超低分子量肝素抑制神经细胞内钙释放保护谷氨酸损伤原代培养大鼠神经元

目的探讨超低分子量肝素(ultra low molecular weight heparin,ULMWH)对谷氨酸(Glu)诱导原代培养大鼠大脑皮层神经细胞损伤的保护作用及其作用机制。方法采用体外培养大鼠大脑皮层神经细胞,建立谷氨酸(Glu)诱导损伤模型,采用MTT法检测细胞活力、Hoechest33258染色法观察凋亡细胞形态改变和检测凋亡细胞数。同时,采用Fura-2/AM双波长荧光分光光度法测定神经细胞内钙离子浓度([Ca2+]i)。结果提前应用ULMWH可提高Glu损伤神经细胞的生存能力,降低Glu诱导的凋亡细胞数。同时,ULM-WH不论是在含Ca2+测量介质还是在无Ca2+测量介质中均可降低神经细胞[Ca2+]i。结论ULMWH对Glu损伤神经细胞具有保护作用,该作用可能与其抑制神经细胞内Ca2+释放而降低胞内[Ca2+]i有关。

Ultra low molecular weight heparin attenuates glutamate-induced neurotoxicity by reducing intracellular calcium release

Aim To investigate the effect of ultra low molecular weight heparin(ULMWH)to protect primarily cultured rat cortical neurons from the deleterious effects of the neurotoxicant glutamate (Glu)and explore the related mechanism.Methods Cortical neurons of fetal rats were cultured carefully in vitro and treated with 100 μmol·L-1 Glu so that the protective effects of ULMWH were observed thoroughly. And then the viability of cortical neurons, morphological change together with the number of apoptotic neurons,and the concentration of intracellular free Ca2+([Ca2+]i)were measured by MTT assay, Hoechst33258 staining and Fura-2/AM double wavelength fluoremetry, respectively.Results Brief exposure of cultures to 100 μmol·L-1Glu led to extensive neuronal death and rapid increase of [Ca2+]i.Pretreatment with ULMWH over the concentration range of 0.01~1 mg·L-1 significantly inhibited the Glu-induced neuronal cell death assessed by a MTT assay and the number of apoptotic nuclei, evidenced by Hoechst 33258 staining.Glu-induced elevation of[Ca2+]i was decreased by pretreatment with 1 mg·L-1 ULMWH, which was indicated by Fura-2/AM not only in assay medium containing Ca2+ but also in Ca2+-free assay medium.Conclusions Those results suggest that ULMWH protects the cultured neurons from Glu-induced neurotoxicity, which may be attributed to its alleviating intracellular free Ca2+ overload via suppressing intracellular Ca2+ release from internal stores induced by Glu.