China Animal Husbandry and Veterinary Medicine ›› 2024, Vol. 51 ›› Issue (10): 4235-4245.doi: 10.16431/j.cnki.1671-7236.2024.10.005

• Physiological and Biochemical • Previous Articles    

Effect of Herbacetin on Macrophage Ferroptosis

SUN Weixiang1,2, ZHANG Wang3, LI Haoda1, ZHANG Ting1, QIN Feng1,2, YUAN Yamei1,2, ZHANG Li1,2, CHEN Yu1, ZHU Shanyuan1   

  1. 1. School of Animal Pharmacy, Jiangsu Agri-Animal Husbandry Vocational College, Taizhou 225300, China;
    2. Jiangsu Key Laboratory for High-Tech Research and Development of Veterinary Biopharmaceuticals, Taizhou 225300, China;
    3. Nanjing University of Chinese Medicine Hanlin College, Taizhou 225300, China
  • Received:2024-04-19 Published:2024-09-30

Abstract: 【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 80 μmol/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 (15 μmol/L),herbacetin (20,40 and 80 μmol/L),ferroptosis inhibitor ferrostatin-1 (10 μmol/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 Fe2+ 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 0 μmol/L herbacetin,40 μmol/L herbacetin could significantly enhance macrophage viability (P<0.05). Compared with 40 μmol/L herbacetin,80 μmol/L herbacetin could significantly decrease macrophage viability (P<0.05). Therefore,5-40 μmol/L could be used as a safe drug concentration. Compared with control group,the LDH activity,the concentrations of total iron ion,Fe2+ and Fe3+,the average fluorescence intensity of Fe2+,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,Fe2+ and Fe3+,the average fluorescence intensity of Fe2+,the content of GSSG,the percentage of GSSG to total glutathione,and MDA level of cells in 40 μmol/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 40 μmol/L herbacetin group were significantly increased (P<0.05). Compared with Fer-1 group,40 μmol/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,40 μmol/L herbacetin could significantly increase the expression of Nrf-2,HO-1 and GPX-4 (P<0.05). Compared with Fer-1 group,40 μmol/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 40 μmol/L herbacetin could significantly improve the ferroptosis of macrophages and its mechanism.

Key words: ferroptosis; herbacetin; macrophage; lipid peroxidation; cell damage

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