方妍妍;万磊;董文哲;文建庭;刘健;
FANG Yan-yan;WAN Lei;DONG Wen-zhe;WEN Jian-ting;LIU Jian;Graduate School of Anhui University of Traditional Chinese Medicine;the First Affiliated Hospital of Anhui University of Traditional Chinese Medicine;

• 1:安徽中医药大学研究生院
• 2:安徽中医药大学第一附属医院

摘要(Abstract)：

通过体外实验研究雷公藤甲素(triptolide,TP)对VEGFA,SDF-1,CXCR4通路的影响,探讨其改善强直性脊柱炎(AS)患者血小板活化的机制。外周血单核细胞(PBMC)用于实验。分为4组:正常组(NC)、模型组(MC)、雷公藤甲素组(TP)、AMD3100组。MTT法检测TP最佳浓度。ELISA法检测TNF-α,IL-1β,IL-4,IL-10,VEGFA,VEGFR的表达,实时荧光定量PCR(RT-qPCR)法检测SDF-1,CXCR4,VEGFA的表达,Western blot法检测SDF-1,CXCR4,VEGFA,VEGFR蛋白的表达,免疫荧光法检测CD62p,CD40L,PDGFA的表达水平。MTT结果显示中剂量TP在24 h时对细胞抑制作用最强。ELISA及PCR法结果显示TP可抑制IL-1β,TNF-α,VEGFA,VEGFR,SDF-1,CXCR4,VEGFA mRNA的表达; Western blot结果说明TP可抑制SDF-1,CXCR4,VEGFA,VEGFR蛋白的表达;免疫荧光结果说明TP可抑制CD62p,CD40L,PDGFA的表达。TP可能通过下调SDF-1,CXCR4,VEGFA,VEGFR mRNA,进而下调IL-1β,TNF-α,上调IL-4,IL-10细胞因子的表达,从而调节血小板活化。
The effect of triptolide( TP) on VEGFA,SDF-1,CXCR4 pathway were investigated in vitro to explore the mechanism in improving platelet activation in patients with ankylosing spondylitis( AS). Peripheral blood mononuclear cells( PBMC) were used for the experiment and divided into 4 groups: normal group( NC),model group( MC),triptolide group( TP),and AMD3100 group. The optimal concentration of TP was measured by the MTT method. The expressions of TNF-α,IL-1β,IL-4,IL-10,VEGFA and VEGFR were detected by ELISA. The expressions of SDF-1,CXCR4 and VEGFA were detected by real-time quantitative PCR( RT-qPCR).The expressions of SDF-1,CXCR4,VEGFA and VEGFR were detected by Western blot. The expression levels of CD62 p,CD40 L and PDGFA were detected by immunofluorescence. MTT results showed that medium-dose TP had the strongest inhibitory effect on cells at24 h. The results of ELISA and PCR showed that TP inhibited mRNA expressions of IL-1β,TNF-α,VEGFA,VEGFR and SDF-1,CXCR4 and VEGFA. The results of Western blot indicated that TP inhibited SDF-1,CXCR4 and VEGFA,VEGFR protein expressions; immunofluorescence results indicate that TP can inhibit the expressions of CD62 p,CD40 L,PDGFA. TP may regulate platelet activation by down-regulating SDF-1,CXCR4,VEGFA and VEGFR mRNA expressions,thereby down-regulating IL-1β and TNF-αexpressions,and up-regulating the expressions of IL-4 and IL-10 cytokines.

关键词(KeyWords)： 强直性脊柱炎;雷公藤甲素;VEGFA;SDF-1;CXCR4;血小板活化
ankylosing spondylitis;triptolide;VEGFA;SDF-1;CXCR4;platelet activation

Abstract:

Keywords:

基金项目(Foundation): 安徽省科技攻关项目(1604A0802085);; 2016年中央引导地方科技发展专项(财教[2016]1188);; 现代中医内科应用基础与开发研究安徽省重点实验室项目(财教[2016]518);; 2015年技术创新服务体系专项(财教[2015]1551);; 中医药行业科研专项(201307001);; 安徽高校自然科学研究重点项目(KJ2017A281)

作者(Authors): 方妍妍;万磊;董文哲;文建庭;刘健;
FANG Yan-yan;WAN Lei;DONG Wen-zhe;WEN Jian-ting;LIU Jian;Graduate School of Anhui University of Traditional Chinese Medicine;the First Affiliated Hospital of Anhui University of Traditional Chinese Medicine;

DOI: 10.19540/j.cnki.cjcmm.20181113.001

参考文献(References)：

• [1]刘健.风湿病中医临床实践[M].合肥:安徽科学技术出版社,2013.
• [2]Zeng Q Y,Chen R,Darmawan J,et al.Rheumatic diseases in China[J/OL].Arthritis Res Ther,2008,doi:10.1186/ar2368.
• [3]Bowness P.HLA-B27[J].Annu Rev Immunol,2015,33(10):29.
• [4]Faham M,Carlton V,Moorhead M,et al.Discovery of T cell receptorβmotifs specific to HLA-B27 positive ankylosing spondylitis by deep repertoire sequence analysis[J].Arthritis Rheum,2017,69(4):774.
• [5]方利,刘健,朱福兵,等.基于细胞因子/NF-κB信号通路探讨强直性脊柱炎患者血液高凝状态形成的机制[J].中华中医药杂志,2016,31(9):3756.
• [6]李春杏,李太生,朱珠,等.雷公藤抗炎免疫调节活性单体的研究进展[J].中国中药杂志,2014,39(21):4159.
• [7]徐文莉,罗艺,李康,等.强直性脊柱炎患者外周血T淋巴细胞亚群及NKT细胞的表达及意义[J].检验医学与临床,2018,15(2):192.
• [8]Vander L S,Valkenburg H A,Cats A.Evaluation of diagnostic criteria for ankylosing spondylitis[J].Arthritis Rheum,1984,27(4):361.
• [9]彭桉平,黄宪章,刘瑞萍,等.雷公藤内酯醇调控miR-155抑制类风湿性关节炎患者单核细胞促炎反应[J].细胞与分子免疫学杂志,2014,30(6):635.
• [10]武彦琴,李达.藤类中药在血液病中的应用现状[J].中华中医药杂志,2016,31(10):4151.
• [11]陶玲,肖芳,朱卫丰,等.雷公藤减毒研究进展[J].中国实验方剂学杂志,2017,23(5):229.
• [12]方利,刘健,朱福兵,等.基于细胞因子/NF-κB信号通路探讨强直性脊柱炎患者血瘀状态形成的机制[J].中华中医药学刊,2016,34(12):2913.
• [13]方妍妍,刘健,万磊,等.基于关联规则对423例强直性脊柱炎患者血小板参数及免疫炎症代谢指标的数据挖掘研究[J].中国免疫学杂志,2018,34(7):1039.
• [14]Kisacik B,Tufan A,Kalyoncu U,et al.Mean platelet volume(MPV)as an inflammatory marker in ankylosing spondylitis and rheumatoid arthritis[J].Joint Bone Spine,2008,75(3):291.
• [15]Sakellariou G T,Anastasilakis A D,Bisbinas I,et al.Circulating periostin levels in patients with AS:association with clinical and radiographic variables,inflammatory markers and molecules involved in bone formation[J].Rheumatology(Oxford),2015,54(5):908.
• [16]Falco D E,Porcelli D.SDF-1 involvement in endothelial phenotype and ischemia-induced recruitment of bone marrow progenitor cells[J].Blood,2004,104(12):3482.
• [17]Romain B,Hachet H M,Rohr S,et al.Hypoxia differentially regulated CXCR4 and CXCR7 signaling incolon cancer[J].Mol Cancer,2014,21(13):58.
• [18]涂然,王寿勇.SDF-1/CXCR4与炎性疾病的相关研究进展[J].医学综述,2016,22(4):653.
• [19]Lu F,Liu L,Yu D H,et al.Therapeutic effect of Rhizoma Diosco-reae Nipponicae on gouty arthritis based on the SDF-1/CXCR4and p38 MAPK pathway:an in vivo and in vitro study[J].Phyto-ther Res,2014,28(2):280.
• [20]刘强,邓华聪,何辉,等.银丹心脑通软胶囊对2型糖尿病患者血小板活化因子、血液流变学和血管内皮细胞功能的影响[J].中国现代应用药学,2015,32(1):86.
• [21]谢则金,王厚照,林一.CD62p,IL-6和hs-CRP检测在类风湿性关节炎中的临床价值[J].检验医学,2017,32(11):966.
• [22]Zhou X W,Li W Y,Ding Q.Up-regulation of soluble P-selectin predicates its prognostic value in patients with ankylosing spondylitis[J].Int J Clin Exp Pathol,2015,8(6):7272.
• [23]高伟.合并脑外伤肢体骨折患者血清中VEGF,PDGF的含量分析[D].长沙:中南大学,2011.
• [24]朱晋坤,毛华,尹扬光,等.血小板源性生长因子和血小板源性内皮细胞生长因子在内皮细胞和血管平滑肌细胞中的作用研究[J].中国全科医学,2015,18(9):1023.
• [25]文利,王国良,盛桐亮,等.PDGF,IL-11在男性强直性脊柱炎骨代谢中的作用研究[J].医学研究杂志,2014,43(11):134.
• [26]谭军,陈建国.类风湿性关节炎患者血清CD40L及IL-6 hsCRP表达及临床意义[J].重庆医学,2013,42(36):4402.