GPX4失活通过JNK通路缓解LPS诱导巨噬细胞炎症反应

doi:10.16431/j.cnki.1671-7236.2022.11.008

中国畜牧兽医 ›› 2022, Vol. 49 ›› Issue (11): 4178-4186.doi: 10.16431/j.cnki.1671-7236.2022.11.008

GPX4失活通过JNK通路缓解LPS诱导巨噬细胞炎症反应

朱家威^1,2, 赵潇晗^1,2,3, 马翠^1,2, 汤超华^1,2, 司玮^1,2, 赵青余^1,2, 张军民^1,2, 秦玉昌¹

1. 中国农业科学院北京畜牧兽医研究所, 动物营养学国家重点实验室, 北京 100193;
2. 中国农业科学院北京畜牧兽医研究所, 农业农村部华北动物遗传资源与营养科学观测实验站, 北京 100193;
3. 青岛农业大学动物科技学院, 青岛 266109

修回日期:2022-08-01 出版日期:2022-11-05 发布日期:2022-11-04
通讯作者: 秦玉昌 E-mail:qinyuchang@caas.cn
作者简介:朱家威,E-mail:zhujiaweisemail@163.com。
基金资助:
中国富硒产业研究院富硒专项研发计划项目（2019QCY-3.3）；中国农业科学院科技创新工程协同创新任务（CAAS-XTCX20190025-8）；中国农业科学院科技创新工程（ASTIP-IAS-12）

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

ZHU Jiawei^1,2, ZHAO Xiaohan^1,2,3, MA Cui^1,2, TANG Chaohua^1,2, SI Wei^1,2, ZHAO Qingyu^1,2, ZHANG Junmin^1,2, QIN Yuchang¹

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

摘要/Abstract

摘要： 【目的】研究硒蛋白谷胱甘肽过氧化物酶4（glutathione peroxidases 4，GPX4）失活如何参与调控脂多糖（lipopolysaccharide，LPS）诱导的RAW264.7巨噬细胞炎症反应及其潜在的分子机制。【方法】体外培养RAW264.7巨噬细胞，以DMSO为对照，使用0.1~5.0 μmol/L GPX4抑制剂FIN56处理，通过CCK-8法检测细胞活力和Western blotting检测GPX4蛋白表达水平，确定抑制剂最适浓度。将RAW264.7巨噬细胞分为4组：对照组，添加DMSO培养24 h；FIN56（GPX4抑制剂）组，添加0.5 μmol/L FIN56培养24 h；DMSO-LPS组，DMSO培养24 h后使用LPS （100 ng/mL）刺激3 h；FIN56-LPS组，FIN56培养24 h后使用LPS刺激3 h。各组细胞经培养后，利用荧光探针2'，7'-二氯二氢荧光素二乙酸酯（2'，7'-dichlorodi-hydrofluorescein diacetate，H₂DCFDA）检测细胞内活性氧（reactive oxygen species，ROS）水平，使用试剂盒法检测细胞内丙二醛（malondialdehyde，MDA）水平，分别使用ELISA和荧光定量PCR检测促炎细胞因子白细胞介素1β（interleukin-1β，IL-1β）、白细胞介素6（interleukin-6，IL-6）、肿瘤坏死因子α（tumor necrosis factor-α，TNF-α）的分泌水平和基因表达量，使用Western blotting法检测Toll样受体4（toll like receptor 4，TLR4）信号通路相关蛋白表达水平。【结果】与DMSO处理相比，0.5 μmol/L FIN56处理巨噬细胞24 h对细胞活性无显著影响（P>0.05），且显著降低GPX4蛋白表达水平（P<0.05），故选用0.5 μmol/L FIN56用于后续实验。与对照组相比，FIN56和LPS均显著增加了ROS积累（P<0.05），而与DMSO-LPS组相比，FIN56-LPS组ROS水平显著升高（P<0.05）。与对照组相比，LPS可显著增加小鼠巨噬细胞IL-1β、IL-6和TNF-α的mRNA表达量和IL-6含量，以及c-Jun氨基末端蛋白激酶（c-Jun N-terminal protein kinase，JNK）和c-Jun磷酸化水平（P<0.05），而FIN56可显著下调LPS诱导的IL-6的mRNA表达量和含量及JNK和c-Jun磷酸化水平（P<0.05），但对p38蛋白（p38 MAPK，p38）、细胞外调节蛋白激酶（phosphorylate extracellular regulated protein kinases1/2，Erk1/2）蛋白表达水平无显著影响（P>0.05）。【结论】 GPX4失活可有效阻断JNK和c-Jun磷酸化，从而特异性抑制LPS诱导的巨噬细胞的炎症反应。该结果可为以GPX4为靶点研发抗炎治疗策略提供理论依据。

关键词: 谷胱甘肽过氧化物酶4(GPX4); RAW264.7; JNK信号通路; 炎症反应

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

中图分类号:

Q554

朱家威, 赵潇晗, 马翠, 汤超华, 司玮, 赵青余, 张军民, 秦玉昌. GPX4失活通过JNK通路缓解LPS诱导巨噬细胞炎症反应[J]. 中国畜牧兽医, 2022, 49(11): 4178-4186.

ZHU Jiawei, ZHAO Xiaohan, MA Cui, TANG Chaohua, SI Wei, ZHAO Qingyu, ZHANG Junmin, QIN Yuchang. GPX4 Inactivation Alleviates LPS-induced Macrophage Inflammatory Response via JNK Pathway[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(11): 4178-4186.

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GPX4失活通过JNK通路缓解LPS诱导巨噬细胞炎症反应

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

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