半枝莲碱B抑制胶质瘤U251细胞增殖并诱导DNA损伤与凋亡的实验研究

Scutebarbatine B suppresses proliferation and induces apoptosis of hu⁃man glioma U251 cells

目的:探讨半枝莲碱B(SBT-B)对人胶质瘤U251细胞增殖、DNA损伤和凋亡的影响。方法:U251细胞经SBT-B(0.05、0.1和0.2μmol/L)处理24 h,台盼蓝染色检测细胞活力;参照半数抑制浓度(IC50),将U251细胞分为对照(control)组和SBT-B处理组(处理浓度分别为0.05、0.1和0.2μmol/L);5-乙炔基-2'-脱氧尿苷(EdU)掺入法检测SBT-B对U251细胞增殖的影响;免疫荧光染色检测DNA损伤;TUNEL细胞凋亡检测试剂盒检测细胞凋亡;同时,SBT-B处理U251细胞24 h,Western blot检测细胞凋亡、DNA损伤修复及丝裂原活化蛋白激酶(MAPK)通路相关蛋白表达水平。结果:SBT-B显著抑制U251细胞活力和增殖(P<0.05)。U251细胞经SBT-B作用24 h后,磷酸化组蛋白H2AX(γ-H2AX)灶点形成及表达水平显著增加(P<0.05),DNA损伤修复蛋白Rad51的表达水平则下调(P<0.05);SBT-B显著诱导U251细胞凋亡(P<0.05),同时促进caspase-8、caspase-9和聚(ADP-核糖)聚合酶(PARP)的剪切(P<0.05)。SBT-B处理24 h后,细胞外信号调节激酶(ERK)、c-Jun氨基末端激酶(JNK)和p38 MAPK的磷酸化水平显著增加(P<0.05)。结论:SBT-B抑制U251细胞增殖,并诱导细胞DNA损伤和凋亡,其可能通过激活MAPK信号通路而对胶质瘤细胞发挥抑制作用。

AIM:To investigate the effect of scutebarbatine B(SBT-B)on the proliferation,DNA damage and apoptosis of human glioma U251 cells.METHODS:The U251 cells were treated with various concentrations(0.05,0.1 and 0.2μmol/L)of SBT-B for 24 h,and the cell viability was detected by Trypan blue staining assay.The U251 cells were divided into control group,0.05μmol/L SBT-B group,0.1μmol/L SBT-B group and 0.2μmol/L SBT-B group ac⁃cording to the value of IC50.EdU incorporation assay was used to determine the inhibitory effect of SBT-B(0.1 and 0.2μmol/L)on the cell proliferation.DNA damage was measured by immunofluorescence assay.Apoptosis was detected by TUNEL method.The expression levels of apoptosis-related proteins,DNA damage repair-related proteins and mitogen-activated protein kinase(MAPK)signaling pathway-related proteins were detected by Western blot.RESULTS:Treatment with SBT-B inhibited the viability and proliferation of U251cells(P<0.05).Treatment with SBT-B for 24 h significantly increasedγ-H2AX foci formation and protein levels,wihile the expression of Rad51 was down-regulated(P<0.05).Treat⁃ment with SBT-B induced cell apoptosis and cleavage of caspase-8,caspase-9 and poly(ADP-ribose)polymerase(PARP)in U251 cells(P<0.05).In addition,SBT-B increased the phosphorylation of extracellular signal-regulated kinase(ERK),c-Jun N-terminal kinase(JNK)and p38 MAPK(P<0.05).CONCLUSION:Treatment with SBT-B inhibits cell proliferation and triggers DNA damage and apoptosis through MAPK signaling pathway in U251 cells.