参苓白术散通过TLR4/NFκB/Nrf2通路和自噬对脂多糖诱导的GES-1细胞炎症反应的影响  

Effects of Shenling Baizhu Powder on lipopolysaccharide-induced inflammatory response of GES-1 cells through TLR4/NFκB/Nrf2 pathway and autophagy

作　　者：范春兰 张润 王洪平 尹琼 李品 马全涛 杨涵 李晋生[1] 王志斌 FAN Chunlan;ZHANG Run;WANG Hongping;YIN Qiong;LI Pin;MA Quantao;YANG Han;LI Jinsheng;WANG Zhibin(Beijing Tongrentang Co.,Ltd.Scientific Research Institute,Beijing 100079,China)

机构地区：[1]北京同仁堂股份有限公司科学研究所,北京100079

出　　处：《现代药物与临床》2024年第9期2193-2203,共11页Drugs & Clinic

基　　金：国家科技重大专项(2018ZX09201-011)。

摘　　要：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、12.5μg/mL组可显著降低细胞上清NO含量(P<0.01);参苓白术散6.25、12.5μg/mL组能显著降低IL-6和IL-1β含量,参苓白术散12.5μg/mL组能显著降低TNF-α含量(P<0.05、0.01);参苓白术散6.25、12.5μg/mL组ROS生成显著减少(P<0.05、0.01);参苓白术散12.5μg/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12.5μg/mL组Caspase 3/7绿色荧光强度显著降低(P<0.05);参苓白术散12.5μg/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、12.5μg/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12.5μg/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, ShenlingBaizhu 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 12.5 μg/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

关 键 词：参苓白术散 人胃黏膜上皮细胞 脂多糖 炎症反应 Toll样受体4/核因子E2相关因子2信号通路 自噬 

分 类 号：R285[医药卫生—中药学]