天名精根提取物调控TLRs信号抑制RAW264.7细胞炎症的研究
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摘要
[目的]阐明天名精根提取物对RAW264.7细胞炎症反应的影响及其机制。[方法]采集野生天名精全株,处理得天名精根粉末,制备天名精根乙醇和石油醚提取物,并以高效液相色谱法(high performance liquid chromatography,HPLC)检测其中主要成分;采用脂多糖(lipopolysaccharide,LPS)、灭活的金黄色葡萄球菌(heat-inactivated preparations of Staphylococcus aureus pathogens,SAC)刺激RAW264.7细胞,构建体外炎症模型。以MTT法检测细胞增殖,qPCR检测白细胞介素-1β(interleukin-1β,IL-1β)、IL-6、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、Toll样受体-2(Toll-like receptor-2,TLR-2)、TLR-4、髓样分化因子88(myeloid differentiation primary response 88,MyD88)、核转录因子-κB(nuclear factor-κB,NF-κB)m RNA表达,Western blot检测NF-κB p65蛋白表达。[结果]与正常对照组比较,LPS或SAC刺激后RAW264.7细胞IL-1β、IL-6、TNF-αmRNA表达增加(P<0.01),NF-κB蛋白与mRNA表达增加(P<0.01);LPS刺激后TLR-4、MyD88 m RNA表达增加(P<0.01,P<0.05),SAC刺激后TLR-2、MyD88 m RNA表达增加(P<0.01)。天名精根提取物干预后,与LPS组或SAC组比较,RAW264.7细胞IL-1β、IL-6、TNF-αmRNA表达减低(P<0.01),同时NF-κB m RNA和NF-κB p65蛋白表达也减低(P<0.01);TLR-4和MyD88 m RNA表达明显低于LPS组(P<0.01,P<0.05),TLR-2和MyD88 mRNA表达表达明显低于SAC组(P<0.01,P<0.05)。[结论]天名精根提取物可以抑制由LPS和灭活的金黄色葡萄球菌引起的RAW264.7细胞炎症反应,其机制可能与抑制TLRs-NF-κB信号通路有关。
[Objective] To elucidate the effect and mechanism of the root extract of Carpesium abrotanoides Linn.(CA) on the inflammatory response of RAW264.7 cells. [Methods] The effective components of CA were separated, and the constituents were detected by high performance liquid chromatography(HPLC).In vitro inflammatory model was constructed by using lipopolysaccharide(LPS) and heat-inactivated preparations of Staphylococcus aureus pathogens(SAC) to stimulate RAW264.7 cells. MTT assay was used for cell proliferation detection; m RNA expression of interleukin-1 β(IL-1β),IL-6, TNF-α, Toll-like receptor-2(TLR-2), TLR-4, myeloid differentiation primary response 88(MyD88), nuclear factor-κB(NF-κB) was detected by qPCR; protein expression of NF-κB p65 was detected by Western blot. [Results] Compared with normal control group, the expression of IL-1 β, IL-6,TNF-α mRNA and NF-κB protein and mRNA increased(P <0.01), after treated with LPS or SAC. After treated with LPS, the expression of TLR-4,MyD88 m RNA increased(P<0.01,P<0.05); and the expression of TLR-2, MyD88 mRNA increased after treated with SAC(P<0.01). Compared with LPS group or SAC group, the expression of IL-1β, IL-6, TNF-α mRNA and NF-κB protein and m RNA decreased after treated with the root extract of CA(P<0.01).The expression of TLR-4 and MyD88 mRNA were lower than LPS group(P<0.01,P<0.05), and the expression of TLR-2 and MyD88 m RNA were lower than SAC group(P<0.01,P<0.05). [Conclusion]The root extract of CA can inhibit the in vitro inflammatory response of RAW264.7 cells caused by LPS and SAC, which may be due to inhibition of TLRs-NF-κB signaling pathway.
[Objective] To elucidate the effect and mechanism of the root extract of Carpesium abrotanoides Linn.(CA) on the inflammatory response of RAW264.7 cells. [Methods] The effective components of CA were separated, and the constituents were detected by high performance liquid chromatography(HPLC).In vitro inflammatory model was constructed by using lipopolysaccharide(LPS) and heat-inactivated preparations of Staphylococcus aureus pathogens(SAC) to stimulate RAW264.7 cells. MTT assay was used for cell proliferation detection; m RNA expression of interleukin-1 β(IL-1β),IL-6, TNF-α, Toll-like receptor-2(TLR-2), TLR-4, myeloid differentiation primary response 88(MyD88), nuclear factor-κB(NF-κB) was detected by qPCR; protein expression of NF-κB p65 was detected by Western blot. [Results] Compared with normal control group, the expression of IL-1 β, IL-6,TNF-α mRNA and NF-κB protein and mRNA increased(P <0.01), after treated with LPS or SAC. After treated with LPS, the expression of TLR-4,MyD88 m RNA increased(P<0.01,P<0.05); and the expression of TLR-2, MyD88 mRNA increased after treated with SAC(P<0.01). Compared with LPS group or SAC group, the expression of IL-1β, IL-6, TNF-α mRNA and NF-κB protein and m RNA decreased after treated with the root extract of CA(P<0.01).The expression of TLR-4 and MyD88 mRNA were lower than LPS group(P<0.01,P<0.05), and the expression of TLR-2 and MyD88 m RNA were lower than SAC group(P<0.01,P<0.05). [Conclusion]The root extract of CA can inhibit the in vitro inflammatory response of RAW264.7 cells caused by LPS and SAC, which may be due to inhibition of TLRs-NF-κB signaling pathway.
引文
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[12]汪蕾,田丽,程凡,等.天名精萜类化学成分及其细胞毒活性研究[J].中草药,2018,49(3):530-535.WANG Lei,TIAN Li,CHENG Fan,et al.Terpenes from Carpesium abrotanoides and their cytotoxic activities[J].Chinese Traditional and Herbal Drugs,2018,49(3):530-535.
[13]沈冰冰,唐雪阳,陈林,等.天名精全草挥发油化学成分的SPME-GC/MS分析[J].中国现代中药,2017,19(7):965-971.SHEN Bingbing,TANG Xueyang,CHEN Lin,et al.Analysis of volatile constituents from aerial part of Carpesium abrotanoides by SPME-GC/MS[J].Mod Chin Med,2017,19(7):965-971.
[14]杨勇勋,张建平,王群,等.贵州天名精的化学成分研究[J].中草药,2017,48(15):3037-3041.YANG Yongxun,ZHANG Jianping,WANG Qun,et al.Study on chemical constituents from Carpesium faberi[J].Chinese Traditional and Herbal Drugs,2017,48(15):3037-3041.
[15]Hornung V,Rothenfusser S,Britsch S,et al.Quantitative expression of toll-like receptor 1-10 mRNA in cellular subsets of human peripheral blood mononuclear cells and sensitivity to CpG oligodeoxynucleotides[J].J Immunol,2002,168(9):4531-4537.
[16]Lu Y C,Yeh W C,Ohashi P S,et al.LPS/TLR4 signal transduction pathway[J].Cytokine,2008,42(2):145-151.
[17]Takeda K,Akira S.Toll-like receptors[J].Curr Protoc Immunol,2015,109:1-10.
[18]Kawai T,Akira S.The role of pattern-recognition receptors in innate immunity:update on Toll-like receptors[J].Nat Immunol,2010,11(5):373-384.
[19]Holden N S,Squires P E,Kaur M,et al.Phorbol esterstimulated NF-κB-dependent transcription:roles for isoforms of novel protein kinase C[J].Cell Signal,2008,20(7):1338-1348.
[20]Cao S,Zhang X,Edwards J P,et al.NF-kappaB1(p50)homodimers differentially regulate pro-and anti-inflammatory cytokines in macrophages[J].J Biol Chem,2006,281(36):26041-26050.
[21]Son Y H,Jeong Y T,Lee K A,et al.Roles of MAPKand NF-kappaB in interleukin-6 induction by lipopolysaccharide in vascular smooth muscle cells[J].J Cardiovasc Pharmacol,2008,51(1):71-77.
[22]Smale S T.Hierarchies of NF-κB target-gene regulation[J].Nat Immunol,2011,12(8):689-694.
[2]刘平安,刘敏,潘微薇,等.天名精化学成分研究[J].中药材,2014,37(12):2213-2215.LIU Ping’an,LIU Min,PAN Weiwei,et al.Study on chemical constituents from Carpesium abrotanoides[J].Journal of Chinese Medicinal Materials,2014,37(12):2213-2215.
[3]Lee E K,Jeong D W,Lim S J.Carpesium abrotanoides,extract inhibits inducible nitric oxide synthase expression induced by toll-like receptor agonists[J].Food Sci Biotechnol,2014,23(5):1637-1641.
[4]Zhong Y,Chiou Y S,Pan M H,et al.Anti-inflammatory activity of lipophilic epigallocatechin gallate(EGCG)derivatives in LPS-stimulated murine macrophages[J].Food Chem,2012,134(2):742-748.
[5]Kwon O K,Lee M Y,Yuk J E,et al.Anti-inflammatory effects of methanol extracts of the root of Lilium Lancifolium on LPS-stimulated Raw264.7 cells[J].Ethnopharmacol,2010,130(1):28-34.
[6]Fairweather D,Rose N R.Inflammatory heart disease:a role for cytokines[J].Lupus,2005,14(9):646-651.
[7]Kretschmer D,Hanzelmann D,Peschel A,et al.Lipoprotein immunoproteomics question the potential of Staphylococcus aureus TLR2 agonists as vaccine antigens[J].Proteomics,2016,16(20):2603-2604.
[8]Wang F,Yang K,Ren F C,et al.Sesquiterpene lactones from Carpesium abrotanoides[J].Fitoterapia,2009,80(1):21-24.
[9]Lee J,Hwang K H,Kim G H,et al.In vitro evaluation of anti-inflammatory activity for salad-food material Carpesium abrotanoides[J].J Food Biochem,2013,37(1):18-25.
[10]Li X W,Weng L,Gao X,et al.Antiproliferative and apoptotic sesquiterpene lactones from Carpesium faberi[J].Bioorg Med Chem Lett,2011,21(1):366-372.
[11]Ko Y E,Oh S R,Song H H,et al.The effect of 4α,5α-epoxy-10α,14-dihydro-inuviscolide,a novel immunosuppressant isolated from Carpesium abrotanoides,on the cytokine profile in vitro and in vivo[J].Int Immunopharmacol,2015,25(1):121-129.
[12]汪蕾,田丽,程凡,等.天名精萜类化学成分及其细胞毒活性研究[J].中草药,2018,49(3):530-535.WANG Lei,TIAN Li,CHENG Fan,et al.Terpenes from Carpesium abrotanoides and their cytotoxic activities[J].Chinese Traditional and Herbal Drugs,2018,49(3):530-535.
[13]沈冰冰,唐雪阳,陈林,等.天名精全草挥发油化学成分的SPME-GC/MS分析[J].中国现代中药,2017,19(7):965-971.SHEN Bingbing,TANG Xueyang,CHEN Lin,et al.Analysis of volatile constituents from aerial part of Carpesium abrotanoides by SPME-GC/MS[J].Mod Chin Med,2017,19(7):965-971.
[14]杨勇勋,张建平,王群,等.贵州天名精的化学成分研究[J].中草药,2017,48(15):3037-3041.YANG Yongxun,ZHANG Jianping,WANG Qun,et al.Study on chemical constituents from Carpesium faberi[J].Chinese Traditional and Herbal Drugs,2017,48(15):3037-3041.
[15]Hornung V,Rothenfusser S,Britsch S,et al.Quantitative expression of toll-like receptor 1-10 mRNA in cellular subsets of human peripheral blood mononuclear cells and sensitivity to CpG oligodeoxynucleotides[J].J Immunol,2002,168(9):4531-4537.
[16]Lu Y C,Yeh W C,Ohashi P S,et al.LPS/TLR4 signal transduction pathway[J].Cytokine,2008,42(2):145-151.
[17]Takeda K,Akira S.Toll-like receptors[J].Curr Protoc Immunol,2015,109:1-10.
[18]Kawai T,Akira S.The role of pattern-recognition receptors in innate immunity:update on Toll-like receptors[J].Nat Immunol,2010,11(5):373-384.
[19]Holden N S,Squires P E,Kaur M,et al.Phorbol esterstimulated NF-κB-dependent transcription:roles for isoforms of novel protein kinase C[J].Cell Signal,2008,20(7):1338-1348.
[20]Cao S,Zhang X,Edwards J P,et al.NF-kappaB1(p50)homodimers differentially regulate pro-and anti-inflammatory cytokines in macrophages[J].J Biol Chem,2006,281(36):26041-26050.
[21]Son Y H,Jeong Y T,Lee K A,et al.Roles of MAPKand NF-kappaB in interleukin-6 induction by lipopolysaccharide in vascular smooth muscle cells[J].J Cardiovasc Pharmacol,2008,51(1):71-77.
[22]Smale S T.Hierarchies of NF-κB target-gene regulation[J].Nat Immunol,2011,12(8):689-694.