Pyroptosis is an inflammatory and lysis type of programmed cell death. In the canonical pyroptosis signaling pathway, the NLRP3 inflammasome activates inflammatory caspase-1, which then shears cut the executor protein GSDMD. The N domains of GSDMD move to heterogeneous membranes, form pores, and release inflammatory cytokines IL-1β and IL-18, causing cell membrane swelling and rupture. Pyroptosis is mainly regulated by the key proteins in the signaling pathway, including inflammasome, caspase-1, GSDMD, IL-1β, and IL-18, as well as their agonists and inhibitors. Appropriate pyroptosis can improve host defense mechanisms, while excessive pyroptosis would derive pathological effects on central nervous system, leding to neuroinflammatory response, blood-brain barrier damage, and cognitive disfunction.

Purpose: Gastrointestinal heat retention syndrome (GHRS) often occurs in adolescents, resulting into nervous system injury. Realgar, an arsenic mineral with neuroprotective effect, has been widely used to treat GHRS. However, its mechanism of action remains unknown. Methods: A GHRS rat model was established using a high protein and high calorie diet. We performed macroscopic characterization by assessing bowel sounds, hot/cold preference, anal temperature, and fecal features. Atomic fluorescence spectroscopy was employed to evaluate brain arsenic level while hippocampal ultrastructural changes were analyzed using transmission electron microscopy. In addition, inflammatory cytokines and BBB breakdown were analyzed by western blotting, immunofluorescence assays, and immunohistochemistry staining. We also evaluated hippocampal metabolites by LC-MS while fecal microorganisms were assessed by 16S rDNA sequencing. Results: Our data showed that the high protein and high calorie diet induced GHRS. The rat model depicted decreased bowel sounds, increased fecal characteristics score, preference for low temperature zone, and increased anal temperature. In addition, there was increase in inflammatory factors IL-6, Iba-1, and NF-κB p65 as well as reduced BBB structural protein Claudin-5 and Occludin. The data also showed appearance of hippocampus metabolites disorder and fecal microbial imbalance. Realgar treatment conferred a neuroprotective effect by inhibiting GHRS-specific characteristics, neuroinflammatory response, BBB impairment, metabolites disorder, and microbial imbalance in the GHRS rat model. Conclusion: Taken together, our analysis demonstrated that realgar confers a neuroprotective effect in GHRS rats through modulation of the microbiota-gut-brain axis.