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NLRP3 Inflammasome Activation Is Involved in LPA₁-Mediated Brain Injury after Transient Focal Cerebral Ischemia.

ABSTRACT: Lysophosphatidic acid receptor 1 (LPA₁) contributes to brain injury following transient focal cerebral ischemia. However, the mechanism remains unclear. Here, we investigated whether nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome activation might be an underlying mechanism involved in the pathogenesis of brain injury associated with LPA₁ following ischemic challenge with transient middle cerebral artery occlusion (tMCAO). Suppressing LPA₁ activity by its antagonist attenuated NLRP3 upregulation in the penumbra and ischemic core regions, particularly in ionized calcium-binding adapter molecule 1 (Iba1)-expressing cells like macrophages of mouse after tMCAO challenge. It also suppressed NLRP3 inflammasome activation, such as caspase-1 activation, interleukin 1? (IL-1?) maturation, and apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) speck formation, in a post-ischemic brain. The role of LPA₁ in NLRP3 inflammasome activation was confirmed in vitro using lipopolysaccharide-primed bone marrow-derived macrophages, followed by LPA exposure. Suppressing LPA₁ activity by either pharmacological antagonism or genetic knockdown attenuated NLRP3 upregulation, caspase-1 activation, IL-1? maturation, and IL-1? secretion in these cells. Furthermore, nuclear factor-?B (NF-?B), extracellular signal-regulated kinase 1/2 (ERK1/2), and p38 were found to be LPA₁-dependent effector pathways in these cells. Collectively, results of the current study first demonstrate that LPA₁ could contribute to ischemic brain injury by activating NLRP3 inflammasome with underlying effector mechanisms.

SUBMITTER: Lee CH

PROVIDER: S-EPMC7697439 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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NLRP3 Inflammasome Activation Is Involved in LPA1-Mediated Brain Injury after Transient Focal Cerebral Ischemia.

Lee Chi-Ho CH Sapkota Arjun A Gaire Bhakta Prasad BP Choi Ji Woong JW

International journal of molecular sciences 20201114 22

Lysophosphatidic acid receptor 1 (LPA1) contributes to brain injury following transient focal cerebral ischemia. However, the mechanism remains unclear. Here, we investigated whether nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome activation might be an underlying mechanism involved in the pathogenesis of brain injury associated with LPA1 following ischemic challenge with transient middle cerebral artery occlusio ...[more]

PMID: 33202644

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Project description:Ischemic stroke is a devastating disease with long-term disability. However, the pathogenesis is unclear and treatments are limited. Meisoindigo, a second-generation derivative of indirubin, has general water solubility and is well-tolerated. Previous studies have shown that meisoindigo reduces inflammation by inhibiting leukocyte chemotaxis and migration. In the present study, we investigated the hypothesis that meisoindigo was also protective against ischemic stroke, then evaluated its underlying mechanisms. In vivo, adult male C57BL/6J wild-type mice were used to produce a middle cerebral artery occlusion (MCAO) stroke model. On day three after reperfusion, obvious improvement in neurological scores, infarct volume reduction and cerebral edema amelioration were observed in meisoindigo treatment. Moreover, immunofluorescence staining and western-blot showed that the expression of NLRP3 inflammasome and its associated proteins in neurons and microglia was inhibited by meisoindigo. The effects of Meisoindigo on NLRP3 inflammasome inactivation and increased the M2 phenotype of microglia/macrophage through shifting from a M1 phenotype, which was possibly mediated by inhibition of TLR4/NF-κB. Furthermore, we verified the inhibitory effect of meisoindigo on TLR4/NF-κB signaling pathway, and found that meisoindigo treatment could significantly suppressed the expression of TLR4/NF-κB pathway-associated proteins in a dose-dependent manner, meanwhile, which resulted in downregulation of HMGB1 and IL-1β. Next, we established an in vitro oxygen glucose deprivation/Reperfusion (OGD/R) model in HT-22 and BV2 cells to simulate ischemic conditions. Cytotoxicity assay showed that meisoindigo substantially improved relative cell vitality and in HT-22 and BV2 cells following OGD/R in vitro. After suffering OGD/R, the TLR4/NF-κB pathway was activated, the expression of NLRP3 inflammasome-associated proteins and M1 microglia/macrophage were increased, but meisoindigo could inhibit above changes in both HT-22 and BV2 cells. Additionally, though lipopolysaccharide stimulated the activation of TLR4 signaling in OGD/R models, meisoindigo co-treatment markedly reversed the upregulation of TLR4 and following activation of NLRP3 inflammasome and polarization of M1 microglia/macrophages mediated by TLR4. Overall, we demonstrate for the first time that meisoindigo post-treatment alleviates brain damage induced by ischemic stroke in vivo and in vitro experiments through blocking activation of the NLRP3 inflammasome and regulating the polarization of microglia/macrophages via inhibition of the TLR4/NF-κB signaling pathway.

Project description:BackgroundMyocardial ischemia-reperfusion injury (MIRI) is the most common cause of death worldwide. The NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome plays an important role in the inflammatory response to MIRI. Dexmedetomidine (DEX), a specific agonist of α2-adrenergic receptor, is commonly used for sedation and analgesia in anesthesia and critically ill patients. Several studies have shown that dexmedetomidine has a strong anti-inflammatory effect in many diseases. Here, we investigated whether dexmedetomidine protects against MIRI by inhibiting the activation of the NLRP3 inflammasome in vitro.MethodsWe established an MIRI model in cardiomyocytes (CMs) alone and in coculture with cardiac fibroblasts (CFs) by hypoxia/reoxygenation (H/R) in vitro. The cells were treated with dexmedetomidine with or without MCC950 (a potent selective NLRP3 inhibitor). The beating rate and cell viability of cardiomyocytes, NLRP3 localization, the expression of inflammatory cytokines and NLRP3 inflammasome-related proteins, and the expression of apoptosis-related proteins, including Bcl2 and BAX, were determined.ResultsDexmedetomidine treatment increased the beating rates and viability of cardiomyocytes cocultured with cardiac fibroblasts. The expression of the NLRP3 protein was significantly upregulated in cardiac fibroblasts but not in cardiomyocytes after H/R and was significantly attenuated by dexmedetomidine treatment. Expression of the inflammatory cytokines IL-1β, IL-18 and TNF-α was significantly increased in cardiac fibroblasts after H/R and was attenuated by dexmedetomidine treatment. NLRP3 inflammasome activation induced the increased expression of cleaved caspase1, mature IL-1β and IL-18, while dexmedetomidine suppressed H/R-induced NLRP3 inflammasome activation in cardiac fibroblasts. In addition, dexmedetomidine reduced the expression of Bcl2 and BAX in cocultured cardiomyocytes by suppressing H/R-induced NLRP3 inflammasome activation in cardiac fibroblasts.ConclusionDexmedetomidine treatment can suppress H/R-induced NLRP3 inflammasome activation in cardiac fibroblasts, thereby alleviating MIRI by inhibiting the inflammatory response.

Project description:Nod-like receptor protein 3 (NLRP3) inflammasome is a crucial contributor in the inflammatory process during cerebral ischemia/reperfusion (I/R) injury. ATF4 plays a pivotal role in the pathogenesis of cerebral I/R injury, however, its function and underlying mechanism are not fully characterized yet. In the current study, we examined whether ATF4 ameliorates cerebral I/R injury by inhibiting NLRP3 inflammasome activation and whether mitophagy is involved in this process. In addition, we explored the role of parkin in ATF4-mediated protective effects. Method: To address these issues, healthy male adult Sprague-Dawley rats were exposed to middle cerebral artery occlusion for 1 h followed by 24 h reperfusion. Adeno-associated virus (AAV) and siRNA were injected into rats to overexpress and knockdown ATF4 expression, respectively. After pretreatment with AAV, mdivi-1(mitochondrial division inhibitor-1) was injected into rats to block mitophagy activity. Parkin expression was knockdown using specific siRNA after AAV pretreatment. Result: Data showed that ATF4 overexpression induced by AAV was protective against cerebral I/R injury, as evidenced by reduced cerebral infraction volume, decreased neurological scores and improved outcomes of HE and Nissl staining. In addition, overexpression of ATF4 gene was able to up-regulate Parkin expression, enhance mitophagy activity and inhibit NLRP3 inflammasome-mediated inflammatory response. ATF4 knockdown induced by siRNA resulted in the opposite effects. Furthermore, ATF4-mediated inhibition of NLRP3 inflammasome activation was strongly affected by mitophagy blockage upon mdivi-1 injection. Besides, ATF4-mediated increase of mitophagy activity and inhibition of NLRP3 inflammasome activation were effectively reversed by Parkin knockdown using siRNA. Conclusion: Our study demonstrated that ATF4 is able to alleviate cerebral I/R injury by suppressing NLRP3 inflammasome activation through parkin-dependent mitophagy activity. These results may provide a new strategy to relieve cerebral I/R injury by modulating mitophagy-NLRP3 inflammasome axis.

Dataset Information

NLRP3 Inflammasome Activation Is Involved in LPA₁-Mediated Brain Injury after Transient Focal Cerebral Ischemia.

Publications

NLRP3 Inflammasome Activation Is Involved in LPA<sub>1</sub>-Mediated Brain Injury after Transient Focal Cerebral Ischemia.

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