风湿祛痛胶囊对VEGF诱导的人脐静脉内皮细胞功能的影响
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摘要
目的:观察风湿祛痛胶囊对血管内皮细胞生长因子(VEGF)诱导的人脐静脉内皮细胞(HUVEC)的增殖、迁移、黏附、侵袭和管腔形成能力的影响,以及对VEGF受体2(VEGFR2)的干预作用。方法:VEGF体外诱导HUVEC,加入风湿祛痛胶囊低、中、高质量浓度(0. 02,0. 1,0. 5μg·L-1)作用后,分别采用噻唑蓝(MTT)比色法、转移小室(transwell)法、黏附实验、细胞侵袭及管腔形成实验检测HUVEC的增殖活性、迁移、黏附、侵袭及管腔形成能力,蛋白免疫印迹法(Western blot)检测细胞中VEGFR2磷酸化水平和蛋白含量,实时荧光定量聚合酶链式反应(Real-time PCR)检测VEGFR2 mRNA表达水平。结果:与正常组比较,VEGF诱导24,48 h后均能显著升高HUVEC的增殖活性(P <0. 01),诱导24 h能明显升高HUVEC的迁移、黏附、侵袭和管腔形成能力(P <0. 01),显著升高HUVEC中VEGFR2磷酸化及蛋白和mRNA表达水平(P <0. 01);与VEGF组比较,风湿祛痛胶囊低、中、高质量浓度组作用48 h均能明显抑制HUVEC增殖活性(P <0. 05,P <0. 01),作用24 h对VEGF诱导的HUVEC迁移、黏附、侵袭和管腔形成能力有明显抑制作用(P <0. 05),也能下调VEGFR2磷酸化及蛋白和mRNA表达水平(P <0. 05)。结论:风湿祛痛胶囊能降低VEGF诱导的HUVEC细胞增殖、迁移、黏附、侵袭及管腔形成能力,这一作用可能与其抑制VEGFR2的磷酸化、蛋白和mRNA表达水平有关。
Objective: To study the effect of Fengshi Qutong capsule on the migration,adhesion,invasion and tube formation of human synovial cells and the phosphorylation and protein expression of vascular endothelial growth factor receptor 2(VEGFR2). Method: With human umbilical vein endothelial cells(HUVEC)as the research object,low,middle and high-dose Fengshi Qutong capsule(0. 02,0. 1,0. 5 μg · L-1) on HUVEC was determined by methye thiazolye telrazlium(MTT) colorimetric assay for the follow-up experiment. The transwell migration,adhesion and transwell invasion test were used to detect the migration and adhesion of the different concentrations of Fengshi Qutong capsule in HUVEC. The expression of VEGFR2 in HUVEC was detected by Western blot,and Real-time PCR was used to detect the content of VEGFR2 mRNA in cells. Result: Compared with normal group,the proliferation of HUVEC was significantly increased after 24 h and 48 h of VEGF induction(P < 0. 01),the migration,adhesion,invasion and lumen formation of HUVEC were significantly increased within24 h(P < 0. 01),and VEGFR2 phosphorylation protein and mRNA expression levels in HUVEC were significantly elevated in HUVEC(P < 0. 01). Compared with VEGF group,0. 02,0. 1 and 0. 5 μg·L-1 Fengshi Qutong capsule were administered in vitro for 48 h to inhibit HUVEC proliferation activity in a dose-dependent manner(P < 0. 05,P < 0. 01). Within 24 h,VEGF-induced HUVEC migration,adhesion,invasion and lumen formation were significantly inhibited(P < 0. 05),and negatively regulated VEGFR2 phosphorylation protein and gene expression levels(P < 0. 05). Conclusion: Fengshi Qutong capsule can inhibit the migration,adhesion,invasion and tube formation of HUVEC. This effect may be related to the inhibition of phosphorylation,and protein and mRNA expression level of VEGFR2.
Objective: To study the effect of Fengshi Qutong capsule on the migration,adhesion,invasion and tube formation of human synovial cells and the phosphorylation and protein expression of vascular endothelial growth factor receptor 2(VEGFR2). Method: With human umbilical vein endothelial cells(HUVEC)as the research object,low,middle and high-dose Fengshi Qutong capsule(0. 02,0. 1,0. 5 μg · L-1) on HUVEC was determined by methye thiazolye telrazlium(MTT) colorimetric assay for the follow-up experiment. The transwell migration,adhesion and transwell invasion test were used to detect the migration and adhesion of the different concentrations of Fengshi Qutong capsule in HUVEC. The expression of VEGFR2 in HUVEC was detected by Western blot,and Real-time PCR was used to detect the content of VEGFR2 mRNA in cells. Result: Compared with normal group,the proliferation of HUVEC was significantly increased after 24 h and 48 h of VEGF induction(P < 0. 01),the migration,adhesion,invasion and lumen formation of HUVEC were significantly increased within24 h(P < 0. 01),and VEGFR2 phosphorylation protein and mRNA expression levels in HUVEC were significantly elevated in HUVEC(P < 0. 01). Compared with VEGF group,0. 02,0. 1 and 0. 5 μg·L-1 Fengshi Qutong capsule were administered in vitro for 48 h to inhibit HUVEC proliferation activity in a dose-dependent manner(P < 0. 05,P < 0. 01). Within 24 h,VEGF-induced HUVEC migration,adhesion,invasion and lumen formation were significantly inhibited(P < 0. 05),and negatively regulated VEGFR2 phosphorylation protein and gene expression levels(P < 0. 05). Conclusion: Fengshi Qutong capsule can inhibit the migration,adhesion,invasion and tube formation of HUVEC. This effect may be related to the inhibition of phosphorylation,and protein and mRNA expression level of VEGFR2.
引文
[1]郭炜,刘春芳,林娜.类风湿性关节炎滑膜血管新生与中医药[J].中国实验方剂学杂志,2012,18(10):308-312.
[2] LIU Y,LI Y,XU Y. Inhibitory effects of novel integrintargeting peptides on angiogenesis activity in HUVEC cells in vitro[J]. Cell Biochem Funct,2011,29(5):429-435.
[3] QI X,LIU G,QIU L,et al. Marine bromophenol bis(2,3-dibromo-4,5-dihydroxybenzyl)ether,represses angiogenesis in HUVEC cells and in zebrafish embryos via inhibiting the VEGF signal systems[J]. Biomed Pharmacother,2015,75(2015):58-66.
[4] LIN J M,ZHAO J Y,ZHUANG Q C,et al. Xiongshao capsule promotes angiogenesis of HUVEC via enhancing cell proliferation and up-regulating the expression of b FGF and VEGF[J]. Chin J Integr Med,2011,17(11):840-846.
[5] LI Y,ZHU H,WEI X,et al. LPS induces HUVEC angiogenesis in vitro through miR-146a-mediated TGF-β1inhibition[J]. Am J Transl Res,2017,9(2):591-600.
[6]姚晖.风湿祛痛胶囊结合穴位注射治疗类风湿性关节炎的临床研究[J].世界中医药,2016,11(1):52-54.
[7]郭燕芬,吴宽裕,赵钟文,等.中医药优化方案治疗湿热痹阻型类风湿关节炎的不良反应观察[J].风湿病与关节炎,2014,3(11):15-19.
[8]考希良,董嘉琪.二妙散不同配伍对大鼠佐剂性关节炎的抗炎效应[J].中医药学报,2013,41(3):107-109.
[9]王靖霞,刘春芳,何莲花,等.风湿祛痛胶囊对大鼠胶原诱导性关节炎的影响[J].中国实验方剂学杂志,2019,25(2):89-95.
[10] Stefanini M O,WU F T,Mac Gabhann F,et al. The presence of VEGF receptors on the luminal surface of endothelial cells affects VEGF distribution and VEGF signaling[J]. PLo S Comput Biol, 2009, 5(12):e1000622.
[11] Ferrara N,Houck K,Jakeman L,et al. Molecular and biological properties of the vascular endothelial growth factor family of proteins[J]. Endocr Rev,1992,13(1):18-32.
[12] AI N,CHONG C M,CHEN W,et al. Ponatinib exerts anti-angiogenic effects in the zebrafish and human umbilical vein endothelial cells via blocking VEGFR signaling pathway[J]. Oncotarget, 2018,9(62):31958-31970.
[13] Gliki G. Signaling transduction mechanisms mediating biological actions of the vascular endothelial growth factor family[J]. Cardiovasc Res,2001,49(3):568-581.
[14] Wise L M,Veikkola T,Mercer A A,et al. Vascular endothelial growth factor(VEGF)2 like protein from orf virus NZ2 binds to VEGFR2and neuropilin21[J]. Proc Natl Acad Sci USA,1999,96(6):3071-3076.
[15] Otrock Z K,Makarem J A,Shamseddine A I. Vascular endothelial growth factor family of ligands and receptors:review[J]. Blood Cells Mol Dis, 2007,38(3):258-268.
[16]张三印,陈钢,黄秀深,等.血管生成的中医理论初探[J].中医杂志,2007,48(9):773.
[17]陶方方,汪丽佩,储利胜,等.蕲蛇蛇毒对胶原诱导性关节炎大鼠血管生成的影响[J].中国实验方剂学杂志,2014,20(14):168-172.
[18]任海霞,肖诚,孔丽,等.海蛇乙醇提取物对胶原诱导关节炎大鼠滑膜血管生成的抑制作用[J].中国中医基础医学杂志,2007(2):115-116.
[19]王志文,丁春菊,张爱国,等.麝香乌龙丸对佐剂性关节炎大鼠滑膜病理形态及血管内皮生长因子、内皮抑素表达的影响[J].中国实验方剂学杂志,2011,17(19):241-244.
[2] LIU Y,LI Y,XU Y. Inhibitory effects of novel integrintargeting peptides on angiogenesis activity in HUVEC cells in vitro[J]. Cell Biochem Funct,2011,29(5):429-435.
[3] QI X,LIU G,QIU L,et al. Marine bromophenol bis(2,3-dibromo-4,5-dihydroxybenzyl)ether,represses angiogenesis in HUVEC cells and in zebrafish embryos via inhibiting the VEGF signal systems[J]. Biomed Pharmacother,2015,75(2015):58-66.
[4] LIN J M,ZHAO J Y,ZHUANG Q C,et al. Xiongshao capsule promotes angiogenesis of HUVEC via enhancing cell proliferation and up-regulating the expression of b FGF and VEGF[J]. Chin J Integr Med,2011,17(11):840-846.
[5] LI Y,ZHU H,WEI X,et al. LPS induces HUVEC angiogenesis in vitro through miR-146a-mediated TGF-β1inhibition[J]. Am J Transl Res,2017,9(2):591-600.
[6]姚晖.风湿祛痛胶囊结合穴位注射治疗类风湿性关节炎的临床研究[J].世界中医药,2016,11(1):52-54.
[7]郭燕芬,吴宽裕,赵钟文,等.中医药优化方案治疗湿热痹阻型类风湿关节炎的不良反应观察[J].风湿病与关节炎,2014,3(11):15-19.
[8]考希良,董嘉琪.二妙散不同配伍对大鼠佐剂性关节炎的抗炎效应[J].中医药学报,2013,41(3):107-109.
[9]王靖霞,刘春芳,何莲花,等.风湿祛痛胶囊对大鼠胶原诱导性关节炎的影响[J].中国实验方剂学杂志,2019,25(2):89-95.
[10] Stefanini M O,WU F T,Mac Gabhann F,et al. The presence of VEGF receptors on the luminal surface of endothelial cells affects VEGF distribution and VEGF signaling[J]. PLo S Comput Biol, 2009, 5(12):e1000622.
[11] Ferrara N,Houck K,Jakeman L,et al. Molecular and biological properties of the vascular endothelial growth factor family of proteins[J]. Endocr Rev,1992,13(1):18-32.
[12] AI N,CHONG C M,CHEN W,et al. Ponatinib exerts anti-angiogenic effects in the zebrafish and human umbilical vein endothelial cells via blocking VEGFR signaling pathway[J]. Oncotarget, 2018,9(62):31958-31970.
[13] Gliki G. Signaling transduction mechanisms mediating biological actions of the vascular endothelial growth factor family[J]. Cardiovasc Res,2001,49(3):568-581.
[14] Wise L M,Veikkola T,Mercer A A,et al. Vascular endothelial growth factor(VEGF)2 like protein from orf virus NZ2 binds to VEGFR2and neuropilin21[J]. Proc Natl Acad Sci USA,1999,96(6):3071-3076.
[15] Otrock Z K,Makarem J A,Shamseddine A I. Vascular endothelial growth factor family of ligands and receptors:review[J]. Blood Cells Mol Dis, 2007,38(3):258-268.
[16]张三印,陈钢,黄秀深,等.血管生成的中医理论初探[J].中医杂志,2007,48(9):773.
[17]陶方方,汪丽佩,储利胜,等.蕲蛇蛇毒对胶原诱导性关节炎大鼠血管生成的影响[J].中国实验方剂学杂志,2014,20(14):168-172.
[18]任海霞,肖诚,孔丽,等.海蛇乙醇提取物对胶原诱导关节炎大鼠滑膜血管生成的抑制作用[J].中国中医基础医学杂志,2007(2):115-116.
[19]王志文,丁春菊,张爱国,等.麝香乌龙丸对佐剂性关节炎大鼠滑膜病理形态及血管内皮生长因子、内皮抑素表达的影响[J].中国实验方剂学杂志,2011,17(19):241-244.