As an organ in contact with the outside world, the intestines are particularly susceptible to a wide range of external stimuli. These stimuli include bacteria and lipopolysaccharide (LPS), which can lead to the disruption of the intestinal barrier and severe intestinal inflammation. [1,2]. Persistent intestinal inflammation is a key trigger for the development of gastrointestinal diseases such as inflammatory bowel disease (IBD) which is mainly comprised of Crohn's disease (CD) and ulcerative colitis (UC) [3,4]. So far, the incidence of IBD is still reported to be on the rise globally, causing great physical and mental harm to patients [5]. However, due to the complexity of the inflammatory response mechanisms in organisms, there are still no effective therapies to block the progression of IBD [6]. Accordingly, it is of utmost importance to explore the underlying mechanisms that can inhibit the intestinal inflammatory response, thereby preventing the development of IBD.

A substantial body of research demonstrates that when pathogens or irritants invade the animal intestine, on the one hand, they trigger multiple inflammatory signaling pathways, including Toll-like receptor 4 (TLR4), NF-κB, and mitogen-activated protein kinase (MAPK). This activation induces the release of pro-inflammatory cytokines such as tumor necrosis factor (TNF)-α, interleukin-6 (IL-6), IL-18, and IL-1β. The accumulation of these cytokines contributes to intestinal inflammation and tissue damage. [[7], [8], [9]]. On the other hand, these pathogens or stimuli can also activate various important receptors in the intestine, such as NOD-like receptor protein 3 (NLRP3) inflammasome [10], through danger-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs), which subsequently induce the shearing of caspase-1; the sheared caspase-1 (cleaved-caspase-1) can promote the maturation and release of IL-1β and IL-18, leading to inflammatory responses; meanwhile, it can induce pyroptosis, referred to as cellular inflammatory necrosis, by promoting the activation of the gasdermin D (GSDMD) execution protein, which subsequently worsens the intestinal inflammatory injury [[11], [12], [13]]. Beyond that, chronic and acute intestinal inflammation are strongly associated with necroptosis, a type of programmed cell death that is independent on caspase [14]. Research has established that necroptosis can be triggered exclusively by Receptor-Interacting Protein Kinase 1 (RIPK1), RIPK3, and their downstream effector, Mixed Lineage Kinase Domain-Like (MLKL) [15]. Necroptosis and pyroptosis are recognized as a crucial role in homeostasis and inflammation in the gut caused by a variety of factors [16]. Therefore, pharmacologically targeting the TLR4/NF-κB/MAPK signaling cascade and its downstream cell death pathways (pyroptosis and necroptosis) could alleviate intestinal inflammation, offering a mechanistic basis for innovative IBD therapies.

To date, pregnane X receptor (PXR), an important member of the nuclear receptor superfamily and encoded by nuclear receptor subfamily 1 group I member 2 (NR1I2), is a key regulator against exogenous toxins and is mainly found in tissues such as the small intestine, liver, and kidneys, where it can inhibit the inflammatory response by regulating multiple inflammatory signaling pathways [17,18]. In the present study, we identified that pregnenolone 16α‑carbonitrile (PCN), a kind of PXR agonist, improved intestinal barrier dysfunction, intestinal inflammation, pyroptosis and necroptosis in EHEC-infected mice and LPS-induced intestinal epithelial cells, which were reversed by deficiency or knockdown of PXR. It has been shown that PXR forms a heterodimer with retinoid X receptor (RXR), which can bind directly to the p65 subunit of NF-κB [19], thereby limiting the TLR4 signaling cascade response [20]. Overexpression of PXR inhibits endotoxin-induced phosphorylation of MAPK subunits (JNK) and improves the barrier function of small intestinal epithelial cells [21]. In addition, PXR is also closely related to NLRP3 activation, but whether it positively or negatively regulates NLRP3? There is still considerable controversy. Hudson et al. [22] found that PXR activation in macrophages can trigger the rapid release of cellular ATP through pannexin-1 channels, leading to P2X7 receptor activation and subsequent activation of NLRP3. Interestingly, Wang et al. [23] showed that overexpression or activation of PXR significantly inhibited the oxidized LDL-induced NLRP3 activation in vascular endothelial cells. Nevertheless, few investigations have explored PXR's ability to suppress intestinal inflammation by modulating necroptotic pathway [24,25].

Although significant efforts to understand the anti-inflammatory role of PXR in IBD pathogenesis, the detailed molecular mechanism remains to be further explored. Consequently, this research constructed enterohaemorrhagic Escherichia coli O157:H7 (EHEC)- and lipopolysaccharide (LPS)-induced intestinal inflammation models in vivo and in vitro, which are widely recognized models of intestinal inflammation [26], with the aim of exploring whether PXR-regulated inflammatory responses are associated with the NLRP3/caspase-1/GSDMD and RIPK3/RIPK1/MLKL signaling pathways.