Gracillin Suppresses Intestinal Epithelial Inflammation and Improves Intestinal Barrier in IBD via the SDHB/NF-κB Axis

Abstract

Background: Gracillin has been shown to have anti-inflammatory activity, but its role in inflammatory bowel disease (IBD) remains unclear. This study aimed to examine the effects of Gracillin on IBD and its possible regulatory mechanisms. Methods: NCM460 cell viability was measured using cell counting kit-8 assay after treatment with different concentrations of Gracillin or lipopolysaccharide (LPS). The levels of inflammatory cytokines (interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α)), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tight junction proteins (zonula occludens-1 (ZO-1), Occludin, Claudin-1), succinate dehydrogenase subunit B (SDHB) and nuclear factor kappa B (NF-κB) pathway (p-P65, P65) were determined by quantitative real-time Polymerase Chain Reaction (qRT-PCR) or western blot. The monolayer integrity of the cells was measured using trans-epithelial electrical resistance. Additionally, the production of reactive oxygen species (ROS) and lactate dehydrogenase (LDH) release were examined using specific assay kits. After silencing SDHB via cell transfection, rescue experiments were carried out. Results: Gracillin did not show any cytotoxicity to NCM460 cells. Upon administration of Gracillin, the cell viability, monolayer integrity, tight junction proteins (ZO-1, Occludin, and Claudin-1) and SDHB protein level were increased, whereas the mRNA level of inflammatory cytokines (IL-6 and TNF-α), mRNA and protein levels of iNOS and COX-2 were down-regulated in LPS-treated cells (p < 0.05). Furthermore, the activation of NF-κB pathway and the increased ROS production and LDH release induced by LPS, were suppressed by Gracillin in the cells (p < 0.05). Additionally, SDHB silencing reversed the regulatory effects of Gracillin on cell viability, inflammation, tight junction and NF-κB pathway in LPS-treated cells (p < 0.05). Conclusion: Gracillin can block NF-κB pathway to reduce inflammation and improve intestinal barrier in IBD in vitro by up-regulating SDHB.