白花蛇舌草-半枝莲药对组分对结肠腺癌Lovo细胞生物学行为的影响

摘要:目的　探讨白花蛇舌草-半枝莲药对组分对结肠腺癌Lovo细胞增殖、侵袭、迁移和凋亡的影响及作用机制。方法　将白花蛇舌草、半枝莲按质量1∶1进行3次煎煮，获得水提物，后取适量浸膏用石油醚回流脱脂，再以乙酸乙酯进行多次萃取，获得白花蛇舌草-半枝莲药对组分，并计算得率。实验分为对照组（正常培养Lovo细胞）、白花蛇舌草-半枝莲药对组分低剂量组（10 mg/L）、中剂量组（30 mg/L）及高剂量组（50 mg/L）。通过噻唑蓝比色法（MTT）检测各组细胞培养24、48、72 h后的增殖抑制率。各组细胞培养48 h后，流式细胞仪检测细胞周期分布；Transwell实验检测细胞侵袭能力；划痕实验检测细胞迁移能力；TUNEL法检测细胞凋亡情况；Western blot法检测Grb2相关结合蛋白1（Gab1）、血管内皮生长因子受体2（VEGFR-2）、磷脂酰肌醇3-激酶（PI3K）、苏氨酸激酶（Akt）、基质金属蛋白酶-9（MMP-9）、B淋巴细胞瘤-2基因（Bcl-2）、Bcl-2相关X蛋白（Bax）蛋白表达情况。结果　化学萃取后的白花蛇舌草-半枝莲药对中主要含有对羟基苯乙酮、野黄芩苷、木犀草素和芹菜素4种化合物，组分得率为0.61%。与对照组相比，低、中、高剂量组细胞增殖抑制率升高，G1期肿瘤细胞比例增加，细胞凋亡指数增高，侵袭细胞数和划痕闭合率明显减小（均P＜0.05），细胞中Gab1、VEGFR-2、PI3K、Akt、MMP-9、Bcl-2蛋白表达降低，Bax表达升高（均P＜0.05），且存在剂量依赖性。结论　白花蛇舌草-半枝莲药对组分可抑制结肠腺癌Lovo细胞的增殖，降低其迁移和侵袭能力，诱导细胞凋亡，其机制可能与抑制Gab1/VEGFR-2/PI3K/Akt信号通路活化有关。

Effects of Hedyotis diffusa-sculellaria barbata components on biological behavior of adenocarcinoma of colon Lovo cells

Abstract: Objective To investigate the effect and mechanism of Hedyotis diffusa-sculellaria barbata components on the proliferation, invasion, migration and apoptosis of adenocarcinoma of colon Lovo cells. Methods The Hedyotis diffusa and scutellaria barbata were decocted for three times according to the quality of 1∶1 to obtain the water extract. Then, a proper amount of extract was taken and degreased with petroleum ether, and then extracted with ethyl acetate for many times to obtain the components of Hedyotis diffusa and scutellaria barbata and calculated the yield. The experimental cells were divided into control group (normal cultured Lovo cells), Hedyotis diffusa-sculellaria barbata component low dose group (10 mg/L), medium dose group (30 mg/L), and high dose group (50 mg/L). The proliferation inhibition rates were detected by thiazole blue colorimetric (MTT) assay. The invasion ability and migration ability were checked by Transwell and cell scratch test. Flow cytometry was used to detect cell cycle distribution, and TUNEL was used to check apoptosis rate. The expression of Grb2-associated binding protein 1 (Gab1)/Vascular endothelial growth factor receptor 2 (VEGFR-2)/Phosphatidylinositol 3-kinase (PI3K)/Kthreonine kinase (Akt) signaling pathway protein, apoptotic protein Bcl-2 and Bax and metalloproteinase-9 (MMP-9) were detected by Western blot assay. Results Four compounds, p-hydroxyacetophenone, scutellarin, luteolin and apigenin, were found in the pair of Hedyotis diffusa and Scutellaria barbata. The yield was 0.61% after ethyl acetate extraction. Compared with the control group, the cell proliferation inhibition rate, apoptosis index (AI) value, the proportion of G1 phase of tumor cells increased in the low, middle and high dose groups, the number of cell penetrating membrane and the rate of scratch closure decreased significantly (P＜0.05). The expressions of Gab1, VEGFR-2, PI3K, Akt, MMP-9 and Bcl-2 proteins decreased, the expression of Bax increased with a dose-dependent manner (P＜0.05). Conclusion Hedyotis diffusa-sculellaria barbata components can inhibit the proliferation of adenocarcinoma of colon Lovo cells in G1 phase, reduce the migration and invasion and induce the apoptosis. The mechanism might be related to the inhibition of Gab1/VEGFR-2/PI3K/Akt signaling pathway.