珠子参皂苷对人胃癌细胞HGC-27的抑制作用及机制研究  

Inhibition and Mechanism of Saponins from Rhizoma Panacis Majoris Against HGC-27 Human Gastric Cancer Cells

作　　者：张媛媛[1] 陈烨韬 石孟琼[2] 朱丽金 江伟杰 张继红[3] 陈茂华[3] 许登清[1] 邹坤[1] Zhang Yuanyuan;Chen Yetao;Shi Mengqiong;Zhu Lijin;Jiang Weijie;Zhang Jihong;Chen Maohua;Xu Dengqing;Zou Kun(Hubei Key Laboratory of Natural Products Research and Development&Yichang Key Laboratory of Development and Utilization of Health Products with Drug Food Homology,College of Biological and Pharmaceutical Sciences,China Three Gorges University,Yichang 443002;Medical College,China Three Gorges University,Yichang 443002;Chinese Medicine Clinical Medical College,China Three Gorges University,Yichang 443002)

机构地区：[1]三峡大学天然产物研究与利用湖北省重点实验室&药食同源大健康产品开发利用宜昌市重点实验室生物与制药学院,宜昌443002 [2]三峡大学医学院,宜昌443002 [3]三峡大学中医临床医学院,宜昌443002

出　　处：《中药药理与临床》2021年第4期23-31,共9页Pharmacology and Clinics of Chinese Materia Medica

基　　金：国家自然科学基金委员会项目(编号:81341141);湖北省卫生和计划生育委员会项目(鄂卫生计生通[2017]20号);三峡大学硕士学位论文培优基金项目(编号:2017YPY086、2018SSPY140);湖北省生物酵素工程研究技术研究中心项目(编号:JS2018-06)。

摘　　要：目的:探讨珠子参皂苷对胃癌细胞HGC-27抑制作用及可能的机制。方法:采用MTT法和乳酸脱氢酶(LDH)释放实验检测珠子参皂苷10、20、40、60、80、100、120μg/mL对肿瘤细胞(HGC-27、A549、MCF-7)和正常细胞(MCF10a、HL-7702)增殖的影响;划痕试验和Transwell试验检测细胞迁移、侵袭能力,平板克隆和Hoechst 33258染色试验检测细胞增殖和凋亡,流式细胞术检测线粒体活性,双荧光素酶报告试验证实miR-10a靶向抑制磷脂酰肌醇-3-激酶催化亚单位α(Pik3ca)的表达;Real-time PCR法检测miR-10a、Bcl-2、Bcl-xl、Bax、Bad mRNA表达,Western blot法检测p-PIK3CA、PIK3CA、p-AKT、AKT、非甾体类抗炎药相关基因-1(NAG-1)、BCL-2、BCL-XL、BAX、BAD、细胞色素c(Cytochrome c)、pro-Caspase-3、pro-Caspase-9、Cleaved-caspase-3、Cleaved-caspase-9、凋亡蛋白酶活化因子-1(Apaf-1)、X连锁凋亡抑制蛋白(XIAP)、凋亡诱导因子(AIF)蛋白表达。结果:珠子参皂苷对HGC-27细胞的增殖具有较强抑制作用(IC_(50)=29.77μg/mL),呈浓度依赖性显著抑制HGC-27细胞增殖、迁移和侵袭,明显降低线粒体活性、增加LDH释放率、诱导细胞凋亡(P<0.05或P<0.01),证实miR-10a可靶向抑制Pik3ca表达,珠子参12.5、25、50μg/mL可上调miR-10a、Bax、Bad mRNA表达,下调Bcl-2、Bcl-xl mRNA表达,上调Cytochrome c、Cleaved-caspase-3、Cleaved-caspase-9、Apaf-1、AIF蛋白表达和增加BAX/BCL-2、BAD/BCL-XL蛋白及基因比率,下调NAG-1、pro-Caspase-3、pro-Caspase-9、XIAP蛋白表达和降低p-PIK3CA/PIK3CA、p-AKT/AKT比率。结论:珠子参皂苷能够抑制HGC-27胃癌细胞增殖、迁移和侵袭,并诱导其凋亡,其机制可能与上调miR-10a表达、抑制PI3K/AKT/NAG-1信号通路激活,进而参与调控BCL-2及Caspase家族途径有关。Objective:To investigate the inhibitory effect of saponins from Rhizoma Panacis Majoris on HGC-27 human gastric cancer cells and its possible mechanism. Methods: The effects of saponins from Rhizoma Panacis Majoris at different concentrations(10, 20, 40, 60, 80, 100, and 120 μg/mL) on the proliferation of tumor cells(HGC-27, A549, and MCF-7) and normal cells(MCF10 a and HL-7702) were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) and lactate dehydrogenase(LDH) release assays. The scratch assay and Transwell test were conducted to evaluate cell migration and invasion, followed by the determination of cell proliferation and apoptosis by colony-forming assay and Hoechst 33258 staining. Mitochondrial activity was detected by flow cytometry, and the expression of phosphatidylinositol-3-kinase catalytic subunit α(PIK3 CA) was confirmed by dual luciferase reporter assay. The mRNA expression levels of miR-10 a, Bcl-2, Bcl-xl, Bax and Bad were assayed by real-time PCR. The protein expression levels of phosphorylated PIK3 CA(p-PIK3 CA), PIK3 CA, p-Akt, Akt, nonsteroidal anti-inflammatory drug-activated gene-1(NAG-1), Bcl-2, Bcl-xl, Bax, Bad, cytochrome c, pro-Caspase-3, pro-Caspase-9, cleaved-Caspase-3, cleaved-Caspase-9, apoptotic protease activating factor-1(Apaf-1), X-linked inhibitor of apoptosis protein(XIAP) and apoptosis-inducing factor(AIF) were measured by Western blotting. Results: Saponins from Rhizoma Panacis Majoris had a strong inhibitory effect against the proliferation of HGC-27 cells(IC_(50)=29.77 μg/mL). Apart from significantly inhibiting the proliferation, migration and invasion of HGC-27 cells in a concentration dependent manner, it also decreased the mitochondrial activity, increased the LDH release rate, and induced cell apoptosis(P<0.05 or P<0.01). It was confirmed that miR-10 a inhibited the expression of PIK3 CA. Saponins from Rhizoma Panacis Majoris at 12.5, 25, and 50 μg/mL up-regulated the mRNA expression levels of miR-10 a, Bax, Bad, Bax/Bcl-2, and Bad/Bcl-xl

关 键 词：珠子参皂苷 HGC-27细胞 miR-10a 磷脂酰肌醇-3-激酶/蛋白激酶B/非甾体类抗炎药相关基因-1通路 线粒体凋亡通路 

分 类 号：R285.5[医药卫生—中药学]