Timosaponin AIII

Objective: To investigate the mechanism through which timosaponin AⅢ (TAⅢ)-based liposomes loaded with auranofin (AUF) (T-AUF-LPs) induce ferroptosis in anaplastic thyroid carcinoma cells. Methods: T-AUF-LPs were prepared using the thin-film hydration method, and their physicochemical properties and stability were characterized. Additionally, conventional liposomes (C-AUF-Lips) were prepared using cholesterol as the membrane material. Cellular uptake efficiency was evaluated in CAL-62 cells using coumarin-6-labeled liposomes. Cell viability was assessed via CCK-8 assay. The role of ferroptosis was confirmed using the inhibitor Ferrostatin-1 (Fer-1) and flow cytometric analysis of cell death. The expression of ferroptosis-related markers (ACSL4, NCOA4, GPX4) were detected using RT-qPCR and Western blot. Levels of intracellular iron, glutathione (GSH), and lipid peroxidation were measured. A nude mouse tumor xenograft model was established to evaluate the in vivo antitumor efficacy of T-AUF-LPs, and hematoxylin-eosin (HE) staining was performed to assess potential systemic toxicity. Results: T-AUF-LPs appeared as uniformly distributed spherical particles with an average size of (119.40±3.11) nm, which was significantly smaller than that of the conventional liposomes C-AUF-LPs (P＜0.001). The zeta potential of T-AUF-LPs was (-12.07±0.65) mV, lower than that of C-AUF-LPs (P=0.002). Furthermore, CAL-62 cells exhibited significantly enhanced cellular uptake of T-AUF-LPs compared to C-AUF-LPs. Cell experiments demonstrated that among the established groups, i.e., Control, TAⅢ, AUF, AUF+TAⅢ, C-AUF-LPs, and T-AUF-LPs, the T-AUF-LPs group exhibited the lowest half-maximal inhibitory concentration (IC₅₀=0.66 μmol/L) and the highest CAL-62 cell mortality (14.7±1.3)%. Furthermore, this group showed significantly elevated lipid peroxidation (2.07±0.28), increased iron content (4.64±0.17), and markedly reduced glutathione (GSH) levels (0.11±0.05). At the molecular level, mRNA expression of ACSL4 and NCOA4 was up-regulated (13.10±0.94 and 7.52±0.49, respectively), while GPX4 mRNA was down-regulated (0.16±0.21). Consistently, ACSL4 and NCOA4 protein expression was increased (1.30±0.06 and 1.13±0.31, respectively), whereas GPX4 protein expression was decreased (0.31±0.18).Notably, pretreatment with the ferroptosis inhibitor Fer-1 significantly reversed T-AUF-LPs-induced cell death, reducing mortality to (8.8±0.8)%. Animal studies demonstrated that among all groups, tumor growth was most significantly suppressed in the T-AUF-LPs group, which exhibited the smallest tumor volume on day 15 post-inoculation. No significant body weight loss was observed in nude mice, and histopathological assessment of the heart, liver, lungs, and kidneys revealed no apparent toxic damage. In tumor tissues of the T-AUF-LPs group, mRNA expression of ACSL4 and NCOA4 was significantly up-regulated (1.59±0.29 and 8.65±3.48, respectively), while GPX4 mRNA expression was markedly down-regulated (0.11±0.01). Consistently, ACSL4 and NCOA4 protein levels were increased (1.26±0.31 and 1.14±0.39, respectively), whereas GPX4 protein expression was significantly reduced (0.56±0.12). Conclusion: T-AUF-LPs inhibited the growth of anaplastic thyroid carcinoma cells by activating the ferroptosis pathway.

The increasing global prevalence of attention deficit hyperactivity disorder (ADHD) and its substantial societal burden have intensified research efforts to elucidate its pathophysiology and therapeutic interventions. Xiaoer Huanglong Granule (XHG), a well-established traditional Chinese herbal formulation, has demonstrated clinical efficacy in ADHD management and has been approved by the China National Medical Products Administration and successfully launched on the market. However, its pharmacological mechanisms remain incompletely understood. This study aims to systematically evaluate the therapeutic potential of the herbal formulation XHG in ADHD using an integrated approach that combines network pharmacology, behavioral assays, and molecular analyses.

We employed network pharmacology-based screening to identify bioactive compounds and target proteins associated with ADHD. Molecular docking simulations were conducted to assess binding affinities. Behavioral effects were evaluated using open-field and elevated plus maze tests, and protein expression levels were analyzed via Western blot.

Network analysis identified six key bioactive compounds (methyl-L-phenylalanine, 1,4-β-Dmannotriose, aurantiamide acetate, N-cis-feruloyltyramine, timosaponin A-III, and apigenin) and five critical targets (TH, ADORA2A, SLC6A3, SLC6A4, COMT). Molecular docking confirmed strong binding interactions. Behavioral tests showed that XHG significantly reduced hyperlocomotion and impulsivity-like behaviors. Western blot analyses further revealed regulatory effects on key neurotransmitter-related proteins.

This integrated study elucidates the multi-target neuroprotective mechanisms of XHG in ADHD and provides a modern scientific foundation for the application of traditional herbal medicine in treating neuropsychiatric disorders.

This study provides the first comprehensive evidence supporting the neuroprotective properties of XHG in ADHD through multi-target mechanisms, offering a scientific basis for its clinical application as a potential therapeutic agent.