Hepatotoxicity of Huangdujuangen mediated by mTOR/p53/FOXO pathways in zebrafish; insights from Integrated multi-omics and network pharmacology.

BACKGROUND

"Huangdujuangen (HDJG)", composed of the roots of Rhododendron molle G. Don, is a traditional Chinese medicine that is widely known for its promising therapeutic potential for a wide range of diseases. Nevertheless, the potentially hepatotoxic properties of this substance have raised considerable public health concerns and warrant more in-depth investigation. Currently, the specific toxic constituents and the underlying mechanisms are still poorly understood.

PURPOSE: This experiment aimed to investigate the potential mechanisms underlying hepatotoxicity induced by HDJG.

METHODS

Zebrafish at 4 days post-fertilization were employed as an experimental model, wherein water extracts (WE) and alcoholic extracts (AE) of HDJG were administered at varying concentrations. Hepatotoxic effects were evaluated through a comprehensive assessment of liver phenotypes, histopathological alterations, and biochemical parameters. To unravel the principal chemical constituents, potential targets, and associated signaling pathways related to hepatotoxicity, a combination of Ultra-high performance liquid chromatography coupled with quadrupole electrostatic field orbitrap mass spectrometry (UHPLC-Q-Exactive Orbitrap MS) and network pharmacology approaches was systematically employed. Moreover, integrated transcriptomic, proteomic, and molecular biological techniques were harnessed to explore the underlying toxic mechanisms at both the gene and protein expression levels.

RESULTS

The findings of acute toxicity studies revealed that the AE of HDJG exhibited markedly greater toxicity than its water-based counterpart. This heightened toxicity was predominantly manifested through elevated mortality rates, pronounced morphological changes, abnormal fluctuations in liver enzyme levels, and substantial alterations in tissue architecture. A total of 169 bioactive constituents were identified within the AE. Of these, 103 compounds were confirmed to be absorbed into the circulatory system, where they interacted with 558 molecular targets and influenced 204 distinct signaling pathways. Multi-omics in-depth analysis indicated that HDJG primarily perturbed the mTOR signaling pathway, the p53 signaling cascade, and the FOXO signaling network, ultimately inducing significant hepatic cellular injury.

CONCLUSION

This study demonstrated that HDJG has the notable capacity to activate the mTOR/p53/FOXO signaling pathway, thereby intricately disrupting key biological processes such as autophagy, apoptosis, and oxidative stress, which collectively contribute to the development of hepatotoxic effects. These findings offer a solid theoretical foundation and meaningful reference for the safety evaluation and appropriate clinical application of HDJG.