalized hepatocyte cell line, we identified Icariside Ⅱ as the key hepatotoxic component among five tested flavonoids, with an IC50 value of 15.67 μM in the cell line model. Icariside Ⅱ induces ROS overproduction, triggers mitochondrial dysfunction, and sequentially activated the intrinsic apoptotic pathway and p38 MAPK signaling, ultimately leading to hepatocyte apoptosis.

CONCLUSION: To our knowledge, this study represents one of the first applications of a liver microfluidic organ chip model for screening hepatotoxic components in Epimedium brevicornu Maxim., establishing a novel methodological framework for traditional Chinese medicine safety evaluation. By systematically comparing the screening results with those obtained from traditional immortalized cell line models, we have further validated the reliability of the identified hepatotoxic components. The mechanistic verification of these components' toxic effects, including their impact on cellular redox homeostasis and mitochondrial function, provides a comprehensive understanding of Epimedium brevicornu Maxim. induced hepatotoxicity.