Mechanisms by which stress hormones promote 5-HT release from enterochromaffin cells and exacerbate inflammatory responses in macrophages

Objective To investigate the effects of stress hormones on intestinal function, and explore the mechanism by which stress hormones modulate the inflammatory response of macrophages via 5-hydroxytryptamine (5-HT), thereby providing a theoretical basis for 5-HT signaling in alleviating stress-induced intestinal inflammatory injury.

Method Using RIN14B enterochromaffin cells and RAW264.7 macrophages as in vitro models, we systematically examined the regulatory effects of stress hormones on 5-HT metabolism and macrophage function through in vitro hormone intervention, enzyme-linked immunosorbent assay (ELISA), calcium imaging, immunofluorescence, RT-qPCR, and Western blot, combined with network pharmacology analysis.

Result Norepinephrine (NE) and dexamethasone (DEX) significantly promoted 5-HT release from RIN14B cells and elicited intracellular calcium signaling. Specifically, NE promoted calcium influx by binding to the Adrb2 receptor, whereas DEX specifically upregulated the expression of tryptophan hydroxylase 1 (TPH1), the rate-limiting enzyme in 5-HT synthesis, through the glucocorticoid receptor. Network pharmacology analysis of 5-HT and the stress response revealed that 5-HT stably binds to the Drd2, Maoa, Maob, and Htr1a proteins to exert its effects, and activates neuroinflammatory responses through the neuroactive ligand-receptor interaction, calcium signaling, cAMP signaling, and inflammatory signaling pathways. In vitro cell culture experiments confirmed that 5-HT significantly upregulated the expression of TNF-α in RAW264.7 cells, induced intracellular accumulation of reactive oxygen species (ROS), and suppressed the transcription and protein expression of Nrf2 and its downstream target gene HO-1. Furthermore, 5-HT promoted macrophage proliferation via an internalization mechanism.

Conclusion This study reveals that stress hormones promote 5-HT release from enterochromaffin cells by activating calcium signaling and upregulating TPH1 expression. Excessive 5-HT disrupts the antioxidant defense mechanism of macrophages and promotes their proliferation, suggesting that it may exacerbate intestinal inflammatory responses. These findings provide an important scientific basis for elucidating the mechanisms underlying stress-induced intestinal inflammation.