摘要
目的:蛋白聚糖TPG-1具有良好的抗肝癌活性,本研究进一步探究蛋白聚糖TPG-1抗肝癌的分子作用机制。方法:用槐耳蛋白聚糖TPG-1(0,0.05,0.1,0.25,0.5,1 g·L^(-1))处理人肝癌SK-HEP-1细胞,采用噻唑蓝(MTT)比色法检测槐耳蛋白聚糖TPG-1对SK-HEP-1细胞增殖的影响,采用流式细胞术检测TPG-1对SK-HEP-1细胞凋亡的影响。对经TPG-1处理过的人肝癌SK-HEP-1细胞进行基因芯片检测分析,探讨TPG-1抗肝癌的分子作用机制。结果:与空白组比较,槐耳蛋白聚糖TPG-1能够显著抑制人肝癌SK-HEP-1细胞的增殖能力(P<0.01)。TPG-1(1 g·L^(-1))作用于SK-HEP-1细胞48 h,SK-HEP-1细胞凋亡率显著增加(P<0.01),且TPG-1给药能够显著促进细胞凋亡重要执行者含半胱氨酸的天冬氨酸蛋白水解酶(Caspase)-3和Caspase-7的剪切水平(P<0.01)。与空白组比较,TPG-1(0.1 g·L^(-1))能够抑制SK-HEP-1细胞的迁移能力(P<0.05)。基因芯片检测分析结果显示,TPG-1处理后,SK-HEP-1细胞中差异表达基因有971个,其中表达上调基因486个,表达下调基因485个。基因本体(GO)和通路(Pathway)分析结果显示表达差异基因的功能主要涉及白细胞介素-6(IL-6)生物合成、抗原加工与呈递、超氧化物歧化酶活性、丝裂原活化蛋白激酶激酶激酶(MAPKKK)级联反应的正调控、自然杀伤(NK)细胞趋化、趋化因子生物合成等。此外,表达差异基因所涉及的信号通路主要包括核苷酸寡聚化结构域(NOD)样受体信号通路,凋亡,Toll样受体信号通路,维甲酸诱导基因-Ⅰ(RIG-Ⅰ)样受体信号通路,T细胞受体信号通路及趋化因子信号通路等。蛋白免疫印迹法结果显示,TPG-1(1 g·L^(-1))能够显著激活SK-HEP-1细胞中丝裂原活化蛋白激酶(MAPK)信号通路(P<0.01)。结论:槐耳蛋白聚糖TPG-1能够抑制人肝癌SK-HEP-1细胞增殖及迁移能力并促进其发生凋亡,MAPK信号通路的激活可能与TPG-1发挥抑制人肝癌SK-HEP-1细胞生长作用有关。
Objective: Proteoglycan TPG-1 isolated from Trametes robiniophila(Huaier)has proved to have anti-hepatoma activity, and this paper aims to explore the molecular mechanism. Method: Human hepatoma SK-HEP-1 cells were treated with TPG-1(0,0.05,0.1,0.25,0.5,1 g·L^(-1)). Then cell survival was detected by methyl thiazolyl tetrazolium(MTT)and apoptosis by flow cytometry. In addition,expression of genes in SK-HEP-1 cells treated with or without TPG-1 was examined by DNA microarray to preliminarily explore the anti-hepatoma molecular mechanism of TPG-1. Result: TPG-1 inhibited the proliferation of SKHEP-1 cells as compared with the blank group(P<0.01). After treatment with 1 g·L^(-1)TPG-1 for 48 h,the apoptosis rate of SK-HEP-1 cells increased(P<0.01),and TPG-1 promoted the cleavage of cysteinyl aspartate specific proteinase(Caspase)-3 and Caspase-7,the key mediators of apoptosis(P<0.01). Additionally,TPG-1(1 g·L^(-1))suppressed the migration of SK-HEP-1 cells(P<0.05). A total of 971 differentially expressed genes(DEGs)were identified in SK-HEP-1 cells after treatment with TPG-1,with 486 up-regulated and 485 downregulated. These DEGs were mainly involved in the Gene Ontology(GO) terms of interleukin-6(IL-6)biosynthesis,antigen processing and presentation,superoxide dismutase activity,positive regulation of mitogenactivated protein kinase kinase kinase(MAPKKK)cascade,nature killer(NK)cell chemotaxis,and chemokine biosynthesis,and the Kyoto Encyclopedia of Genes and Genomes(KEGG)pathways of nucleotide-binding oligomerization domain(NOD)-like receptor signaling pathway, apoptosis, Toll-like receptor signaling pathway,retinoic acid-inducible gene-Ⅰ(RIG-Ⅰ )-like receptor signaling pathway,T-cell receptor signaling pathway,and chemokine signaling pathway. Western blot results showed that TPG-1(1 g·L^(-1))activated mitogenactivated protein kinase(MAPK)signaling pathway in SK-HEP-1 cells(P<0.01). Conclusion: Proteoglycan TPG-1 inhibited the proliferation and migration,and induced apoptosis of human hepatoma SK-HEP-1 cells. Upregulation of MAPK signaling pathway may be responsible for the growth inhibition of human hepatoma SKHEP-1 cells by TPG-1.
作者
杨爱琳
黄惠铭
刘亚鑫
欧阳里山
范海涛
屠鹏飞
胡仲冬
YANG Ai-lin;HUANG Hui-ming;LIU Ya-xin;OUYANG Li-shan;FAN Hai-tao;TU Peng-fei;HU Zhong-dong(Modern Research Center for Traditional Chinese Medicine,School of Chinese Materia Medica,Beijing University of Chinese Medicine,Beijing 100029,China;College of Bioengineering,Beijing Polytechnic,Beijing\00029,China)
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2022年第1期72-78,共7页
Chinese Journal of Experimental Traditional Medical Formulae
基金
北京市科技新星计划项目(Z191100001119083)
中华中医药学会青年人才托举工程项目[CACM-2018-(QNRC2-B05)]。
