紫花牡荆素对脂多糖诱导的BEAS-2B细胞损伤和NF-κB-Keap1-Nrf2/ARE通路的影响

Effects of casticin on lipopolysaccharide-induced BEAS-2B cell damage and NF-κB-Keap1-Nrf2/ARE pathway

目的:探讨紫花牡荆素(CAS)对脂多糖诱导的人正常肺上皮细胞(BEAS-2B)损伤和NF-κB-Keap1-Nrf2/ARE通路的影响。方法:利用不同浓度的CAS预处理BEAS-2B细胞1 h、2 h、4 h,用1μg/ml脂多糖处理BEAS-2B细胞,构建细胞损伤模型,ELISA检测细胞上清炎症因子含量,筛选CAS最适作用浓度和时间;再将细胞分为正常组、模型组、Casticin组、ML385组、Casticin+ML385组、地塞米松组;流式细胞术检测细胞凋亡,ELISA检测细胞上清炎症因子的浓度,Western blot检测细胞NF-κB p65、p-NF-κB p65、Keap1、Nrf2以及细胞核中的Nrf2蛋白水平。结果:CAS最适作用条件为10μmol/L、2 h;与正常组相比,模型组的细胞凋亡率、炎症因子含量、p-NF-κB p65和Keap1蛋白表达水平显著升高(P<0.01),细胞和细胞核中Nrf2蛋白的表达水平显著降低(P<0.01);与模型组相比,Casticin组和地塞米松组的细胞凋亡率和炎症因子含量显著降低(P<0.01),Casticin组p-NF-κB p65和Keap1蛋白表达水平显著降低(P<0.01),细胞和细胞核中Nrf2蛋白的表达水平显著升高(P<0.01);与ML385组相比,Casticin+ML385组的细胞凋亡率,炎症因子含量,p-NF-κB p65和Keap1蛋白表达水平显著降低(P<0.01),细胞和细胞核中Nrf2蛋白的表达水平显著升高(P<0.01)。结论:CAS通过调节NF-κB-Keap1-Nrf2/ARE信号通路,抑制脂多糖诱导的细胞炎症。

Objective:To investigate the effects of casticin(CAS)on lipopolysaccharide-induced injury of human normal lung epithelial cells(BEAS-2B)and NF-κB-Keap1-Nrf2/ARE pathway.Methods:BEAS-2B cells were pretreated with different concentra-tions of casticin for 1 h,2 h and 4 h,and then treated with 1μg/ml liposolysaccharide to construct cell damage model.The contents of inflammatory factors in cell supernatant was detected by ELISA to screen the optimal concentration and time of casticin.The cells were divided into normal group,model group,Casticin group,ML385 group,Casticin+ML385 group and dexamethasone group.Cell apoptosis was detected by flow cytometry,the concentrations of inflammatory factors were detected by ELISA,and the levels of NF-κB p65,p-NF-κB p65,Keap1,Nrf2 and Nrf2 protein in cell nucleus were detected by Western blot.Results:The optimal concentration of CAS was 10μmol/L and the optimal time was 2 h.Compared with normal group,the apoptosis rate,the contents of inflammatory fac-tors,p-NF-κB p65 and Keap1 protein expression levels in model group were significantly increased(P<0.01),and Nrf2 protein expression levels in cells and nuclei were significantly decreased(P<0.01).Compared with model group,apoptosis rate and contents of inflammatory factors in casticin group and dexamethasone group were significantly decreased(P<0.01),protein expression levels of p-NF-κB p65 and Keap1 in Casticin group were significantly decreased(P<0.01).The expression level of Nrf2 protein in cells and nuclei was significantly increased(P<0.01).Compared with ML385 group,apoptosis rate,inflammatory factors contents,p-NF-κB p65 and Keap1 protein expression levels in Casticin+ML385 group were significantly decreased(P<0.01),the expression level of Nrf2 protein in cells and nuclei were significantly increased(P<0.01).Conclusion:CAS can inhibit cell inflammation induced by lipo-polysaccharide by regulating NF-κB-Keap1-Nrf2/ARE signaling pathway.