高白鲑鱼油通过抑制核因子κB、信号传导转录激活因子1和琥珀酸盐_缺氧诱导因子-1α信号通路的抗炎机制.pdf
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1、营养卫生 食品科学 2023,Vol.44,No.17 67Anti-inflammatory Mechanism of Coregonus peled Oil via the Inhibition of Nuclear Factor Kappa-B(NF-B),Signal Transducer and Activator of Transcription 1(STAT1),and Succinate/Hypoxia-Inducible Factor-1(HIF-1)Signaling PathwaysLIU Hongxu,SONG Guoku,WANG Zhen,XIA Xiaodong,
2、QIN Ningbo*(College of Food Science and Technology,Dalian Polytechnic University,Dalian 116034,China)Abstract:Objective:To investigate the chemical composition and anti-inflammatory mechanism of Coregonus peled oil(CPO).Methods:CPO was extracted by ultrasonic homogenization and its major chemical co
3、mponents were detected.In addition,its fatty acid(FA)composition was analyzed by gas chromatography-mass spectrometry(GC-MS).Macrophage RAW264.7 cells were cultured as an inflammation model and stimulated with lipopolysaccharides(LPS).Cytotoxicity was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,
4、5-diphenyltetrazolium bromide(MTT)assay.The accumulation of succinate was detected by liquid chromatography tandem mass spectrometry(LC-MS/MS).The concentration of nitric oxide(NO)and the activity of inducible nitric oxide synthase(iNOS)in the supernatant were measured by commercial kits.Enzyme-link
5、ed immunosorbent assay(ELISA)was used to determine the levels of interleukin-6(IL-6),interleukin-1(IL-1)and tumor necrosis factor-(TNF-)in the supernatant.The expression levels of CD68,CD86,Arg1,CD206,YM1 and HIF-1 were detected by quantitative real-time polymerase chain reaction(qPCR),and their pro
6、tein expression levels were determined by Western blotting.Results:The major chemical constituents of CPO were palmitoleic acid,oleic acid,eicosapntemacnioc acid(EPA),and docosahexaenoic acid(DHA).CPO significantly inhibited the production of NO,iNOS,IL-6,TNF-,and IL-1 as well as their gene expressi
7、on levels.CPO blocked NF-B activation by inhibiting the degradation of inhibitor of NF-B(IB)and decreasing p65 nuclear translocation in LPS-induced RAW264.7 cells.In addition,CPO promoted macrophage polarization from M1 to M2 types by inhibiting the phosphorylation of signal transducer and activator
8、 of transcription 1(STAT1).The content of succinate and the expression levels of hypoxia-inducible factor-1(HIF-1)gene and protein indicated that CPO could inhibit the production of IL-1 by inhibiting the accumulation of succinate.Conclusion:The anti-inflammatory mechanism of CPO may be due to the i
9、nhibition of the NF-B,STAT1,and succinate/HIF-1 signaling pathways.Keywords:Coregonus peled;fish oil;inflammation;polarization;succinate/hypoxia-inducible factor-1高白鲑鱼油通过抑制核因子B、信号传导转录激活因子1和琥珀酸盐/缺氧诱导因子-1信号通路的抗炎机制刘宏旭,宋国库,王 珍,夏效东,秦宁波*(大连工业大学食品学院,辽宁 大连 116034)摘 要:目的:探讨高白鲑鱼油的化学成分和抗炎作用机制。方法:采用匀浆萃取超声法从高白鲑中
10、提取鱼油,并对其主要化学成分进行检测。采用气相色谱-质谱联用仪对高白鲑鱼油进行脂肪酸成分分析。构建巨噬细胞RAW264.7炎症细胞模型,用脂多糖(lipopolysaccharide,LPS)刺激后,通过MTT法检测细胞毒性,采用液相色谱串联质谱联用仪对琥珀酸盐含量进行测定;采用试剂盒法测定上清液中一氧化氮(nitric oxide,NO)含量 收稿日期:2022-09-28基金项目:国家自然科学基金青年科学基金项目(32101963);辽宁省教育厅高校基本科研项目(面上项目)(LJKMZ20220890)第一作者简介:刘宏旭(1998)(ORCID:0000-0001-8377-9520),
11、女,硕士研究生,研究方向为食品微生物与健康。E-mail:*通信作者简介:秦宁波(1987)(ORCID:0000-0003-3384-064X),男,副教授,博士,研究方向为食品微生物与健康。E-mail:68 2023,Vol.44,No.17 食品科学 营养卫生和一氧化氮合酶(inducible nitric oxide synthase,iNOS)活力;采用酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)测定细胞上清液中白细胞介素-6(interleukin-6,IL-6)、白细胞介素-1(interleukin-1,IL-1)和肿瘤坏
12、死因子-(tumor necrosis factor-,TNF-)质量浓度。采用实时荧光定量聚合酶链式反应法测定细胞上清液中CD68、CD86、Arg1、CD206、YM1、HIF-1基因的相对表达量,并通过蛋白免疫印迹法测定其蛋白的相对表达量。结果表明:高白鲑鱼油的主要化学成分为棕榈油酸、油酸、二十碳五烯酸和二十二碳六烯酸。其可以显著抑制NO、iNOS、IL-6、TNF-、IL-1的产生及相关基因的表达。鱼油可通过抑制LPS诱导RAW264.7细胞中的核因子-B(nuclear factor kappa-B,NF-B)抑制剂的降解和减少p65核易位来抑制NF-B的激活。此外,高白鲑鱼油可通过
13、抑制信号传导转录激活因子1(signal transducer and activator of transcription 1,STAT1)的磷酸化,将巨噬细胞从M1型极化为M2型。另外,通过测定琥珀酸盐的含量、缺氧诱导因子-1(hypoxia-inducible factor-1,HIF-1)基因和蛋白的表达量发现,高白鲑鱼油可以通过抑制琥珀酸盐的积累来抑制IL-1的产生。结论:高白鲑鱼油可能通过抑制NF-B、STAT1和琥珀酸盐/HIF-1信号通路抑制炎症。关键词:高白鲑;鱼油;炎症;极化;琥珀酸盐/缺氧诱导因子-1DOI:10.7506/spkx1002-6630-20220928-3
14、07中图分类号:TS201.4 文献标志码:A 文章编号:1002-6630(2023)17-0067-12引文格式:LIU Hongxu,SONG Guoku,WANG Zhen,et al.Anti-inflammatory mechanism of Coregonus peled oil via the inhibition of nuclear factor kappa-B(NF-B),signal transducer and activator of transcription 1(STAT1),and succinate/hypoxia-inducible factor-1(HI
15、F-1)signaling pathwaysJ.食品科学,2023,44(17):67-78.DOI:10.7506/spkx1002-6630-20220928-307.http:/LIU Hongxu,SONG Guoku,WANG Zhen,et al.Anti-inflammatory mechanism of Coregonus peled oil via the inhibition of nuclear factor kappa-B(NF-B),signal transducer and activator of transcription 1(STAT1),and succin
16、ate/hypoxia-inducible factor-1(HIF-1)signaling pathwaysJ.Food Science,2023,44(17):67-78.(in English with Chinese abstract)DOI:10.7506/spkx1002-6630-20220928-307.http:/During inflammatory processes,macrophages play an important role in inflammatory and immune responses by acting as an internal defens
17、e against microbial infections1-3.Macrophages are a diverse group of immune cells that initiate an innate immune response through phagocytosis and cytokine release.Despite the fact that macrophage phenotypes in our bodies are plastic and adaptable,they can be simply divided into two extremes:classic
18、ally activated macrophages(M1)associated with pro-inflammatory responses and alternatively activated macrophages(M2)associated with anti-inflammatory responses4.M1 macrophages contribute to the pro-inflammatory process by activating the signal transducer and activator of transcription 1(STAT1)and nu
19、clear factor kappa-B(NF-B)signaling pathways,as well as regulating the expression of cytokines5-6.M2 macrophages,on the other hand,play an important role in the resolution of inflammation by activating STAT3 and STAT6 and regulating the expression of interleukin-10(IL-10),interleukin-4(IL-4)and mann
20、ose receptor(CD206)7-8.Once activated,macrophages could rapidly respond by producing cytokines and chemokines to clear the foreign material,indicating that dramatic changes in cell metabolism influence macrophage phenotypic and functional changes.When macrophages are stimulated,oxidative phosphoryla
21、tion(OXPHOS)decreases and glycolysis and pentose-phosphate pathways increase.The rapid increase in glycolysis is linked to macrophage inflammatory phenotypes9-10.Numerous studies have revealed that M1 macrophages rely on glycolysis to produce adenosine triphosphate(ATP)and contribute to the accumula
22、tion of tricarboxylic acid(TCA)cycle metabolites.M2 macrophages,on the other hand,rely primarily on OXPHOS and the TCA cycle is intact11-12.Succinate,as a TCA cycle intermediate,is essential for ATP generation in mitochondria13-14.Succinates new role as a metabolic signal in inflammation has recentl
23、y emerged13.Succinate,accumulated in activated macrophages,has been shown to stimulate the production of pro-inflammatory cytokines and stabilize the transcription factor hypoxia-inducible factor-1(HIF-1)during inflammation10-15.Furthermore,glycolysis in inflammatory macrophages is strongly linked t
24、o HIF-1.As a result,succinate has spreaded beyond metabolism into inflammatory signaling.营养卫生 食品科学 2023,Vol.44,No.17 69Natural products have gained popularity as anti-inflammatory agents with few side effects16-18.Previous researches indicated fish oil is well known for their anti-inflammatory prope
25、rties in sever chronic diseases19-21.Coregonus peled is a freshwater species from cold regimes that belongs to the Salmonidae family and is widespread in Europe and Asia.C.peled is currently cultured in Sayram Lake(Xinjiang,China)and primarily processed into fillets for market sales in China.The fis
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