红花中羟基红花黄色素A的提取及对肝癌细胞的抑制作用

Extraction of hydroxy safflor yellow A and the inhibitory effects on liver cancer cells

目的探讨闪式提取技术提取红花中羟基红花黄色素A(hydroxysafflor yellow A,HSYA)及其对肝癌细胞生长、侵袭和迁移能力的影响和机制。方法以提取含量为指标,考察提取时间、提取电压、最佳料液比对提取的影响,并通过正交试验优选最佳提取工艺。将不同质量浓度的HSYA分别作用于肝癌SMMC-7721和Hep G2细胞,在作用24、48、72 h后,采用MTT法、Transwell法和流式细胞术分别检测细胞的增殖能力、迁移能力、侵袭能力和细胞周期与凋亡情况。用免疫印迹法(Western blot)分析细胞中VEGF、PI3K、AKT及Snail蛋白的表达结果。结果闪式提取HSYA最佳工艺为料液比40∶1 g·mL-1,3 min和80 V电压。HSYA可以抑制肝癌SMMC-7721和Hep G2细胞的增殖,且有剂量和时间相关性(P <0. 05),并可以显著抑制其侵袭和迁移(P <0. 05),促进细胞凋亡,且随着HSYA浓度的增加,细胞的侵袭和迁移能力逐渐降低,细胞凋亡率逐渐增加,细胞G0/G1期比例显著增加(P均<0. 05),S期细胞比例显著降低(P均<0. 05)。HSYA使VEGF、PI3K、AKT及Snail蛋白表达下降(P均<0. 05)。结论闪式提取HSYA工艺简单,提取率高,可以用于红花中HSYA的提取。同时HSYA能抑制肝癌细胞的增殖、侵袭和迁移,阻滞细胞周期停留在G0/G1期,诱导肝癌细胞凋亡,其机制可能与抑制PI3K/AKT/Snail信号通路的激活有关,为HSYA治疗肝癌的临床应用提供理论依据。

Objective To investigate the extraction of hydroxy safflor yellowA( HSYA) from Carthamus tinctorius L. by smashing tissue extraction method and the effects of HSYA on the growth,migration and invasion in liver cancer cells and its mechanism. Methods The extract content of HSYA as index of evaluation was used to explore the effects of extraction time,extraction voltage and optimum liquid-material ratio on extraction content and orthogonal test was to choose optimum extracting conditions. The different doses of HSYA were used to treat SM M C-7721 and HepG2 cells at 24,48,72 h. M TT assay was used to measure the cell proliferation,transwell assay was used to determine the invasion and migration,flowcytometry was used to determine the cell cycle and cell apoptosis,and the expression of VEGF,PI3 K,AKT and Snail were detected by Western blot. Results The optimum extracting conditions of smashing tissue extraction in HSYA were liquid-material ratio of 40 ∶ 1,3 min extraction time,and 80 V voltage. HSYA inhibited SM M C-7721 and HepG2 cells growth proliferation,and the inhibition has time-dose dependent effect( P < 0. 05). HSYA significantly inhibited the invasion and migration of cell( P < 0. 05),promoted the cell apoptosis( P < 0. 05),increased the percentage of cells in G0-G1 phase and decreased the percentage of cells in S phase( P all < 0. 05). HSYA significantly down-regulated the expression of VEGF,PI3 K,AKT and Snail( P all < 0. 05). Conclusion The smashing tissue extraction of HSYA is easy and effective,which can be used in the extraction of HSYA from Carthamus tinctorius L. M eanwhile,HSYA can suppress the SM M C-7721 and HepG2 cells growth,migration and invasion,block the cell cycle in G0/G1 phase and induce the cell apoptosis through inhibiting PI3 K/AKT/Snail signal pathway. It provides a theoretical support of HSYA in clinical application.