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目的探索半胱氨酰白三烯(CysLT)受体在大鼠皮层神经元缺氧缺糖损伤中的调节作用及作用机制。方法培养大鼠原代皮层神经元或大鼠原代皮层混合培养细胞,以缺糖缺氧(OGD)模拟脑缺血损伤,以MTT还原法/LDH释放检测确定神经元存活/损伤,以免疫组化做神经元(抗MAP2)、星形胶质细胞(抗GFAP)、小胶质细胞(抗Iba1)特异性染色,观察各细胞数量变化,以PI/Hoechst染色确定细胞凋亡/坏死。以受体激动剂LTD4,NMLTC4,CysLT1拮抗剂孟鲁司特、CysLT2拮抗剂HAMI3379/CysLT1-siRNA及CysLT2-shRNA为药理学干预/基因干扰工具。结果 LTD4(0.1～1000 nmol·L-1),NMLTC4(0.1～1000 nnmol·L-1)不能诱导单纯神经元损伤;CysLT1-siRNA及CysLT2-shRNA不能减轻OGD诱导的神经元损伤;LTD4(1～1000 nnmol·L-1),NMLTC4(0.1～1000 nnmol·L-1)能诱导皮层混合细胞损伤;HAMI3379(0.001～1μmol·L-1)和CysLT2-shRNA能减轻OGD,LTD4或NMLTC4诱导的皮层混合细胞损伤及其诱导的小胶质细胞激活,CysLT1-siRNA无此作用。结论 CysLT2受体可能参与OGD诱导神经元损伤的调节,CysLT2拮抗剂可通过减轻小胶质细胞激活而减轻OGD诱导的神经元损伤。
Objective To investigate the regulatory effect and mechanism of CysLT receptor on hypoxia and hypoglycemia in rat cortical neurons. Methods Cultured primary cortical neurons or primary cultured rat cortical neurons were incubated with simulated hypoxia and hypoxia (OGD) to simulate cerebral ischemic injury. MTT reduction / LDH release assay was used to determine the survival and injury of neurons. The neurons (anti-MAP2), astrocytes (anti-GFAP) and microglia (anti-Iba1) were stained specifically to observe the changes of the number of cells. PI / Hoechst staining was used to determine the apoptosis / necrosis. Receptor agonists LTD4, NMLTC4, CysLT1 antagonist montelukast, CysLT2 antagonists HAMI3379 / CysLT1-siRNA and CysLT2-shRNA were used as pharmacological intervention / gene interference tools. Results Single neuronal injury was induced by LTD4 (0.1-1000 nmol·L-1) and NMLTC4 (0.1-1000 nnmol·L-1). CysLT1-siRNA and CysLT2-shRNA did not attenuate OGD-induced neuronal injury. LTD4 ~100 nmol·L-1) and NMLTC4 (0.1-1000 nnmol·L-1) could induce cortical mixed cell injury. HAMI3379 (0.001-1 μmol·L-1) and CysLT2-shRNA could reduce the induction of OGD, LTD4 or NMLTC4 CysLT1-siRNA has no effect on cortical mixed cell injury and its induced microglial activation. Conclusion CysLT2 receptors may be involved in the regulation of OGD-induced neuronal injury. CysLT2 antagonists may reduce OGD-induced neuronal damage by reducing microglial activation.