灵芝提取物对神经元缺氧复氧损伤的保护作用及其机制的离体研究

Ganoderma lucidum extract protects rat cerebral cortical neurons from hypoxia/reoxygenation injury in vitro

目的:研究灵芝提取物(GLE)对原代大鼠皮层神经元氧糖剥夺-再复氧(OGD/R)损伤模型的影响,在细胞水平探讨GLE神经保护作用的机制。方法:新生SD大鼠的皮层神经元原代培养至第7d,用GLE预处理神经元OGD/R模型。选取OGD/R后的0h、3h、6h、12h、24h、48h和72h作为观察点,观察GLE干预后对各个时点的细胞形态学、细胞毒作用、细胞线粒体活性、细胞凋亡率及凋亡相关蛋白表达量的影响。结果:GLE(0.1、1、10mg/L)能明显减少神经元LDH的释放,提高线粒体活性。流式细胞术结果证实了GLE能抑制神经元OGD/R损伤导致的凋亡,这种作用甚至能持续至OGD/R后的72h。GLE(0.1、1、10mg/L)下调caspase-3、caspase-8蛋白的表达,GLE(10mg/L)还能下调caspase-9蛋白表达。结论:GLE对OGD/R诱导的神经元损伤有一定的保护作用,可能是有效防治急性缺血性卒中的神经保护药物。

AIM： To investigate the neuroprotective effect of Ganoderma lucidum extract （GLE） in an in vitro model of primary cultured neurons with oxygen and glucose deprivation （OGD）. METHODS： Neuronal injury was induced by oxygen and glucose deprivation/reoxygenation （OGD/R）. The neuronal injury and viability were determined by LDH leakage and XTT assay at 0 h,3 h,6 h,12 h,24 h,48 h and 72 h after OGD/R. Neuronal apoptosis was detected by flow cytometry （FCM）. The expression of apoptosis-related proteins was analyzed by Western blotting.RESULTS： The viability of the neurons increased with exposure to GLE （0.1 mg/L,1 mg/L and 10 mg/L）after OGD/R. The LDH releases were also significantly reduced. GLE significantly inhibited OGD/R-induced apoptosis of cultured rat cortical neurons in a concentration-dependent and time-dependent manner（P〈0.05）. GLE at concentrations of 0.1 mg/L,1 mg/L and 10 mg/L inhibited the expression of caspase-3 and caspase-8 proenzyme. Additionally,GLE at concentration of 10 mg/L suppressed the expression of caspase-9 proenzyme.CONCLUSION： Our findings provide the evidence that the GLE has neuroprotective effect on cerebral ischemia. The mechanisms are related to the inhibition of caspase-3,-8 and-9 activations. GLE may be a novel and effective reagent for treating ischemic stroke.