萝卜硫素抑制幽门螺杆菌诱导胃黏膜上皮细胞NLRP3炎症小体活化的研究

Study on Sulforaphane Suppresses NLRP3 Inflammasome Activation in Gastric Mucosa Epithelial Cells Triggered by Helicobacter Pylori

目的研究萝卜硫素(sulforaphane,SFP)对幽门螺杆菌感染的胃黏膜上皮GES-1细胞线粒体和氧化应激损伤及NLRP3炎症小体活化的影响。方法 SFP(100 ng·mL^(-1))预处理GES-1细胞12 h,然后109 CFU·mL^(-1)的幽门螺杆菌感染细胞6 h;CCK8检测细胞活力,qRT-PCR分析PGC1α、COX-4、NRF1、TFAM、SOD1和HO-1 mRNA表达水平,Western blotting检测NLRP3、CASP1 p20、CASP1 p10、pro-IL-1β和IL-1β蛋白表达水平,并对GES-1细胞线粒体膜电位、氧耗量和ROS水平进行检测。结果幽门螺杆菌感染可明显诱导NLRP3、CASP1 p20、CASP1 p10、IL-1β蛋白和PGC1α、COX-4、NRF1 mRNA表达,促使氧消耗量和ROS水平增高,而细胞活力、SOD1、HO-1、TFAM mRNA表达和线粒体膜电位降低(P<0.05);SFP处理后能明显抑制NLRP3、IL-1β蛋白和PGC1α、COX-4、NRF1 mRNA表达,降低氧消耗量和ROS水平,而细胞活力、SOD1、HO-1、TFAM mRNA表达和线粒体膜电位增高(P<0.05);另外,ROS抑制剂NAC也能显著抑制NLRP3炎症小体和IL-1β表达水平。结论 SFP可通过降低线粒体损伤和ROS水平改善幽门螺杆菌感染诱导GES-1细胞炎症反应。

OBJECTIVE To study the effects of sulforaphane(SFP) on the mitochondrial damage, oxidative stress injury and NLRP3 inflammasome activation in gastric mucosa epithelial cells(GES-1) induced by Helicobacter pylori infection. METHODS GES-1 cells pre-treated with 100 ng·mL^(-1) of SFP for 12 h, then cells were infected with 109 CFU·mL^(-1) of Helicobacter pylori for 6 h. CCK8 was used to detect the cell viability. qRT-PCR was performed to measure the expression levels of PGC1α, COX-4, NRF1, TFAM, SOD1 and HO-1 m RNA. Western blotting was used to determine the expressions of NLRP3, CASP1 p20, CASP1 p10, pro-IL-1β and IL-1β. In addition, the mitochondrial membrane potential, oxygen consumption and ROS level were detected. RESULTS Helicobacter pylori infection could evidently induce the expression levels of NLRP3, CASP1 p20, CASP1 p10, IL-1β protein and PGC1α, COX-4, NRF1 m RNA, and promote the oxygen consumption and ROS level, whereas significantly inhibit the cell viability, SOD1, HO-1, TFAM mRNA expression and the mitochondrial membrane potential(P<0.05). Furthermore, the expression levels of NLRP3, IL-1β protein and PGC1α, COX-4, NRF1 mRNA, and the oxygen consumption and ROS level were greatly decreased after treatment with SFP in GES-1 cells induced with Helicobacter pylori infection, whereas significantly induced the cell viability, SOD1, HO-1, TFAM mRNA expression and the mitochondrial membrane potential(P<0.05). In addition, ROS inhibitor(NAC) also could suppress the expressions of NLRP3 inflammasome and IL-1β. CONCLUSION SFP can improve the inflammatory response of GES-1 cells triggered by Helicobacter pylori infection by reducing mitochondrial damage and ROS levels.