知母皂苷元对高糖引起的体外培养大鼠海马神经元损伤的保护作用

摘要

Aim To investigate whether Sarsasapoge-nin ( Sar ) can protect hippocampal neurons from hyper-glycemia induced neurotoxicity. Methods Primary cultures were obtained from hippocampi of 0-to 24-h-old Sprague-Dawley rats. After 7 d in culture, the cells were used for the treatment. The cultured neurons were divided into control group, hyperglycemia treatment group, hyperglycemia ( glucose 50 mmol·L -1 ) + Sar ( 10, 30,100 μmol·L-1 )groups. In control group, cultured hippocampal neurons were incubated with 0. 1% DMSO for 48 h. In hyperglycemia treatment group, cultured neurons were incubated with glucose ( 30, 50, 100 mmol·L-1 ) for 48 h. In hyperglycemia + Sar group, Sar was added to the neurons 1 h prior to incubation with glucose 50 mmol·L-1. Cell viability was determined by MTT. Immunostaining and Western blot were used for synaptophysin ( SYP ) protein expression. Neuronal apoptosis was quantified by scoring the percentage of cells with apoptotic nuclear morphology after Hoechst 33258 staining. The cellular extracts were prepared for Western blot of active caspase-3 and poly ADP-ribose polymerase ( PARP ) protein expression. Results Cultured hippocampal neurons treated with hyperglycemia resulted in the decrease in cell viability, the decrease in SYP expression, and the increase in the percentage of apoptotic neurons, active caspase-3 and PARP expressions. Sar prevented the hyperglycemia induced decrease in SYP, and reduced hyperglycemia induced apoptotic morphology and active caspase-3 and PARP protein levels. Conclusion These results indicate that Sar can protect hippocampal neurons against hyperglycemia neurotoxicit.u0000 the%目的 探讨知母皂苷元(sarsasapogenin,Sar)对高糖引起的海马神经元损伤是否有保护作用.方法选用出生0~24 h Sprague-Dawley(SD)大鼠乳鼠,取海马,进行海马神经元体外培养,培养7 d用于实验.实验分为正常对照组、高糖组、高糖+Sar(10、30、100 μmol·L-1)组;对照组:加入等量含0.1% DMSO培养基;高糖处理组:分别加入葡萄糖(30、50、100 mmol·L-1)作用48 h;高糖+Sar (10、30、100 μmol·L-1)组:先加入不同浓度Sar(10、30、100 μmol·L-1)作用1 h,然后加入葡萄糖(50 mmol·L-1)作用48 h;应用MTT方法观察细胞活力,免疫荧光和Western免疫印迹方法观察神经元突触素表达的改变.应用Hoechst 33258 核染色检测神经元凋亡.应用Western免疫印迹方法观察caspase-3和多聚ADP核糖聚合酶(PARP)蛋白表达改变.结果加入葡萄糖(30、50、100 mmol·L-1)作用48 h可使培养神经元细胞活力明显降低,神经元突触素蛋白表达明显降低.另外高糖也可引起神经元凋亡细胞百分比和活性caspase-3、PARP蛋白表达水平也较对照组明显提高.在加入高糖前加入不同浓度Sar (10、30、100 μmol·L-1)可明显对抗高糖引起的这些变化.培养海马神经元突触素蛋白表达较高糖组(50 mmol·L-1)明显增加,神经元凋亡细胞百分比和活性caspase-3、PARP蛋白表达水平也较高糖组明显降低.结论 Sar对高糖引起的海马神经元损伤具有明显的保护作用.