新风胶囊含药血清通过抑制VEGFA/SDF-1/CXCR4通路缓解强直性脊柱炎患者血小板活化的机制
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摘要
目的通过体外实验研究新风胶囊(XFC)对VEGFA/SDF-1/CXCR4通路的影响,探讨其改善强直性脊柱炎(AS)患者血小板活化的的机制。方法分离外周血单核细胞(PBMC),分为正常组(NC)、模型组(MC)、新风胶囊组(XFC)、AMD3100组,MTT法检测XFC最佳浓度,免疫荧光法检测CD62p、CD40L、PDGFA的表达,ELISA法检测TNF-ɑ、IL-1β、IL-4、IL-10、VEGFA及VEGFR的表达,实时荧光定量PCR(RT-qPCR)法检测SDF-1、CXCR4、VEGFA mRNA的表达,Western blot法检测SDF-1、CXCR4、VEGFA、VEGFR蛋白的表达。结果中剂量XFC在24 h时对细胞抑制作用最强。与正常组相比,模型组CD62p、CD40L、PDGFA、IL-1β、TNF-α、VEGFA、VEGFR、SDF-1、CXCR4表达均升高,IL-4、IL-10降低(P<0.05,P<0.01)。与模型组相比,XFC组CD62p、CD40L、PDGFA、IL-1β、TNF-α、VEGFA、VEGFR、SDF-1、CXCR4表达均降低,IL-4、IL-10升高(P<0.05,P<0.01)。结论 XFC具有类AMD3100样作用,通过抑制VEGFA/SDF-1/CXCR4通路的表达,下调IL-1β、TNF-α,上调IL-4、IL-10,调节免疫炎症反应,从而降低AS患者血小板活化。
This study was designed to investigate the effect of Xinfeng Capsule(XFC) on VEGFA/SDF-1/CXCR4 pathway in vitro and to explore the mechanism of relieving platelet activation in patients with ankylosing spondylitis(AS). Peripheral blood mononuclear cells(PBMC) from normal subjects and AS patients were divided into normal group(NC), model group(MC), Xinfeng capsule group(XFC) and AMD3100 group. The optimal concentration of XFC was detected by MTT assay; the expression levels of CD62 p, CD40 L and PDGFA were detected by fluorescence method; the expression levels of TNF-ɑ, IL-1β, IL-4, IL-10, VEGFA and VEGFR were detected by ELISA. The mRNA and protein expression of SDF-1, CXCR4 and VEGFA were detected by real-time fluorescent quantitative PCR(RT-qPCR) and Western blotting, respectively. MTT results showed that medium dose XFC had the strongest inhibitory effect on cells at 24 h. Compared with the normal group, the expression of CD62 p,CD40 L, PDGFA, IL-1β, TNF-α, VEGFA, VEGFR, SDF-1 and CXCR4 increased, while IL-4 and IL-10 decreased in model group(P<0.05, P<0.01), and XFC could reverse all these changes(P<0.05, P<0.01). In conclusion, XFC may relieve platelet activation in AS patients by inhibiting the expression of VEGFA/SDF-1/CXCR4 pathway, downregulating IL-1β, TNF-α, up-regulating IL-4 and IL-10, and regulating immune inflammatory response.
This study was designed to investigate the effect of Xinfeng Capsule(XFC) on VEGFA/SDF-1/CXCR4 pathway in vitro and to explore the mechanism of relieving platelet activation in patients with ankylosing spondylitis(AS). Peripheral blood mononuclear cells(PBMC) from normal subjects and AS patients were divided into normal group(NC), model group(MC), Xinfeng capsule group(XFC) and AMD3100 group. The optimal concentration of XFC was detected by MTT assay; the expression levels of CD62 p, CD40 L and PDGFA were detected by fluorescence method; the expression levels of TNF-ɑ, IL-1β, IL-4, IL-10, VEGFA and VEGFR were detected by ELISA. The mRNA and protein expression of SDF-1, CXCR4 and VEGFA were detected by real-time fluorescent quantitative PCR(RT-qPCR) and Western blotting, respectively. MTT results showed that medium dose XFC had the strongest inhibitory effect on cells at 24 h. Compared with the normal group, the expression of CD62 p,CD40 L, PDGFA, IL-1β, TNF-α, VEGFA, VEGFR, SDF-1 and CXCR4 increased, while IL-4 and IL-10 decreased in model group(P<0.05, P<0.01), and XFC could reverse all these changes(P<0.05, P<0.01). In conclusion, XFC may relieve platelet activation in AS patients by inhibiting the expression of VEGFA/SDF-1/CXCR4 pathway, downregulating IL-1β, TNF-α, up-regulating IL-4 and IL-10, and regulating immune inflammatory response.
引文
[1]刘健.风湿病中医临床实践[M].合肥:安徽科学技术出版社,2013:103-125.
[2]方利,刘健,朱福兵,等.基于细胞因子/NF-kB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):3756-3759.
[3]Shibuya M.VEGF-VEGFR system as a target for suppressing inflammation and other diseases[J].Endocr Metab Immune Disord Drug Targets,2015,15(2):135-144.
[4]Sakellariou GT,Anastasilakis AD,Bisbinas I,et al.Circulating periostin levels in patients with AS:association with clinical and radiographic variables,inflammatory markers and molecules involved in bone formations[J].Rheumatology(Oxford),2015,54(5):908-914.
[5]Falco DE,Porcelli D.SDF-1 involvement in endothelial phenotype and ischemia-induced recruitment of bone marrow progenitor cells[J].Blood,2004,104(12):3482-3483.
[6]Romain B,Hachet Haas M,Rohr S,et al.Hypoxia differentially regulated CXCR4 and CXCR7 signaling incolon cancer[J].Mol Cancer,2014,21(13):58.
[7]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):653-657.
[8]Zheng X,Zhao FC,Pang Y,et al.Downregulation of miR-221-3p contributes to IL-1β-induced cartilage degradation by directly targeting the SDF1/CXCR4signaling pathway[J].J Mol Med,2017,95(6):615-627.
[9]Shi H,Lu R,Wang S,et al.Effects of SDF-1/CXCR4 on acute lung injury induced by cardiopulmonary bypasss[J].Inflammation,2017,40(3):937-945.
[10]Wang F,Meng M,Chen L,et al.Development and validation of a high-performance thin-layer chromatographic fingerprint method for the evaluation of Qiyi Capsule with the reference of myotonin[J].J Planar Chromat,2014,27(3):199-203.
[11]方利,刘健,万磊,等.新风胶囊通过抑制mi R155/NF-κB信号通路改善强直性脊柱炎活动期患者高凝状态[J].细胞与分子免疫学杂志,2016,32(8):1094-1099.
[12]Vander Linden S,Valkenburg HA,Cats A.Evaluation of diagnostic criteria for ankylosing spondylitis.A proposal for modification of the New York criterias[J].Arthritis Rheum,1984,27(4):361-368.
[13]文建庭,刘健,万磊,等.基于Notch和PKC/NF-κB通路串话研究新风胶囊改善佐剂性关节炎大鼠肺功能机制[J].免疫学杂志,2018,34(7):553-561.
[14]孟楣,王芳,王晓玉,等.新风胶囊中水溶性蛋白的SDS-PAGE分析方法研究[J].中药材,2014,37(1):141-143.
[15]柳晓娜,朱宁,魏国峰,等.糖尿病合并急性冠状动脉综合征患者血小板表面PAC-1和CD62P表达水平[J].中国动脉硬化杂志,2015,23(2):161-164.
[16]李云婷,张雷雷,张巧丽.血小板活化标志物CD62P、CD63在糖尿病患者早期肾脏微血管病变中的表达[J].河北医科大学学报,2015,36(5):551-553.
[17]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):966-969.
[18]Zhou X,Li W,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):7272-7276.
[19]高伟.(2011)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.
[20]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):1023-1028.
[21]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):134-137.
[22]谭军,陈建国.类风湿性关节炎患者血清CD40L及IL-6 hs-CRP表达及临床意义[J].重庆医学,2013,42(36):4402-4404.
[23]Kisacik B,Tufan A,Kalyoncu U,et al.Mean platelet volume(MPV)as an inflam-matory marker in ankylosing spondylitis and rheumatoid arthritis[J].Joint Bone Spine,2008,75(3):291-294.
[2]方利,刘健,朱福兵,等.基于细胞因子/NF-kB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):3756-3759.
[3]Shibuya M.VEGF-VEGFR system as a target for suppressing inflammation and other diseases[J].Endocr Metab Immune Disord Drug Targets,2015,15(2):135-144.
[4]Sakellariou GT,Anastasilakis AD,Bisbinas I,et al.Circulating periostin levels in patients with AS:association with clinical and radiographic variables,inflammatory markers and molecules involved in bone formations[J].Rheumatology(Oxford),2015,54(5):908-914.
[5]Falco DE,Porcelli D.SDF-1 involvement in endothelial phenotype and ischemia-induced recruitment of bone marrow progenitor cells[J].Blood,2004,104(12):3482-3483.
[6]Romain B,Hachet Haas M,Rohr S,et al.Hypoxia differentially regulated CXCR4 and CXCR7 signaling incolon cancer[J].Mol Cancer,2014,21(13):58.
[7]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):653-657.
[8]Zheng X,Zhao FC,Pang Y,et al.Downregulation of miR-221-3p contributes to IL-1β-induced cartilage degradation by directly targeting the SDF1/CXCR4signaling pathway[J].J Mol Med,2017,95(6):615-627.
[9]Shi H,Lu R,Wang S,et al.Effects of SDF-1/CXCR4 on acute lung injury induced by cardiopulmonary bypasss[J].Inflammation,2017,40(3):937-945.
[10]Wang F,Meng M,Chen L,et al.Development and validation of a high-performance thin-layer chromatographic fingerprint method for the evaluation of Qiyi Capsule with the reference of myotonin[J].J Planar Chromat,2014,27(3):199-203.
[11]方利,刘健,万磊,等.新风胶囊通过抑制mi R155/NF-κB信号通路改善强直性脊柱炎活动期患者高凝状态[J].细胞与分子免疫学杂志,2016,32(8):1094-1099.
[12]Vander Linden S,Valkenburg HA,Cats A.Evaluation of diagnostic criteria for ankylosing spondylitis.A proposal for modification of the New York criterias[J].Arthritis Rheum,1984,27(4):361-368.
[13]文建庭,刘健,万磊,等.基于Notch和PKC/NF-κB通路串话研究新风胶囊改善佐剂性关节炎大鼠肺功能机制[J].免疫学杂志,2018,34(7):553-561.
[14]孟楣,王芳,王晓玉,等.新风胶囊中水溶性蛋白的SDS-PAGE分析方法研究[J].中药材,2014,37(1):141-143.
[15]柳晓娜,朱宁,魏国峰,等.糖尿病合并急性冠状动脉综合征患者血小板表面PAC-1和CD62P表达水平[J].中国动脉硬化杂志,2015,23(2):161-164.
[16]李云婷,张雷雷,张巧丽.血小板活化标志物CD62P、CD63在糖尿病患者早期肾脏微血管病变中的表达[J].河北医科大学学报,2015,36(5):551-553.
[17]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):966-969.
[18]Zhou X,Li W,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):7272-7276.
[19]高伟.(2011)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.
[20]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):1023-1028.
[21]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):134-137.
[22]谭军,陈建国.类风湿性关节炎患者血清CD40L及IL-6 hs-CRP表达及临床意义[J].重庆医学,2013,42(36):4402-4404.
[23]Kisacik B,Tufan A,Kalyoncu U,et al.Mean platelet volume(MPV)as an inflam-matory marker in ankylosing spondylitis and rheumatoid arthritis[J].Joint Bone Spine,2008,75(3):291-294.
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