NOD-like Receptor Pyrin Domain-containing Protein Research Articles

The PGC-1α/ERRα/ULK1 pathway contributes to Perioperative neurocognitive disorders by inducing mitochondrial dysfunction and activating NLRP3 inflammasome in aged mice

Perioperative neurocognitive disorders (PND) are intractable, indistinct, and considerably diminish the postoperative quality of life of patients. It has been proved that Peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) was involved in neurodegenerative diseases by regulating mitochondrial biogenesis. The underlying mechanisms of PGC-1α and Nod-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome in PND are not well understood. In this study, we constructed a model of laparotomy in aged mice, and then examined the cognition changes with novel object recognition tests and fear condition tests. The protein levels of PGC-1α and NLRP3 in the hippocampus were detect after surgery. Our results showed that NLRP3 and downstream PI3K/AKT pathway expressions were augmented in the hippocampus after surgery, whereas, the expressions of PGC-1α/estrogen-related receptor α (ERRα)/Unc-51-like autophagy activating kinase 1 (ULK1) pathway were diminished after surgery. In addition, we found that NLRP3 was mainly co-localized with neurons in the hippocampus, and synaptic-related proteins were reduced after surgery. At the same time, transmission electron microscopy (TEM) showed that mitochondria were impaired after surgery. Pharmacological treatment of MCC950, a selective NLRP3 inhibitor, effectively alleviated PND. Activation of PGC-1α with ZLN005 significantly ameliorated PND by enhancing the PGC-1α/ERRα/ULK1 signaling pathway, and further suppressing NLRP3 activation. As a result, we conclude that suppression of the PGC-1α/ERRα/ULK1 signaling pathway is the primary mechanism of PND which caused mitochondrial dysfunction, and activated NLRP3 inflammasome and downstream PI3K/AKT pathway, eventually improved cognitive dysfunction.

Lycopene alleviates zearalenone-induced oxidative stress, apoptosis, and NLRP3 inflammasome activation in mice kidneys

The aim of this study was to investigate the protective effects of lycopene on renal damage caused by zearalenone (ZEN). Male Kunming mice were treated daily for 4 weeks by intragastric administration with 40 mg/kg ZEN in the presence or absence of lycopene (2.5 or 5 mg/kg). The results showed that lycopene markedly alleviated the damage of renal structure and function in mice induced by ZEN, as indicated by the reduced degree of pathological damage and the decreased levels of urea nitrogen and creatinine. Meanwhile, results of dihydroethidine (DHE) staining and biochemical markers revealed that ZEN exposure notably increased the levels of reactive oxygen species (ROS) and malondialdehyde (MDA), decreased the level of GSH, and reduced the activities of catalase (CAT) and superoxide dismutase (SOD). Administration of lycopene alleviated the increased oxidative stress induced by ZEN. Moreover, ZEN ingestion notably resulted in apoptosis, increased the protein levels of BCL2 associated X protein (Bax) and cleaved caspase-3, and decreased the protein levels of apoptosis regulator Bcl-2 (Bcl-2), which were reversed by lycopene intervention. Results of immunofluorescence demonstrated that lycopene reversed ZEN-induced the upregulation of NOD-like receptor pyrin domain-containing protein 3 (NLRP3), Caspase-1, and interleukin-1 beta (IL-1β) in mice kidneys. Lycopene supplementation could alleviate ZEN-induced renal toxicity by inhibiting oxidative stress, apoptosis, and NLRP3 inflammasome activation.

Anti-pneumoconiosis effect of schisantherin A in PMA-induced A549 cells and SiO2/TiO2nanoparticles-induced acute pulmonary injury in mice

There has been significant global interest in respiratory health driven by the coronavirus disease (COVID-19) and severe environmental pollution. This study explored the potential of schisantherin A (SchA), a compound derived from Schisandra chinensis, to protect against acute pneumoconiosis. We assessed the effects of SchA on phorbol 12-myristate 13-acetate (PMA)-stimulated A549 alveolar epithelial cells and SiO2/TiO2-induced pulmonary injury in mice. In A549 cells, SchA significantly decreased pro-inflammatory mediators such as inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and interleukin (IL)-8 levels. SchA-mediated reduction in inflammatory mediators was associated with the downregulation of PMA-stimulated nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and mitogen-activated protein kinase (MAPK) signaling activation. In SiO2/TiO2-induced lung-injured mice, SchA administration significantly reduced MUC5AC production in lung tissue. SchA administration significantly downregulated the overexpression of NK-κB and the subsequent production of COX-2, iNOS, and NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasomes. It significantly suppressed expected increases in total cell numbers and pro-inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α) and IL-1β in the bronchoalveolar lavage fluid (BALF) in SiO2/TiO2-stimulated mice. In contrast, the SiO2/TiO2-mediated decrease in IL-10 levels was significantly improved by SchA treatment. These fundamental results can be used to develop potential treatments involving SchA for acute pneumoconiosis.

Inflammatory markers and noncoding-RNAs responses to low and high compressions of HIIT with or without berberine supplementation in middle-aged men with prediabetes.

This study compared the capacity of two different models of HIIT [high-(HC) and low-(LC) compression], with or without the use of berberine (BBR), on NOD-like receptor pyrin domain-containing protein-3 (NLRP3), H19, interleukin (IL)-1β, high-sensitivity C-reactive protein (hs-CRP), and insulin resistance markers. Fifty-four middle-aged men with overweight or obesity and prediabetes [fasting blood glucose (FBG) 110-180 mg/dL] were randomly and equally assigned to the HC, LC, HC + BBR, LC + BBR, BBR, and non-exercising control (CON) groups. The HC (2:1 work-to-rest) and LC (1:1 work-to-rest) home-based training programs included 2-4 sets of 8 exercises at 80%-95% HRmax, twice a week for 8 weeks. Participants in the berberine groups received approximately 1000 mg daily. All exercise interventions led to a significant reduction in hs-CRP, IL-1β, insulin, FBG, and insulin resistance index (HOMA-IR) versus CON. Notably, there was a significant reduction in FBG and HOMA-IR with the BBR group compared to the baseline. Both NLRP3 and H19 experienced a significant drop only with LC in comparison to the baseline. While both exercise protocols were beneficial overall, LC uniquely exhibited more anti-inflammatory effects, as indicated by reductions in H19 and NLRP3. However, the addition of berberine to the exercise programs did not demonstrate additional benefits.

Evaluation of inflammasomes as biomarker following non-surgical periodontal treatment

ObjectiveThe purpose of this study was to investigate interleukin (IL)-1β, IL-18, nod-like receptor pyrin domain-containing protein 3 (NLRP3), apoptosis-related speck-like protein containing a caspase activation and recruitment domain (ASC), and caspase-1 levels in saliva and serum in different periodontal diseases and to evaluate the changes after non-surgical periodontal treatment (NSPT). DesignA total of 45 participants, 15 healthy, 15 gingivitis, and 15 stage III grade C (SIIIGC) periodontitis patients, were included in the study. Periodontal parameters were assessed, and salivary and serum samples were collected at baseline in all groups and one and three months after NSPT in gingivitis and periodontitis groups. An enzyme-linked immunosorbent assay was used to analyse IL-1β, IL-18, NLRP3, ASC, and caspase-1 levels. ResultsAfter NSPT, improvement was observed in all clinical parameters, along with periodontal inflamed surface area (PISA) in gingivitis and periodontitis groups. PISA scores were positively correlated with IL-1β, NLRP3, and caspase-1 at baseline (p < 0.05). Salivary and serum IL-1β, NLRP3 levels were higher in periodontitis compared to healthy controls at baseline and reduced after treatment (p < 0.05). Receiver operating characteristic analysis revealed that salivary IL-1β, NLRP3, and caspase-1 had the ability to discriminate SIIIGC periodontitis patients from healthy subjects (p < 0.05). ConclusionIn conclusion, salivary IL-1β, NLRP3, and caspase-1 are at aberrantly high levels in SIIIGC periodontitis and are remarkably decreased following NSPT; these inflammasome biomarkers may show potential utility in diagnosing and monitoring periodontitis.

Interaction of obesity and proteins associated with the NLRP3 inflammasome following mild traumatic brain injury

The NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome has been associated with worse outcomes from severe traumatic brain injury (TBI). The NLRP3 inflammasome is also strongly associated with other pro-inflammatory conditions, such as obesity. Little is known about the potential effect of mild TBI (mTBI) on the NLRP3 inflammasome and the extent to which modifying factors, such as obesity, may augment the inflammatory response to mTBI. The purpose of this study was to evaluate the association of NLRP3 inflammasome proteins with obese body mass index (BMI ≥ 30) within 24 h of mTBI after presenting to a level 1 trauma center emergency department. This is a secondary analysis of prospectively enrolled patients with mTBI who presented to the emergency department of one U.S. Level 1 trauma center from 2013 to 2018 (n = 243). A series of regression models were built to evaluate the association of NLRP3 proteins obtained from blood plasma within 24 h of injury and BMI as well as the potential interaction effect of higher BMI with NLRP3 proteins (n = 243). A logistic regression model revealed a significant association between IL-18 (p < 0.001) in mTBI patients with obese BMI compared to mTBI patients with non-obese BMI (< 30). Moderation analyses revealed statistically significant interaction effects between apoptotic speck-like protein (ASC), caspase-1, IL-18, IL-1β and obese BMI which worsened symptom burden, quality of life, and physical function at 2 weeks and 6 months post-injury. Higher acute concentrations of IL-1β in the overall cohort predicted higher symptoms at 6-months and worse physical function at 2-weeks and 6-months. Higher acute concentrations of IL-18 in the overall cohort predicted worse physical function at 6-months. In this single center mTBI cohort, obese BMI interacted with higher acute concentrations of NLRP3 inflammasome proteins and worsened short- and long-term clinical outcomes.

Pharmacological Analysis of NLRP3 Inflammasome Inhibitor Sodium [(1,2,3,5,6,7-Hexahydro-s-indacen-4-yl)carbamoyl][(1-methyl-1H-pyrazol-4-yl)({[(2S)-oxolan-2-yl]methyl})sulfamoyl]azanide in Cellular and Mouse Models of Inflammation Provides a Translational Framework.

Interleukin (IL)-1β is an apex proinflammatory cytokine produced in response to tissue injury and infection. The output of IL-1β from monocytes and macrophages is regulated not only by transcription and translation but also post-translationally. Release of the active cytokine requires activation of inflammasomes, which couple IL-1β post-translational proteolysis with pyroptosis. Among inflammasome platforms, NOD-like receptor pyrin domain-containing protein 3 (NLRP3) is implicated in the pathogenesis of numerous human disorders in which disease-specific danger-associated molecular patterns (DAMPS) are positioned to drive its activation. As a promising therapeutic target, numerous candidate NLRP3-targeting therapeutics have been described and demonstrated to provide benefits in the context of animal disease models. While showing benefits, published preclinical studies have not explored dose-response relationships within the context of the models. Here, the preclinical pharmacology of a new chemical entity, [(1,2,3,5,6,7-hexahydro-s-indacen-4-yl)carbamoyl][(1-methyl-1H-pyrazol-4-yl)({[(2S)-oxolan-2-yl]methyl})sulfamoyl]azanide (NT-0249), is detailed, establishing its potency and selectivity as an NLRP3 inhibitor. NT-0249 also is evaluated in two acute in vivo mouse challenge models where pharmacodynamic/pharmacokinetic relationships align well with in vitro blood potency assessments. The therapeutic utility of NT-0249 is established in a mouse model of cryopyrin-associated periodic syndrome (CAPS). In this model, mice express a human gain-of-function NLRP3 allele and develop chronic and progressive IL-1β-dependent autoinflammatory disease. NT-0249 dose-dependently reduced multiple inflammatory biomarkers in this model. Significantly, NT-0249 decreased mature IL-1β levels in tissue homogenates, confirming in vivo target engagement. Our findings highlight not only the pharmacological attributes of NT-0249 but also provide insight into the extent of target suppression that will be required to achieve clinical benefit.

Peficitinib alleviated acute lung injury by blocking glycolysis through JAK3/STAT3 pathway

Peficitinib is a selective Janus kinase (JAK3) inhibitor recently developed and approved for the treatment of rheumatoid arthritis in Japan. Glycolysis in macrophages could induce NOD-like receptor (NLR) family and pyrin domain-containing protein 3 (NLRP3) inflammasome activation, thus resulting in pyroptosis and acute lung injury (ALI). The aim of our study was to investigate whether Peficitinib could alleviate lipopolysaccharide (LPS)-induced ALI by inhibiting NLRP3 inflammasome activation. Wild type C57BL/6J mice were intraperitoneally injected with Peficitinib (5 or 10 mg·kg−1·day−1) for 7 consecutive days before LPS injection. The results showed that Peficitinib pretreatment significantly relieved LPS-induced pulmonary edema, inflammation, and apoptosis. NLRP3 inflammasome and glycolysis in murine lung tissues challenged with LPS were also blocked by Peficitinib. Furthermore, we found that the activation of JAK3/signal transducer and activator of transcription 3 (STAT3) was also suppressed by Peficitinib in mice with ALI. However, in Jak3 knockout mice, Peficitinib did not show obvious protective effects after LPS injection. In vitro experiments further showed that Jak3 overexpression completely abolished Peficitinib-elicited inhibitory effects on pyroptosis and glycolysis in LPS-induced RAW264.7 macrophages. Finally, we unveiled that LPS-induced activation of JAK3/STAT3 was mediated by toll-like receptor 4 (TLR4) in RAW264.7 macrophages. Collectively, our study proved that Peficitinib could protect against ALI by blocking JAK3-mediated glycolysis and pyroptosis in macrophages, which may serve as a promising candidate against ALI in the future.

Therapeutic Effect of Levetiracetam Against Thioacetamide-Induced Hepatic Encephalopathy Through Inhibition of Oxidative Stress and Downregulation of NF-κB, NLRP3, iNOS/NO, Pro-Inflammatory Cytokines and Apoptosis.

Hepatic encephalopathy (HE) is a serious brain disorder which associated with neurological and psychiatric manifestations. Oxidative stress and neuroinflammation and apoptosis play main roles in the development of brain damage in HE. Levetiracetam is an antiseizure drug with established antioxidant and anti-inflammatory activities. In the present study we investigated the therapeutic effects of levetiracetam against brain injury in HE and its underlying mechanisms of action. Male C57BL/6 mice were subjected to the induction of HE by the injection of thioacetamide (200 mg/kg) for 2 days. Mice were treated with levetiracetam at two doses (50 or 100 mg/kg/day) for 3 days in the treatment groups. Animals were subjected to a behavioral test and the brain tissues were dissected for histopathological, biochemical, gene expression and immunofluorescence analysis. The results showed that levetiracetam alleviated body weight loss and improved locomotor activity of mice with HE. Levetiracetam treatment decreased the histopathological changes, lipid peroxidation and protein carbonylation while restored the antioxidants (GSH, SOD and CAT) in the brain. Levetiracetam decreased the expression and activity of NF-κB, NOD-like receptor pyrin domain-containing protein 3 (NLRP3) and pro-inflammatory cytokines (TNF-α, IL-1β, IL-6, and IFN-γ) in the brain tissue. Administration of levetiracetam inhibited iNOS/NO pathway and myeloperoxidase (MPO) activity in the brain. Moreover, caspase-3 was decreased and the ratio of Bcl2/Bax was increased in the brain of mice treated with levetiracetam. These findings suggest that levetiracetam may be a promising therapeutic agent for brain injury in HE through inhibiting the oxidative, inflammatory and apoptotic pathways.

Target Cell Activation of a Structurally Novel NOD-Like Receptor Pyrin Domain-Containing Protein 3 Inhibitor NT-0796 Enhances Potency.

The NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome is a central regulator of innate immunity, essential for processing and release of interleukin-1β and pyroptotic cell death. As endogenous NLRP3 activating triggers are hallmarks of many human chronic inflammatory diseases, inhibition of NLRP3 has emerged as a therapeutic target. Here we identify NDT-19795 as a novel carboxylic acid-containing NLRP3 activation inhibitor in both human and mouse monocytes and macrophages. Remarkably, conversion of the carboxylate to an isopropyl-ester (NT-0796) greatly enhances NLRP3 inhibitory potency in human monocytes. This increase is attributed to the ester-containing pharmacophore being more cell-penetrant than the acid species and, once internalized, the ester being metabolized to NDT-19795 by carboxylesterase-1 (CES-1). Mouse macrophages do not express CES-1, and NT-0796 is ineffective in these cells. Mice also contain plasma esterase (Ces1c) activity which is absent in humans. To create a more human-like model, we generated a mouse line in which the genome was modified, removing Ces1c and replacing this segment of DNA with the human CES-1 gene driven by a mononuclear phagocyte-specific promoter. We show human CES-1 presence in monocytes/macrophages increases the ability of NT-0796 to inhibit NLRP3 activation both in vitro and in vivo. As NLRP3 is widely expressed by monocytes/macrophages, the co-existence of CES-1 in these same cells affords a unique opportunity to direct ester-containing NLRP3 inhibitors precisely to target cells of interest. Profiling NT-0796 in mice humanized with respect to CES-1 biology enables critical modeling of the pharmacokinetics and pharmacodynamics of this novel therapeutic candidate. SIGNIFICANCE STATEMENT: Inhibition of NLRP3 represents a desirable therapeutic strategy for the treatment of multiple human disorders. In this study pharmacological properties of a structurally-novel, ester-containing NLRP3 inhibitor NT-0796 are characterized. To study pharmacodynamics of NT-0796 in vivo, a mouse line was engineered possessing more human-like traits with respect to carboxylesterase biology. In the context of these hCES-1 mice, NT-0796 serves as a more effective inhibitor of NLRP3 activation than the corresponding acid, highlighting the full translational potential of the ester strategy.

METTL14 contributes to acute lung injury by stabilizing NLRP3 expression in an IGF2BP2-dependent manner

Acute lung injury (ALI) as well as its more severe form, acute respiratory distress syndrome (ARDS), frequently leads to an uncontrolled inflammatory response. N6-methyladenosine (m6A) modification was associated with the progression of several inflammatory diseases. However, the role of methyltransferase-like 14 (METTL14)-mediated m6A methylation in ALI/ARDS remains unclear. Here, we reported an increase in overall expression levels of m6A and METTL14 in circulating monocyte-derived macrophages recruited to the lung following ALI, which is correlated with the severity of lung injury. We further demonstrated the critical function of METTL14 in activating NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome in vitro and in mouse models of ALI/ARDS, and validated NLRP3 as the downstream target of METTL14 by the m6A RNA immunoprecipitation (MeRIP) and RIP assays. Mechanistically, METTL14-methylated NLRP3 transcripts were subsequently recognized by insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2), an m6A reader, which stabilized NLRP3 mRNA. Furthermore, we observed that IGF2BP2 knockdown diminished LPS-induced ALI in mice by downregulating NLRP3 expression. In summation, our study revealed that the molecular mechanism underlying the pathogenesis of ALI/ARDS involves METTL14-mediated activation of NLRP3 inflammasome in an IGF2BP2 dependent manner, thereby demonstrating the potential of METTL14 and IGF2BP2 as promising biomarkers and therapeutic targets for ALI/ARDS treatment.

The roles of ubiquitination and deubiquitination of NLRP3 inflammasome in inflammation-related diseases: A review.

The inflammatory response is a natural immune response that prevents microbial invasion and repairs damaged tissues. However, excessive inflammatory responses can lead to various inflammation-related diseases, posing a significant threat to human health. The NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome is a vital mediator in the activation of the inflammatory cascade. Targeting the hyperactivation of the NLRP3 inflammasome may offer potential strategies for the prevention or treatment of inflammation-related diseases. It has been established that the ubiquitination and deubiquitination modifications of the NLRP3 inflammasome can provide protective effects in inflammation-related diseases. These modifications modulate several pathological processes, including excessive inflammatory responses, pyroptosis, abnormal autophagy, proliferation disorders, and oxidative stress damage. Therefore, this review discusses the regulation of NLRP3 inflammasome activation by ubiquitination and deubiquitination modifications, explores the role of these modifications in inflammation-related diseases, and examines the potential underlying mechanisms.

HFD-exacerbated Metabolic Side Effects of Olanzapine Are Suppressed by ER Stress Inhibitor.

Numerous schizophrenic patients are suffering from obesity primarily attributed to antipsychotic medication and poor dietary habits. This study investigated the progressive deterioration of olanzapine-induced metabolic disorders in the presence of a high-fat diet (HFD) and explored the involvement of endoplasmic reticulum (ER) stress. Female Sprague-Dawley rats fed on a standard chow diet or HFD were treated with olanzapine (3 mg/kg/day) and the ER stress inhibitor 4-phenylbutyric acid (4-PBA, 1 and 0.5 g/kg/day) for 8 days. Changes in body weight, food intake, and plasma lipids were assessed. Hepatic fat accumulation was evaluated using oil red O staining. Western blotting and immunofluorescence assays were employed to examine the expression of ER stress markers, NOD-like receptor pyrin domain-containing protein 3 (NLRP3), and proopiomelanocortin (POMC) in the hypothalamus or liver. Compared to olanzapine alone, olanzapine+HFD induced greater weight gain, increased hyperlipidemia, and enhanced hepatic fat accumulation (P<0.05). Co-treatment with 4-PBA exhibited a dose-dependent inhibition of these effects (P<0.05). Further mechanistic investigations revealed that olanzapine alone activated ER stress, upregulated NLRP3 expression in the hypothalamus and liver, and downregulated hypothalamic POMC expression. The HFD exacerbated these effects by 50%-100%. Moreover, co-administration of 4-PBA dose-dependently attenuated the olanzapine+HFD-induced alterations in ER stress, NLRP3, and POMC expression in the hypothalamus and liver (P<0.05). HFD worsened olanzapine-induced weight gain and lipid metabolic disorders, possibly through ER stress-POMC and ER stress-NLRP3 signaling. ER stress inhibitors could be effective in preventing olanzapine+HFD-induced metabolic disorders.

Takinib inhibits microglial M1 polarization and oxidative damage after subarachnoid hemorrhage by targeting TAK1-dependent NLRP3 inflammasome signaling pathway.

Transforming growth factor-β-activated kinase 1 (TAK1) positively regulates oxidative stress and inflammation in different diseases. Takinib, a novel and specific TAK1 inhibitor, has beneficial effects in a variety of disorders. However, the effects of takinib on early brain injury (EBI) after subarachnoid hemorrhage (SAH) and the underlying molecular mechanisms remain unknown. Our study showed that takinib administration significantly inhibited phosphorylated TAK1 expression after SAH. In addition, takinib suppressed M1 microglial polarization and promoted M2 microglial polarization. Furthermore, blockade of TAK1 by takinib reduced neuroinflammation, oxidative damage, brain edema, and neuronal apoptosis, and improved neurological behavior after SAH. Mechanistically, we revealed that TAK1 inhibition by takinib mitigated reactive oxygen species (ROS) production and ROS-mediated nod-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome activation. In contrast, NLRP3 activation by nigericin abated the neuroprotective effects of takinib against EBI after SAH. In general, our study demonstrated that takinib could protect against EBI by targeting TAK1-ROS-NLRP3 inflammasome signaling. Inhibition of TAK1 might be a promising option in the management of SAH.

Prostaglandin D2 regulates Escherichia coli-induced inflammatory responses through TLR2, TLR4, and NLRP3 in macrophages

Prostaglandin D2 (PGD2) synthesis is closely associated with the innate immune response mediated by pattern recognition receptors (PPRs). We determined PGD2 synthesis whether mediated by Toll-like receptor 2 (TLR2), TLR4 and Nod-like receptor pyrin domain-containing protein 3 (NLRP3) in Escherichia coli (E. coli)-, lipopolysaccharide (LPS)- and Braun lipoprotein (BLP)-stimulated macrophages. Our data demonstrate that TLR2, TLR4, and NLRP3 could regulate the synthesis of PGD2 through cyclo-oxygenase-2 (COX-2) and hematopoietic PGD synthase (H-PGDS) in E. coli-, LPS- or BLP-stimulated macrophages, suggesting that TLR2, TLR4, and NLRP3 are critical in regulating PGD2 secretion by controlling PGD2 synthetase expression in E. coli-, LPS- or BLP-stimulated macrophages. The H-PGDS (a PGD2 specific synthase) inhibitor pre-treatment could down-regulate the secretion of TNF-α, RANTES and IL-10 in LPS- and E. coli-stimulated macrophage. Meanwhile, H-PGDS inhibitor could down-regulate the secretion of TNF-α, while up-regulated RANTES and IL-10 secretion in BLP-stimulated macrophages, suggesting that PGD2 could regulate the secretion of cytokines and chemokines in E. coli-, LPS- or BLP-stimulated macrophages. Furthermore, exogenous PGD2 regulates the secretion of cytokines and chemokines through activation of MAPK and NF-κB signaling pathways after E. coli-, LPS- or BLP stimulation in macrophages. Taken together, PGD2 is found able to regulate E. coli-induced inflammatory responses through TLR2, TLR4, and NLRP3 in macrophages.

CXCL5 promotes lipotoxicity of hepatocytes through upregulating NLRP3/Caspase-1/IL-1β signaling in Kupffer cells and exacerbates nonalcoholic steatohepatitis in mice

Immune-inflammatory responses play a key role in the development of nonalcoholic steatohepatitis (NASH). Previous studies have demonstrated that CXC motif chemokine ligand 5 (CXCL5) correlates positively with obesity and type 2 diabetes. This study is to explore the functional role of CXCL5 in the pathogenesis of NASH. To establish a NASH model, mice were fed with methionine-and choline-deficient high-fat diet for 6 weeks and anti-CXCL5 mAb was injected during the same period. An in vitro NASH model was established by treating palmitic acid (PA), using a trans-well co-culture system of mouse primary hepatocytes and Kupffer cells (KCs), and recombinant mouse (rm) CXCL5 was treated after PA administration. Our data showed that hepatic CXCL5 levels were highly expressed in the NASH mouse model. CXCL5 neutralization significantly alleviated the severity of NASH livers, demonstrated by pathological analysis, decreased biochemicals, and inflammation. Besides, neutralizing CXCL5 reduced lipid accumulation, cell death, and fibrosis in injured livers. In vitro, rmCXCL5 could not affect the activation of hepatic stellate cells. Also, rmCXCL5 exacerbated PA-induced hepatotoxicity and lipid deposition in hepatocytes co-cultured with KCs rather than in single-cultured hepatocytes. Mechanistically, rmCXCL5 not only promoted NOD-like receptor pyrin domain-containing protein 3 (NLRP3) expression, Cleaved caspase-1 expression, and interleukin 1 beta (IL-1β) secretion in single-cultured and co-cultured KCs but also increased lipid deposition in co-cultured hepatocytes. In addition, MCC950, an inhibitor of NLRP3, almost abolished the effects of rmCXCL5 on PA-treated co-culture system. Therefore, CXCL5 could exacerbate NASH by promoting lipotoxicity of hepatocytes via upregulating NLRP3/Caspase-1/IL-1β signaling in KCs.

Pharmacology of a Potent and Novel Inhibitor of the NOD-Like Receptor Pyrin Domain-Containing Protein 3 (NLRP3) Inflammasome that Attenuates Development of Nonalcoholic Steatohepatitis and Liver Fibrosis.

The NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome is a multiprotein complex and component of the innate immune system that is activated by exogenous and endogenous danger signals to promote activation of caspase-1 and the maturation and release of the proinflammatory cytokines interleukin (IL)-1β and IL-18. Inappropriate activation of NLRP3 has been implicated in the pathophysiology of multiple inflammatory and autoimmune diseases, including cardiovascular disease, neurodegenerative diseases, and nonalcoholic steatohepatitis (NASH), thus increasing the clinical interest of this target. We describe in this study the preclinical pharmacologic, pharmacokinetic, and pharmacodynamic properties of a novel and highly specific NLRP3 inhibitor, JT001 (6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazine-3-sulfonylurea). In cell-based assays, JT001 potently and selectively inhibited NLRP3 inflammasome assembly, resulting in the inhibition of cytokine release and the prevention of pyroptosis, a form of inflammatory cell death triggered by active caspase-1. Oral administration of JT001 to mice inhibited IL-1β production in peritoneal lavage fluid at plasma concentrations that correlated with mouse in vitro whole blood potency. Orally administered JT001 was effective in reducing hepatic inflammation in three different murine models, including the Nlrp3A350V /+CreT model of Muckle-Wells syndrome (MWS), a diet-induced obesity NASH model, and a choline-deficient diet-induced NASH model. Significant reductions in hepatic fibrosis and cell damage were also observed in the MWS and choline-deficient models. Our findings demonstrate that blockade of NLRP3 attenuates hepatic inflammation and fibrosis and support the use of JT001 to investigate the role of NLRP3 in other inflammatory disease models. SIGNIFICANCE STATEMENT: Persistent inflammasome activation is the consequence of inherited mutations of NLRP3 and results in the development of cryopyrin-associated periodic syndromes associated with severe systemic inflammation. NLRP3 is also upregulated in nonalcoholic steatohepatitis, a metabolic chronic liver disease currently missing a cure. Selective and potent inhibitors of NLRP3 hold great promise and have the potential to overcome an urgent unmet need.

Dopamine Activates the D1R-Zn2+ Signaling Pathway to Trigger Inflammatory Response in Primary-Cultured Rat Embryonic Cortical Neurons.

Neuroinflammation is an early event during the pathogenesis of neurodegenerative disorders. Most studies focus on how the factors derived from pathogens or tissue damage activate the inflammation-pyroptosis cell death pathway. It is unclear whether endogenous neurotransmitters could induce inflammatory responses in neurons. Our previous reports have shown that dopamine-induced elevation of intracellular Zn2+ concentration via the D1-like receptor (D1R) is a prerequisite for autophagy and cell death in primary cultured rat embryonic neurons. Here we further examined that this D1R-Zn2+ signaling initiates the transient inflammatory response leading to cell death in cultured cortical neurons. Pretreating the cultured neurons with Zn2+ chelator and inhibitors against inflammation could enhance the cell viability in neurons treated with dopamine and dihydrexidine, an agonist of D1R. Both dopamine and dihydrexidine greatly enhanced inflammasome formation; a Zn2+ chelator, N,N,N',N'-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine, suppressed this increment. Dopamine and dihydrexidine increased the expression levels of NOD-like receptor pyrin domain-containing protein 3 and enhanced the maturation of caspase-1, gasdermin D, and IL-1β; these changes were all Zn2+-dependent. Dopamine treatment did not recruit the N-terminal of the gasdermin D to the plasma membrane but enhanced its localization to the autophagosomes. Pretreating the neurons with IL-1β could increase the viability of neurons challenged with dopamine. These results demonstrate a novel D1R-Zn2+ signaling cascade activating neuroinflammation and cell death. Therefore, maintaining a balance between dopamine homeostasis and inflammatory responses is an important therapeutic target for neurodegeneration. Dopamine elicits transient inflammatory responses in cultured cortical neurons via the D1R-Zn2+ signaling pathway. Dopamine elevates [Zn2+]i to induce the formation of inflammasomes, which activates caspase-1, resulting in the maturation of IL-1β and gasdermin D (GSDMD). Therefore, the homeostasis of dopamine and Zn2+ are critical therapeutic targets for inflammation-derived neurodegeneration.

Gentiopicroside enhances the protective effect of trimetazidine against myocardial ischemia-reperfusion injury via the AMPK/NLRP3 inflammasome signaling.

Myocardial ischemia-reperfusion injury (MI/R) leads to the inevitable clinical consequences of myocardial infarction and subsequent heart failure. Trimetazidine (TMZ), an anti-ischemic agent, exerts protective potential in MI/R but had limited efficacy for some patients. Here we sought to investigate the single and combined application of gentiopicroside (GPS) and TMZ in MI/R. Notably, GPS had little cytotoxicity to cardiomyocytes. GPS attenuated hypoxia/reoxygenation (H/R)-induced cell death, reactive oxygen species production, lactate dehydrogenase and malondialdehyde releases, and antioxidant stress enzyme superoxide dismutase activity, indicating the protective efficacy of GPS against H/R-induced oxidative injury. Importantly, GPS enhanced the protective efficacy of TMZ against H/R-mediated cardiomyocyte injury. Additionally, GPS mitigated the transcription and releases of pro-inflammatory cytokine interleukin-6 and tumor necrosis factor-α in H/R-treated cardiomyocytes, which were enhanced after co-treatment with TMZ. Mechanistically, GPS activated the AMP-activated protein kinase (AMPK) signaling to inhibit H/R-induced NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome, which was further enhanced after costimulation with TMZ. Importantly, blocking the AMPK signaling reversed the protective roles of GPS and its combination with TMZ in H/R-induced oxidative insult and inflammation. In vivo, both GPS and TMZ alleviated the abnormal cardiac structure, cardiomyocyte apoptosis, and cardiac dysfunction in MI/R rats, which were further enhanced after administration with GPS and TMZ together. Furthermore, GPS intensified TMZ-mediated inhibition of oxidative injury, inflammation, and the AMPK/NLRP3 signaling in MI/R rats. Collectively, GPS enhances the protective efficacy of TMZ against MI/R injury through AMPK activation-mediated inhibition of NLRP3 inflammasome signaling, implying a promising therapeutic agent for the treatment of MI/R.

AMPK activation attenuates cancer-induced bone pain by reducing mitochondrial dysfunction-mediated neuroinflammation.

Bone metastasis of cancer cells leads to severe pain by disrupting bone structure and inducing central sensitization. Neuroinflammation in the spinal cord plays a decisive role in the maintenance and development of pain. In the current study, male Sprague-Dawley (SD) rats are used to establish a cancer-induced bone pain (CIBP) model by intratibial injection of MRMT-1 rat breast carcinoma cells. Morphological and behavioral analyses verify the establishment of the CIBP model, which represents bone destruction, spontaneous pain and mechanical hyperalgesia in CIBP rats. Activation of astrocytes marked by upregulated glial fibrillary acidic protein (GFAP) and enhanced production of the proinflammatory cytokine interleukin-1β (IL-1β) are accompanied by increased inflammatory infiltration in the spinal cord of CIBP rats. Furthermore, activation of the NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome is consistent with increased neuroinflammation. Adenosine monophosphate-activated protein kinase (AMPK) activation is involved in attenuating inflammatory pain and neuropathic pain. Intrathecal injection of the AMPK activator AICAR in the lumbar spinal cord reduces dynamin-related protein 1 (Drp1) GTPase activity and suppresses NLRP3 inflammasome activation. This effect consequently alleviates pain behaviors in CIBP rats. Cell research on C6 rat glioma cells indicates that AICAR treatment restores IL-1β-induced impairment of mitochondrial membrane potential and elevation of mitochondrial reactive oxygen species (ROS). In summary, our findings indicate that AMPK activation attenuates cancer-induced bone pain by reducing mitochondrial dysfunction-mediated neuroinflammation in the spinal cord.