To investigate the protective effect and mechanism of Chimonanthus nitens Oliv. essential oil (CEO) on lipopolysaccharide (LPS)-induced inflammatory injury in intestinal epithelioid cell line No.6 (IEC-6) cells.

IEC-6 cells were stimulated with LPS to establish an inflammatory injury model, and CEO treatment was applied as an intervention. The cell counting kit-8 (CCK-8) assay was used to evaluate cell viability and determine the appropriate concentration for model induction. IEC-6 cells were divided into four groups: the control, CEO, LPS, and CEO + LPS groups. The CEO group and CEO + LPS group were pre-incubated with 20 mg / L CEO for 24 h, while the LPS group and CEO + LPS group were treated with 10 mg / L LPS to induce inflammatory injury. Interleukin-1 beta (IL-1β) secretion levels in the IEC-6 cell supernatants were measured using enzyme linked immunosorbent assay (ELISA). Reactive oxygen species (ROS) generation was assessed using flow cytometry. The expression levels of P65, phosphorylated P65, Cleaved caspase-1, and vesicle-associated membrane protein-associated protein B (VAPB) in IEC-6 cells were determined by western-blotting. Mitochondrial membrane potential changes, mitochondrial calcium levels, mitochondria-associated endoplasmic reticulum membranes (MAMs) formation, and co-localization of NOD-like receptor protein 3 (NLRP3) with MAMs were examined using confocal microscopy.

Significant differences were observed among the four groups in IL-1β secretion, P65 phosphorylation, Cleaved caspase-1 expression, ROS levels, mitochondrial membrane potential, mitochondrial calcium levels, and VAPB protein expression (F = 15.860, 22.260, 11.340, 65.220, 32.210, 15.800, 7.210; all P < 0.05). Compared with the control group, the IL-1β secretion, ROS production, P65 phosphorylation, Cleaved caspase-1 expression, mitochondrial calcium accumulation, and VAPB protein expression were significantly increased, while the mitochondrial membrane potential was decreased in the LPS group (all P < 0.05). Compared with the LPS group, the CEO + LPS group exhibited a significant increase in mitochondrial membrane potential, along with a reduction in IL-1β secretion, ROS production, P65 phosphorylation, Cleaved caspase-1 expression, mitochondrial calcium accumulation, and VAPB protein expression (all P < 0.05). Confocal microscopy showed that the formation of MAMs was less in the control group and CEO group, and the co-localization of NLRP3 with MAMs was also less. In the LPS group, the formation of MAMs increased, and the co-localization of NLRP3 with MAMs also increased. However, in the CEO + LPS group, the formation of MAMs and their co-localization with NLRP3 were improved.

CEO exerts a protective effect against LPS-induced inflammatory injury in IEC-6 cells. The underlying mechanism may involve the inhibition of nuclear factor kappa-B pathway activation and the regulation of VAPB protein expression, thereby influencing MAMs formation and function to mitigate mitochondrial damage.