血小板活化因子通过N-甲基-D-天(门)冬氨酸/突触后密度蛋白93途径致神经元损伤

Neuronal injury induced by platelet activating factor involved in NMDA/PSD_(93) signaling pathway

目的 探讨血小板活化因子(PAF)所致的神经元损伤是否涉及N 甲基 D 天(门)冬氨酸/突触 后密度蛋白93(NMDA/PSD93)信号通路。方法 细胞培养系统培养原代野生型和PSD93基因敲除型小鼠皮 质神经元;0.3μmol/LPAF处理神经元24h或5μmol/LPAF受体拮抗剂(BN52021),10μmol/L非竟争性 NMDA受体拮抗剂(MK 801)和60μmol/L神经性一氧化氮合成酶(nNOS)抑制剂(L NAMA)预处理,碘化 物/钙黄绿素染色检测细胞凋亡;免疫印迹检测野生型和基因敲除型小鼠皮质神经元中的多种蛋白表达;细 胞免疫组化和共聚焦显微镜观察在同一神经轴突上共同表达多种蛋白;放免法测定神经元细胞蛋白中环鸟 苷磷酸(cGMP)活性。结果 (1)PSD93基因敲除型神经元不表达PSD93外,与野生型一样表达PSD95、N 甲基 D 天(门)冬氨酸受体(NR2A)和nNOS。(2)神经轴突共同表达PSD93、NR2A和nNOS。(3)PSD93基因敲除型 小鼠皮质神经元减少PAF对神经的毒性作用,并降低其cGMP活性。结论 PAF通过NMDA/PSD93途径致神 经细胞损伤。

Objective To study whether neuronal injury induced by platelet activating factor (PAF) was involved in NMDA /PSD 93 signaling pathway.Methods Primary cortex neurons culture were from wild type and PSD 93 knockout mice.Then the neurons were pretreated with 0.3 μmol/L PAF for 24 hours, 5 μmol/L BN52021, 10 μmol/L MK-801, or 60 μmol/L L-NAMA, respectively. The cells were stained with PI/Calcein for apoptosis detection. Varied protein expressions were also observed in the neurons by western blot. Proteins colocalized on neuraxon were detected by cell immunochemistry and confocol microscopy, and cGMP activity was measured by radioimmunoassay.Results (1) PSD 95, NR2A, and nNos except PSD 93 expressed in the cortex neurons from PSD 93 knockout mice. (2) PSD 93, NR2A and nNos colocalized on neurite. (3) Neurotoxicity and cGMP avtivity induced by PAF decreased in PSD 93 knockout cortex neurons.Conclusion NMDA/PSD 93 signaling pathway is involved in neuronal injury induced by PAF.