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目的 3,6’-二芥子酰基蔗糖(DISS)是远志中的寡糖酯类成分,通过研究DISS对人神经母细胞瘤细胞SH-SY5Y的增殖作用和对环磷酸腺苷反应元件结合蛋白(CREB)通路的影响,探究其发挥神经保护作用的分子机制。方法采用四甲基噻唑蓝(MTT)法测定SH-SY5Y细胞生长曲线,筛选最佳种板密度和给药时间,并检测不同浓度DISS对细胞增殖的影响;采用Western blotting法检测细胞给予DISS后不同时间点CREB、磷酸化CREB(p-CREB)和脑源性神经生长因子(BDNF)的表达水平;给予调节CREB活性的相关上游通路拮抗剂U0126(MEK拮抗剂)、H89(PKA拮抗剂)、KN93(CaMK拮抗剂)拮抗CREB上游通路,并给予DISS保护,用Western blotting法检测DISS对拮抗前后细胞内CREB,p-CREB,BDNF蛋白表达的影响。结果 SH-SY5Y最佳中板密度为2×108L-1,DISS(60μmol·L-1,30μmol·L-1)对SH-SY5Y细胞具有明显的促增殖作用(P<0.05);与正常细胞相比,DISS给药15 min时可显著增加细胞内p-CREB,BDNF的表达(P<0.05);DISS可明显增加拮抗后SH-SY5Y细胞内CREB,p-CREB,BDNF的表达水平(P<0.05)。结论 DISS可以调节CREB活性并促进下游BDNF等相关因子的表达,其神经保护作用的机制可能与活化Ca2+/CaM/CaMK-CREB-BDNF信号通路有关。
Aim To investigate the effect of DISS on the proliferation of human neuroblastoma cell line SH-SY5Y and the effect of cyclic adenosine monophosphate reactive element binding protein ( CREB) pathway to explore its molecular mechanism of neuroprotection. Methods The growth curve of SH-SY5Y cells was determined by MTT method. The optimal density and administration time of SH-SY5Y cells were determined, and the effects of different concentrations of DISS on the proliferation of SH-SY5Y cells were detected. The cells were treated with DISS by Western blotting (CREB), phosphorylated CREB (p-CREB) and brain derived neurotrophic factor (BDNF) at different time points. Relevant upstream pathway antagonist U0126 (MEK antagonist), H89 (PKA antagonist) , KN93 (CaMK antagonist) antagonized CREB upstream pathway, and given DISS protection, using Western blotting assay DISS on CREB, p-CREB, BDNF protein expression before and after antagonism. Results The optimal platelet density of SH-SY5Y was 2 × 108 L-1. DISS (60 μmol·L-1, 30 μmol·L-1) significantly enhanced the proliferation of SH-SY5Y cells (P <0.05) DISS significantly increased the expression of CREB, p-CREB and BDNF in SH-SY5Y cells after treated with DISS for 15 min (P <0.05) <0.05). Conclusion DISS can regulate the activity of CREB and promote the expression of downstream factors such as BDNF. The mechanism of its neuroprotection may be related to the activation of Ca2 + / CaM / CaMK-CREB-BDNF signaling pathway.