China Animal Husbandry and Veterinary Medicine ›› 2022, Vol. 49 ›› Issue (11): 4178-4186.doi: 10.16431/j.cnki.1671-7236.2022.11.008

• Physiological and Biochemical • Previous Articles     Next Articles

GPX4 Inactivation Alleviates LPS-induced Macrophage Inflammatory Response via JNK Pathway

ZHU Jiawei1,2, ZHAO Xiaohan1,2,3, MA Cui1,2, TANG Chaohua1,2, SI Wei1,2, ZHAO Qingyu1,2, ZHANG Junmin1,2, QIN Yuchang1   

  1. 1. State Key Laboratory of Animal Nutrition, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China;
    2. Scientific Observing and Experiment Station of Animal Genetic Resources and Nutrition in North China of Ministry of Agriculture and Rural Affairs, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China;
    3. College of Animal Science and Technology, Qingdao Agricultural University, Qingdao 266109, China
  • Revised:2022-08-01 Online:2022-11-05 Published:2022-11-04

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Abstract: 【Objective】 This study aimed to determine whether selenoprotein glutathione peroxidases 4 (GPX4) could regulate inflammatory responses affect in RAW264.7 macrophages upon lipopolysaccharide (LPS) stimulation and the underlying molecular mechanisms.【Method】 RAW264.7 macrophages were cultured in vitro.DMSO was used as the control, and 0.1-5.0 μmol/L GPX4 inhibitor FIN56 was used to treat RAW264.7 macrophages.The optimal concentration of GPX4 inhibitor FIN56 on RAW264.7 was determined by CCK-8 method and Western blotting.RAW264.7 was divided into 4 groups:Control group, RAW264.7 was treated with DMSO for 24 h;FIN56 group, RAW264.7 was treated with 0.5 μmol/L FIN56 and cultured for 24 h;DMSO-LPS group, RAW264.7 was treated with DMSO and then stimulated with 100 ng/mL LPS for 3 h;FIN56-LPS group, RAW264.7 was treated with FIN56 for 24 h then stimulated with 100 ng/mL LPS for 3 h.After cultured, the reactive oxygen species (ROS) level was detected by H2DCFDA, the cellular malondialdehyde (MDA) level was detected by the kit method, the expression of IL-1β, IL-6, and TNF-α mRNA was analyzed by Real-time PCR and the levels of cytokines in supernatants were detected by ELISA.Toll-like receptor 4 (TLR4) relative protein expression levels was detected by Western blotting.【Result】 Compared with DMSO treatment, cell viability had no significantly varied (P>0.05), while GPX4 protein level was significantly downregulated by 0.5 μmol/L FIN56 pretreatment for 24 h on macrophages (P<0.05).Therefore 0.5 μmol/L FIN56 was used for subsequent assay.Compared with control group, both FIN56 and LPS significantly increased ROS production (P<0.05), while compared with DMSO-LPS group, FIN56 resulted in a remarkable increase in ROS production(P<0.05).Compared with control group, LPS could significantly increase the gene expression of IL-1β, IL-6, TNF-α and up-regulated IL-6 production in macrophages, and the phosphorylation levels of JNK and c-Jun (P<0.05).While FIN56 could significantly down-regulated LPS-induced IL-6 gene expression and production (P<0.05), and decreased JNK and c-Jun phosphorylation levels (P<0.05), but had no significant effect on the protein expression levels of p38 and Erk1/2(P>0.05).【Conclusion】 Treatment of GPX4 inhibitor significantly suppressed LPS induced-cinflammatory response via JNK1/3 and c-Jun phosphorylation in macrophages.The findings of the study would provide a basis for anti-inflammatory or cytoprotective therapy.

Key words: selenoprotein glutathione peroxidases 4 (GPX4); RAW264.7; JNK signal pathway; inflammation response

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