莲心碱通过抑制NF-κB激活Nrf2/HO-1通路发挥抗炎抗氧化作用  

Liensinine exerts anti-inflammatory and antioxidant effects by inhibiting nuclear factor-κB and activating the nuclear factor-erythroid factor 2-related factor 2/heme oxygenase-1 pathway

作　　者：张锐虎 姚茹 史泽雅 郭民 霍怡彤 侯佳楠 陈朝阳 ZHANG Ruihu;YAO Ru;SHI Zeya;GUO Min;HUO Yitong;HOU Jianan;CHEN Zhaoyang(Laboratory Animal Center of Shanxi Medical University,Taiyuan 030001,China;Shanxi Key Laboratory of Experimental Animal Science and Animal Model of Human Disease,Taiyuan 030001)

机构地区：[1]山西医科大学实验动物中心,太原030001 [2]山西省实验动物与人类疾病动物模型重点实验室,太原030001

出　　处：《中国实验动物学报》2022年第2期191-197,共7页Acta Laboratorium Animalis Scientia Sinica

基　　金：山西省实验动物专项资金项目(2015K02)。

摘　　要：目的 探讨莲心碱(liensinine, lie)在脂多糖(lipid polysaccharides, lPS)诱导的RAW264.7细胞炎症模型中的抗炎抗氧化作用机制。方法 将细胞分为对照组(Control group),LPS组(LPS group),莲心碱(0.5、1、2μmol/L)组。采用CCK-8法筛选莲心碱的无细胞毒性浓度,Griess法检测一氧化氮(NO)的浓度,酶联免疫法(ELISA)检测前列腺素2(PGE2)的浓度以及促炎因子(TNF-α、IL-6和IL-1β)的含量、实时荧光定量PCR(qRT-PCR)检测COX-2和iNOS mRNA的表达,DCFH-DA探针检测细胞中活性氧(ROS)含量,试剂盒检测细胞中超氧化物歧化酶(SOD)的活性以及谷胱甘肽(GSH)和丙二醛(MDA)的含量,蛋白质免疫印迹法(Western Blot)检测NF-κB、MAPK和Nrf2/HO-1通路中关键蛋白的表达情况。结果 0~4μmol/L莲心碱对细胞活力无影响,与LPS组相比,莲心碱组(0.5、1、2μmol/L)细胞中的促炎因子含量下降(P<0.05),NO和PGE2浓度下降(P<0.05),COX-2和iNOS mRNA表达降低(P<0.05),NF-κB和MAPK通路被抑制(P<0.05),且具有剂量依赖性,莲心碱处理后ROS和MDA的含量显著低于LPS组(P<0.01),SOD的活性和GSH的含量显著高于LPS组(P<0.01),Nrf2/HO-1通路活性也较于LPS组显著增加(P<0.05)。结论 莲心碱通过抑制NF-κB和MAPK通路与激活Nrf2/HO-1通路发挥抗炎抗氧化作用。Objective To investigate the anti-inflammatory and antioxidant effects of liensinine(LIE) derived from lotus on a lipopolysaccharide(LPS)-induced RAW264.7 macrophage inflammatory model, and to study the molecular mechanisms responsible for these effects in relation to the nuclear factor(NF-κB), mitogen-activated protein kinase(MAPK), and nuclear factor-erythroid factor 2-related factor 2(Nrf2)/heme oxygenase(HO)-1 pathways. Methods RAW264.7 macrophages were divided into Control group, LPS group and LIE(0.5, 1, 2 μmol/L) groups. Cells were subjected to Cell Counting Kit 8 screening of non-cytotoxic concentrations of LIE, nitric oxide(NO) was detected by the Griess method, prostaglandin 2(PGE2) and pro-inflammatory factors(TNF-α,IL-6 and IL-1β) were detected by enzyme-linked immunoassay, cyclooxygenase-2(COX-2) and inducible nitric oxide(iNOS) synthase gene expression were detected by real-time polymerase chain reaction, and reactive oxygen species(ROS) were detected by dichloro-dihydro-fluorescein diacetate assay(which detects superoxide dismutase(SOD) activity and glutathione(GSH) and malondialdehyde(MDA) contents), and NF-κB, MAPK, and Nrf2/HO-1 pathway protein expression were detected by Western Blot. Results LIE 0 ~ 4 μmol/L had no effect on cell viability. Levels of pro-inflammatory factors levels were significantly lower in the LIE(0.5, 1, 2 μmol/L) group compared with the LPS group, NO and PGE2 concentrations and COX-2 and iNOS mRNA expression levels were all decreased, and NF-κB and MAPK pathways were inhibited( P < 0.05) in dose-dependent manners. The ROS and MDA contents were also significantly lower and SOD activity and GSH were significantly higher compared with the LPS group(P < 0.01), and the activity of the Nrf2/HO-1 pathway was also significantly higher than that of the LPS group(P < 0.05). Conclusions LIE exerts anti-inflammatory and antioxidant effects by inhibiting the NF-κB and MAPK pathways and activating the Nrf2/HO-1 pathway.

关 键 词：莲心碱 脂多糖 RAW264.7巨噬细胞 炎症 氧化应激 

分 类 号：Q95-33[生物学—动物学]