骨科祛痰逐瘀方经MAPK/ERK通路诱导血管内皮细胞迁移与血管管腔形成
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摘要
目的探究祛痰逐瘀方(Qutan Zhuyu Decoction,QZD)经MAPK/ERK通路诱导血管内皮细胞迁徙与血管管腔形成的作用,明确QZD在骨血管化重建中的机制。方法对大鼠进行QZD灌胃,并采集和分离获得含药血清;培养SVEC和10T1/2细胞,分别将二者分为空白组、bFGF组(阳性对照组)、QZD中药组(实验组),其中实验组为将QZD含药血清加入细胞培养基中。对干预后的SVEC进行迁徙实验,另外对SVEC进行管腔形成实验,对10T1/2进行免疫荧光染色观察中药促管腔形成效果,运用Western blot检测ERK1/2、p38等蛋白表达,研究QZD对SVEC的作用机制。结果 (1)QZD含药血清较空白组促进SVEC细胞的迁徙(P<0.05);(2)QZD含药血清较空白组可促进SVEC血管管腔形成,包括管腔面积(P<0.05)和管腔腔周长度(P<0.05);(3)荧光染色实验提示,QZD含药血清较空白组促进10T1/2细胞呈规律性聚集,并形成管腔状状态;(4)Western blot实验提示,QZD含药血清可促进SVEC中ERK1/2和p38的磷酸化(P<0.05)。结论 QZD经MARK/ERK通路调控和诱导血管内皮细胞的迁徙和血管官腔的形成,提示其可运用于骨血管化重建中。
Objective Qutan Zhuyu Decoction(QZD) is an important prescription for the treatment of orthopedic diseases. The purpose of our study is to investigate the role of QZD in inducing vascular endothelial cell migration and vascular lumen formation via MAPK/ERK pathway, and to clarify the mechanism of QZD in bone vascularization. Methods QZD was administered to rats, and serum containing QZD was collected and isolated. SVEC and 10 T1/2 cells were cultured and divided into blank group, bFGF group(positive control group) and QZD group(test group). QZD containing serum was added into the cell culture medium of the test group. The migration experiment was carried out on the SVEC after intervention; the SVEC was subjected to lumen formation experiment; the immunofluorescence staining of 10 T1/2 was used to observe the effect of QZD on lumen formation; the expression of ERK1/2, p38 was detected by Western blot for measuring its mechanism. Results(1) QZD containing serum promoted the migration of SVEC cells compared with the blank group(P<0.05);(2) QZD-containing serum could promote the formation of SVEC vascular lumen, including lumen area(P<0.05) and length of cavity(P<0.05);(3) Fluorescence staining experiment suggested that QZD-containing serum promotes regular aggregation of 10 T1/2 cells and forms a luminal state compared with the blank group;(4) Western blot test suggested that QZD containing serum promoted phosphorylation of ERK1/2 and p38 in SVEC(P<0.05). Conclusion QZD regulates and induces vascular endothelial cell migration and lumen formation via MARK/ERK pathway, indicating that it can be used in bone vascular remodeling.
Objective Qutan Zhuyu Decoction(QZD) is an important prescription for the treatment of orthopedic diseases. The purpose of our study is to investigate the role of QZD in inducing vascular endothelial cell migration and vascular lumen formation via MAPK/ERK pathway, and to clarify the mechanism of QZD in bone vascularization. Methods QZD was administered to rats, and serum containing QZD was collected and isolated. SVEC and 10 T1/2 cells were cultured and divided into blank group, bFGF group(positive control group) and QZD group(test group). QZD containing serum was added into the cell culture medium of the test group. The migration experiment was carried out on the SVEC after intervention; the SVEC was subjected to lumen formation experiment; the immunofluorescence staining of 10 T1/2 was used to observe the effect of QZD on lumen formation; the expression of ERK1/2, p38 was detected by Western blot for measuring its mechanism. Results(1) QZD containing serum promoted the migration of SVEC cells compared with the blank group(P<0.05);(2) QZD-containing serum could promote the formation of SVEC vascular lumen, including lumen area(P<0.05) and length of cavity(P<0.05);(3) Fluorescence staining experiment suggested that QZD-containing serum promotes regular aggregation of 10 T1/2 cells and forms a luminal state compared with the blank group;(4) Western blot test suggested that QZD containing serum promoted phosphorylation of ERK1/2 and p38 in SVEC(P<0.05). Conclusion QZD regulates and induces vascular endothelial cell migration and lumen formation via MARK/ERK pathway, indicating that it can be used in bone vascular remodeling.
引文
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[3] Zhang M,Malik AB,Rehman J.Endothelial progenitor cells and vascular repair [J].Curr Opin Hematol,2014,21(3):224-228.
[4] Schrimpf C,Teebken OE,Wilhelmi M,et al.The role of pericyte detachment in vascular rarefaction[J].J Vasc Res,2014,51(4):247-258.
[5] Stegen S,van Gastel N,Carmeliet G.Bringing new life to damaged bone:the importance of angiogenesis in bone repair and regeneration[J].Bone,2015,70:19-27.
[6] Segar CE,Ogle ME,Botchwey EA.Regulation of angiogenesis and bone regeneration with natural and synthetic small molecules[J].Curr Pharm Des,2013,19(19):3403-3419.
[7] 杨建军.祛痰逐瘀汤治疗不稳定型心绞痛的疗效观察[J].中国医药指南,2012,10(23):311-312.
[8] 曹银洲,赵敏.祛痰逐瘀法治疗血管性痴呆临床研究[J].中医学报,2015,30(1):127-129.
[9] 陈镇秋,何伟,魏秋实,等.祛痰逐瘀法对激素性股骨头坏死兔骨髓基质干细胞生物活性的影响[J].中华中医药杂志,2016,31(11):2732-2735.
[10] 陈镇秋,魏秋实,吴淮,等.祛痰逐瘀法预防家兔激素性股骨头坏死的病理学评价[J].中医正骨,2011,23(7):17-19.
[11] Kusumbe AP,Ramasamy SK,Adams RH.Coupling of angiogenesis and osteogenesis by a specific vessel subtype in bone[J].Nature,2014,507(7492):323-328.
[12] Ramasamy SK,Kusumbe AP,Wang L,et al.Endothelial Notch activity promotes angiogenesis and osteogenesis in bone[J].Nature,2014,507(7492):376-380.
[13] Stratman AN,Davis GE.Endothelial cell-pericyte interactions stimulate basement membrane matrix assembly:influence on vascular tube remodeling,maturation,and stabilization[J].Microsc Microanal,2012,18(1):68-80.
[14] Udagawa A,Sato S,Hasuike A,et al.Micro-CT observation of angiogenesis in bone regeneration [J].Clin Oral Implants Res,2013,24(7):787-792.
[15] Chim SM,Tickner J,Chow ST,et al.Angiogenic factors in bone local environment[J].Cytokine Growth Factor Rev,2013,24(3):297-310.
[16] Dirckx N,Van Hul M,Maes C.Osteoblast recruitment to sites of bone formation in skeletal development,homeostasis,and regeneration[J].Birth Defects Res C Embryo Today,2013,99(3):170-191.
[17] Wang S,Amato KR,Song W,et al.Regulation of endothelial cell proliferation and vascular assembly through distinct mTORC2 signaling pathways[J].Mol Cell Biol,2015,35(7):1299-1313.
[18] Chao CY,Lii CK,Ye SY,et al.Docosahexaenoic acid inhibits vascular endothelial growth factor (VEGF)-induced cell migration via the GPR120/PP2A/ERK1/2/eNOS signaling pathway in human umbilical vein endothelial cells[J].J Agric Food Chem,2014,62(18):4152-4158.
[19] Li P,Guo X,Lei P,et al.PI3K/Akt/uncoupling protein 2 signaling pathway may be involved in cell senescence and apoptosis induced by angiotensin II in human vascular endothelial cells[J].Mol Biol Rep,2014,41(10):6931-6937.
[20] Chim SM,Kuek V,Chow ST,et al.EGFL7 is expressed in bone microenvironment and promotes angiogenesis via ERK,STAT3,and integrin signaling cascades[J].J Cell Physiol,2015,230(1):82-94.
[21] Kuek V,Yang Z,Chim SM,et al.NPNT is expressed by osteoblasts and mediates angiogenesis via the activation of extracellular signal-regulated kinase[J].Sci Rep,2016,6:36210.