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目的观察竹节香附素A对人肝癌细胞株HepG_2细胞血管内皮生长因子(VEGF)表达的影响。方法体外培养HepG_2细胞,将不同浓度的竹节香附素A与HepG_2细胞共培养,采用酶联免疫吸附(ELISA)方法检测不同浓度竹节香附素A处理HepG_2细胞24 h后细胞培养上清液中VEGF蛋白含量的变化;半定量逆转录-聚合酶链反应(RT-PCR)法和蛋白免疫印迹(Western blot)法分别检测竹节香附素A对HepG_2细胞VEGF mRNA和蛋白相对表达水平(阳性条带灰度值/β-actin带灰度值)的影响。结果 (1)不同浓度(5、10、20μg/ml)竹节香附素A处理HepG_2细胞24 h后,细胞培养上清液中VEGF水平呈剂量依赖性降低(P=0.000)。(2)竹节香附素A对HepG_2细胞VEGF mRNA表达的影响:(1)不同浓度竹节香附素A(0、5、10、20μg/ml)处理HepG_2细胞24 h后,VEGF mRNA的相对表达量呈剂量依赖性减少(0.96±0.07、0.75±0.09、0.55±0.10、0.32±0.11,P=0.000)。(2)10μg/ml竹节香附素A分别处理HepG_2细胞0、6、12、24 h后,VEGF mRNA的相对表达水平呈时间依赖性下降(0.77±0.05、0.53±0.09、0.32±0.12、0.15±0.04,P=0.000)。(3)竹节香附素A抑制HepG_2细胞VEGF蛋白的表达:(1)不同浓度竹节香附素A(0、5、10、20μg/ml)处理HepG_2细胞24 h后,VEGF蛋白相对表达水平呈剂量依赖性下降(2.08±0.28、1.43±0.22、0.72±0.15、0.17±0.10,P=0.000)。(2)10μg/ml竹节香附素A分别处理HepG_2细胞0、6、12、24 h后VEGF蛋白相对表达水平呈时间依赖性减少(2.47±0.05、1.36±0.02、0.30±0.02、0.07±0.02,P=0.000)。结论竹节香附素A能够下调HepG_2细胞VEGF表达,这可能是其抑制肝癌细胞生长增殖的重要机制之一。
Objective To observe the effect of sarcandrosin A on the expression of vascular endothelial growth factor (VEGF) in human hepatocellular carcinoma cell line HepG_2. Methods HepG_2 cells were cultured in vitro. Different concentrations of sarcandrine A and HepG_2 were co-cultured. The HepG_2 cells were treated with different concentrations of sarcaxin A for 24 h by enzyme-linked immunosorbent assay (ELISA) VEGF protein levels in HepG2 cells were detected by semi-quantitative RT-PCR and Western blot, respectively. The relative expression levels of VEGF mRNA and protein (Positive band gray value / β-actin with gray value). Results (1) After treated with different concentrations of sarcaxin A (5, 10, and 20μg / ml) for 24 h, the VEGF level in the cell culture supernatant decreased in a dose-dependent manner (P = 0.000). (2) The effect of sarcandrosin A on the expression of VEGF mRNA in HepG_2 cells: (1) After treatment with different concentrations of sarcadroside A (0, 5, 10, 20μg / ml) for 24 h, The relative expression decreased in a dose-dependent manner (0.96 ± 0.07,0.75 ± 0.09,0.55 ± 0.10,0.32 ± 0.11, P = 0.000). (2) At 0, 6, 12, and 24 h after treatment with 10 μg / ml sarcaxin, the relative expression of VEGF mRNA decreased in a time-dependent manner (0.77 ± 0.05, 0.53 ± 0.09, 0.32 ± 0.12, 0.15 ± 0.04, P = 0.000). (3) Scutellamentin A inhibited the expression of VEGF protein in HepG_2 cells: (1) The expression of VEGF protein in HepG_2 cells treated with different concentrations of sarcaxin A (0, 5, 10, 20μg / ml) The levels decreased in a dose-dependent manner (2.08 ± 0.28, 1.43 ± 0.22, 0.72 ± 0.15, 0.17 ± 0.10, P = 0.000). (2) The relative expression level of VEGF protein in HepG2 cells treated with 10μg / ml SJA for 0, 6, 12 and 24 hours decreased in a time-dependent manner (2.47 ± 0.05, 1.36 ± 0.02, 0.30 ± 0.02 and 0.07 ± 0.02, P = 0.000). Conclusion Scutellamentin A can down-regulate the expression of VEGF in HepG_2 cells, which may be one of the important mechanisms of its inhibition on the growth and proliferation of HepG2 cells.