URB597 protects against NLRP3 inflammasome activation by inhibiting autophagy dysfunction in a rat model of chronic cerebral hypoperfusion
URB597 protects against NLRP3 inflammasome activation by inhibiting autophagy dysfunction in a rat model of chronic cerebral hypoperfusion
复制标题
URB597 通过抑制慢性脑灌注不足大鼠模型中的自噬功能障碍来防止 NLRP3 炎症小体激活
DOI：
10.1186/s12974-019-1668-0
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发表时间：
2019-12-09
影响因子：
9.3
通讯作者：
Hai, Jian
中科院分区：
文献类型：
--
作者：
Su, Shao-Hua;Wu, Yi-Fang;Hai, Jian
背景资料：先前的研究报道，URB 597（URB）具有治疗慢性脑灌注不足（CCH）诱导的神经炎症和自噬功能障碍的治疗潜力。然而，CCH诱导的异常过度自噬和神经炎症的相互作用机制仍然未知。在本研究中，我们研究了在诱导CCH的条件下，自噬受损在大鼠海马中含核巧酸结合寡聚化结构域样受体家族pyrin domain-containing（NLRP）3炎性小体激活中的作用和潜在的机制，以及URB治疗的影响。采用双侧颈总动脉结扎（BCCAo）建立CCH大鼠模型，随机分为11组：（1）假手术组;（2）BCCAo组;（3）BCCAo+自噬抑制剂3-甲基腺嘌呤（3-MA），（4）BCCAo+溶酶体抑制剂氯喹（CQ），（5）BCCAo+小胶质细胞活化抑制剂米诺环素，（6）BCCAo+ROS清除剂N-乙酰半胱氨酸（NAC），（7）BCCAo+URB，（8）BCCAo +URB+3-MA，（9）BCCAo + URB+CQ，（10）BCCAo + URB+米诺环素，（11）BCCAo+URB+NAC。通过免疫荧光染色评估LC 3、p62、LAMP 1、TOM 20和NLRP 3的细胞定位。免疫印迹法检测自噬相关蛋白（LC 3、p62、LAMP 1、BNIP 3和parkin）、NLRP 3炎性小体相关蛋白（NLRP 3、CASP 1和IL-1 β）、小胶质细胞标志物（OX-42）和促炎细胞因子（iNOS和考克斯-2）的水平，ELISA法检测促炎细胞因子（IL-1 β和TNF-α）的水平。通过二氢乙锭染色评估活性氧（ROS）。结果：CCH可诱导小胶质细胞过度活化和ROS积聚，促进NLRP 3炎性小体的活化和IL-1 β的释放。阻断的自噬和线粒体自噬通量增强了NLRP 3-CASP 1炎性体通路的激活。然而，URB通过降低线粒体活性氧和小胶质细胞过度活化，恢复溶酶体功能，从而进一步抑制NLRP 3-CASP 1炎性小体通路的激活，减轻了受损的自噬和线粒体自噬。结论：URB在慢性脑缺血损伤中的作用是对已有研究的补充。
Background: Previous studies reported that URB597 (URB) had therapeutic potential for treating chronic cerebral hypoperfusion (CCH)-induced neuroinflammation and autophagy dysfunction. However, the interaction mechanisms underlying the CCH-induced abnormal excessive autophagy and neuroinflammation remain unknown. In this study, we investigated the roles of impaired autophagy in nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing (NLRP) 3 inflammasome activation in the rat hippocampus and the underlying mechanisms under the condition of induced CCH as well as the effect of URB treatment.Methods: The CCH rat model was established by bilateral common carotid artery occlusion (BCCAo), and rats were randomly divided into 11 groups as follows: (1) sham-operated, (2) BCCAo; (3) BCCAo+autophagy inhibitor 3-methyladenine (3-MA), (4) BCCAo+lysosome inhibitor chloroquine (CQ), (5) BCCAo+microglial activation inhibitor minocycline, (6) BCCAo+ROS scavenger N-acetylcysteine (NAC), (7) BCCAo+URB, (8) BCCAo+URB+3-MA, (9) BCCAo+URB+CQ, (10) BCCAo+URB+minocycline, (11) BCCAo+URB+NAC. The cell localizations of LC3, p62, LAMP1, TOM20 and NLRP3 were assessed by immunofluorescence staining. The levels of autophagy-related proteins (LC3, p62, LAMP1, BNIP3 and parkin), NLRP3 inflammasome-related proteins (NLRP3, CASP1 and IL-1 beta), microglial marker (OX-42) and proinflammatory cytokines (iNOS and COX-2) were evaluated by western blotting, and proinflammatory cytokines (IL-1 beta and TNF-a) were determined by ELISA. Reactive oxygen species (ROS) were assessed by dihydroethidium staining. The mitochondrial ultrastructural changes were examined by electron microscopy.Results: CCH induced microglial overactivation and ROS accumulation, promoting the activation of the NLRP3 inflammasome and the release of IL-1 beta. Blocked autophagy and mitophagy flux enhanced the activation of the NLRP3-CASP1 inflammasome pathway. However, URB alleviated impaired autophagy and mitophagy by decreasing mitochondrial ROS and microglial overactivation as well as restoring lysosomal function, which would further inhibit the activation of the NLRP3-CASP1 inflammasome pathway.Conclusion: These findings extended previous studies indicating the function of URB in the mitigation of chronic ischemic injury of the brain.