目的 探讨参苓白术散对幽门螺旋杆菌脂多糖诱导人胃黏膜上皮细胞（GES-1）炎症反应的影响及作用机制。方法 建立幽门螺旋杆菌脂多糖诱导的GES-1细胞炎症模型。采用Griess法检测一氧化氮（NO）释放水平；CCK8法检测细胞活力；ELISA检测炎性细胞因子[（肿瘤坏死因子-α（TNF-α）、白细胞介素（IL）-6和IL-1β]的水平；荧光探针染色检测细胞活性氧（ROS）和Caspase 3/7含量；荧光探针JC-1观察GES-1细胞线粒体膜电位的变化；免疫荧光技术检测核因子-κB（NF-κB）p65的核转位情况；荧光定量PCR检测Toll样受体4（TLR4）和核因子E2相关因子2（Nrf2）的基因表达水平；透射电镜观察细胞中自噬小体数量；蛋白质免疫印迹法检测TLR4、Nrf2、LC3II和p62蛋白的表达水平。结果 与模型组比较，参苓白术散6.25、12.5 μg/mL组可显著降低细胞上清NO含量（P＜0.01）；参苓白术散6.25、12.5 μg/mL组能显著降低IL-6和IL-1β含量，参苓白术散12.5 μg/mL组能显著降低TNF-α含量（P＜0.05、0.01）；参苓白术散6.25、12.5 μg/mL组ROS生成显著减少（P＜0.05、0.01）；参苓白术散12.5 μg/mL组细胞线粒体膜电位水平显著提高（P＜0.05）；参苓白术散可以显著抑制细胞NF-κB p65核位移（P＜0.05）；参苓白术散12.5 μg/mL组Caspase 3/7绿色荧光强度显著降低（P＜0.05）；参苓白术散12.5 μg/mL组细胞TLR4蛋白相对表达量显著降低、Nrf2相对表达量显著升高（P＜0.05），参苓白术散各剂量组TLR4 mRNA相对表达量均显著降低（P＜0.01），参苓白术散6.25、12.5 μg/mL组Nrf2 mRNA相对表达量均显著升高（P＜0.05、0.01）。参苓白术散能够提高脂多糖环境下GES-1细胞内自噬小体的数量。与模型组比较，参苓白术散12.5 μg/mL组细胞内LC3II表达显著升高，p62表达显著降低（P＜0.05）。结论 参苓白术散能改善脂多糖诱导的GES-1细胞炎症反应，其作用机制可能与调节TLR4/NF-κB/Nrf2信号通路和促进细胞自噬有关。

Objective To study the anti-inflammatory effect of Shenling Baizhu Power on human gastric mucosal epithelial cell (GES-1) induced by Helicobacter pylori lipopolysaccharide, and its mechanism. Methods The lipopolysaccharide-induced GES-1 cell inflammation model was established. NO production was assessed by Griess reagent. Cell viability was determined by CCK8 assay. The levels of TNF-α, IL-1β, and IL-6 in cell culture supernatant were measured by ELISA. ROS production were detected through fluorescent probe assay. Caspase 3/7 activation was evaluated using Caspase 3/7 Green Ready Probe. JC-1 was used for the detection of the mitochondrial membrane potential. NF-κB was detected by immunofluorescence. TLR4 and Nrf2 mRNA expression were detected by qRT-PCR. Transmission electron microscopy was applied to observe the number of autophagosomes in GES-1 cells. The expression levels of TLR4, Nrf2, LC3II and p62 were determined by Western blotting. Results Compared with model groups, Shenling Baizhu Powder 6.25 and 12.5 μg/mL groups could significantly reduce the content of NO in cell supernatant (P < 0.01). The contents of IL-6 and IL-1β in Shenling Baizhu Powder 6.25 and 12.5μg/mL groups were significantly decreased, and the contents of TNF-α in Shenling Baizhu Powder 12.5 μg/mL groups were significantly decreased (P < 0.05, 0.01). Compared with model group, ROS production in Shenling Baizhu Powder 6.25 and 12.5 μg/mL groups was significantly decreased (P < 0.05, 0.01). The level of mitochondrial membrane potential in Shenling Baizhu Powder 12.5 μg/mL group was significantly increased (P < 0.05), Shenling Baizhu Powder could significantly inhibit NF-κB p65 nuclear shift (P < 0.05). Compared with model group, the green fluorescence intensity of Caspase 3/7 in Shenling Baizhu Powder 12.5 μg/mL group was significantly decreased (P < 0.05). The relative expression levels of TLR4 in Shenling Baizhu Powder 12.5 μg/mL group were significantly decreased, while Nrf2 expression levels significantly increased (P < 0.05), and the relative expression levels of TLR4 mRNA in each dose group were significantly decreased (P < 0.01). The relative expression of Nrf2 mRNA in Shenling Baizhu Powder groups 6.25 and 12.5 μg/mL were significantly increased (P < 0.05, 0.01). Shenling Baizhu Powder can increase the number of autophagosomes in GES-1 cells in lipopolysaccharide environment. Compared with model group, LC3II expression in Shenling Baizhu Powder 12.5 μg/mL group was significantly increased, and p62 expression was significantly decreased (P < 0.05). Conclusion Shenling Baizhu Power mitigated lipopolysaccharide-induced inflammatory response in GES-1 cells, which may be associated with the TLR4/NFκB/Nrf2 signaling pathway and activating autophagy.

R285

国家科技重大专项(2018ZX09201-011)