Tsamba-stir-fried Tiebangchui: Attenuates the toxicity of raw Tiebangchui without compromising its analgesic efficacy in Caenorhabditis elegans through TRPV channel regulation.

ETHNOPHARMACOLOGICAL RELEVANCE: Tiebangchui (TBC) is a commonly used Tibetan medicine. However, its narrow therapeutic safety window necessitates strategies to reduce toxicity while preserving therapeutic efficacy. At present, tsamba-stir-fried TBC (TS-TBC) has become a characteristic pretreatment approach for TBC.

AIM OF THE STUDY: This study aimed to preliminary explore the material basis and mechanism underlying toxicity reduction and efficacy retention in tsamba-stir-fried TBC.

MATERIALS AND METHODS: First, UV, UPLC-Q-Exactive-Orbitrap-MS and HPLC were employed to characterize chemical compounds of raw TBC and TS-TBC. Subsequently, we conducted comprehensive evaluation in analgesia efficacy, basic toxicity, overall toxicity, neurotoxicity and cardiotoxic-like toxicity of using C. elegans N2. Further, network pharmacology and molecular docking approaches were adopted to screen for the potential targets of TS-TBC. To clarify the molecular mechanism of TS-TBC, we performed assays using C. elegans N2 and five mutant strains, combined with qRT-PCR detection. Finally, a compounds-phenotype-gene network was constructed via correlation analysis.

RESULTS: We preliminarily identified 77 alkaloids in raw TBC and 80 alkaloids in TS-TBC. The contents of aconitine, 3-deoxyaconitine and 3-acetylaconitine decreased after processing, while the level of benzoylaconine increased. TS-TBC treatment showed to reduce toxicity of raw TBC in C. elegans N2 without compromising its analgesic efficacy. According to the result of network pharmacology and molecular docking, TRPV channel proteins were identified as potential targets of TS-TBC. In C. elegans mutant strains, deficiency in ocr-1, ocr-2, ocr-3, ocr-4, or osm-9 genes led to the loss of TS-TBC efficacy. Similarly, deletion of ocr-2 or osm-9 genes enhanced TS-TBC-induced apoptosis, while ablation of ocr-4 or osm-9 abolished its antioxidant effect. qRT-PCR validation showed that TS-TBC could downregulate the expression of ocr-2, ocr-3, and ocr-4 genes, while it exerted no significant effect on osm-9 gene.

CONCLUSIONS: Our findings revealed that changes in the contents of characteristic alkaloids, rather than total alkaloids, constitute the material basis for the toxicity reduction and efficacy retention of TS-TBC, and TRPV channel proteins serve as its potential targets. These findings provide a scientific basis for the rational clinical application of processed TBC.