双丹明目胶囊抑制人脐静脉内皮细胞增殖有效成分筛选研究
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摘要
目的从双丹明目胶囊24个入血成分中筛选出抑制人脐静脉内皮细胞(HUVEC)增殖的有效成分。方法将双丹明目胶囊24个入血成分标准品均稀释成终浓度为1、10、100μg/mL的溶液,分别干预体外培养的HUVEC,采用MTT法检测细胞存活率,对各成分进行初筛,再对有效成分进行浓度细化,确定有效成分的最佳作用浓度。在此基础上,采用Hoechst33258荧光染色和流式细胞术检测各有效成分对HUVEC凋亡的影响。结果 MTT检测结果显示,双丹明目胶囊24个入血成分中4个成分能有效抑制HUVEC增殖(P<0.05),分别为3-乙酸齐墩果酸、丹参酮ⅡA、芹菜素和大波斯菊苷,其最佳作用浓度分别为75、75、50、75μg/mL;进一步研究发现,上述4个成分均能使HUVEC细胞核固缩、碎裂,数量减少,细胞凋亡率显著增加(P<0.05),其中以丹参酮ⅡA和芹菜素效果尤为明显。结论 3-乙酸齐墩果酸、丹参酮ⅡA、芹菜素和大波斯菊苷是双丹明目胶囊抑制HUVEC增殖、发挥治疗糖尿病视网膜病变疗效的有效成分。
Objective To screen out the effective components inhibiting the proliferation of human umbilical vein endothelial cells(HUVEC) from 24 blood components of Shuangdan Mingmu Capsules. Methods All the 24 standards were diluted to a final concentration of 1, 10, and 100 μg/mL, and the HUVEC were cultured in vitro. The cell proliferation inhibition rate was measured by MTT method, and the components were screened. Concentration refinement was used to determine the optimal concentration of effective components. Hoechst33258 fluorescence staining and flow cytometry were used to detect the effects of various effective components on apoptosis of HUVEC.Results The results of MTT experiment showed that among the 24 blood components of Shuangdan Mingmu Capsules, 4 components could effectively inhibit the proliferation of HUVEC(P<0.05), that were 3-acetate oleanolic acid, tanshinone ⅡA, apigenin and cosmosin, and the optimal concentrations were 75, 75, 50, and 75 μg/mL,respectively. Further studies found that the above four components could make the nucleus of HUVEC pyknosis,fragmentation, number decreased, and the apoptotic rate increased significantly(P<0.05), especially the effect of tanshinone ⅡA and apigenin. Conclusion 3-acetyl oleanolic acid, tanshinone ⅡA, apigenin and cosmosin are effective components of Shuangdan Mingmu Capsules for inhibiting the proliferation of HUVEC and treating diabetic retinopathy.
Objective To screen out the effective components inhibiting the proliferation of human umbilical vein endothelial cells(HUVEC) from 24 blood components of Shuangdan Mingmu Capsules. Methods All the 24 standards were diluted to a final concentration of 1, 10, and 100 μg/mL, and the HUVEC were cultured in vitro. The cell proliferation inhibition rate was measured by MTT method, and the components were screened. Concentration refinement was used to determine the optimal concentration of effective components. Hoechst33258 fluorescence staining and flow cytometry were used to detect the effects of various effective components on apoptosis of HUVEC.Results The results of MTT experiment showed that among the 24 blood components of Shuangdan Mingmu Capsules, 4 components could effectively inhibit the proliferation of HUVEC(P<0.05), that were 3-acetate oleanolic acid, tanshinone ⅡA, apigenin and cosmosin, and the optimal concentrations were 75, 75, 50, and 75 μg/mL,respectively. Further studies found that the above four components could make the nucleus of HUVEC pyknosis,fragmentation, number decreased, and the apoptotic rate increased significantly(P<0.05), especially the effect of tanshinone ⅡA and apigenin. Conclusion 3-acetyl oleanolic acid, tanshinone ⅡA, apigenin and cosmosin are effective components of Shuangdan Mingmu Capsules for inhibiting the proliferation of HUVEC and treating diabetic retinopathy.
引文
[1]涂良钰,王文长,王育良,等.双丹明目胶囊治疗糖尿病视网膜病变的临床研究[J].中国新药杂志,2009,24(12):2331-2336.
[2]符超君,凌艳君,颜家朝,等.双丹明目胶囊对糖尿病视网膜病变大鼠血糖血脂及血液流变学的影响[J].湖南中医药大学学报,2018,38(5):489-492.
[3]符超君,凌艳君,颜家朝,等.双丹明目胶囊对糖尿病视网膜病变大鼠视网膜组织低氧诱导因子-1α和核因子-κB表达的影响[J].中国中医药信息杂志,2018,25(6):44-47.
[4]龙红萍,秦裕辉,刘峥嵘,等.UPLC-Q-TOF法分析双丹明目胶囊化学成分[J].中成药,2017,39(7):1527-1531.
[5]龙红萍,秦裕辉,刘峥嵘,等.UPLC-Q-TOF-MS法分析双丹明目胶囊入血成分[J].中成药,2017,39(10):2204-2206.
[6]WANG J J,ZHU M,LE Y Z.Functions of Müller cell-derived vascular endothelial growth factor in diabetic retinopathy[J].World JDiabetes,2015,6(5):726-733.
[7]ZHOU Y,YUAN J,QI C,et al.Calcium dobesilate may alleviate diabetes-induced endothelial dysfunction and inflammation[J].Mol Med Rep,2017,16(6):8635-8642.
[8]SAHIN M,SAHIN E,GüMü?LüS.Effects of lycopene and apigenin on human umbilical vein endothelial cells in vitro under angiogenic stimulation[J].Acta Histochem,2012,114(2):94-100.
[9]ZOU Y,CHIOU G C.Apigenin inhibits laser-induced choroidal neovascularization and regulates endothelial cell function[J].J Ocul Pharmacol Ther,2006,22(6):425-430.
[2]符超君,凌艳君,颜家朝,等.双丹明目胶囊对糖尿病视网膜病变大鼠血糖血脂及血液流变学的影响[J].湖南中医药大学学报,2018,38(5):489-492.
[3]符超君,凌艳君,颜家朝,等.双丹明目胶囊对糖尿病视网膜病变大鼠视网膜组织低氧诱导因子-1α和核因子-κB表达的影响[J].中国中医药信息杂志,2018,25(6):44-47.
[4]龙红萍,秦裕辉,刘峥嵘,等.UPLC-Q-TOF法分析双丹明目胶囊化学成分[J].中成药,2017,39(7):1527-1531.
[5]龙红萍,秦裕辉,刘峥嵘,等.UPLC-Q-TOF-MS法分析双丹明目胶囊入血成分[J].中成药,2017,39(10):2204-2206.
[6]WANG J J,ZHU M,LE Y Z.Functions of Müller cell-derived vascular endothelial growth factor in diabetic retinopathy[J].World JDiabetes,2015,6(5):726-733.
[7]ZHOU Y,YUAN J,QI C,et al.Calcium dobesilate may alleviate diabetes-induced endothelial dysfunction and inflammation[J].Mol Med Rep,2017,16(6):8635-8642.
[8]SAHIN M,SAHIN E,GüMü?LüS.Effects of lycopene and apigenin on human umbilical vein endothelial cells in vitro under angiogenic stimulation[J].Acta Histochem,2012,114(2):94-100.
[9]ZOU Y,CHIOU G C.Apigenin inhibits laser-induced choroidal neovascularization and regulates endothelial cell function[J].J Ocul Pharmacol Ther,2006,22(6):425-430.