在长爪沙鼠全脑缺血再灌注模型中半胱氨酰白三烯受体拮抗剂通过调节神经元自噬减轻损伤机制研究

Global cerebral ischemia is a problem of increasing clinical significance, but it has not been successfully dealed with pharmacologically. Cysteinyl leukotrienes (CysLTs) are potent inflammatory mediators that are involved in brain injury after cerebral ischemia. The actions of CysLTs are mediated by at least two G protein-coupled receptors, CysLT1R and CysLT2R. Pranlukast is one of CysLT1R selective antagonists and HAMI 3379 is an CysLT2R selective antagonist. Cysteinyl leukotriene receptor (CysLTR) expression are up-regulated in injured neurons, proliferated astrocytes and microglia after rats suffer cerebral ischemia. Autophagy is activated following cerebral ischemia. The cited references and preliminary tests results showed that pranlukast protects against brain injury after global cerebral ischemia in mongolian gerbils as well as HAMI 3379, and CysLTs induce autophagy, so we hypothesized that CysLTR antagonists protect against brain injury after global cerebral ischemia through regulating autophagy. To test this hypothesis, we will investigate the effect of CysLTR antagonists on autophagy in global cerebral ischemia mongolian gerbils and primary hippocampal neurons or the mixed culture of cortical cells with ischemic injury induced by oxygen-glucose deprivation (OGD), in the process, molecular biological technique, electron microscope and immunohistochemical methods will be employed, then the regulating effects will be further verified by autophagy inducer rapamycin and autophagy inhibitor 3-methyladenine, then siRNA of autophagy related gene Atg 5 and beclin 1 will be adopted to determine the mechanism behind these effects.The project will clarify how CysLTR antagonists act on cerebral ischemia injury, and CysLTR antagonists will be a new class of therapeutic agents in the treatment of ischemic stroke, in the meanwhile, mongolian gerbils, a featured experimental animal of our country, will be further developed.

全脑缺血是心脏骤停等全身危重疾病的并发症，长爪沙鼠是全脑缺血的最佳模型动物之一。脑缺血后，炎症介质半胱氨酰白三烯（CysLTs）释放增加，其受体（CysLTR）表达上调，参与脑缺血后神经元损伤等病变。自噬是缺血性神经元损伤的机制之一。前期研究表明，CysLTR拮抗剂对全脑缺血再灌注损伤有保护作用，CysLTs可直接诱导自噬的激活，因此，我们推测 CysLTR拮抗剂通过调节自噬减轻长爪沙鼠全脑缺血再灌注损伤。为此，拟在长爪沙鼠全脑缺血模型、原代海马神经元，神经元-胶质细胞混合培养系统中，诱导缺血再灌注损伤，采用分子生物学、电镜等技术，观察 CysLTR拮抗剂对自噬的调节，并用自噬诱导剂及抑制剂对结果作进一步验证，最后采用 siRNA干扰技术探讨CysLTR拮抗剂对自噬形成过程的影响，明确其作用机制。本项目将促进长爪沙鼠的开发和应用，并为CysLTR拮抗剂在脑缺血治疗中的应用提供新的理论依据。

全脑缺血是心脏骤停等全身危重疾病的并发症，长爪沙鼠是全脑缺血的最佳模型动物之一。脑缺血后，炎症介质半胱氨酰白三烯（CysLTs）释放增加，其受体（CysLTR）表达上调，参与脑缺血后神经元损伤等病变。自噬是缺血性神经元损伤的机制之一。.我们研究表明，长爪沙鼠全脑缺血再灌注损伤后CysLT1R，CysLT2R蛋白表达并呈动态变化，并在皮层、海马区均有明显表达上调，提示CysLTR介导缺血后急性神经元损伤、亚急性期小胶质细胞的激活及慢性期星形胶质细胞细胞的增生。.进一步研究表明，半胱氨酰白三烯受体拮抗剂（普鲁司特、HAMI 3379）腹腔注射对长爪沙鼠全脑缺血再灌注损伤具有保护作用，可提高神经症状评分，减少神经功能损伤，减轻皮层及海马区神经元损伤及丢失，减少自噬相关蛋白Beclin-1及 LC3的表达及海马区自噬泡的数量，因此其神经保护作用与下调皮层、海马区的自噬有关。.普鲁司特和HAMI 3379在缺血后给药对长爪沙鼠全脑缺血再灌注慢性损伤亦有保护作用，能促进长爪沙鼠全脑缺血后长期神经功能的恢复，并可抑制缺血后慢性期的胶质疤痕形成有关细胞的增生，这一发现有力地证实CysLTR拮抗剂对全脑缺血损伤具有持久的神经保护作用。.本研究证实HAMI 3379腹腔注射给药对大鼠局灶性脑缺血急性、亚急性损伤有保护作用，并可减轻炎症反应，尤其能较强抑制小胶质细胞激活，其有效剂量是0.1 ~ 0.4 mg/kg，治疗时间窗为术后1 h；对脑缺血慢性损伤亦有保护作用。另在小鼠小胶质细胞株BV2细胞，HAMI 3379可抑制OGD/R诱导BV2细胞适度激活后的炎症因子合成及释放的增加以及吞噬功能、细胞自噬的增强。HAMI 3379还可抑制OGD/R诱导的CysLT1R-CysLT2R相互作用以及细胞内移动变化，且可增强CysLT1R表达上调。.本项目首先以长爪沙鼠为实验动物，系统的观察长爪沙鼠全脑缺血再灌注后CysLT1R和CysLT2R的时间和空间分布特征，证实CysLTR拮抗剂对全脑缺血损伤的保护作用，为长爪沙鼠在脑缺血研究中的广泛应用提供坚实可靠的基础，本研究还证实了HAMI 3379通过抑制CysLT2R介导的小胶质细胞激活，并为CysLTR拮抗剂在脑缺血治疗中的应用提供新的理论依据。