基于PI3K/Akt/NF-κB信号通路探讨商陆果发酵液体外抗肝癌作用

Exploring the Anti-Hepatoma Effect of Fermentation Liquor of Fresh Fruit of Phytolacca acinosa Roxb. in Vitro Based on PI3K/Akt/NF-κB Signaling Pathway

关键词:  商陆果发酵液  Bel-7402肝癌细胞  周期阻滞  PI3K/Akt/NF-κB信号通路

Key words:  Fermentation liquor of fresh fruit of Phytolacca acinosa Roxb.  Bel-7402 hepatoma cells  cell cycle arrest  PI3K/Akt/NF-κB signaling pathway

摘要:

目的 基于磷脂酰肌醇3-激酶（PI3K）/蛋白激酶B（Akt）和核转录因子（NF）-κB信号通路探讨商陆果发酵液及其组分对Bel-7402肝癌细胞的作用。方法 采用超声提取、乙醇醇沉、732阳离子交换树脂以及D101大孔吸附树脂柱色谱分离技术的联合使用将商陆果发酵液进行分离。以人肝癌细胞Bel-7402为研究对象，采用细胞活力检测试剂盒（CCK8）法考察商陆果发酵液及其组分对其增殖能力的影响，以获得主要药效物质以及合适剂量用于后续实验。利用平板克隆形成实验、细胞划痕实验、流式细胞术以及蛋白质印迹法（Western Blot）检测生物碱组分对Bel-7402肝癌细胞克隆形成能力、迁移能力、凋亡和周期的影响及其作用机制。结果 相同浓度梯度下，商陆果发酵液生物碱组分抑制Bel-7402肝癌细胞增殖效果最佳，IC50浓度在0.75 mg·mL-1左右，即选择0.375 mg·mL-1、0.75 mg·mL-1、1.5 mg·mL-1的生物碱作为后续实验低、中、高剂量组。后续实验发现生物碱组分可以降低Bel-7402肝癌细胞的克隆形成能力和迁移能力，促进细胞凋亡，阻滞细胞于S期和G2/M期。进一步研究发现，生物碱组分能上调Bel-7402肝癌细胞中p53蛋白和促凋亡蛋白Bax表达水平，下调PI3K、Akt、NF-κB P65、环氧化酶2（COX-2）、血管内皮生长因子（VEGF）和抗凋亡蛋白Bcl-2的表达水平，导致Bax/Bcl-2比率增加。结论 商陆果发酵液体外治疗肝癌主要药效物质基础是生物碱组分，其可能通过抑制PI3K/Akt/NF-κB信号通路活化，进而增加Bax/Bcl-2比值来促进细胞凋亡和下调VEGF抑制细胞增殖，从而发挥抗肿瘤作用。

Abstract:

OBJECTIVE To explore the effect of fermentation liquor of fresh fruit of Phytolacca acinosa Roxb. on Bel-7402 hepatoma cells based on phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) / nuclear factor (NF)-κB signaling pathway. METHODS Fermentation liquor of fresh fruit of Phytolacca acinosa Roxb.was isolated by the combined use of ultrasonic extraction, ethanol precipitation, 732 cation exchange resin and D101 macroporous adsorption resin. Human hepatoma cell line Bel-7402 was used as the research object. The effects of fermentation liquor of fresh fruit of Phytolacca acinosa Roxb. and its components on its proliferation were investigated by Cell Counting Kit-8 (CCK8), in order to obtain the main active substances and appropriate doses for follow-up experiments. Using plate clone formation test, cell scratch test, flow cytometry and western blot to detect the effects of alkaloid components on clone formation ability, migration ability, apoptosis and cell cycle of Bel-7402 hepatoma cells and its mechanism. RESULTS Under the same concentration gradient, the alkaloid component of fermentation liquor of fresh fruit of Phytolacca acinosa Roxb. had the best inhibitory effect on the proliferation of Bel-7402 hepatoma cells, and the concentration of IC50 was about 0.75 mg·mL-1, that is, the alkaloids of 0.375 mg·mL-1, 0.75 mg·mL-1 and 1.5mg·mL-1 were selected as the low, middle and high dose groups in the follow-up experiment. In follow-up experiments, it was found that alkaloid components could reduce the clone formation and migration ability of Bel-7402 hepatoma cells, promote apoptosis, and block cells in S phase and G2/M phase. Further studies showed that alkaloids could up-regulate the expression levels of p53 protein and pro-apoptotic protein Bax in Bel-7402 hepatoma cells, and down-regulate the expression levels of PI3K, Akt, NF- κB P65, Cyclooxygenase 2 (COX-2), vascular endothelial growth factor (VEGF) protein and anti-apoptotic protein Bcl-2, resulting in an increase in Bax/Bcl-2 ratio. CONCLUSION The main active substances of fermentation liquor of fresh fruit of Phytolacca acinosa Roxb. in the treatment of hepatocellular carcinoma in vitro is alkaloid components, which may exert its anti-tumor effect by inhibiting the expression of proteins related to PI3K/Akt/NF- κB signaling pathway. and then increasing the ratio of Bax/Bcl-2 to promote apoptosis and decrease VEGF to inhibit cell proliferation.