特女贞苷对脂多糖诱导的巨噬细胞促炎反应的抑制作用及机制

Protection against LPS-induced pro-inflammatory response by nuezhenide in macrophages

目的:探讨特女贞苷(NED)抑制细菌脂多糖(LPS)诱导巨噬细胞促炎反应的机制。方法:用50μmol/L NED处理RAW264.7巨噬细胞12 h后,采用CCK-8法测定细胞活力,Annexin V-FITC/PI法测定细胞凋亡率,转录组测序分析异常基因表达,免疫荧光检测Nrf2蛋白表达定位。进一步使用100 ng/mL LPS(模型组)与不同浓度(0、12.5、25和50μmol/L)NED联合处理RAW264.7细胞干预12 h后,采用ELISA测定细胞上清液中TNF-α和IL-6含量;实时荧光定量PCR(qPCR)检测细胞TNF-α和IL-6 mRNA的表达;分别采用DCFH-DA、MitoTracker-Green和JC-1染色测定细胞中活性氧、线粒体含量和膜电位;试剂盒测定细胞内抗氧化酶活力、线粒体复合物I和III活性以及ATP合成的变化。结果:与空白对照组相比,50μmol/L NED对巨噬细胞存活和凋亡无明显影响(P>0.05),但可抑制模型组LPS诱导促炎因子TNF-α、IL-6的表达和分泌(P<0.05),并增强抗炎因子转录表达(P<0.05),呈现明显抗炎效应。与模型组比较,联合NED干预组增强Nrf2核转位及下游抗氧化因子转录表达(P<0.05),提高细胞还原型谷胱甘肽(GSH)含量(P<0.05),并降低活性氧(ROS)和氧化型谷胱甘肽(GSSG)水平(P<0.05),减轻LPS刺激巨噬细胞氧化应激(P<0.05)。此外,NED刺激线粒体生物合成基因表达升高(P<0.05),逆转LPS诱导线粒体膜电位、复合物I和III活性下降(P<0.05),促进线粒体数量增多和ATP合成(P<0.05)。结论:特女贞苷能够激活Nrf2抗氧化系统,增强线粒体生物合成和代谢功能,抑制LPS诱导巨噬细胞促炎分化,具有成为抗炎症疾病候选药物的潜力。

OBJECTIVE:This study aimed to investigate effects of nuezhenide(NED)on LPS-induced proinflammatory response in macrophages.METHODS:RAW264.7 cells were treated with 50μmol/L NED for 12 hours.Differential gene expressions were detected via transcriptome,cell viability using the CCK8 kit,and apoptosis rates measured using Annexin V-FITC/PI staining,and expression and localization of Nrf2 protein were detected by immunofluorescence.The cells were treated with LPS(100 ng/mL)and NED(12.5,25 and 50μmol/L)for 12 hours.Then,TNF-αand IL-6 contents in the supernatant of cultured cells were determined by ELISA,and expressions of TNF-αand IL-6 by RT-PCR.Moreover,ROS,mitochondrial mass,and membrane potential were measured by flow cytometry after staining with DCFH-DA,MitoTracker-Green and JC-1,respectively.Enzyme activities of SOD and CAT,activities of mitochondrial complexes I andⅢ,as well as ATP contents were determined using commercial kits.RESULTS:Our results show that 50μmol/L NED had no significant effects on survival and apoptosis of the RAW264.7 macrophages(P>0.05).However,NED treatments blunted LPS-induced expression of TNF-αand IL-6(P<0.05),and enhanced expression of anti-inflammatory cytokines(P<0.05).Meanwhile,NED significantly promoteed Nrf2 nuclear translocation and downstream antioxidant transcription(P<0.05),which contributed to inhibition of LPS-induced accumulation of ROS and GSSG(P<0.05).In addition,NED reversed the LPS-induced decreased of mitochondrial membrane potential,mitochondrial complexes I and III activities,as well as ATP contents through promoting mitochondrial biogenesis(P<0.05).CONCLUSION:NED inhibited LPS-induced macrophages pro-inflammatory differentiation through enhancing Nrf2 antioxidant system and mitochondrial biogenesis.Application of NED may be a potential strategy for treatment of inflammatory diseases.