双去甲氧基姜黄素对小鼠脑神经母瘤细胞的促神经分化作用及机制研究

Effects of bisdemethoxycurcumin promoting neuronal differentiation of neuroblastoma cells in mice and its mechanism

目的研究姜黄素衍生物双去甲氧基姜黄素(BC)对小鼠脑神经母瘤细胞Neuro-2a(N2a)的促神经分化作用及机制。方法采用MTT法检测BC(1、2、4、6、8、10μmol/L)对N2a细胞存活率的影响,确定药物处理浓度范围。设对照组、视黄酸(RA)组(10μmol/L)和BC组(1、2、4μmol/L),培养48、72 h后,对分化细胞的神经突起长度进行测量并计算细胞分化率;采用Western blot法检测4μmol/L BC作用5、15、30、60、120 min后细胞中蛋白激酶B(Akt)、细胞外调节蛋白激酶1/2(ERK1/2)、p38丝裂原活化蛋白激酶(p38)蛋白的磷酸化水平。以抑制剂LY294002(LY)和PD98059(PD)干预后,进一步验证BC对Akt和ERK蛋白磷酸化水平及促神经分化的影响。结果根据MTT实验确定后续诱导细胞分化的BC浓度为1、2、4μmol/L。分化48 h后,与对照组比较,RA组和BC 1、2、4μmol/L组细胞分化率及BC 4μmol/L组细胞神经突起长度均显著升高/增加(P<0.05或P<0.01);BC继续诱导分化至72 h后,与对照组比较,RA组细胞分化率和神经突起长度、BC 4μmol/L组细胞分化率和BC 2μmol/L组细胞神经突起长度均显著升高/增加(P<0.05或P<0.01)。与0 min组比较,BC 4μmol/L作用5、15、30、60、120 min组细胞中Akt、ERK1/2、p38蛋白的磷酸化水平均有不同程度升高,部分差异有统计学意义(P<0.05或P<0.01)。加入抑制剂LY/PD后,与BC组比较,PD+BC组细胞中ERK1/2蛋白的磷酸化水平显著降低(P<0.01),LY组、LY+BC组、PD组、PD+BC组细胞分化率均显著降低(P<0.01)。结论BC可以促进N2a细胞分化,增加细胞分化率和神经突起长度,其机制可能与激活MEK/ERK和PI3K/Akt信号通路有关。

OBJECTIVE To study the effects of the curcumin derivative bisdemethoxycurcumin(BC)promoting neuronal differentiation of neuroblastoma cells Neuro-2a(N2a)in mice and its mechanism.METHODS The effects of BC(1,2,4,6,8,10μmol/L)on the viability of N2a cells were detected by MTT assay to determine the concentration range of drug treatment.The control group,retinoic acid(RA)group(10μmol/L)and BC groups(1,2 and 4μmol/L)were set up,and the length of neural protrusions of the differentiated cells was measured and the cell differentiation rate was calculated after 48 h and 72 h of culture.Compared with 0 min group,Western blot was used to detect the phosphorylation levels of protein kinase B(Akt),extracellularsignal regulated kinase 1/2(ERK1/2),and p38 mitogen-activated protein kinase(p38)proteins in cells treated by 4μmol/L BC for 5,15,30,60,120 min.After intervention with inhibitors LY294002(LY)and PD98059(PD),the effects of BC on Akt and ERK1/2 protein phosphorylation levels and promoting neural differentiation were further validated.RESULTS According to the MTT experiment,the BC concentrations for subsequent induction of cell differentiation were determined to be 1,2,and 4μmol/L.After 48 hours of differentiation,compared with the control group,the cell differentiation rate in RA group and BC 1,2 and 4μmol/L groups,the length of cellular neural processes in the BC 4μmol/L group significantly increased(P<0.05 or P<0.01);after inducing differentiation of BC for 72 hours,compared with the control group,the cell differentiation rate and the length of cellular neural processes in the RA group,the cell differentiation rate in the BC 4μmol/L group,and the length of cellular neural processes in the BC 2μmol/L group all significantly increased(P<0.05 or P<0.01).Compared with the 0 min group,the phosphorylation levels of Akt,ERK1/2,and p38 proteins in cells of the 5,15,30,60 and 120 min groups increased to varying degrees after treated by 4μmol/L BC,and some differences were statistically significant(P<0.05 or P<0.01).After adding the inhibitor LY/PD,compared with the BC group,the phosphorylation level of ERK1/2 protein in the PD+BC group cells were significantly reduced(P<0.01),and the cell differentiation rates in the LY group,LY+BC group,PD group,and PD+BC group was significantly reduced(P<0.01).CONCLUSIONS BC promotes N2a cell differentiation mainly by increasing cell differentiation rate and neural protrusion length.The mechanism may be related to the activation of mitogen-activated protein kinase/ERK and PI3K/Akt signaling pathways.