探讨山蜡梅挥发油（CEO）对脂多糖（LPS）所致小肠隐窝上皮细胞（IEC-6）炎症损伤的保护作用及机制。

使用LPS刺激IEC-6细胞构建炎症损伤模型，并采用CEO进行干预，通过细胞计数试剂盒8（CCK-8）法测定各组细胞活力，明确建立模型的最佳药物浓度。随后将IEC-6细胞将分为对照组、CEO组、LPS组和CEO + LPS组。CEO组和CEO + LPS组采用20 mg / L CEO预孵育24 h，LPS组和CEO + LPS组使用10 mg / L LPS构建炎症损伤模型。酶联免疫吸附法（ELISA）检测各组IEC-6细胞上清白细胞介素1β（IL-1β）分泌水平，流式细胞仪检测细胞内活性氧（ROS）生成情况。蛋白印记法检测IEC-6细胞内P65、磷酸化P65、Cleaved caspase-1、囊泡相关膜蛋白相关蛋白B（VAPB）蛋白表达水平。共聚焦显微镜检测细胞内线粒体膜电位、线粒体钙离子水平、线粒体相关内质网膜（MAMs）形成情况及核苷酸结合寡聚化结构域样受体蛋白3（NLRP3）蛋白与MAMs的共定位关系。

4组细胞IL-1β、P65蛋白磷酸化、Cleaved caspase-1蛋白、ROS含量、线粒体膜电位、线粒体钙离子水平及VAPB蛋白表达水平比较，差异均有统计学意义（F = 15.860、22.260、11.340、65.220、32.210、15.800、7.210，P均< 0.05）。与对照组比较，LPS组IL-1β分泌增加，ROS生成增加，线粒体膜电位下降，P65蛋白磷酸化水平和Cleaved caspase-1蛋白表达上升，线粒体钙离子蓄积，VAPB蛋白表达量上升（P均< 0.05）；与LPS组比较，CEO + LPS组细胞IL-1β分泌减少，ROS生成减少，线粒体膜电位上升，P65蛋白磷酸化水平和Cleaved caspase-1蛋白表达下降，线粒体内钙离子蓄积减少，VAPB蛋白表达量下降（P均< 0.05）。共聚焦显微镜显示对照组和CEO组MAMs形成较少，且NLRP3与MAMs共定位较少；LPS组MAMs形成增多，且与NLRP3蛋白共定位增加；CEO + LPS组MAMs形成及与NLRP3共定位情况则有所改善。

CEO对LPS所致IEC-6细胞炎症损伤具有保护作用，其机制可能是通过抑制核因子κB通路激活和调节VAPB蛋白表达，影响MAMs的形成和功能，从而减轻线粒体损伤。

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.