穿心莲内酯调控Nrf2/HO-1通路减轻百草枯诱导的Ⅱ型肺泡上皮细胞氧化损伤

Andrographolide alleviates paraquat-induced oxidative stress injury of alveolar epithelial typeⅡcells via Nrf2/HO-1 pathway

目的探讨穿心莲内酯(Andrographolide)减轻百草枯(PQ)诱导的小鼠Ⅱ型肺泡细胞(MLE-12)氧化应激损伤。方法常规培养MLE-12细胞,分为对照组、PQ组、0.1%DMSO+PQ组和穿心莲内酯+PQ组;给予25μmol·L^(-1)穿心莲内酯预处理6 h,后给予800μmol·L^(-1) PQ干预24 h。CCK-8检测细胞活性;RT-qPCR和Western blot检测Nrf2和HO-1 mRNA和蛋白水平;试剂盒检测细胞内超氧化物歧化酶(SOD)、丙二醛(MDA)和过氧化氢酶(CAT)水平;流式细胞仪和荧光显微镜检测细胞内的活性氧(ROS)水平。结果PQ能够降低MLE-12细胞活性,并呈剂量依赖性;PQ为200μmol·L^(-1)、400μmol·L^(-1)、800μmol·L^(-1)、1200μmol·L^(-1)和2000μmol·L^(-1)对应细胞活性为89.5%、74.27%、58.65%、37.65%和26.83%。浓度为25μmol·L^(-1)及以下的穿心莲内酯对MLE-12细胞活性无明显抑制作用。800μmol·L^(-1) PQ显著降低MLE-12细胞活性(P<0.01),穿心莲内酯(5、10、25μmol·L^(-1))显著改善PQ诱导的细胞活性(P<0.01)。25μmol·L^(-1)穿心莲内酯+PQ组SOD、CAT表达水平较PQ组明显升高,而MDA和ROS较PQ组明显下降,差异显著(P<0.05)。与PQ组比较,25μmol·L^(-1)穿心莲内酯干预后Nrf2和HO-1 mRNA和蛋白水平明显升高,差异显著(P<0.05)。结论穿心莲内酯能够对百草枯介导的氧化应激损伤起保护作用,机制可能与增强Nrf2/HO-1抗氧化应激通路有关。

Objective To investigate the effect of Andrographolide on oxidative stress injury of mouse typeⅡalveolar cells(MLE-12)induced by Paraquat(PQ).Methods MLE-12 cells were conventionally cultured and divided into the Control group,PQ group,0.1%DMSO+PQ group,and andrographolide+PQ group.25μmol/L Andrographolide was pretreated for 4 h,followed by 800μmol/L PQ for 24 h.Cell activity was detected by the CCK-8 method.mRNA and protein levels of Nrf2 and HO-1 were detected by RT-qPCR and Western blot.The levels of superoxide dismutase(SOD),malondialdehyde(MDA),and catalase(CAT)were detected by the kits.Intracellular reactive oxygen species(ROS)were detected by flow cytometry and fluorescence microscopy.Results PQ could decrease the activity of MLE-12 cells in a dose-dependent manner.The cell activity of PQ at the concentration of 200μmol/L,400μmol/L,800μmol/L,1200μmol/L,and 2000μmol/L were 89.5%,74.27%,58.65%,37.65%,and 26.83%,respectively.Andrographolide at a concentration of no more than 25μmol/L had no significant inhibition on MLE-12 cell activity.PQ at the concentration of 800μmol/L significantly decreased the activity of MLE-12 cells(P<0.01),and andrographolide(5,10,25μmol/L)significantly improved the activity of MLE-12 cells induced by PQ(P<0.01).The expression levels of SOD and CAT in the 25μmol/L Andrographolide+PQ group were significantly higher than those in the PQ group,while MDA and ROS were significantly lower than those in the PQ group(P<0.05).Compared with the PQ group,mRNA and protein levels of Nrf2 and HO-1 were significantly increased after treatment with andrographolide at 25μmol/L(P<0.05).Conclusion Andrographolide can protect against paraquat-mediated oxidative stress injury,and the mechanism may be related to the enhancement of the Nrf2/HO-1 antioxidant stress pathway.