洋葱槲皮素对巨噬细胞HO-1表达影响及相关信号通路的研究
|
摘要
目的洋葱槲皮素具有抗炎作用,但具体机制尚不明确。本研究主要探讨其对RAW 264.7巨噬细胞HO-1表达影响及相关信号通路的作用。方法采用Griess法检测NO水平;用Western blot法检测HO-1、Nrf2、Keap1的表达水平;免疫共沉淀法检测Nrf2和Keap1相互作用;用双荧光素酶报告检测考察Nrf2与ARE的结合水平。结果 Western blot结果显示洋葱槲皮素能显著提高LPS诱导的HO-1表达水平,增加Nrf2的表达水平,且降低Keap1的表达水平。免疫共沉淀法结果表明,无论是否存在LPS诱导,洋葱槲皮素均可显著降低Keap1/Nrf2比值。双荧光素酶报告实验的结果表明,洋葱槲皮素能增强ARE介导的荧光素酶活性。SnPP实验结果表明,SnPP能减弱洋葱槲皮素对NO、iNOS和NF-κB的抑制作用。结论洋葱槲皮素可能通过诱导HO-1的表达产生抗炎作用,这种诱导作用可能与Keap1/Nrf2/ARE信号通路有关。
Onion quercetin has anti-inflammatory effect, but its mechanism is still unclear. This study was to investigate its effect on HO-1 expression in RAW 264.7 macrophages and its related pathway. The levels of HO-1,Nrf2, and Keap1 were detected by Western blot. The interaction between Nrf2 and Keap1 was detected by coimmunoprecipitation. The binding level of Nrf2 and ARE was detected by double luciferase report. Western blot showed that onion quercetin could significantly increase the expression level of HO-1 and Nrf2 induced by LPS, and decrease the level of Keap1. The results of co-immunoprecipitation showed that onion quercetin could significantly reduce the Keap1/Nrf2 ratio regardless of the LPS induction. The results of double luciferase report test showed that onion quercetin could enhance the ARE-mediated luciferase activities. The results of SnPP test showed that SnPP could attenuate the inhibitory effects of onion quercetin on NO, iNOS and NF-κB. The anti-inflammatory effect of onion quercetin could be attributed to the induction of the HO-1 expression. This induction may be related to Keap1/Nrf2/ARE pathway.
Onion quercetin has anti-inflammatory effect, but its mechanism is still unclear. This study was to investigate its effect on HO-1 expression in RAW 264.7 macrophages and its related pathway. The levels of HO-1,Nrf2, and Keap1 were detected by Western blot. The interaction between Nrf2 and Keap1 was detected by coimmunoprecipitation. The binding level of Nrf2 and ARE was detected by double luciferase report. Western blot showed that onion quercetin could significantly increase the expression level of HO-1 and Nrf2 induced by LPS, and decrease the level of Keap1. The results of co-immunoprecipitation showed that onion quercetin could significantly reduce the Keap1/Nrf2 ratio regardless of the LPS induction. The results of double luciferase report test showed that onion quercetin could enhance the ARE-mediated luciferase activities. The results of SnPP test showed that SnPP could attenuate the inhibitory effects of onion quercetin on NO, iNOS and NF-κB. The anti-inflammatory effect of onion quercetin could be attributed to the induction of the HO-1 expression. This induction may be related to Keap1/Nrf2/ARE pathway.
引文
[1]Kuprash DV,Nedospasov SA.Molecular and cellular mechanisms of inflammation[J].Biochemistry,2016,81(11):1237-1239.
[2]Aktan F.iNOS-mediated nitric oxide production and its regulation[J].Life Sciences,2004,75(6):639-653.
[3]李俊,冯丽洁,刘一平.有氧运动对2型糖尿病大鼠血管炎症及Nrf2/ARE信号通路的影响[J].免疫学杂志,2019,35(2):93-98.
[4]Gozzelino R,Jeney V,Soares MP.Mechanisms of cell protection by heme oxygenase-1[J].Annu Rev Pharmacol Toxicol,2010,50(1):323-354.
[5]Poss KD,Tonegawa S.Reduced stress defense in heme oxygenase 1-deficient cells[J].Proc Nat Acad Sci U S A,1997,94(20):10925-10930.
[6]Ryter SW,Choi AMK.Targeting heme oxygenase-1 and carbon monoxide for therapeutic modulation of inflammation[J].Transl Res,2016,167(1):7.
[7]Sharma JN,Al Omran A,Parvathy SS.Role of nitric oxide in inflammatory diseases[J].Inflammopharmacology,2007,15(6):252-259.
[8]Yong W,Liang Z,Wang C,et al.Protective effect of quercetin and chlorogenic acid,two polyphenols widely present in edible plant varieties,on visible light-induced retinal degeneration in vivo[J].J Funct Foods,2017,33:103-111.
[9]Nile SH,Nile AS,Keum YS,et al.Utilization of quercetin and quercetin glycosides from onion(Allium cepa L.)solid waste as an antioxidant,urease and xanthine oxidase inhibitors[J].Food Chem,2017,235:119-126.
[10]倪湾,李敬双,于洋.洋葱槲皮素对脂多糖诱导的小鼠腹腔巨噬细胞炎症反应抑制作用[J].食品工业科技,2017,38(23):284-288.
[11]Tsikas D.Analysis of nitrite and nitrate in biological fluids by assays based on the Griess reaction:Appraisal of the Griess reaction in the l-arginine/nitric oxide area of research[J].J Chromatogr B Analyt Technol Biomed Life Sci,2007,851(1):51-70.
[12]Alcaraz MJ,Fernández P,Guillén MI.Anti-inflammatory actions of the heme oxygenase-1 pathway[J].Curr Pharm Des,2003,9(30):2541-2551.
[13]Pahl HL.Activators and target genes of Rel/NF-κBtranscription factors[J].Oncogene,1999,18(49):6853-6866.
[14]Tak PP,Firestein GS.NF-kappaB:a key role in inflammatory diseases[J].J Clin Invest,2001,107(1):7-11.
[15]Oh GS,Pae HO,Lee BS,et al.Hydrogen sulfide inhibits nitric oxide production and nuclear factor-κB via heme oxygenase-1 expression in RAW264.7 macrophages stimulated with lipopolysaccharide[J].Free Radical Biol Med,2006,41(1):106-119.
[16]Yu MH,Sun AH,Kim YM,et al.β-Adrenergic receptormediated HO-1 induction,via PI3K and p38 MAPK,by isoproterenol in RAW 264.7 cells leads to inhibition of HMGB1 release in LPS-activated RAW 264.7 cells and increases in survival rate of CLP-induced septic mice[J].Biochem Pharmacol,2011,82(7):769-777.
[17]Kiichi N,Pyo KH,Hui GX,et al.Carbon monoxide differentially inhibits TLR signaling pathways by regulating ROS-induced trafficking of TLRs to lipid rafts[J].J Exp Med,2006,203(10):2377-2389.
[18]Wang X,Kim HK,Ryter S,et al.The heme oxygenase-1/carbon monoxide pathway suppresses TLR4 signaling by regulating the interaction of TLR4 with caveolin-1[J].JImmunol,2009,182(6):3809-3818.
[19]Ananta P,Britta EV,Rainer B,et al.Signaling to heme oxygenase-1 and its anti-inflammatory therapeutic potential[J].Biochem Pharmacol,2010,80(12):1895-1903.
[20]Ma Q.Role of nrf2 in oxidative stress and toxicity[J].Annu Rev Pharmacol Toxicol,2013,53(1):401.
[21]胡流芳,王迎,任汝静,等.Keap1-Nrf2/ARE信号通路的抗氧化应激作用及其调控机制[J].国际药学研究杂志,2016,43(1):146-152.
[22]段家翔,甯交琳,鲁开智.Nrf2出核转运机制及意义的研究进展[J].医学研究生学报,2014,27(8):874-877.
[23]Makoto K,Li L,Noriko I,et al.The antioxidant defense system Keap1-Nrf2 comprises a multiple sensing mechanism for responding to a wide range of chemical compounds[J].Mol Cell Biol,2009,29(2):493-502.
[24]Niture SK,Raju K,Jaiswal AK.Regulation of Nrf2-an update[J].Free Radical Biol Med,2014,66(1):36-44.
[25]刘宇彤,车楠,李莉.花青素通过Nrf-2/HO-1信号通路调控哮喘气道炎症[J].免疫学杂志,2019,35(1):36-41.
[2]Aktan F.iNOS-mediated nitric oxide production and its regulation[J].Life Sciences,2004,75(6):639-653.
[3]李俊,冯丽洁,刘一平.有氧运动对2型糖尿病大鼠血管炎症及Nrf2/ARE信号通路的影响[J].免疫学杂志,2019,35(2):93-98.
[4]Gozzelino R,Jeney V,Soares MP.Mechanisms of cell protection by heme oxygenase-1[J].Annu Rev Pharmacol Toxicol,2010,50(1):323-354.
[5]Poss KD,Tonegawa S.Reduced stress defense in heme oxygenase 1-deficient cells[J].Proc Nat Acad Sci U S A,1997,94(20):10925-10930.
[6]Ryter SW,Choi AMK.Targeting heme oxygenase-1 and carbon monoxide for therapeutic modulation of inflammation[J].Transl Res,2016,167(1):7.
[7]Sharma JN,Al Omran A,Parvathy SS.Role of nitric oxide in inflammatory diseases[J].Inflammopharmacology,2007,15(6):252-259.
[8]Yong W,Liang Z,Wang C,et al.Protective effect of quercetin and chlorogenic acid,two polyphenols widely present in edible plant varieties,on visible light-induced retinal degeneration in vivo[J].J Funct Foods,2017,33:103-111.
[9]Nile SH,Nile AS,Keum YS,et al.Utilization of quercetin and quercetin glycosides from onion(Allium cepa L.)solid waste as an antioxidant,urease and xanthine oxidase inhibitors[J].Food Chem,2017,235:119-126.
[10]倪湾,李敬双,于洋.洋葱槲皮素对脂多糖诱导的小鼠腹腔巨噬细胞炎症反应抑制作用[J].食品工业科技,2017,38(23):284-288.
[11]Tsikas D.Analysis of nitrite and nitrate in biological fluids by assays based on the Griess reaction:Appraisal of the Griess reaction in the l-arginine/nitric oxide area of research[J].J Chromatogr B Analyt Technol Biomed Life Sci,2007,851(1):51-70.
[12]Alcaraz MJ,Fernández P,Guillén MI.Anti-inflammatory actions of the heme oxygenase-1 pathway[J].Curr Pharm Des,2003,9(30):2541-2551.
[13]Pahl HL.Activators and target genes of Rel/NF-κBtranscription factors[J].Oncogene,1999,18(49):6853-6866.
[14]Tak PP,Firestein GS.NF-kappaB:a key role in inflammatory diseases[J].J Clin Invest,2001,107(1):7-11.
[15]Oh GS,Pae HO,Lee BS,et al.Hydrogen sulfide inhibits nitric oxide production and nuclear factor-κB via heme oxygenase-1 expression in RAW264.7 macrophages stimulated with lipopolysaccharide[J].Free Radical Biol Med,2006,41(1):106-119.
[16]Yu MH,Sun AH,Kim YM,et al.β-Adrenergic receptormediated HO-1 induction,via PI3K and p38 MAPK,by isoproterenol in RAW 264.7 cells leads to inhibition of HMGB1 release in LPS-activated RAW 264.7 cells and increases in survival rate of CLP-induced septic mice[J].Biochem Pharmacol,2011,82(7):769-777.
[17]Kiichi N,Pyo KH,Hui GX,et al.Carbon monoxide differentially inhibits TLR signaling pathways by regulating ROS-induced trafficking of TLRs to lipid rafts[J].J Exp Med,2006,203(10):2377-2389.
[18]Wang X,Kim HK,Ryter S,et al.The heme oxygenase-1/carbon monoxide pathway suppresses TLR4 signaling by regulating the interaction of TLR4 with caveolin-1[J].JImmunol,2009,182(6):3809-3818.
[19]Ananta P,Britta EV,Rainer B,et al.Signaling to heme oxygenase-1 and its anti-inflammatory therapeutic potential[J].Biochem Pharmacol,2010,80(12):1895-1903.
[20]Ma Q.Role of nrf2 in oxidative stress and toxicity[J].Annu Rev Pharmacol Toxicol,2013,53(1):401.
[21]胡流芳,王迎,任汝静,等.Keap1-Nrf2/ARE信号通路的抗氧化应激作用及其调控机制[J].国际药学研究杂志,2016,43(1):146-152.
[22]段家翔,甯交琳,鲁开智.Nrf2出核转运机制及意义的研究进展[J].医学研究生学报,2014,27(8):874-877.
[23]Makoto K,Li L,Noriko I,et al.The antioxidant defense system Keap1-Nrf2 comprises a multiple sensing mechanism for responding to a wide range of chemical compounds[J].Mol Cell Biol,2009,29(2):493-502.
[24]Niture SK,Raju K,Jaiswal AK.Regulation of Nrf2-an update[J].Free Radical Biol Med,2014,66(1):36-44.
[25]刘宇彤,车楠,李莉.花青素通过Nrf-2/HO-1信号通路调控哮喘气道炎症[J].免疫学杂志,2019,35(1):36-41.