白扁豆多糖对神经细胞缺氧性凋亡的保护机制

摘要

目的:模拟体内环境,研究白扁豆多糖(dolichos bean seed polysaccharide,DBSP)对胎鼠大脑皮层神经细胞缺氧性凋亡的保护机制.方法:原代培养胎鼠大脑皮层神经细胞;建立缺氧/复氧(anoxia/reoxygenation,A/R)损伤模型;实验分4 组:对照组、A/R组、白扁豆多糖预处理组(DBSP+A/R组)、阻断剂组(LY294002+DBSP+A/R组);用比色法检测细胞活性与乳酸脱氢酶的释放水平;流式细胞仪分析测定Ca2+相对浓度、活性氧、线粒体膜电位和细胞凋亡的变化;Western blot法检测总蛋白激酶B(total protein kinase B, T-Akt)和磷酸化蛋白激酶B(phosphorylated protein kinase B,p-Akt)蛋白表达.结果:从细胞活性来看,A/R组比对照组明显降低(P<0.01),表明A/R对神经细胞产生了损伤.同时,与A/R组相比,DBSP+A/R组细胞活性显著上升、细胞凋亡明显减少(P<0.01),乳酸脱氢酶的释放水平下降,表明DBSP对受A/R损伤的神经细胞产生保护作用,进一步结果显示,与A/R组相比,DBSP+A/R组中Ca2+相对浓度和活性氧生成显著降低、显著增加线粒体膜电位和p-Akt蛋白表达(P<0.01).而与DBSP+A/R组相比,LY294002+DBSP+A/R组中p-Akt蛋白表达显著减少(P<0.01),同时细胞活性与线粒体膜电位下降,且LDH的释放水平、Ca2+相对浓度、活性氧、细胞凋亡明显增加(P<0.05),表明DBSP对神经细胞的保护作用受到PI3K-Akt信号通路抑制剂LY294002的抑制作用而被削弱.结论:DBSP可通过PI3K-Akt信号转导通路抑制神经细胞的缺氧性凋亡.%Objective: The aim of this study was to explore the protective effect of dolichos bean seed polysaccharides (DBSP) against anoxia/reoxygenation (A/R) injury and its potential mechanism of action in neurons. Methods: Primary cultured cerebral cortical neurons were randomly divided into four groups including control group, A/R group, DBSP + A/R group and DBSP + LY294002 + A/R group. Cell survival rate and lactate dehydrogenase (LDH) release were determined. Apoptosis, mitochondrial membrane potential and Ca2+concentration were determined by flow cytometry analysis. The expression of T-Akt and p-Akt protein was evaluated by Western blot. Results: Compared with the control group, cell survival rate of the A/R group was decreased (P < 0.01) and LDH release, Ca2+concentration and apoptotic rate were significantly increased (P < 0.01). Compared with the A/R group, cell survival rate of the DBSP + A/R group was increased, and LDH release, Ca2+concentration and apoptotic rate were significantly decreased (P < 0.01), suggesting that DBSP could protect neurons against A/R injury. Mitochondrial membrane potential and the expression of p-Akt protein increased significantly in the DBSP +A/R group when compared with the A/R group (P < 0.01). The expression of p-Akt protein after treatment with LY294002 (a potent inhibitor of the PI3K-Akt signaling pathway) + DBSP + A/R was attenuated in neurons when compared with A/R + DBSP treatment (P < 0.05). Further experimental results showed that the treatment of cells with LY294002 completely blocked the expression of p-Akt protein, and significantly attenuated the protective effect of DBSP on neurons subjected to A/R. Conclusion: The protective mechanism of polysaccharides from dolichos bean seeds on neuronal cells is closely related to the PI3K-Akt signaling pathway.