基于Nrf2/HO-1通路抑制铁死亡探究甘草含药血清对LPS诱导的Caco2细胞炎症的影响

Effect of Glycyrrhizae Radix et Rhizoma-containing Serum on LPS-induced Inflammation in Caco2 Cells Based on Inhibition of Ferroptosis by Nrf2/HO-1 Pathway

目的:探讨甘草含药血清激活核因子E2相关因子2(Nrf2)/血红素加氧酶-1(HO-1)通路抑制铁死亡对脂多糖(LPS)诱导的人结肠上皮腺癌细胞(Caco2)炎症的影响。方法:将Caco2细胞分为正常组、模型组(LPS,200μg·L^(-1))、甘草含药血清低、中、高浓度组(5%、10%、20%)、铁死亡抑制剂组(3-氨基-4-环己基氨基苯甲酸乙酯,Fer-1,10μmol·L^(-1))。正常组细胞正常培养,其他组建立炎症模型,甘草含药血清低、中、高浓度组分别加入5%、10%、20%含药血清处理24 h,铁死亡抑制剂组给予Fer-1处理24 h。透射电镜观察各组线粒体形态;流式细胞术检测细胞内Fe^(2+)水平;微板法检测超氧化物歧化酶(SOD)活性、丙二醛(MDA)和谷胱甘肽过氧化物酶(GSH-Px)水平,酶联免疫吸附测定法(ELISA)检测白细胞介素-1β(IL-1β)、IL-6、IL-10、肿瘤坏死因子-α(TNF-α)含量;蛋白免疫印迹法(Western blot)检测Nrf2、HO-1、铁蛋白重链1(FTH1)、谷胱甘肽过氧化物酶4(GSH-Px4)蛋白的表达水平。运用小干扰核糖核酸(siRNA)的方式来观察Nrf2在铁死亡调控中的作用。将干扰后细胞分为空载体组(NC)、Si-Nrf2组、含药血清(20%)+Si-Nrf2组、含药血清(20%)+NC组。微板法检测MDA、SOD、GSH-Px水平;Western blot检测Nrf2、HO-1、FTH1、GSH-Px4蛋白表达水平。结果:与正常组比较,模型组线粒体固缩,线粒体膜增厚,线粒体形态变小;模型组Fe2+含量显著增加(P<0.01);SOD活性显著降低(P<0.01),GSH-Px表达显著下降(P<0.01),MDA含量显著增高(P<0.01);Nrf2、HO-1表达降低(P<0.05),FTH1表达显著性下降(P<0.01),GSH-Px4表达显著降低(P<0.01)。甘草含药血清处理组,中、高浓度组Fe2+含量显著降低(P<0.01),SOD活性和GSH-Px活性显著升高(P<0.01),MDA水平显著降低(P<0.01);高浓度组Nrf2表达明显升高(P<0.05),中浓度组HO-1与GSH-Px4蛋白的表达有所增高(P<0.05),低、中、高浓度组FTH1水平皆显著增加(P<0.01)。机制研究发现,与NC组比较,转染Nrf2 siRNA的细胞中MDA含量增加(P<0.01),SOD活性下降(P<0.01),GSH-Px活性减少(P<0.01);Nrf2、HO-1表达下降(P<0.01),FTH1和GSH-Px4蛋白含量降低(P<0.01);与Si-Nrf2组比较,在甘草含药血清干预后,细胞内MDA含量降低(P<0.01),SOD活性提高(P<0.01),GSH-Px活性上升(P<0.01);Nrf2、FTH1蛋白表达增加(P<0.05),HO-1和GSH-Px4蛋白表达水平明显升高(P<0.01)。结论:甘草含药血清可降低LPS诱导的Caco2细胞炎症因子及氧化应激水平,其机制与促进Nrf2/HO-1信号通路的表达,减轻细胞内脂质过氧化从而抑制铁死亡的发生有关。

Objective:To investigate the effect of Glycyrrhizae Radix et Rhizoma(GR)-containing serum on lipopolysaccharide(LPS)-induced inflammation in human colon epithelial adenocarcinoma cells(Caco2)based on inhibition of ferroptosis by the nuclear factor erythroid 2-related factor 2(Nrf2)/heme oxygenase-1(HO-1)pathway.Method:Caco2 cells were divided into a normal group,a model group(LPS,200μg·L^(-1)),low-,medium-,and high-dose GR-containing serum groups(5%,10%,20%),and a ferroptosis inhibitor group(3-amino-4-cyclohexylamino-benzoic acid ethyl ester,Fer-1,10μmol·L^(-1)).The cells in the normal group were cultured normally,while those in other groups underwent the induction of an inflammation model.The cells in the low-,medium-,and high-dose GR-containing serum groups were treated with 5%,10%,and 20%GR-containing serum for 24 hours,respectively,and the cells in the ferroptosis inhibitor group were treated with Fer-1 for 24 hours.Transmission electron microscopy was used to observe mitochondrial morphology in each group.Flow cytometry was used to detect intracellular Fe^(2+)levels.Microplate assays were performed to measure superoxide dismutase(SOD)activity,malondialdehyde(MDA)and glutathione peroxidase(GSH-Px)levels.Enzyme-linked immunosorbent assay(ELISA)was used to measure interleukin-1β(IL-1β),IL-6,IL-10,and tumor necrosis factor-α(TNF-α)levels.Western blot was used to measure the expression levels of Nrf2,HO-1,ferritin heavy chain 1(FTH1),and glutathione peroxidase 4(GSH-Px4)proteins.Small interfering RNA(siRNA)was used to investigate the role of Nrf2 in ferroptosis regulation.The cells after interference were divided into a negative control(NC)group,a Si-Nrf2 group,a GR-containing serum(20%)+Si-Nrf2 group,and a GR-containing serum(20%)+NC group.Microplate assays were performed to measure MDA,SOD,and GSH-Px levels,and Western blot was used to measure the expression levels of Nrf2,HO-1,FTH1,and GSH-Px4 proteins.Result:Compared with the normal group,the model group showed mitochondrial contraction,increased mitochondrial membrane thickness,and smaller mitochondrial morphology,increased Fe2+content(P<0.01),blunted SOD activity(P<0.01),decreased GSHPx expression(P<0.01),increased MDA content(P<0.01),reduced expression levels of Nrf2 and HO-1(P<0.05),reduced FTH1 expression(P<0.01),and down-regulated GSH-Px4 expression(P<0.01).In the GRcontaining serum groups,the medium-and high-dose groups showed a significant decrease in Fe2+content(P<0.01),potentiated SOD and GSH-Px activities(P<0.01),and decreased MDA levels(P<0.01).The high-dose group showed a significant increase in Nrf2 expression(P<0.05),and the medium-dose group showed increased expression of HO-1 and GSH-Px4 proteins(P<0.05).The expression levels of FTH1 significantly increased in the low-,medium-,and high-dose groups(P<0.01).The study on mechanism revealed that compared with the NC group,the cells transfected with Nrf2 siRNA showed increased MDA content(P<0.01),blunted SOD activity(P<0.01),decreased GSH-Px activity(P<0.01),decreased expression of Nrf2 and HO-1(P<0.01),and reduced levels of FTH1 and GSH-Px4 proteins(P<0.01).Compared with the Si-Nrf2 group,the cells treated with GR-containing serum showed a decrease in MDA content(P<0.01),an increase in SOD activity(P<0.01),an increase in GSH-Px activity(P<0.01),increased expression of Nrf2 and FTH1 proteins(P<0.05),and higher expression levels of HO-1 and GSH-Px4 proteins(P<0.01).Conclusion:GR-containing serum can reduce the inflammatory cytokines and oxidative stress levels in LPS-induced Caco2 cells.Its mechanism is related to the promotion of Nrf2/HO-1 signaling pathway expression,alleviating intracellular lipid peroxidation and inhibiting ferroptosis.