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-1β), microglial marker (OX-42) and proinflammatory cytokines (iNOS and COX-2) were evaluated by western blotting, and proinflammatory cytokines (IL-1β 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-1β. 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.

中文翻译：

---

URB597通过抑制慢性脑灌注不足的大鼠模型中的自噬功能障碍来保护NLRP3炎性体活化。

背景技术以前的研究报道，URB597（URB）具有治疗慢性脑灌注不足（CCH）引起的神经炎症和自噬功能障碍的治疗潜力。但是，CCH诱导的异常过度自噬和神经炎症潜在的相互作用机制仍然未知。在这项研究中，我们研究了受损的自噬在大鼠海马中核苷酸结合的寡聚化域样受体家族含吡喃域的炎症小体活化中的作用及其潜在机制，以及诱导CCH的作用。的URB治疗。方法采用双侧颈总动脉闭塞（BCCAo）建立CCH大鼠模型，将大鼠随机分为11组，分别为：（1）假手术；（2）BCCAo；（2）假手术；（2）假手术；（2）假手术。（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LC3，p62，LAMP1，TOM20和NLRP3的细胞定位。自噬相关蛋白（LC3，p62，LAMP1，BNIP3和帕金蛋白），NLRP3炎性体相关蛋白（NLRP3，CASP1和IL-1β），小胶质标记物（OX-42）和促炎细胞因子（iNOS和COX-2）的水平通过蛋白质印迹法评估），并通过ELISA确定促炎细胞因子（IL-1β和TNF-α）。通过二氢乙啶染色评估活性氧（ROS）。通过电子显微镜检查线粒体的超微结构变化。结果CCH诱导小胶质细胞过度活化和ROS积累，促进NLRP3炎性小体的活化和IL-1β的释放。自噬和线粒体通量受阻可增强NLRP3-CASP1炎性体途径的激活。但是，URB通过降低线粒体ROS和小胶质细胞过度活化以及恢复溶酶体功能来减轻自噬和线粒体受损，这将进一步抑制NLRP3-CASP1炎性体途径的激活。结论这些发现扩展了先前的研究，表明了URB在缓解慢性缺血性脑损伤中的作用。促进NLRP3炎性小体的活化和IL-1β的释放。自噬和线粒体通量受阻可增强NLRP3-CASP1炎性体途径的激活。但是，URB通过减少线粒体ROS和小胶质细胞过度活化以及恢复溶酶体功能来减轻自噬和线粒体受损，这将进一步抑制NLRP3-CASP1炎性体途径的激活。结论这些发现扩展了先前的研究，表明了URB在缓解慢性缺血性脑损伤中的作用。促进NLRP3炎性小体的活化和IL-1β的释放。自噬和线粒体通量受阻可增强NLRP3-CASP1炎性体途径的激活。但是，URB通过减少线粒体ROS和小胶质细胞过度活化以及恢复溶酶体功能来减轻自噬和线粒体受损，这将进一步抑制NLRP3-CASP1炎性体途径的激活。结论这些发现扩展了先前的研究，表明了URB在缓解慢性缺血性脑损伤中的作用。这将进一步抑制NLRP3-CASP1炎性体途径的激活。结论这些发现扩展了先前的研究，表明了URB在缓解慢性缺血性脑损伤中的作用。这将进一步抑制NLRP3-CASP1炎性体途径的激活。结论这些发现扩展了先前的研究，表明了URB在缓解慢性缺血性脑损伤中的作用。