Abstract
BackgroundPrevious 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.MethodsThe 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.ResultsCCH 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.ConclusionThese findings extended previous studies indicating the function of URB in the mitigation of chronic ischemic injury of the brain.
Highlights
A substantial amount of evidence indicates that chronic cerebral hypoperfusion (CCH), a state of chronic cerebral blood flow reduction, is associated with several cerebrovascular and neurodegenerative disorders including Alzheimer’s disease (AD), carotid stenosis/occlusion, cerebral arteriovenous malformation, dural arteriovenous fistula, moyamoya disease and cerebral small vessel disease [1,2,3,4,5,6]
CCH-induced autophagy dysfunction may be correlated with IL-1β secretion in rat hippocampus In our previous studies [9, 11], we found that CCH caused the release of proinflammatory cytokines and induced lysosomal dysfunction, which may promote the accumulation of autophagosomes, resulting in abnormal excessive autophagy
Since it has been reported that autophagy dysfunction may contribute to the induction of neuroinflammation [29, 30], we examined whether there was a link between abnormal excessive autophagy and neuroinflammation in the presence of CCH
Summary
A substantial amount of evidence indicates that chronic cerebral hypoperfusion (CCH), a state of chronic cerebral blood flow reduction, is associated with several cerebrovascular and neurodegenerative disorders including Alzheimer’s disease (AD), carotid stenosis/occlusion, cerebral arteriovenous malformation, dural arteriovenous fistula, moyamoya disease and cerebral small vessel disease [1,2,3,4,5,6]. Nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing (NLRP) 3 inflammasome is a member of the NLR family of innate immune cell sensors. It is a cytosolic multi-protein complex composed of NLRs, adaptor protein apoptosis-associated speck-like protein (ASC) and caspase-1 (CASP1) [15]. 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
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