Glycosides from Cistanche tubulosa suppress intestinal ACSL4-mediated ferroptosis to ameliorate depression with intestinal dysfunction symptoms through regulating arachidonic acid metabolism

Abstract Background Depression is highly comorbid with intestinal dysfunction, yet antidepressant therapies are limited by suboptimal efficacy and frequent gastrointestinal adverse effects. Existing cellular and animal data support the potentially dual protective effects of total glycosides (TG) from Cistanche tubulosa on stress-triggered central depression and intestinal dysfunction symptoms. However, their efficacy and underlying mechanism against depression with intestinal dysfunction symptoms remain unconfirmed to date. Methods A chronic restraint stress mouse model of depression with intestinal dysfunction symptoms was established. Mice were divided into five groups (control, model, fluoxetine, low/high-dose TG) and treated intragastrically for 5 weeks. Behavioral tests and intestinal function assays were performed to evaluate therapeutic effects. Histological staining, immunofluorescence, biochemical analyses, ELISA, untargeted metabolomics, molecular docking, and western blotting were used to explore the underlying mechanism. Results TG administration significantly ameliorated chronic stress-induced depressive-like behaviors, as evidenced by restored sucrose preference, increased locomotor activity and central exploration, and reduced immobility time. Concurrently, TG improved multi-segment intestinal dysmotility, including shortening total gut transit time, restoring colonic propulsion, and enhancing gastric emptying and intestinal transit rates. Meanwhile, TG could preserve intestinal barrier integrity by mitigating epithelial damage and restoring tight junction proteins (ZO-1, occludin) and MUC2 expression, while reducing pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) in the intestine, serum and hippocampus. Furthermore, metabolomic analysis identified arachidonic acid metabolism as the key altered pathway, in which TG decreased the cecal levels of arachidonic acid and 20-hydroxy-leukotriene B4, and increased the level of dinoprost that were associated with ferroptosis. Correspondingly, TG were found to suppress intestinal oxidative stress, lipid peroxidation and Fe²⁺ accumulation, reverse chronic stress-induced ACSL4 upregulation and GPX4 downregulation, and its major constituents (e.g., echinacoside) showed strong binding affinity with ACSL4, which indicated that TG could modulate the intestinal ACSL4-mediated ferroptosis. Conclusion The present study demonstrates that TG exert a novel pharmacological effect in ameliorating comorbid depressive-like behaviors and intestinal dysfunction symptoms in stressed mice, suggesting that TG act via a gut-targeted pathway by suppressing intestinal ACSL4-mediated ferroptosis through regulating arachidonic acid metabolism. This novel pharmacological mechanism highlights the potential of TG as a natural product-based intervention for comorbid depression and intestinal dysfunction symptoms.