, 2021). In Gram-negative bacteria, lipopolysaccharide (LPS) is not only a element on the cell wall but one of several major causes in the pathogenesis of septic shock and endotoxemia (Wang et al., 2019). Toll-like receptors (TLRs) play a important part in inflammation provoked by diverse stimuli as the major receptors of innate immunity, resulting in activating the inflammatory pathway, for example TLR2, TLR4, and so on (Wang et al., 2017). Amongst them, TLR4 was the initial TLR found in humans, that is accountable for recognizing bacterial LPS, initiating endotoxinmediated inflammation (Miyake, 2004). Numerous research have located that LPS-induced low-grade chronic systemic inflammatory will lead to a substantial alteration inside the function and key TJ proteins expression (Tao et al., 2017; Feng et al., 2022). Inhibiting LPS-mediated activation of TLR4 and subsequent inflammation pathway might be a valuable strategy to against the impairment of intestinal barrier. Excessive reactive oxygen species (ROS) production and cell redox imbalance have already been shown to possess a crucial part within the pathophysiology with the inflammatory response (Veith et al., 2019). NADPH oxidase (NOX) was firstly recognized as a main source of ROS in immune cells; and recently has been revealed to possess a important part in inflammation-related-cellular signalling, like NOX1, NOX2, NOX3, NOX4, NOX5, and DOUX1 and DOUX2 (Vermot et al., 2021). Amongst all these homologs, NOX1 is abundantly expressed in the gastrointestinal tract and has been implicated in inflammatory responses and nearby innate immune (Geiszt et al., 2003a; Juhasz et al., 2017). Recent research demonstrated a pivotal function of NOX1-derived ROS in gut intestinal epithelial homeostasis and barrier functions regulation, indicating NOX1, a reactive oxygen species (ROS)generating oxidase may well be critical for endotoxin-induced intestinal barrier dysfunction (Geiszt et al.Siglec-10 Protein web , 2003b; Yasuda et al.SOD2/Mn-SOD Protein Gene ID , 2012; Yokota et al., 2017). Rhizoma alisamatis (Ze xie in Chinese, Takusha in Japanese, and Taeksa in Korean, AR) is 1 of a broadly utilized medicinal and dietary plants in some East Asian countries (Jang and Lee, 2021). Alisol B 23-acetate (AB23A) was isolated in the rhizome of Rhizoma alisamatis as a natural triterpenoid and has been established to show outstanding bioactivity, comprising anti-hyperlipidemia, hepatoprotective, and anti-inflammatory (Meng et al., 2015; Wang et al., 2019). In addition, A novel study indicated that AB23A enhances intestinal permeability and microecological disorders in mice with colitis-associated cancer (CAC) (Zhu et al.PMID:23600560 , 2021). Our prior study also has confirmed that AB23A could inhibit high fat diet-induced down-regulation of TJ-related proteins in NAFLD mice (Xia et al., 2021). Even so, AB23A mechanism on intestinal permeability is poorly recognized. Therefore, assessing the protective effects and basic AB23Amechanism on intestinal barrier function in monolayers was the objective of this investigation.Caco-MATERIALS AND Approaches ReagentChengdu Have to Biotechnology Co., Ltd. (Chengdu, China) and Beyotime Biotechnology (Jiangsu, China) provided AB23A (purity 98 ) (Supplementary Figure S1) and LPS from E. coli (0111: B4), respectively. AB23A stock solutions were made in 0.1 DMSO, and operating solutions were made in a culture medium followed by filter sterilization (0.2 mm) just before utilization. Gibco Life Technologies supplied Dulbecco’s Modified Eagle’s Medium (DMEM) culture medium and fetal bovine serum (FBS). All b.