Effects of Euphorbiasteroid on gene expression in lung cancer cells based on gene chip

Objective Using gene chip technology to explore the molecular mechanism of euphorbiasteroid in the treatment of non-small cell lung cancer(NSCLC).Methods A549 cells were used as the in vitro model,and they were randomly divided into control group and different concentrations of euphorbiasteroid administration groups.Each group had 3 duplicate wells,after the cells were cultured in vitro,the cell viability was evaluated by CCK-8 method.Gene chip technology was used to screen the differentially expressed genes(DEGs)between the control group and the euphorbiasteroid administration group.The differential genes were further analyzed for Gene Ontology(GO)function enrichment and Kyoto Encyclopedia of Genes and Genome(KEGG)pathway enrichment analysis.The STRING online analysis platform combined with Cytoscape software to construct a target protein interaction(PPI)network and perform topological analysis to screen key targets,and use Real-time PCR(RT-PCR)and molecular docking technology to verify key targets.Results According to the analysis of gene chip data,276 differentially expressed genes were screened,including 117 up-regulated genes and 159 down-regulated genes.GO analysis showed that differentially expressed genes were mainly involved in cell division,cell proliferation,cell cycle and other processes,involving protein binding,protein kinase binding and other functions,and were mainly distributed in nucleoplasm,chromosomes and other parts.KEGG signaling pathway analysis showed that differentially expressed genes were involved in cell cycle,pyrimidine metabolism,p53 signaling pathway and other pathways.PPI network analysis showed that CCNA2,TOP2A,CCNB1,CDC20,and RRM2 may be the key targets of euphorbiasteroid in the treatment of NSCLC.RT-PCR results showed that the expressions of CCNA2,TOP2A,CCNB1,CDC20,and RRM2 were significantly down-regulated in the euphorbiasteroid administered group,which was consistent with the gene chip results.Molecular docking results showed that euphorbiasteroid had good affinity with key targets and could bind spontaneously and stably.Conclusion The combination of gene chip,RT-PCR technology and molecular docking technology can find out the differential genes after the intervention of euphorbiasteroid,which can be used to explore the mechanism of euphorbiasteroid in the treatment of NSCLC.

HPLC测定千金子和千金子霜中两种泻下成分的含量

目的:对不同地区千金子药材及其炮制品千金子霜中泻下成分续随二萜酯和千金二萜醇二乙酸苯甲酸酯的含量进行测定。方法:采用高效液相色谱法,流动相为甲醇-水(68∶32),kromasil C18柱(150mm×4.6mm,5μm),柱温:40℃,检测波长:280nm。结果:续随二萜酯和千金二萜醇二乙酸苯甲酸酯分别在0.200μg～1.200μg、0.206μg～1.651μg范围内,线性关系良好,r=0.9997和r=0.9999。不同产地千金子中所含两种成分的量略有差别,续随二萜酯的质量分数在0.4508%～0.6251%,多数在0.4500%以上。千金二萜醇二乙酸苯甲酸酯的质量分数在0.6923%～0.7529%,多数在0.7200%以上。千金子霜中两种成分的质量分数分别在0.1568%～0.2211%(多数在0.2000%以下)和0.2322%～0.2830%(多数在0.3000%以下)。结论:建立了高效液相色谱法测定千金子脂肪油中两种泻下成分含量的方法,该方法准确、可靠,可用于毒性中药千金子及其炮制品的质量控制。

基于网络药理学和分子对接技术研究千金子引发腹泻的作用机制

目的基于网络药理学和分子对接技术探讨千金子引发腹泻的作用机制。方法通过TCMSP数据库收集千金子化学成分信息,在PubChem、SwissTargetPrediction和GeneCards等数据库收集其化学成分作用靶点及腹泻相关靶点,利用DAVID数据库对千金子与腹泻的共有靶点进行基因本体(gene ontology,GO)功能富集及京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes,KEGG)通路富集;使用Cytoscape软件构建PPI网络图及“活性成分-靶点-疾病”网络,筛选出主要活性成分与核心靶点进行分子对接。培养人结肠癌细胞系HCT116,采用实时荧光定量PCR(real time quantitative PCR,RT-qPCR)验证计算模拟结果。结果经筛选得到千金子潜在活性成分12个及其对应靶点389个,腹泻相关靶点570个,二者共同靶点43个。GO功能富集分析发现共同靶点主要参与ATP结合和ERBB2信号传导途径等生物过程,KEGG信号通路分析发现千金子主要影响Rap1信号通路、PI3K-AKT信号通路、Ras信号通路、ERBB信号通路、HIF-1信号通路、HTLV-I感染和FoxO信号通路。分子对接结果显示,各活性成分与SRC、PIK3CA、AKT1和PIK3R1等靶点结合良好。千金子甾醇可抑制细胞活性,使EGFR和mTOR基因表达异常。结论千金子可能通过beta-sitosterol、stigmasterol、artemetin和euphorbiasteroid等关键活性成分作用于SRC、PIK3CA、AKT1和PIK3R1等靶点和通路,从而引发腹泻。

Fas／FasL信号通路在千金子甾醇诱导HL-60细胞凋亡中的作用与机制研究

目的：在明确千金子甾醇诱导HL-60白血病细胞发生凋亡的基础上，从Fas/FasL信号通路的角度探讨其诱导凋亡的分子机制。方法 HL-60细胞培养体系中分别以千金子甾醇终浓度2．5、10、40μg/ml作用24 h后CCK-8法检测千金子甾醇对HL-60细胞增殖的抑制作用，光学及荧光显微镜下观察细胞形态学变化，Annexin Ⅴ/PI 流式细胞术检测细胞凋亡，反转录-聚合酶链反应法检测Fas/FasL、caspase-8和caspase-3 mRNA转录水平，比色法检测caspase-8和caspase-3的相对活性。结果千金子甾醇可明显抑制HL-60细胞的增殖，增殖抑制率分别为（34．9±3．7）％、（54．6±5．2）％、（61．3±4．3）％，细胞呈典型凋亡形态学改变，细胞早期凋亡率分别为（23．4±3．1）％、（35．7±4．3）％、（53．2±3．9）％。Fas、FasL、caspase-8和caspase-3 mRNA转录水平明显上调（P＜0．01），caspase-8和caspase-3活性显著增高（P＜0．01）。结论千金子甾醇可以诱导HL-60细胞发生剂量依赖性细胞凋亡，其机制与上调Fas/FasL凋亡信号通路有关。

千金子甾醇诱导HL-60细胞凋亡机制研究

目的探讨Bcl-2/Bax信号通路在千金子甾醇诱导HL-60白血病细胞发生凋亡中的作用及其分子机制研究。方法HL-60细胞培养体系中分别加入大、中、小剂量千金子甾醇溶液,甾醇溶液作用细胞24h后,采用CCK-8法检测千金子甾醇对HL-60细胞增殖的抑制作用,光镜下观察细胞形态学变化,AnnexinⅤ/PI流式细胞术检测细胞凋亡,RT-PCR法检测Bcl-2/Bax、Caspase-9和Caspase-3mRNA转录水平,ELISA法检测Caspase-9和Caspase-3蛋白活性。结果 CCK-8法检测结果显示,与对照组比较,千金子甾醇能明显抑制HL-60细胞增殖(F=42.97,P<0.001),各个浓度之间进行比较,差异有统计学意义,P<0.05。流式细胞术检测细胞凋亡率显著增加(F=56.74,P<0.001),经10、20和40μg/mL千金子甾醇处理24h后,HL-60细胞早期凋亡率分别为(23.4±3.1)%、(35.7±4.3)%和(53.2±3.9)%,细胞呈典型凋亡形态学改变。千金子甾醇作用后,Bax mRNA转录水平显著升高,Bcl-2mRNA转录水平显著降低,且呈化合物剂量依赖性(F=53.45,P<0.001),Caspase-9和Caspase-3 mRNA转录水平升高,且呈化合物剂量依赖性(F=34.21,P<0.001),千金子甾醇对HL-60细胞作用24h后Caspase-9和Caspase-3蛋白活性显著升高(F=54.33,P<0.001),且随着千金子甾醇浓度增加蛋白活性逐渐升高。结论千金子甾醇通过调节Bcl-2/Bax凋亡信号通路,诱导HL-60细胞凋亡,其作用呈明显剂量依赖性。