摘要
【目的】研究草质素对RAS-选择性致死小分子3(RSL-3)诱导的RAW264.7巨噬细胞铁死亡效应的抑制作用,探究草质素对巨噬细胞铁死亡的影响。【方法】利用0、5、10、20、40、80μmol/L草质素处理RAW264.7巨噬细胞48 h后,用CCK-8法检测细胞活力。将RAW264.7巨噬细胞分为对照组(Control)、RSL-3(15μmol/L)组、草质素(20、40、80μmol/L)组、铁死亡抑制剂铁抑素-1(10μmol/L Fer-1)组,草质素组和Fer-1组分别与RSL-3共孵育48 h,通过乳酸脱氢酶(LDH)试验检测各组细胞损伤效应,利用比色法检测各组细胞裂解液中铁离子、谷胱甘肽及丙二醛(MDA)含量。利用荧光探针法检测各组细胞胞浆内Fe^(2+)荧光强度、线粒体膜电位(MMP)和脂质活性氧(ROS)水平。采用Western blotting检测各组细胞裂解液核因子红细胞系2相关因子2(Nrf-2)、血红素加氧酶-1(HO-1)及谷胱甘肽过氧化物酶(GPX-4)的表达量。【结果】与0μmol/L草质素组相比,40μmol/L草质素可显著增强巨噬细胞活力(P<0.05);与40μmol/L草质素组相比,80μmol/L草质素组细胞活力显著降低(P<0.05),故5~40μmol/L为药物安全浓度。与对照组相比,RSL-3组细胞内LDH活性、总铁离子浓度、Fe^(2+)浓度、Fe^(3+)浓度、Fe^(2+)平均荧光强度、氧化型谷胱甘肽(GSSG)含量、GSSG占总谷胱甘肽的百分比及MDA水平均显著升高(P<0.05);细胞内还原型谷胱甘肽(GSH)含量、总谷胱甘肽含量、GSH占总谷胱甘肽的百分比、MMP相对百分比及脂质ROS荧光比值均显著降低(P<0.05),提示出现铁死亡效应。与RSL-3组相比,40μmol/L草质素组细胞内LDH活性、总铁离子浓度、Fe^(2+)浓度、Fe^(3+)浓度、Fe^(2+)平均荧光强度、GSSG含量、GSSG占总谷胱甘肽的百分比及MDA水平均显著降低(P<0.05);GSH含量、总谷胱甘肽含量、GSH占总谷胱甘肽的百分比、MMP相对百分比及脂质ROS荧光比值均显著升高(P<0.05)。与Fer-1组相比,40μmol/L草质素组的上述所有指标均无显著差异(P>0.05)。Western blotting结果表明,与RSL-3组相比,40μmol/L草质素显著升高了Nrf-2、HO-1和GPX-4的表达量(P<0.05);与Fer-1组相比,40μmol/L草质素组的上述指标均无显著差异(P>0.05)。【结论】草质素通过调控RSL-3诱导的RAW264.7巨噬细胞铁死亡相关分子指标,升高了GPX-4的表达水平,并激活其Nrf-2/HO-1通路发挥抑制RAW264.7巨噬细胞的铁死亡效应。本研究通过离体试验初步揭示了40μmol/L草质素可显著改善巨噬细胞铁死亡损伤效应及其机制。
【Objective】The aim of this study was to investigate the inhibitory effect of herbacetin on RSL-3-induced ferroptosis in RAW264.7 macrophages,and explore the effect of herbacetin on the ferroptosis of macrophages.【Method】After treatment with 0,5,10,20,40 and 80μmol/L herbacetin for 48 h,the viability of RAW264.7 macrophage was detected using CCK-8 assay.The cells were divided into control and RSL-3(15μmol/L),herbacetin(20,40 and 80μmol/L),ferroptosis inhibitor ferrostatin-1(10μmol/L Fer-1)groups.The herbacetin and Fer-1 groups were co-incubated with RSL-3 stimulation for 48 h,and the cell damage effect of each group was detected by lactate dehydrogenase(LDH)assay.The contents of iron ion,glutathione and malondialdehyde(MDA)in cells of each group were measured by colorimetric method.The intracellular Fe^(2+)fluorescence intensity,mitochondrial membrane potential(MMP)and lipid reactive oxygen species(ROS)levels in each group of cells were detected using fluorescence probe method.Western blotting was used to detect the expression of nuclear factor erythroid 2 related factor 2(Nrf-2),heme oxygenase-1(HO-1)and glutathione peroxidase 4(GPX-4)in cells of all groups.【Result】Compared with 0μmol/L herbacetin,40μmol/L herbacetin could significantly enhance macrophage viability(P<0.05).Compared with 40μmol/L herbacetin,80μmol/L herbacetin could significantly decrease macrophage viability(P<0.05).Therefore,5-40μmol/L could be used as a safe drug concentration.Compared with control group,the LDH activity,the concentrations of total iron ion,Fe^(2+)and Fe^(3+),the average fluorescence intensity of Fe^(2+),GSSG content,the percentage of GSSG in total glutathione,and MDA level of cells in RSL-3 group were significantly increased(P<0.05).The contents of GSH and total glutathione,the percentage of GSH to total glutathione,the relative percentage of MMP,and lipid ROS fluorescence ratio of cells in RSL-3 group were significantly decreased(P<0.05),suggesting the occurrence of ferroptosis.Compared with RSL-3 group,the activity of LDH,the concentrations of total iron ion,Fe^(2+)and Fe^(3+),the average fluorescence intensity of Fe^(2+),the content of GSSG,the percentage of GSSG to total glutathione,and MDA level of cells in 40μmol/L herbacetin group were significantly decreased(P<0.05).The contents of GSH and total glutathione,the percentage of GSH to total glutathione,the relative percentage of MMP,and lipid ROS fluorescence ratio of cells in 40μmol/L herbacetin group were significantly increased(P<0.05).Compared with Fer-1 group,40μmol/L herbacetin group showed no significant difference in all the above indicators(P>0.05).Western blotting results showed that compared with RSL-3 group,40μmol/L herbacetin could significantly increase the expression of Nrf-2,HO-1 and GPX-4(P<0.05).Compared with Fer-1 group,40μmol/L herbacetin group showed no significant difference in all the above indicators(P>0.05).【Conclusion】Herbacetin regulated the molecular indicators related to ferroptosis induced by RSL-3 in RAW264.7 macrophages,increased the expression of GPX-4,and activated its Nrf-2/HO-1 pathway to exert an inhibitory effect on ferroptosis in RAW264.7 macrophages.Through in vitro experiments,this study preliminarily revealed that 40μmol/L herbacetin could significantly improve the ferroptosis of macrophages and its mechanism.
作者
孙伟翔
张旺
李昊达
张婷
秦枫
袁亚梅
张力
陈毓
朱善元
SUN Weixiang;ZHANG Wang;LI Haoda;ZHANG Ting;QIN Feng;YUAN Yamei;ZHANG Li;CHEN Yu;ZHU Shanyuan(School of Animal Pharmacy,Jiangsu Agri-Animal Husbandry Vocational College,Taizhou 225300,China;Jiangsu Key Laboratory for High-Tech Research and Development of Veterinary Biopharmaceuticals,Taizhou 225300,China;Nanjing University of Chinese Medicine Hanlin College,Taizhou 225300,China)
出处
《中国畜牧兽医》
CAS
CSCD
北大核心
2024年第10期4235-4245,共11页
China Animal Husbandry & Veterinary Medicine
基金
2023年江苏省高校协同创新中心项目(00000223001)
江苏省高等学校基础科学(自然科学)研究重大项目(21KJA230001)
2021年江苏省水禽产业技术体系集成创新中心项目(00000221015)
2021年江苏高校协同创新中心项目(00000221010)
江苏农牧科技职业学院科研项目(NSF2021CB04)。
