温阳活血解毒复方对人脐静脉内皮细胞VEGFs/VEGFRs通路的影响
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摘要
目的研究温阳活血解毒复方(WHJF)对血管内皮生长因子(VEGFs/VEGFRs)信号通路的影响,从分子层面探讨其治疗动脉粥样硬化(AS)的机制。方法将SD大鼠随机分为空白对照组(C组)、温阳活血解毒复方低、中、高剂量组(L、M、H组,剂量分别为10.35、31.05、93.15 g·kg~(-1)),于第4天首次灌胃2 h后采血制备含药血清,采用免疫印迹法(Western Blot)及逆转录-聚合酶链式反应(q RT-PCR)法分别观察含药血清对人脐静脉内皮细胞(HUVECs)的VEGFs/VEGFRs信号通路中各信号分子及其基因表达的影响。结果温阳活血解毒复方含药血清对VEGFs/VEGFRs信号通路有抑制作用。在蛋白表达方面,与空白对照组比较,高剂量组中血管内皮生长因子受体2(VEGFR-2)和磷酸化的细胞外调节蛋白激酶(pERK)的表达量明显减少,差异有统计学意义(P<0.05);中剂量组中pERK的表达量明显减少,差异有统计学意义(P<0.05);通路中其他信号分子的表达量无明显减少,差异无统计学意义(P>0.05)。在基因表达方面,与空白对照组比较,温阳活血解毒复方组血管内皮生长因子亚型A、B(VEGFA、VEGFB)及血管内皮生长因子受体1、2(VEGFR-1、VEGFR-2)的mRNA表达量无明显减少,差异无统计学意义(P>0.05)。结论温阳活血解毒复方含药血清可能通过下调VEGFR-2及下游分子pERK的表达,对VEGFs/VEGFRs信号通路发挥抑制作用,从而直接影响细胞DNA合成功能,减少血管新生,起到稳定动脉粥样硬化斑块的作用。
Objective To investigate the effect of drug-containing serum of Wenyang Huoxue Jiedu Formula(WHJF) on the vascular endothelial growth factors and their receptors(VEGFs/VEGFRs) pathways; and toexplore its anti-atherosclerosis(AS) mechanism at molecular level. Methods The SD rats were randomly dividedinto four groups,namely blank control group(C),low-dose(L),middle-dose(M) and high-dose(H) WHJFgroups,which were treated with normal saline,WHJF in the dosages of 10.35,31.05,93.15 g·kg~(-1) respectively,twice a day. At 2 h after first administration of the fourth day,the drug-containing serum was taken from the rats.The expression of signaling molecules and their mRNAs of VEGFs/VEGFRs pathways in the human umbilical veinendothelial cells(HUVECs) were assayed by Western Blot and quantitative real-time polymerase chain reaction(qRT-PCR) respectively. Results The drug-containing serum of WHJF exerted an inhibitory effect on the VEGFs/VEGFRs pathways. The H group showed a reduction of the expression of vascular endothelial growth factor receptor 2(VEGFR-2) and extracellular regulated protein kinases(pERK) when compared with C group(P < 0.05),whileM group disturbed the expression of pERK(P < 0.05). The levels of other proteins showed no obviously change(P > 0.05). There was no difference in the mRNA levels of VEGFA,VEGFB,VEGFR-1 and VEGFR-2(P >0.05). Conclusion The drug-containing serum of WHJF inhibits the VEGFs/VEGFRs pathways of HUVECs viadown-regulating the mediate molecules(VEGFR-2 and pERK), which results in the obstruction of DNAsynthesis, the decrease of angiogenesis and the stabilization of atherosclerosis.
Objective To investigate the effect of drug-containing serum of Wenyang Huoxue Jiedu Formula(WHJF) on the vascular endothelial growth factors and their receptors(VEGFs/VEGFRs) pathways; and toexplore its anti-atherosclerosis(AS) mechanism at molecular level. Methods The SD rats were randomly dividedinto four groups,namely blank control group(C),low-dose(L),middle-dose(M) and high-dose(H) WHJFgroups,which were treated with normal saline,WHJF in the dosages of 10.35,31.05,93.15 g·kg~(-1) respectively,twice a day. At 2 h after first administration of the fourth day,the drug-containing serum was taken from the rats.The expression of signaling molecules and their mRNAs of VEGFs/VEGFRs pathways in the human umbilical veinendothelial cells(HUVECs) were assayed by Western Blot and quantitative real-time polymerase chain reaction(qRT-PCR) respectively. Results The drug-containing serum of WHJF exerted an inhibitory effect on the VEGFs/VEGFRs pathways. The H group showed a reduction of the expression of vascular endothelial growth factor receptor 2(VEGFR-2) and extracellular regulated protein kinases(pERK) when compared with C group(P < 0.05),whileM group disturbed the expression of pERK(P < 0.05). The levels of other proteins showed no obviously change(P > 0.05). There was no difference in the mRNA levels of VEGFA,VEGFB,VEGFR-1 and VEGFR-2(P >0.05). Conclusion The drug-containing serum of WHJF inhibits the VEGFs/VEGFRs pathways of HUVECs viadown-regulating the mediate molecules(VEGFR-2 and pERK), which results in the obstruction of DNAsynthesis, the decrease of angiogenesis and the stabilization of atherosclerosis.
引文
[1]洪永敦,吴兴波.温阳解毒活血法对急性心肌梗死患者的临床干预作用[J].广州中医药大学学报,2012,29(5):519-522.
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[13]张路,吴宗贵,廖德宁,等.通心络对实验性家兔主动脉粥样斑块内血管内皮生长因子表达的影响[J].中国动脉硬化杂志,2004,12(2):177-182.
[14]孙璐,韦立新,石怀银,等.冠状动脉粥样硬化斑块内血管新生与斑块稳定的关系[J].中华病理学杂志,2003,32(5):427-431.
[15]渋谷正史.血管新生とその制御―VEGFと受容体を中心に―[J].炎症再生,2004,24(3):144-153.
[16]RIAZY M,CHEN J H,STEINBRECHER U P.VEGF secretion by macrophages is stimulated by lipid and protein components of Ox LDL via PI3-kinase and PKC zeta activation and is independent of Ox LDL uptake[J].Atherosclerosis,2009,204(1):47-54.
[17]HERTUALA S Y,MAREIN J F.Cardiovascular gene therapy[J].Lancet,2000,355:213-222.
[18]TAKAHASHI T,UENO H,SHIBUYA M.VEGF activates protein kinase C-dependent,but Ras-independent Raf-MEK-MAP kinase pathway for DNA synthesis in primary endothelial cells[J].Oncogene,1999,18:2221-2230.
[19]HAN C J,LIU J T,LIU X F,et al.Angiotensin II induces C-reactive protein expression through ERK1/2 and JNK signaling in human aortic endothelial cells[J].Atherosclerosis,2010,212(1):206-212.
[20]胡庆松,聂如琼.血管内皮细胞标志物研究进展[J].岭南心血管病杂志,2012,18(2):196-201.
[21]PROCTPR B M,JIN X,LUPU T S,et al.Requirement for p38mitogen-activated protein kinase activity in neointima formation after vascular injury[J].Circulation,2008,118:658-666.
[2]陈广进,洪永敦,赵静,等.温阳活血解毒方对ApoE-小鼠动脉粥样硬化斑块及斑块内血管新生的影响[J].中药新药与临床药理,2015,26(2):139-144.
[3]洪永敦,黎智文,陈爽,等.温阳活血解毒复方对人脐静脉内皮细胞功能的影响[J].中药新药与临床药理,2016,7(27):519-524.
[4]LIBBY P.Inflammation in atherosclerosis[J].Nature,2002,420:868-874.
[5]FINN A V,NAKANO M,NARULA,et al.Concept of vulnerable/unstable plaque[J].Arterioscler Thromb Vasc Biol,2010(30):1282-1292.
[6]NAGHAVI M,LIBBY P,FALK E,et al.From vulnerable Plaque to vulnerable Patient:a call for new definitions and risk assessment strategies:Part l[J].Circulation,2003,108(14):1664-1672.
[7]JAIN R K,DUDA D G,CLARK J W,et al.Lessons from phase III clinical trials on anti-VEGF therapy for cancer[J].Nature clinical practice.Oncology,2006,3(1):24-40.
[8]CHEN F,ERIKSSON P,KIMURA T,et al.Apoptosis and angiogenesis are induced in the unstable coronary atheroselerotic plaque[J].Coron Artery Dis,2005,16:191-197.
[9]CELLETTI F L,WAUGH J M,AMABILE P G,et al.Vascular endothelial growth factor enhances atherosclerotic plaque progression[J].Nature medicine,2001,7(4):425-429.
[10]张璐,蒋跃绒,薛梅,等.辛伐他汀对兔动脉粥样硬化斑块稳定性及斑块内血管新生的影响[J].科学通报,2009,54(15):2228-2232.
[11]BENTZON J F,OTSUKA F,VIRMANI R,et al.Mechanisms of plaque formation and rupture[J].Circ Res,2014,114:1852-1866.
[12]EPSTEIN S E,EUGENIO S,TIMOTHY K,et al.Janus phenomenon:the interrelated tradeoffs inherent in therapies designed to enhance collateral formation and those designed to inhibit atherogenesis[J].Circulation,2004,109(23):2826-2831.
[13]张路,吴宗贵,廖德宁,等.通心络对实验性家兔主动脉粥样斑块内血管内皮生长因子表达的影响[J].中国动脉硬化杂志,2004,12(2):177-182.
[14]孙璐,韦立新,石怀银,等.冠状动脉粥样硬化斑块内血管新生与斑块稳定的关系[J].中华病理学杂志,2003,32(5):427-431.
[15]渋谷正史.血管新生とその制御―VEGFと受容体を中心に―[J].炎症再生,2004,24(3):144-153.
[16]RIAZY M,CHEN J H,STEINBRECHER U P.VEGF secretion by macrophages is stimulated by lipid and protein components of Ox LDL via PI3-kinase and PKC zeta activation and is independent of Ox LDL uptake[J].Atherosclerosis,2009,204(1):47-54.
[17]HERTUALA S Y,MAREIN J F.Cardiovascular gene therapy[J].Lancet,2000,355:213-222.
[18]TAKAHASHI T,UENO H,SHIBUYA M.VEGF activates protein kinase C-dependent,but Ras-independent Raf-MEK-MAP kinase pathway for DNA synthesis in primary endothelial cells[J].Oncogene,1999,18:2221-2230.
[19]HAN C J,LIU J T,LIU X F,et al.Angiotensin II induces C-reactive protein expression through ERK1/2 and JNK signaling in human aortic endothelial cells[J].Atherosclerosis,2010,212(1):206-212.
[20]胡庆松,聂如琼.血管内皮细胞标志物研究进展[J].岭南心血管病杂志,2012,18(2):196-201.
[21]PROCTPR B M,JIN X,LUPU T S,et al.Requirement for p38mitogen-activated protein kinase activity in neointima formation after vascular injury[J].Circulation,2008,118:658-666.