血府逐瘀胶囊含药血清对人微血管内皮细胞KLF2表达的影响

Effect of Xuefu Zhuyu Capsule(血府逐瘀胶囊)-Containing Serum on the Expression of KLF2 in Human Microvascular Endothelial Cells

目的探讨血府逐瘀胶囊调控血管新生的可能作用机制。方法制备血府逐瘀胶囊含药血清和空白血清。分别以1.25%、2.5%、5%血府逐瘀胶囊含药血清或空白血清处理人微血管内皮细胞(HMEC-1,2.5×10^(5)个/孔)24h、48h和72h,每组设3个复孔。通过体外成血管试验检测各组细胞管腔形成情况,选择对管腔数先促进后抑制的最佳浓度血府逐瘀胶囊含药血清进行后续试验。HMEC-1细胞2.5×10^(5)个/孔分为血府逐瘀胶囊含药血清组和空白血清组,分别用2.5%血府逐瘀胶囊含药血清或空白血清处理12h、24h、48h。采用实时定量PCR和Wesstern Blot法检测Krüppel样因子2(KLF2)蛋白及mRNA水平。结果 1.25%和2.5%血府逐瘀胶囊含药血清干预HMEC-1细胞48h管腔数均显著增多(P<0.05或P<0.01);干预HMEC-1细胞72h, 1.25%血府逐瘀胶囊含药血清组管腔数显著增多,2.5%血府逐瘀胶囊含药血清组管腔数显著减少(P<0.01)。与同浓度血府逐瘀胶囊含药血清干预24h比较,1.25%血府逐瘀胶囊含药血清干预48、72h, 2.5%血府逐瘀胶囊含药血清干预48h,管腔数均显著增多(P<0.01);与同浓度血府逐瘀胶囊含药血清干预48h比较,仅2.5%血府逐瘀胶囊含药血清干预72h管腔数显著减少(P<0.01)。因此,选择2.5%血府逐瘀胶囊含药血清进行后续试验。与空白血清组比较,血府逐瘀胶囊含药血清组12、24h KLF2蛋白及mRNA水平差异无统计学意义(P>0.05),48h KLF2蛋白水平显著下降、KLF2 mRNA水平显著提高(P<0.05或P<0.01)。与干预12h和24h血府逐瘀含药血清组比较,干预48h血府逐瘀胶囊含药血清组KLF2蛋白表达差异无统计学意义(P>0.05),KLF2 mRNA水平均显著提高(P<0.05)。结论血府逐瘀胶囊可通过下调KLF2表达短时促进血管新生。

Objective To explore the possible mechanism of Xuefu Zhuyu Capsule(XZC, 血府逐瘀胶囊) in regulating angiogenesis. Methods XZC-containing serum and blank serum were prepared. Human microvascular endothelial cells(HMEC-1) were given 1.25%, 2.5% and 5% of XZC-containning serum or blank serum for 24 h, 48 h and 72 h each group was provided with 3 multiple wells. In vitro assays of angiogenesis was performed for assessment of cell lumen formation. The optimal concentration of XZC-containing serum which promoted lumen formulation first and inhibited it later was used for further tests;XZC-containing serum or blank serum of optimal concentration were given to HMEC-1 for 12 h, 24 h or 48 h;real-time quantitative PCR and Wesstern Blotting were used to detect Krüppel-like factor 2(KLF2) protein and mRNA levels. Results The number of lumen of HMEC-1 cells significantly increased after administration with 1.25% or 2.5% XZC-containing serums for 48 hours(P<0.05 or P<0.01);after 72 h intervention with XZC-containing serum, the number of lumen significantly increased when the concentration was 1.25% while decreased when it was 2.5%(P<0.01). Compared to XZC-containing serum of the same concentration administered for 24 h, 1.25% XZC-containing serum administered for 48 h and 72 h, as well as 2.5% XZC-containing serum for 48 h significantly increased the number of lumen(P<0.01);compared to XZC-containing serum of the same concentration administered for 48 h, 2.5% XZC-containing serum for 72 h significantly reduced the number of lumen(P<0.01). Therefore, 2.5% XZC-containing serum was selected for further tests. Compared to those of the blank serum group, the 12 h and 24 h KLF2 protein and mRNA levels of the XZC-containing serum group were not significantly different(P>0.05);the 48 h KLF2 protein level significantly decreased while 48 h KLF2 mRNA level increased(P<0.05 or P<0.01). Compared to those of the XZC-containing serum group administered for 12 h and 24 h, the 48 h KLF2 protein expression was not statistically different(P>0.05), and the 48 h KLF2 mRNA level significantly increased(P<0.05). Conclusion The mechanism of XZC in regulating angiogenesis may be related to the decrease of KLF2 protein level and mRNA level.