Macrophage α7nAChR alleviates the inflammation of neonatal necrotizing enterocolitis through mTOR/NLRP3/IL-1β pathway

•Through the detection of α7nAChR, we have identified the involvement of the cholinergic anti-inflammatory pathway (CAP) in NEC for the first time.•This study successfully demonstrated the anti-inflammatory effect of α7nAChR activation in NEC through detailed exploration and validation of clinical samples, cells, and animal experiments.•Our study will lay a theoretical foundation for the neuroimmune direction of NEC. Neonatal necrotizing enterocolitis (NEC) is one of the most prevalent and severe intestinal emergencies in newborns. The inflammatory activation of macrophages is associated with the intestinal injury of NEC. The neuroimmune regulation mediated by α7 nicotinic acetylcholine receptor (α7nAChR) plays an important role in regulating macrophage activation and inflammation progression, but in NEC remains unclear. This study aims to explore the effect of macrophage α7nAChR on NEC. Mice NEC model were conducted with high-osmolarity formula feeding, hypoxia, and cold stimulation. The α7nAChR agonist PNU-282987 and mTOR inhibitor rapamycin were treated by intraperitoneal injections in mice. The expression and distribution of macrophages, α7nAChR, and phospho-mammalian target of rapamycin (p-mTOR) in the intestines of NEC patients and mice was assessed using immunohistochemistry, immunofluorescence, and flow cytometry. The expression of NLRP3, activated caspase-1 and IL-1β in mice intestines was detected by flow cytometry, western blot or ELISA. In vitro, the mouse RAW264.7 macrophage cell line was also cultured followed by various treatments. Expression of p-mTOR, NLRP3, activated caspase-1, and IL-1β in macrophages was determined. Macrophages accumulated in the intestines and the expression of α7nAChR in the mucosal and submucosal layers of the intestines was increased in both the NEC patients and mice. The p-mTOR and CD68 were increased and co-localized in intestines of NEC patients. In vitro, α7nAChR agonist PNU-282987 significantly reduced the increase of NLRP3, activated caspase-1, and IL-1β in macrophages. PNU-282987 also significantly reduced the increase of p-mTOR. The effect was blocked by AMPK inhibitor compound C. The expression of NLRP3, activated caspase-1, and IL-1β was inhibited after mTOR inhibitor rapamycin treatment. In NEC model mice, PNU-282987 reduced the expression of p-mTOR, NLRP3, activated caspase-1, and IL-1β in the intestine. Meanwhile, rapamycin significantly attenuated NLRP3 activation and the release of IL-1β. Moreover, th