常绿钩吻碱抑制人神经胶质瘤U251细胞增殖的体内外作用特征

常绿钩吻碱(sempervirine,SPV)是从钩吻中分离得到的一种育亨宾型生物碱,本研究探讨了常绿钩吻碱抑制人神经胶质瘤细胞体内外增殖的作用特征。以0~16μmol·L^-1不同浓度常绿钩吻碱处理人神经胶质瘤U251细胞24、48和72 h,分别用MTT法和集落形成实验检测细胞增殖,Hoechst细胞核染色和Annexin V-FITC/PI染色法检测细胞凋亡,Western blot法检测PI3K、AKT、p-AKT、Bax、Bcl-2、caspase-3和cleaved caspase-3蛋白。采用体内裸鼠移植神经胶质瘤模型,观察常绿钩吻碱抗神经胶质瘤的体内作用,所有动物实验操作均严格遵守福建医科大学生物医学伦理委员会相关规定。结果显示,常绿钩吻碱在1~8μmol·L^-1能显著抑制U251细胞的增殖和集落形成,引起细胞核固缩,促进细胞凋亡,蛋白检测表明,其促进caspase-3的切割活化,下调Bcl-2/Bax值和PI3K蛋白的表达,抑制AKT的磷酸化;裸鼠移植瘤实验结果表明,剂量4和8 mg·kg^-1·day^-1常绿钩吻碱干预能抑制体内神经胶质瘤的生长,肿瘤重量分别减少了44.76%和61.26%。本研究在体内外水平表明,常绿钩吻碱能明显抑制神经胶质瘤的增殖,为其抗神经胶质瘤的研究开发奠定基础。

Sempervirine inhibits proliferation of human glioma U251 cells in vitro and in vivo

Sempervirine,a yohimbane-type alkaloid isolated from Gelsemium elegans,was found to significantly inhibit the cellular proliferation of U251 cells in vitro and in vivo in a dose-dependent manner.U251 cells were treated with 0-16μmol·L^-1 of sempervirine for 24,48 or 72 h.An MTT assay and clone formation assay were used to investigate cell survival and clone formation.Hoechst staining and Annexin V-FITC/PI staining were used to measure cell apoptosis.The expression of PI3 K,AKT,p-AKT,Bax,Bcl-2,caspase-3 and cleaved caspase-3 was determined by Western blot analysis.The antitumor effect of sempervirine in vivo was investigated by inoculating nude mice with U251 cells.All animal experiments were in strict accordance with the regulations of the Biomedical Ethics Committee of Fujian Medical University(Fujian,China).The results show that sempervirine significantly inhibits the proliferation and induces the apoptosis of U251 cells,promotes cleavage of caspase-3,down-regulates the protein expression of PI3 K and Bcl-2/Bax,and inhibits phosphorylation of AKT in vitro.Intraperitoneal injection of 4 or 8 mg·kg^-1·day^-1 of sempervirine inhibits U251 cells tumor growth in the xenograft nude mice,and tumor weight decreased by 44.76%and 61.26%,respectively.Our study shows that sempervirine significantly inhibits the proliferation of U251 cells in vitro and in vivo,laying a foundation for further research and development of its anti-glioma effect.