活血益气方及其拆方对缺氧人脐静脉内皮细胞增殖和凋亡的影响
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摘要
目的探讨活血益气方及其拆方促进血管新生的可能作用机制。方法建立人脐静脉内皮细胞(HUVEC)缺氧损伤模型,分为模型组、活血益气方组、活血方组及益气方组。另设正常对照组常规培养。活血益气方组、活血方组及益气方组给予含10%相应药物血清培养,模型组及正常对照组给予生理盐水血清培养,干预24 h。采用CCK-8法检测细胞增殖,Annexin V-FITC/PI双染法检测HUVEC凋亡,WST-1法测定细胞内超氧化物歧化酶(SOD)活力,微板法测定丙二醛(MDA)含量及细胞培养上清乳酸脱氢酶(LDH)含量,实时荧光定量PCR法检测细胞蛋白激酶C(PKC)、Ras、Raf-1 mRNA水平。结果与正常对照组比较,模型组HUVEC增殖明显减少,SOD活力及PKC、Ras、Raf-1 mRNA均显著下降,细胞凋亡及MDA、LDH含量明显增加(P<0.05或P<0.01)。与模型组比较,活血益气方组、活血方组及益气方组HUVEC增殖均显著增加,LDH含量均显著降低,活血益气方组SOD活力显著提高,活血益气方组、活血方组细胞凋亡显著减少,活血益气方组、益气方组MDA含量显著降低,PKC、Ras、Raf-1 mRNA水平显著升高(P<0.05或P<0.01)。结论活血益气方及其拆方可不同程度地促进缺氧后HUVEC增殖并抑制其凋亡,可能与增强细胞抗氧化能力和调节PKC/Ras/Raf-1信号通路有关;活血益气方对细胞的保护作用优于活血方和益气方,二者发挥协同作用。
Objective To discuss the potential mechanism of Huoxue Yiqi Fang( 活血益气方) and its decomposed recipes in promoting angiogenesis. Methods The model of human umbilical vein endothelial cell( HUVEC)in hypoxia was built and divided into the model group,Huoxue Yiqi Fang( HYF) group,Huoxue Fang( HF) group and Yiqi Fang( YF) Group. There was also a control group set in conventional cultivation. The three Chinese medicine groups were cultivated by 10% serum containing corresponding herbs,while the model group and the control group were by serum containing normal saline. They were all intervened for 24 hours. The cell proliferation was detected by CCK-8 method,the HUVEC apoptosis was detected by Annexin V-FITC/PI double staining,and the super oxide dismutase( SOD) activity was detected by WST-1 method,the malondialdehyde( MDA) volume and the lactate dehydrogenase( LDH) volume in cell culture supernatant were detected by microplate test,and the mRNA levels of protein kinase C( PKC),Ras and Raf-1 were detected by real-time quantitative PCR. Results Compared with the control group,in the model group,the HUVEC proliferation,the SOD activity and the PKC,Ras,Raf-1 mRNA declined significantly,and the apoptosis and MDA and LDH volume increased significantly( P < 0. 05 or P < 0. 01).Compared with the model group,in the HYF group,HF group and YF group,HUVEC proliferation increased significantly,LDH volume decreased significantly; SOD activity in HYF Group improved significantly; the apoptosis in HYF group and HF group descended significantly; the MDA volume in HYF group and YF group reduced significantly,while the RKC,Ras and Raf-1 mRNA volume ascended significantly( P < 0. 05 or P < 0. 01). Conclusion HYF and its decomposed recipes could promote the proliferation of HUVEC in hypoxia and inhibit its apoptosis in varying degrees. The mechanism may be enhancing the cellular antioxidant activity and regulating PKC/Ras/Raf-1 signaling pathway. HQF could protect cells better than HF and YF,so HF and YF coordinate together.
Objective To discuss the potential mechanism of Huoxue Yiqi Fang( 活血益气方) and its decomposed recipes in promoting angiogenesis. Methods The model of human umbilical vein endothelial cell( HUVEC)in hypoxia was built and divided into the model group,Huoxue Yiqi Fang( HYF) group,Huoxue Fang( HF) group and Yiqi Fang( YF) Group. There was also a control group set in conventional cultivation. The three Chinese medicine groups were cultivated by 10% serum containing corresponding herbs,while the model group and the control group were by serum containing normal saline. They were all intervened for 24 hours. The cell proliferation was detected by CCK-8 method,the HUVEC apoptosis was detected by Annexin V-FITC/PI double staining,and the super oxide dismutase( SOD) activity was detected by WST-1 method,the malondialdehyde( MDA) volume and the lactate dehydrogenase( LDH) volume in cell culture supernatant were detected by microplate test,and the mRNA levels of protein kinase C( PKC),Ras and Raf-1 were detected by real-time quantitative PCR. Results Compared with the control group,in the model group,the HUVEC proliferation,the SOD activity and the PKC,Ras,Raf-1 mRNA declined significantly,and the apoptosis and MDA and LDH volume increased significantly( P < 0. 05 or P < 0. 01).Compared with the model group,in the HYF group,HF group and YF group,HUVEC proliferation increased significantly,LDH volume decreased significantly; SOD activity in HYF Group improved significantly; the apoptosis in HYF group and HF group descended significantly; the MDA volume in HYF group and YF group reduced significantly,while the RKC,Ras and Raf-1 mRNA volume ascended significantly( P < 0. 05 or P < 0. 01). Conclusion HYF and its decomposed recipes could promote the proliferation of HUVEC in hypoxia and inhibit its apoptosis in varying degrees. The mechanism may be enhancing the cellular antioxidant activity and regulating PKC/Ras/Raf-1 signaling pathway. HQF could protect cells better than HF and YF,so HF and YF coordinate together.
引文
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[9]张晶,王大新,何胜虎,等.卡托普利与黄芪对氧化损伤人脐静脉内皮细胞保护作用的研究[J].中国临床药理学与治疗学,2015,20(8):891-895.
[10]黄琦,邓雯雯,许家鸾,等.人参总皂苷对波动性高糖诱导的人脐静脉内皮细胞凋亡的影响[J].中药新药与临床药理,2012,23(5):500-504.
[11]韩秀娟,刘丹,封亮,等.丹酚酸组分中7种酚酸类成分对缺氧损伤的人脐静脉内皮细胞保护作用的研究[J].中国中药杂志,2016,41(6):1107-1112.
[12]WU LW,MAYO LD,DUNBAR JD,et al.Utilization of distinct signaling pathways by receptors for vascular endothelial cell growth factor and other mitogens in the induction of endothelial cell proliferation[J].J Biol Chem,2000,275(7):5096-5103.
[13]FRIEDLANDER M,BROOKS PC,SHAFFER RW,et al.Definition of two angiogenic pathways by distinct alpha v integrins[J].Science,1995,270(5241):1500-1502.
[14]SEYMOUR LW,SHOAIBI MA,MARTIN A,et al.Vascular endothelial growth factor stimulates protein kinase Cdependent phospholipase D activity in endothelial cells[J].Lab Invest,1996,75(3):427-437.
[15]张冬梅,吴爱明,娄利霞,等.活血益气方对心梗后大鼠缺血心肌血管新生及VEGF表达的影响[J].辽宁中医杂志,2010,37(8):1602-1604,1630.
[16]张冬梅,陈萌,吴爱明,等.活血益气方药物血清对转染VEGF165脐静脉内皮细胞分泌NO、e NOS的影响[J].中西医结合心脑血管病杂志,2011,9(10):1225-1227.
[2]FERRARA N,KERBEL RS.Angiogenesis as a therapeutic target[J].Nature,2005,438(7070):967-974.
[3]郑瑞玲,陈萌,吴爱明,等.活血益气方及其拆方对大鼠心肌梗死边缘区血管内皮生长因子及其上游调控因子miR-126的影响[J].中医杂志,2016,57(12):1061-1064.
[4]李娜,吴世政,张淑坤.脉络宁注射液对人血管内皮细胞缺氧损伤的保护作用[J].中国新药杂志,2012,21(11):1273-1277.
[5]吴爱明,赵明镜,张冬梅,等.心梗后心力衰竭模型大鼠中医证候特点及心脏的超声评价[J].中国中西医结合杂志,2007,27(3):227-230.
[6]陈金星,王硕仁,朱陵群,等.心肌梗死后心力衰竭大鼠心脏基因表达谱及活血益气方药对其心脏的影响[J].中国中西医结合杂志,2005,25(1):45-48.
[7]赵明镜,王硕仁,李敏,等.早期应用活血和益气中药抑制心衰大鼠左室重构和凋亡的对比研究[J].中国中药杂志,2007,32(8):710-714.
[8]顾俊菲,封亮,袁嘉瑞,等.赤芍总苷、川芎总酚酸组分组成结构对缺氧损伤人脐静脉内皮细胞的影响[J].中国中药杂志,2015,40(5):920-926.
[9]张晶,王大新,何胜虎,等.卡托普利与黄芪对氧化损伤人脐静脉内皮细胞保护作用的研究[J].中国临床药理学与治疗学,2015,20(8):891-895.
[10]黄琦,邓雯雯,许家鸾,等.人参总皂苷对波动性高糖诱导的人脐静脉内皮细胞凋亡的影响[J].中药新药与临床药理,2012,23(5):500-504.
[11]韩秀娟,刘丹,封亮,等.丹酚酸组分中7种酚酸类成分对缺氧损伤的人脐静脉内皮细胞保护作用的研究[J].中国中药杂志,2016,41(6):1107-1112.
[12]WU LW,MAYO LD,DUNBAR JD,et al.Utilization of distinct signaling pathways by receptors for vascular endothelial cell growth factor and other mitogens in the induction of endothelial cell proliferation[J].J Biol Chem,2000,275(7):5096-5103.
[13]FRIEDLANDER M,BROOKS PC,SHAFFER RW,et al.Definition of two angiogenic pathways by distinct alpha v integrins[J].Science,1995,270(5241):1500-1502.
[14]SEYMOUR LW,SHOAIBI MA,MARTIN A,et al.Vascular endothelial growth factor stimulates protein kinase Cdependent phospholipase D activity in endothelial cells[J].Lab Invest,1996,75(3):427-437.
[15]张冬梅,吴爱明,娄利霞,等.活血益气方对心梗后大鼠缺血心肌血管新生及VEGF表达的影响[J].辽宁中医杂志,2010,37(8):1602-1604,1630.
[16]张冬梅,陈萌,吴爱明,等.活血益气方药物血清对转染VEGF165脐静脉内皮细胞分泌NO、e NOS的影响[J].中西医结合心脑血管病杂志,2011,9(10):1225-1227.