目的 探討參苓白術(shù)散對幽門螺旋桿菌脂多糖誘導(dǎo)人胃黏膜上皮細(xì)胞（GES-1）炎癥反應(yīng)的影響及作用機(jī)制。方法 建立幽門螺旋桿菌脂多糖誘導(dǎo)的GES-1細(xì)胞炎癥模型。采用Griess法檢測一氧化氮（NO）釋放水平；CCK8法檢測細(xì)胞活力；ELISA檢測炎性細(xì)胞因子[（腫瘤壞死因子-α（TNF-α）、白細(xì)胞介素（IL）-6和IL-1β]的水平；熒光探針染色檢測細(xì)胞活性氧（ROS）和Caspase 3/7含量；熒光探針JC-1觀察GES-1細(xì)胞線粒體膜電位的變化；免疫熒光技術(shù)檢測核因子-κB（NF-κB）p65的核轉(zhuǎn)位情況；熒光定量PCR檢測Toll樣受體4（TLR4）和核因子E2相關(guān)因子2（Nrf2）的基因表達(dá)水平；透射電鏡觀察細(xì)胞中自噬小體數(shù)量；蛋白質(zhì)免疫印跡法檢測TLR4、Nrf2、LC3II和p62蛋白的表達(dá)水平。結(jié)果 與模型組比較，參苓白術(shù)散6.25、12.5 μg/mL組可顯著降低細(xì)胞上清NO含量（P＜0.01）；參苓白術(shù)散6.25、12.5 μg/mL組能顯著降低IL-6和IL-1β含量，參苓白術(shù)散12.5 μg/mL組能顯著降低TNF-α含量（P＜0.05、0.01）；參苓白術(shù)散6.25、12.5 μg/mL組ROS生成顯著減少（P＜0.05、0.01）；參苓白術(shù)散12.5 μg/mL組細(xì)胞線粒體膜電位水平顯著提高（P＜0.05）；參苓白術(shù)散可以顯著抑制細(xì)胞NF-κB p65核位移（P＜0.05）；參苓白術(shù)散12.5 μg/mL組Caspase 3/7綠色熒光強度顯著降低（P＜0.05）；參苓白術(shù)散12.5 μg/mL組細(xì)胞TLR4蛋白相對表達(dá)量顯著降低、Nrf2相對表達(dá)量顯著升高（P＜0.05），參苓白術(shù)散各劑量組TLR4 mRNA相對表達(dá)量均顯著降低（P＜0.01），參苓白術(shù)散6.25、12.5 μg/mL組Nrf2 mRNA相對表達(dá)量均顯著升高（P＜0.05、0.01）。參苓白術(shù)散能夠提高脂多糖環(huán)境下GES-1細(xì)胞內(nèi)自噬小體的數(shù)量。與模型組比較，參苓白術(shù)散12.5 μg/mL組細(xì)胞內(nèi)LC3II表達(dá)顯著升高，p62表達(dá)顯著降低（P＜0.05）。結(jié)論 參苓白術(shù)散能改善脂多糖誘導(dǎo)的GES-1細(xì)胞炎癥反應(yīng)，其作用機(jī)制可能與調(diào)節(jié)TLR4/NF-κB/Nrf2信號通路和促進(jìn)細(xì)胞自噬有關(guān)。

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

國家科技重大專項(2018ZX09201-011)