NLRP3炎性体加重小鼠脑缺血再灌注损伤的机制探讨

摘要

目的：探讨Nod样受体蛋白3（NLRP3）炎性体（NLRP3蛋白、IL-1β、IL-8）在小鼠脑缺血再灌注损伤中的作用和机制。方法将C57BL／6小鼠随机分为对照组和格列本脲（ NLRP3抑制剂）组各20只，每组中一半小鼠通过大脑中动脉栓塞法构建缺血再灌注（ IR）模型，其余接受假手术处理。格列本脲组术前30 min腹腔注射500 mg／kg格列本脲。造模成功后对小鼠进行神经学评分，2，3，5，-氯化三苯基四氮唑染色，并计算缺血梗死面积，HE染色观察病理变化，TUNEL染色观察各组神经元凋亡情况，Western Blot检测小鼠脑组织NLRP3蛋白表达，ELISA检测小鼠脑组织中IL-1β、IL-18浓度。结果各组IR小鼠脑损伤程度均重于假手术小鼠，但是对照组IR小鼠脑损伤程度比格列本脲组严重。 HE染色和TUNEL染色显示，IR小鼠脑组织出现明显损伤，神经元凋亡数量明显增多，格列本脲可减轻IR造成的病理损伤程度。与对照组相比，格列本脲组的脑组织NLRP3蛋白表达及IL-1β、IL-18升高程度明显降低（ P均＜0．05）。结论 NLRP3炎性体可促进小鼠脑IR损伤，主要通过增加细胞因子释放和促进神经元凋亡实现的。%Objective To investigate the effect of NLRP 3 inflammasome in the development of cerebral ischemia reperfusion injury in mice and the mechanism .Methods All of the C57 BL/6 mice were randomly divided into two groups , 20 in each group:the control group and glyburide group .Glyburide was used as inhibitor of NLRP 3 inflammasome and a mouse model of cerebral ischemia reperfusion was established by middle cerebral artery occlusion ( MCAO ) .Ten mice of each group were subjected to MCAO to make the ischemia reperfusion models , and others underwent sham opera-tion.Glyburide 500 mg/kg was given to the mice in the glyburide group by intraperitoneal injection 30 min before surgery. After MCAO, neurological scoring and TTC staining were conducted and the ischemic infarction area was calculated .The pathological change and neuronal apoptosis in brain tissues were observed with HE staining and TUNEL staining , respec-tively.The expression of NLRP3 and cytokines ( IL-1β, IL-18) was identified by Western blotting and ELISA , respective-ly.Results The degree of brain injury in IR mice of each group was more serious than that of the sham operation group , but the IR mouse brain damage of the control group was more serious than that of glyburide group .HE staining and TUNEL staining indicated significant injuries were found in the brain tissues of IR mice , the amount of neuronal apoptosis was in-creased, and the glyburide reduced the degree of pathological injuries caused by reperfusion .Compared with the control group, the increasing degree in expression of NLRP3 protein and cytokines (IL-1β, IL-18) was significantly decreased in the glyburide group (all P<0.05).Conclusion NLRP3 inflammasome promotes the development of cerebral ischemia reperfusion injuries by increasing cytokine release and enhancing the neuron apoptosis .