Nrf2活化正向调控紫草素诱导的HL-60细胞分化

Positive role of Nrf2 in regulating shikonin-induced HL-60 cell differentiation

目的探讨核因子E2相关因子2（Nrf2）在紫草素诱导HL-60细胞分化中的作用。方法取对数生长期HL-60细胞,分别采用不同浓度紫草素、Nrf2激动剂叔丁基对苯二酚（t BHQ）、Nrf2抑制剂鸦胆子苦醇作用。通过Hoechst 33258染色、MTT法观察HL-60细胞形态和增殖情况,NBT试验检测HL-60细胞还原能力,流式细胞术检测HL-60细胞分化率。ELISA法测定HL-60细胞核内Nrf2含量,RT-PCR或real-time PCR法检测HL-60细胞Nrf2、CD11b、CD14 m RNA表达水平及Nrf2下游基因的表达水平。结果经紫草素（50～200μg·L-1）作用,HL-60细胞形态、还原能力均趋向成熟,与空白对照相比细胞抑制率、分化率、Nrf2含量及其下游基因GCLC、NQO1、GCLM m RNA的表达量均升高（P〈0.05或P〈0.01）,且随紫草素浓度增高有上升的趋势。紫草素与t BHQ联合作用,HL-60细胞CD11b、CD14、NQO1、GCLM m RNA表达量高于紫草素组（P〈0.05或P〈0.01）;紫草素与鸦胆子苦醇联合作用,HL-60细胞CD11b、CD14、GCLM m RNA表达量低于紫草素组（P〈0.05或P〈0.01）。结论 Nrf2活化正向调控紫草素诱导的HL-60细胞分化。

AIM To investigate the role of nuclear factor erythroid-2 related factor 2 molecules（Nrf2）in shikonin induced HL-60 differentiation.METHODS The HL-60 cells at logarithmic phase were selected and to study the effects of different concentration of shikonin,t BHQ（Nrf2 agonist） and brusatol（Nrf2 inhibitor）on HL-60 cells.The morphology and proliferation of HL-60 cells were detected by Hoechst 33258 stain and MTT assay.The reducing capacity was detected by NBT reducing test and the differentiation by flow cytometry.The Nrf2 content in HL-60 cell nucleus was analyzed by ELISA and the m RNA expression levels of Nrf2,CD11 b,CD14,and the downstream gene of Nrf2 were determined by RT-PCR and real-time PCR.RESULTS Induced by shikonin（50-200 μg·L-1）,the morphology and reducing capacity of HL-60 cells turned into those of the mature（grain） cells.Compared with the blank control group,the inhibition ratio,differentiation rate,proliferation markedly inhibited proliferation,levels of Nrf2 and its downstream gene GCLC,NQO1,GCLM m RNA expression were higher in the HL-60 cells induced by shikonin（P 〈0.05 or P 〈0.01）.And above indexes have and upward trend with shikonin concentration increased.The CD11 b,CD14,NQO1,GCLM m RNA expression levels of HL-60 cells in the shikonin ＋ t BHQ group were higher than those in the shikonin group（P 〈0.05 or P 〈0.01）.The CD11 b,CD14,GCLM m RNA expression levels of HL-60 cells in the shikonin ＋brusatol group were lower than those in the shikonin group（P 〈0.05 or P〈 0.01）.CONCLUSION Nrf2 activation has a positive regulatory role in the differentiation of HL-60 cells caused by shikonin.