化合物W3D通过调控TLR4-MyD88-NF-κB通路抑制LPS诱导的RAW264.7细胞炎症因子的释放
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摘要
目的建立脂多糖(LPS)诱导的小鼠单核巨噬细胞(RAW264.7)炎症模型,探究新型苯并噁唑酮衍生物4-(5'-二甲氨基)-萘磺酰氧基苯并噁唑酮(W3D)的抗炎活性及其对TLR4-MyD88-NF-κB通路的调控作用。方法 MTT法测定化合物W3D对细胞活力的影响;LPS与不同浓度的化合物W3D共同作用RAW264.7细胞后,ELISA法测定细胞上清液中TNF-α、IL-6、IL-1β、COX-2的含量,Western blot法检测IL-6、TLR4、MyD88、IRAK4、NF-κB的蛋白表达;实时荧光定量PCR法检测细胞中TLR4、MyD88、IL-6 mRNA的表达。结果化合物W3D对LPS诱导的RAW264.7细胞培养液中炎症因子TNF-α、IL-6、IL-1β的分泌有明显的抑制作用,但对COX-2无抑制活性;可明显下调TLR4、MyD88、IL-6的蛋白与mRNA的表达,抑制IRAK4磷酸化和NF-κB的入核活化。结论化合物W3D可通过调控TLR4-MyD88-IRAK4-NF-κB信号通路,抑制TNF-α、IL-6、IL-1β等炎症因子的释放而发挥抗炎活性。
Aim To investigate the effect of the novel benzoxazolone derivative 4-( 5'-dimethylamino)-naphthalenesulfonyl-2( 3H)-benzoxazolone( W3D) on TLR4-MyD88-NF-κB signaling pathway in LPS-induced RAW264.7 cells.Methods The cell viability was detected by MTT assay,and the contents of TNF-α,IL-6,IL-1β and COX-2 in the cell supernatant were analyzed using ELISA methods.The protein expression of IL-6,TLR4,MyD88,p-IRAK4 and NF-κB were investigated by western blot analysis, and the mRNA expressions of TLR4,MyD88 and IL-6 were analyzed by RT-PCR.Results W3D could obviously inhibit the production of TNF-α,IL-6 and IL-1β in LPSinduced RAW264.7 cell supernatant,but it had no effect on the release of COX-2.Compared with the model group,the expressions of TLR4,MyD88 and IL-6 were decreased significantly in a dose dependent manner.Meanwhile,the expressions of p-IRAK4 and nucleus of NF-κB were decrease in W3D treated group compared with the model group.Conclusion The novel compound W3D could inhibit the release of the inflammatory mediators through the regulation of TLR4-MyD88-NF-κB signaling pathway.
Aim To investigate the effect of the novel benzoxazolone derivative 4-( 5'-dimethylamino)-naphthalenesulfonyl-2( 3H)-benzoxazolone( W3D) on TLR4-MyD88-NF-κB signaling pathway in LPS-induced RAW264.7 cells.Methods The cell viability was detected by MTT assay,and the contents of TNF-α,IL-6,IL-1β and COX-2 in the cell supernatant were analyzed using ELISA methods.The protein expression of IL-6,TLR4,MyD88,p-IRAK4 and NF-κB were investigated by western blot analysis, and the mRNA expressions of TLR4,MyD88 and IL-6 were analyzed by RT-PCR.Results W3D could obviously inhibit the production of TNF-α,IL-6 and IL-1β in LPSinduced RAW264.7 cell supernatant,but it had no effect on the release of COX-2.Compared with the model group,the expressions of TLR4,MyD88 and IL-6 were decreased significantly in a dose dependent manner.Meanwhile,the expressions of p-IRAK4 and nucleus of NF-κB were decrease in W3D treated group compared with the model group.Conclusion The novel compound W3D could inhibit the release of the inflammatory mediators through the regulation of TLR4-MyD88-NF-κB signaling pathway.
引文
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[11]陈焕琦,张子恒,周磊,等.OSAHS患者围手术期应用西吡氯铵含片的疗效研究[J].临床耳鼻咽喉头颈外科杂志,2017,31(14):1123-6.[11]Chen H Q,Zhang Z H,Zhou L,et al.Effect of cetylpyridinium chloride Buccal tablets on perioperative application of OSAHS patients[J].J Clin Otorhinolarynglolgy Head Neck Surg,2017,31(14):1123-6.
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[14]Woo H J,Lee J Y,Park H S,et al.Anti-inflammatory action of2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone(CMEP-NQ)suppresses both the MyD88-dependent and TRIF-dependent pathways of TLR4 signaling in LPS-stimulated RAW264.7 cells[J].J Ethnopharmacol,2017,205:103-15.
[15]雷升萍,王靓,龙子江,等.黄精多糖通过TLR4-MyD88-NF-κB通路抑制缺氧/复氧H9c2心肌细胞炎性因子释放[J].中国药理学通报,2017,33(2),255-60.[15]Lei S P,Wang L,Long Z J,et al.Inhibitory effect of polygonatum sibiricum polysaccharides release of inflammatory cytokines of anoxia/reoxygenation H9c2 myocardial cells through TLR4-MyD88-NFκB signaling pathway[J].Chin Pharmacol Bull,2017,33(2),255-60.
[2]Qin J J,Wang W,Zhang R W.Novel natural product therapeutics targeting both inflammation and cancer[J].Chin J Nat Med,2017,15(6):401-16.
[3]于水莲,陶怡,王羿升,等.炎症因素与早期类风湿关节炎心血管病变相关性研究[J].中华风湿病学杂志,2016,20(11):739-45.[3]Yu S L,Tao Y,Wang Y S,et al.The correlation between inflammatory burden with cardiovascular diseases in patients with early rheumatoid arthritis[J].Chin J Rheumatol,2016,20(11):739-45.
[4]Kim Y,Ahn C,Je J.Anti-inflammatory action of high molecular weight Mytilus edulis hydrolysates fraction in LPS-induced RAW264.7 macrophage via NF-κB and MAPK pathways[J].Food Chem,2016,202:9-14.
[5]A Martelli,L Testai,M Anzini,et al.The novel a-inflammatory agent VA694,endowed with both NO-releasing and COX2-selective inhibiting properties,exhibits NO-mediated positive effects on blood pressure,coronary flow and endothelium in an experimental model of hypertension and endothelial dysfunction[J].Pharmacol Res,2013,78:1-9.
[6]Elsaman T,Aldeeb O A A,Aboul-Fadl T,et al.Synthesis,characterization and pharmacological evaluation of certain enzymatically cleavable NSAIDs amide prodrugs[J].Bioorg Chem,2017,70:144-52.
[7]Deng B L,Cullen M D,Zhou Z G,et al.Synthesis and anti-HIV activity of new alkenyldiaryl-methane(ADAM)non-nucleoside reverse transcriptase inhibitors(NNRTIs)incorporating benzoxazolone and benzisoxazole rings[J].Bioorg Med Chem,2006,14(7):2366-74.
[8]Tang L,Ma W,Ma Y,et al.Synthesis and biological activity of4-substituted benzoxazolone derivatives as a new class of s EH inhibitors with high anti-inflammatory activity in vivo[J].Bioorg Med Chem Lett,2013,23(8):2380-3.
[9]唐莉,班树荣,林文瀚,等.天然产物4-O-B-D-葡萄糖-苯并噁唑酮及4-取代苯并噁唑酮类衍生物的合成与其活性研究[J].中国药物化学杂志,2009,19(1):31-5.[9]Tang L,Ban S R,Lin W H,et al.Synthesis and activities of natural product 4-O-β-D-glucopyranosyl-benzoxazolin-2(3H)-one and derivatives of 4-substituted benzoxazolone[J].Chin J Med Chem,2009,19(1):31-5.
[10]罗捷然,唐莉,葛睿,等,新型苯并噁唑酮类衍生物4-邻甲基苯磺酰氧基-苯并噁唑酮(MBB)抗炎作用的研究[A].中国药学会第十三届青年药学科研成果交流会论文集[C].2016:5.[10]Luo J R,Tang L,Ge R,et al.Study on anti-inflammatory effect of 4-(2-methylphenylsulfonyloxy)-2(3H)-benzoxazolone(MBB)[A].Conference proceedings of the thirteenth session of National youth pharmaceutical scientific research achievement exchange of China pharmaceutical association[C].2016:5.
[11]陈焕琦,张子恒,周磊,等.OSAHS患者围手术期应用西吡氯铵含片的疗效研究[J].临床耳鼻咽喉头颈外科杂志,2017,31(14):1123-6.[11]Chen H Q,Zhang Z H,Zhou L,et al.Effect of cetylpyridinium chloride Buccal tablets on perioperative application of OSAHS patients[J].J Clin Otorhinolarynglolgy Head Neck Surg,2017,31(14):1123-6.
[12]Suzuki A,Hanada T,Mitsuyama K,et al.CIS3/SOCS3/SSI3plays a negative regulatory role in STAT3 activation and intestinal inflammation[J].J Exp Med,2001,193(4):471-81.
[13]李德川,鲍秀琦,张德武,等.去氢丹参新酮对神经炎症的抑制作用及机制研究[J].中国药理学通报,2016,32(2):177-83.[13]Li D C,Bao X Q,Zhang D W,et al.The anti-neuroinflammatory effects of dehydromiltirone and related mechanisms[J].Chin Pharmacol Bull,2016,32(2):177-83.
[14]Woo H J,Lee J Y,Park H S,et al.Anti-inflammatory action of2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone(CMEP-NQ)suppresses both the MyD88-dependent and TRIF-dependent pathways of TLR4 signaling in LPS-stimulated RAW264.7 cells[J].J Ethnopharmacol,2017,205:103-15.
[15]雷升萍,王靓,龙子江,等.黄精多糖通过TLR4-MyD88-NF-κB通路抑制缺氧/复氧H9c2心肌细胞炎性因子释放[J].中国药理学通报,2017,33(2),255-60.[15]Lei S P,Wang L,Long Z J,et al.Inhibitory effect of polygonatum sibiricum polysaccharides release of inflammatory cytokines of anoxia/reoxygenation H9c2 myocardial cells through TLR4-MyD88-NFκB signaling pathway[J].Chin Pharmacol Bull,2017,33(2),255-60.