The Potential Mechanisms of Althaea rosea (Linn.) Cavan. Flower in Alleviating Tetrodotoxin Poisoning: An Integrated Metabolomics, Network Pharmacology and Experimental Validation

Renjin Zheng,1,2 Youjia Wu,1 Lingyi Huang,1 Fanxiang Zeng,3 Liying Huang1 1School of Pharmacy, Fujian Medical University, Fuzhou, Fujian, 350122, People’s Republic of China; 2Physical and Chemical Analysis Department, Fujian Center for Disease Control and Prevention, Fujian Provincial Key Laboratory of Zoonosis Research, Fuzhou, Fujian, 350012, People’s Republic of China; 3Department of Pharmacy, Fujian Maternity and Child Health Hospital; China Fujian Key Laboratory of Women and Children’s Critical Diseases Research [Fujian Maternity and Child Health Hospital (Fujian Women and Children’s Hospital)], Fuzhou, Fujian, 350001, People’s Republic of ChinaCorrespondence: Liying Huang, Email [email protected] Fanxiang Zeng, Email [email protected]: Tetrodotoxin (TTX) poisoning manifests rapidly and severely, and there are currently no clinically effective treatments. Althaea rosea (Linn.) Cavan. flower, documented in the “National Compendium of Chinese Herbal Medicines”, is traditionally recognized and clinically applied for its potential to mitigate tetrodotoxin (TTX) poisoning. This study aims to explores the pharmacodynamic components and mechanisms of the ethyl acetate extract of Althaea rosea flower (EAEAR) in a TTX-induced rat model.Methods: Ultra-performance liquid chromatography coupled with quadrupole Orbitrap high-resolution mass spectrometry (UPLC-Q-Orbitrap-HRMS) was used to identify active components in EAEAR. Metabolomics combined with network pharmacology was used to explore the mechanisms underlying the mitigating effects of EAEAR in TTX-intoxicated rats. Experimental validation was performed on key targets of the pathway through Western blotting or enzyme-linked immunosorbent assay. And differential metabolites in key pathways were further validated using ultra-performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry (UPLC-QqQ-MS/MS).Results: A total of 35 active components were identified in EAEAR, 12 core components and 15 core targets were screened in network pharmacology, and metabolomics revealed 15 different metabolites. The arginine and proline metabolism pathway and the arginine biosynthesis pathway were identified as critical pathways for EAEAR’s effect in alleviating TTX poisoning. Validation results indicated that EAEAR treatment led to significant alterations (P < 0.05) in six key targets (MAOA, AOC1, ALDH7A1, NAGS, NOS2, and NOS3) and three differential metabolites (GABA, Pro, and NAG) in TTX-intoxicated rats.Conclusion: EAEAR alleviates TTX poisoning symptoms by modulating targets and metabolites in the arginine and proline metabolism pathways and the arginine biosynthesis pathway. This study provides a theoretical basis for further exploration of its therapeutic potential and mechanisms against TTX poisoning. Keywords: Althaea rosea (Linn.) Cavan. flower, tetrodotoxin, active component, detoxication, metabolomics, network pharmacology