NOD-like Receptor 3 Inflammasome Research Articles

Potential Common Mechanisms of Cytotoxicity Induced by Organophosphorus Pesticides via NLRP3 Inflammasome Activation.

The Multi-Threat Medical Countermeasure (MTMC) technique is crucial for developing common biochemical signaling pathways, molecular mediators, and cellular processes. This study revealed that the Nod-like receptor 3 (NLRP3) inflammasome pathway may be a significant contributor to the cytotoxicity induced by various organophosphorus pesticides (OPPs). The study demonstrated that exposure to six different types of OPPs (paraoxon, dichlorvos, fenthion, dipterex, dibrom, and dimethoate) led to significant cytotoxicity in BV2 cells, which was accompanied by increased expression of NLRP3 inflammasome complexes (NLRP3, ASC, Caspase-1) and downstream inflammatory cytokines (IL-1β, IL-18), in which the order of cytotoxicity was dichlorvos>dipterex>dibrom>paraoxon>fenthion>dimethoate, based on the IC50 values of 274, 410, 551, 585, 2,158, and 1,527,566μM, respectively. The findings suggest that targeting the NLRP3 inflammasome pathway could be a potential approach for developing broad-spectrum antitoxic drugs to combat multi-OPPs-induced toxicity. Moreover, inhibition of NLRP3 efficiently protected the cells against cytotoxicity induced by these six OPPs, and the expression of NLRP3, ASC, Caspase-1, IL-1β, and IL-18 decreased accordingly. The order of NLRP3 affinity for OPPs was dimethoate>paraoxon>dichlorvos>dibrom>(fenthion and dipterex) based on K D values of 89.8, 325, 1,460, and 2,690μM, respectively. Furthermore, the common molecular mechanism of NLRP3-OPPs was clarified by the presence of toxicity effector groups (benzene ring, nitrogen/oxygen-containing functional group); =O, -O-, or =S (active) groups; and combination residues (Gly271, Asp272). This finding provided valuable insights into exploring the common mechanisms of multiple threats and developing effective therapeutic strategies to prevent OPPs poisoning.

Exposure of nonylphenol promoted NLRP3 inflammasome and GSDMD-mediated pyroptosis in allergic rhinitis mice

Studies have confirmed that the intake of nonylphenol (NP) can increase nasal symptoms, eosinophils, and Th2 responses in allergic rhinitis (AR) mice. However, the molecular mechanism of NP exacerbating AR inflammatory response remains unclear. Recent data suggest that NOD-like receptor 3 (NLRP3) inflammasome-mediated pyroptosis contributes to AR development. To investigate the effects of NP on NLRP3 inflammasomes and pyroptosis, an AR mouse model induced by ovalbumin (OVA) was established and treated with 0.5 mg/kg/d NP every other day. Nasal symptoms were evaluated after the final OVA instillation. Mast cells and Eosinophils in the nasal mucosa were observed using toluidine blue and Sirius red staining, respectively. The levels of NLRP3, Caspase-1, ASC, phospho–nuclear factor kappa B (NF-κB) p65, interleukin (IL)-6, TNF-α, IL-18, GSDMD and IL-1β, were assessed by using immunohistochemical staining, ELISA, quantitative real-time PCR, or Western blot. Exposure to NP aggravates AR symptoms and promotes eosinophils, mast cells, and inflammatory factors release, along with significantly increased of NF–κB, NLRP3, Caspase-1, ASC, and GSDMD. It was concluded that NP exposure promotes NLRP3 inflammasome and GSDMD-mediated pyroptosis of the nasal mucosa. Targeted of NLRP3 and GSDMD-mediated pyroptosis may be a novel therapeutic strategy for AR exposed to NP.

NLRP3 inflammasome-mediated pyroptosis involvement in cadmium exposure-induced cognitive deficits via the Sirt3-mtROS axis

Cadmium (Cd), a toxic heavy metal, exerts deleterious effects on neuronal survival and cognitive function. NOD-like receptor 3 (NLRP3) inflammasome-dependent pyroptosis has been linked to Cd-induced cytotoxicity. The current research intended to elucidate the role of NLRP3 inflammasome-mediated pyroptosis in Cd-evoked neuronal death and cognitive impairments and the underlying mechanisms. Exposure to 1 mg/kg Cd for 8 weeks led to hippocampal-dependent cognitive deficits and neural/synaptic damage in mice. NLRP3 inflammasome-related protein expression (NLRP3, ASC, and caspase1 p20) and neuronal pyroptosis were significantly upregulated in Cd-treated hippocampi and SH-SY5Y cells. Moreover, pretreatment with the NLRP3 inhibitor MCC950 mitigated Cd-elicited NLRP3 inflammasome activation and subsequent neuronal pyroptosis in SH-SY5Y cells. Furthermore, exposure to Cd downregulated Sirt3 expression, suppressed SOD2 activity by hyperacetylation, and enhanced mtROS accumulation in vivo and in vitro. Notably, Cd-induced NLRP3 inflammasome-dependent neuronal pyroptosis was attenuated by a mtROS scavenger or Sirt3 overexpression in SH-SY5Y cells. In addition, Cd failed to further suppress SOD activity and activate NLRP3 inflammasome-dependent neuronal pyroptosis in Sirt3 shRNA-treated SH-SY5Y cells. Collectively, our findings indicate that Cd exposure induces neuronal injury and cognitive deficits by activating NLRP3 inflammasome-dependent neuronal pyroptosis and that activation of the NLRP3 inflammasome is partially mediated by the Sirt3-mtROS axis.

Alterations in inflammasome-related immunometabolites in individuals with severe psychiatric disorders

IntroductionPsychiatric disorders are common and significantly impact the quality of life. Inflammatory processes are proposed to contribute to the emergence of psychiatric disorders. In addition to inflammation, disturbances in metabolic pathways have been observed in individuals with different psychiatric disorders. A suggested key player in the interaction between inflammation and metabolism is the Nod-like receptor 3 (NLRP3) inflammasome, and NLRP3 is known to react to a number of specific metabolites. However, little is known about the interplay between these immunometabolites and the NLRP3 inflammasome in mental health disorders.AimTo assess the interplay between immunometabolites and inflammasome function in a transdiagnostic cohort of individuals with severe mental disorders.MethodsMass spectrometry-based analysis of selected immunometabolites, previously known to affect inflammasome function, were performed in plasma from low-functioning individuals with severe mental disorders (n = 39) and sex and aged-matched healthy controls (n = 39) using a transdiagnostic approach. Mann Whitney U test was used to test differences in immunometabolites between psychiatric patients and controls. To assess the relationship between inflammasome parameters, disease severity, and the immunometabolites, Spearman’s rank-order correlation test was used. Conditional logistic regression was used to control for potential confounding variables. Principal component analysis was performed to explore immunometabolic patterns.ResultsAmong the selected immunometabolites (n = 9), serine, glutamine, and lactic acid were significantly higher in the patient group compared to the controls. After adjusting for confounders, the differences remained significant for all three immunometabolites. No significant correlations were found between immunometabolites and disease severity.ConclusionPrevious research on metabolic changes in mental disorders has not been conclusive. This study shows that severely ill patients have common metabolic perturbations. The changes in serine, glutamine, and lactic acid could constitute a direct contribution to the low-grade inflammation observed in severe psychiatric disorders.

Nootkatone Improves Chronic Unpredictable Mild Stress-Induced Depressive-Like Behaviors by Repressing NF-κB/NLRP3-Mediated Neuroinflammation.

To explore the effect of nootkatone (NKT) on chronic unpredictable mild stress (CUMS)-induced depressive-like behaviors and the mechanism underlying NKT improving the depressive-like behaviors. The CUMS-induced depression model was established in mice. Fifty mice were randomized into 5 groups (n=10) in accordance with a random number table: control group, CUMS group, CUMS + NKT (6 mg/kg) group, CUMS + NKT (12 mg/kg) group, and CUMS + ketamine group. From the 22th day, NKT (6 or 12 mg/kg) or ketamine (0.5 mg/kg) was given with intragastric administration every day for 21 days. Behavioral tests including forced swimming test (FST), tail suspension test (TST), sucrose preference test (SPT) and open-field test (OFT) were carried out. The mRNA and protein expressions of interleukin (IL)-1β, IL-18, IL-6, and tumor necrosis factor (TNF)-α in hippocampus were assessed using quantitative realtime polymerase chain reaction (PCR), Western blot analysis, and enzyme linked immunosorbent assay. The nuclear factor-κB (NF-κB)/NOD-like receptor 3 (NLRP3) inflammasome pathway was analyzed using Western blot and immunofluorescence analysis. NKT treatment improved CUMS-induced depressive-like behaviors in mice (P<0.05 or P<0.01). NKT significantly decreased the mRNA and protein levels of IL-1β, IL-18, IL-6, and TNF-α in hippocampus of CUMS mice (P<0.05 or P<0.01). Furthermore, NKT repressed CUMS-induced activation of NF-κB signaling and NLRP3 inflammasome (P<0.01). More important, Nigericin, a NLRP3 activator, destroyed the effect of NKT on repressing neuroinflammation and improving depressive-like behaviors (P<0.05 or P<0.01). NKT ameliorates the depressive-like symptoms, in part by repressing NF-κB/NLRP3-mediated neuroinflammation.

Oxyberberrubine, a novel liver microsomes-mediated secondary metabolite of berberine, alleviates hyperuricemic nephropathy in mice

BackgroundBerberrubine (BRB), one of the major metabolites of berberine (BBR), exerts an anti-hyperuricemic effect even superior to BBR. Liver is an important location for drug transformation. Nevertheless, there are few studies on the bioactivities and metabolites of BRB. PurposeWe investigated whether oxyberberrubine (OBR), a liver metabolite of BRB, exerted urate-lowering and reno-protective effects in hyperuricemic mice. MethodsLiver microsomes were used to incubate BRB for studying its biotransformation. We isolated and identified its new metabolite OBR, and investigated its anti-hyperuricemic and reno-protective effects. In this work, the hyperuricemic mice model was established by receiving potassium oxonate (PO) and hypoxanthine (HX) for 7 consecutive days. 1 h after modeling, different dosages of OBR (5, 10 and 20 mg/kg), BRB (20 mg/kg) or febuxostat (Fex, 5 mg/kg) were given mice by gavage. ResultsResults showed that OBR possessed potent anti-hyperuricemic and reno-protective effects in hyperuricemic mice. Serum uric acid (UA) level was lowered, and the activities of xanthine oxidase (XOD) as well as adenosine deaminase (ADA) in the liver were suppressed after treatment with OBR. Hepatic expressions of XOD were remarkably decreased at mRNA and protein levels by OBR treatment. In addition, OBR prominently alleviated renal injury, embodied in markedly reduced serum creatinine and blood urea nitrogen (BUN) levels, decreased inflammatory mediators (TNF-α, IL-1β, IL-6 and IL-18) levels, mRNA expression of CYP27B1 and repairment of renal tissues damage. Besides, OBR down-regulated renal expression of urate transporter 1 (URAT1), glucose transporter 9 (GLUT9), NOD-like receptor 3 (NLRP3), apoptosis-associated speck-like protein containing CARD (ASC), and caspase-1 at mRNA and protein levels. ConclusionsIn short, our study indicated that OBR possessed superior anti-hyperuricemic and reno-protective effects, at least in part, through the inhibition of XOD, URAT1, GLUT9 and NLRP3 inflammasome signaling pathway in the kidney.

A novel cysteine protease inhibitor in Baylisascaris schroederi migratory larvae regulates inflammasome activation through the TLR4–ROS–NLRP3 pathway

BackgroundGiant pandas (Ailuropoda melanoleuca) are the obligate host of the parasitic roundworm Baylisascaris schroederi. The infection of giant pandas with B. schroederi is very common. At present, little is known about the mechanism of immune interaction between B. schroederi and the host. As an important component of innate immunity, the NOD-like receptor 3 (NLRP3) inflammasome plays an important role in host immune response and the occurrence and development of infectious diseases.MethodsWe analyzed the regulation of NLRP3 inflammasome activation in monocyte-derived macrophages (MDMs) by the recombinant B. schroederi migratory larvae cysteine protease inhibitor rBsCPI-1, knowing from a previous study that the CPI-1 is highly expressed in B. schroederi migratory larvae. We first determined the effects of rBsCPI-1 and excretory–secretory products of B. schroederi migratory larvae on cell proliferation using the CCK-8 and LDH release assays. We then analyzed NLRP3 inflammasome activation, pyroptosis and pro-inflammatory cytokine release by quantitative-PCR, western blotting and enzyme-linked immunosorbent assay. The signaling pathway of rBsCPI-1 to activate NLRP3 inflammasomes was analyzed in activation and inhibition experiments. Finally, the effects of rBsCPI-1 on inflammasome activation in mice immunized with rBsCPI-1 were analyzed.ResultsThe activation and inhibition experiments revealed that rBsCPI-1 induced inflammasome activation through the TLR4–ROS–NLRP3 signaling pathway, with reactive oxygen species (ROS) not only functioning as an activator of the NLRP3 inflammasome, but also an activation product of the NLRP3 inflammasome. rBsCPI-1 promoted the activation and assembly of the NLRP3 inflammasome, which further converted the pro-inflammatory cytokines interleukin (IL)-1β and IL-18 into mature active forms. At the same time, caspase-1 cleaved gasdermin D to trigger cell pyroptosis. The results of animal immunization experiments further confirmed that rBsCPI-1 could induce the activation of the NLRP3 inflammasome.ConclusionsrBsCPI-1 activates the inflammasome through the TLR4–ROS–NLRP3 signaling pathway and further induces the pyroptosis of MDMs and release of pro-inflammatory factors IL-1β and IL-18, thus promoting the occurrence and development of the inflammatory response in the host.Graphical abstract

NLRP3 Inflammasome/Pyroptosis: A Key Driving Force in Diabetic Cardiomyopathy.

Diabetic cardiomyopathy (DCM), a serious diabetic complication, is a kind of low-grade inflammatory cardiovascular disorder. Due to the high risk of morbidity and mortality, DCM has demanded the attention of medical researchers worldwide. The pathophysiological nature of DCM is intricate, and the genesis and development of which are a consequence of the coaction of many factors. However, the exact pathogenesis mechanism of DCM remains unclear. Pyroptosis is a newly identified programmed cell death (PCD) that is directly related to gasdermin D(GSDMD). It is characterized by pore formation on the cell plasma membrane, the release of inflammatory mediators, and cell lysis. The initiation of pyroptosis is closely correlated with NOD-like receptor 3 (NLRP3) activation, which activates caspase-1 and promotes the cleaving of GSDMD. In addition to adjusting the host’s immune defense, NLRP3 inflammasome/pyroptosis plays a critical role in controlling the systemic inflammatory response. Recent evidence has indicated that NLRP3 inflammasome/pyroptosis has a strong link with DCM. Targeting the activation of NLRP3 inflammasome or pyroptosis may be a hopeful therapeutic strategy for DCM. The focus of this review is to summarize the relevant mechanisms of pyroptosis and the relative contributions in DCM, highlighting the potential therapeutic targets in this field.

The Mechanosensitive Ion Channel PIEZO1 in Intestinal Epithelial Cells Mediates Inflammation through the NOD-Like Receptor 3 Pathway in Crohn's Disease.

Crohn's disease (CD) is an incurable chronic intestinal inflammatory disease with no recognized cause. It has been reported that the mechanosensitive ion channel PIEZO1 initiates proinflammatory responses. However, little is known about the role of PIEZO1 in CD. Ileum biopsies were obtained from 30 patients with CD and 15 healthy volunteers. Clinical data were collected to determine the relationship between CD and PIEZO1. First, HT29 cells were incubated with Yoda1 and GsMTx4 (Grammostola spatulata mechanotoxin 4) to activate and inhibit PIEZO1, respectively. Second, PIEZO1 knockdown was performed using small interfering RNA. Third, calcium imaging, flow cytometry, and immunofluorescence were used to detect intracellular calcium and mitochondrial function. Last, real-time quantitative polymerase chain reaction, immunoblotting, and enzyme-linked immunosorbent assay were used to quantify PIEZO1, proinflammatory cytokines, and NLRP3 (NOD-like receptor 3)-related compounds. PIEZO1 was highly expressed in the ileum of patients with CD and correlated positively with the Crohn's Disease Activity Index, platelet count, and hematocrit and fecal calprotectin levels. In HT29 cells, Yoda1 triggered calcium influx, which was inhibited by GsMTx4 treatment and small interfering RNA-mediated PIEZO1 knockdown. Increased calcium concentrations resulted in increased reactive oxygen species accumulation and decreased mitochondrial membrane potential, whereas decreased calcium concentrations caused by GsMTx4 and PIEZO1 knockdown had the opposite effect. Mechanistically, molecules in the NLRP3 pathway were activated in patients with CD and HT29 cells were stimulated by lipopolysaccharide; these effects were reversed by the knockdown of PIEZO1. Finally, PIEZO1 and NLRP3 knockdown decreased proinflammatory cytokine levels in HT29 cells. PIEZO1 in intestinal epithelial cells caused calcium influx, which resulted in mitochondrial dysfunction and activated the NLRP3 inflammasome, mediating intestinal inflammation.

LncRNA ZNF883-Mediated NLRP3 Inflammasome Activation and Epilepsy Development Involve USP47 Upregulation.

The goal of this study was to characterize the mechanisms of long noncoding RNA (lncRNA) ZNF883 regulating NOD-like receptor 3 (NLRP3) inflammasome activation in epilepsy (EP). Rat and cellular EP models were established using pilocarpine and magnesium-free extracellular fluid, respectively, to detect the differential expression of ZNF883, microRNA (miR)-138-5p, ubiquitin-specific peptidase 47 (USP47), and NLRP3. The pathology of the hippocampal neurons was examined by whole-cell patch clamping. The expression of ZNF883, miR-138-5p, and USP47 was modified in epileptic neurons, and the EP rats were injected with sh-ZNF883. Then, alterations in ZNF883, miR-138-5p, and USP47 levels were measured. The histopathology of the hippocampus was detected, along with the detection of IL-6, IL-1β, TNF-α, and NLRP3. Neuronal apoptosis in the rat and cellular EP models was determined. The relationship among ZNF883, miR-138-5p, and USP47 as well as the regulation of NLRP3 ubiquitination by USP47 was determined. ZNF883, USP47, and NLRP3 were increasingly expressed and miR-138-5p was downregulated in epileptic neurons and rats, concurrent with aggravated inflammation and apoptosis. ZNF883 overexpression in epileptic neurons elevated USP47 expression. ZNF883 targeted miR-138-5p and miR-138-5p negatively regulated USP47. In epileptic neurons, inhibiting miR-138-5p or overexpressing USP47 partially reversed the ZNF883 silencing-induced inhibition on NLRP3 inflammasome activation, neuronal apoptosis, and epileptiform activity. ZNF883 silencing in EP rats decreased USP47 and NLRP3, increased miR-138-5p, and inhibited inflammation and apoptosis. USP47 reversed the ubiquitination of NLRP3. ZNF883 inhibits NLRP3 ubiquitination and promotes EP through upregulating USP47 by sponging miR-138-5p.

Modified Citrus Pectin Alleviates Cerebral Ischemia/Reperfusion Injury by Inhibiting NLRP3 Inflammasome Activation via TLR4/NF-ĸB Signaling Pathway in Microglia.

BackgroundGalectin-3 acts as a mediator of microglial inflammatory response following stroke injury. However, it remains unclear whether inhibiting galectin-3 protects against cerebral ischemia/reperfusion injury. We aimed to investigate the neuroprotective effects of modified citrus pectin (MCP, a galectin-3 blocker) in ischemic stroke and underlying mechanisms.MethodsThe middle cerebral artery occlusion/reperfusion (MCAO/R) model in C57BL/6J mice and oxygen-glucose deprivation/reoxygenation (ODG/R) model in neuronal (HT-22) and microglial (BV-2) cells were utilized in the following experiments: 1) the neuroprotective effects of MCP with different concentrations were evaluated in vivo and in vitro through measuring neurological deficit scores, brain water content, infarction volume, cell viability, and cell apoptosis; 2) the mechanisms of its neuroprotection were explored in mice and microglial cells through detecting the expression of NLRP3 (NOD-like receptor 3) inflammasome-related proteins by immunofluorescence staining and Western blotting analyses.ResultsAmong the tested concentrations, 800 mg/kg/d MCP in mice and 4 g/L MCP in cells, respectively, showed in vivo and in vitro neuroprotective effects on all the tests, compared with vehicle group. First, MCP significantly reduced neurological deficit scores, brain water content and infarction volume, and alleviated cell injury in the cerebral cortex of MCAO/R model. Second, MCP increased cell viability and reduced cell apoptosis in the neuronal OGD/R model. Third, MCP blocked galectin-3 and decreased the expression of TLR4 (Toll-like receptor 4)/NF-κBp65 (nuclear factor kappa-B)/NLRP3/cleaved-caspase-1/IL-1β (interleukin-1β) in microglial cells.ConclusionThis is the first report that MCP exerts neuroprotective effects in ischemic stroke through blocking galectin-3, which may be mediated by inhibiting the activation of NLRP3 inflammasome via TLR4/NF-κB signaling pathway in microglia.

Neferine, an Alkaloid from Lotus Seed Embryos, Exerts Antiseizure and Neuroprotective Effects in a Kainic Acid-Induced Seizure Model in Rats.

Current anti-seizure drugs fail to control approximately 30% of epilepsies. Therefore, there is a need to develop more effective anti-seizure drugs, and medicinal plants provide an attractive source for new compounds. This study aimed to evaluate the possible anti-seizure and neuroprotective effects of neferine, an alkaloid from the lotus seed embryos of Nelumbo nucifera, in a kainic acid (KA)-induced seizure rat model and its underlying mechanisms. Rats were intraperitoneally (i.p.) administrated neferine (10 and 50 mg/kg) 30 min before KA injection (15 mg/kg, i.p.). Neferine pretreatment increased seizure latency and reduced seizure scores, prevented glutamate elevation and neuronal loss, and increased presynaptic protein synaptophysin and postsynaptic density protein 95 expression in the hippocampi of rats with KA. Neferine pretreatment also decreased glial cell activation and proinflammatory cytokine (interleukin-1β, interleukin-6, tumor necrosis factor-α) expression in the hippocampi of rats with KA. In addition, NOD-like receptor 3 (NLRP3) inflammasome, caspase-1, and interleukin-18 expression levels were decreased in the hippocampi of seizure rats pretreated with neferine. These results indicated that neferine reduced seizure severity, exerted neuroprotective effects, and ameliorated neuroinflammation in the hippocampi of KA-treated rats, possibly by inhibiting NLRP3 inflammasome activation and decreasing inflammatory cytokine secretion. Our findings highlight the potential of neferine as a therapeutic option in the treatment of epilepsy.

Hederasaponin C Alleviates Lipopolysaccharide-Induced Acute Lung Injury In Vivo and In Vitro Through the PIP2/NF-κB/NLRP3 Signaling Pathway

Gene transcription is governed by epigenetic regulation that is essential for the pro-inflammatory mediators surge following pathological triggers. Acute lung injury (ALI) is driven by pro-inflammatory cytokines produced by the innate immune system, which involves the nod-like receptor 3 (NLRP3) inflammasome and nuclear factor-κB (NF-κB) pathways. These two pathways are interconnected and share a common inducer the phosphatidylinositol 4,5-bisphosphate (PIP2), an epigenetic regulator of (Ribosomal ribonucleic acid (rRNA) gene transcription, to regulate inflammation by the direct inhibition of NF-κB phosphorylation and NLRP3 inflammasome activation. Herein, we report that hederasaponin C (HSC) exerted a therapeutic effect against ALI through the regulation of the PIP2/NF-κB/NLRP3 signaling pathway. In lipopolysaccharide (LPS)/lipopolysaccharide + adenosine triphosphate (LPS+ATP)-stimulated macrophages, our results showed that HSC remarkably inhibited the secretion of interleukin-6 (IL-6), IL-1β, and tumor necrosis factor-α (TNF-α). Moreover, HSC inhibited NF-κB/p65 nuclear translocation and the binding of PIP2 to transforming growth factor-β activated kinase 1 (TAK1). The intracellular calcium (Ca2+) level was decreased by HSC via the PIP2 signaling pathway, which subsequently inhibited the activation of NLRP3 inflammasome. HSC markedly alleviated LPS-induced ALI, restored lung function of mice, and rescued ALI-induced mice death. In addition, HSC significantly reduced the level of white blood cells (WBC), neutrophils, and lymphocytes, as well as pro-inflammatory mediators like IL-6, IL-1β, and TNF-α. Hematoxylin and eosin (H&E) staining results suggested HSC has a significant therapeutic effect on lung injury of mice. Interestingly, the PIP2/NF-κB/NLRP3 signaling pathway was further confirmed by the treatment of HSC with ALI, which is consistent with the treatment of HSC with LPS/LPS+ATP-stimulated macrophages. Overall, our findings revealed that HSC demonstrated significant anti-inflammatory activity through modulating the PIP2/NF-κB/NLRP3 axis in vitro and in vivo, suggesting that HSC is a potential therapeutic agent for the clinical treatment of ALI.

The ethanol extract of flower buds of Tussilago farfara L. attenuates cigarette smoke-induced lung inflammation through regulating NLRP3 inflammasome, Nrf2, and NF-κB

Ethnopharmacological relevanceThe flower buds of Tussilago farfara L. (Abbreviated as FTF) were widely used in traditional Chinese medicine (TCM) to treat respiratory diseases, including asthma, dry throat, great thirst, turbid saliva, stinky pus, and coughs caused by various causes. Aim of studyThe aim of study is to explore the efficiency of FTF in vitro and in vivo for the treatment of lung inflammation, and to illustrate the possible mechanisms of FTF in treating inflammation-related respiratory diseases targeting NOD-like receptor 3 (NLRP3) inflammasome, nuclear factor erythroid 2-related factor 2 (Nrf2), and nuclear transcription factor-κB (NF-κB). MethodsLung inflammation model in vivo was induced by exposure of mice to cigarette smoke (CS) for two weeks. The levels of superoxide dismutase (SOD), malondialdehyde (MDA), inflammatory factors, and histology in lung tissues were investigated in presence or absence of ethanol extract of the flower buds of T. farfara L. (FTF-EtOH). In the cell-based models, nitric oxide (NO) assay, flow cytometry assay, enzyme-linked immunosorbent assay (Elisa), and glutathione (GSH) assay were used to explore the anti-inflammatory and anti-oxidant effects of FTF-EtOH. Possible anti-inflammatory mechanisms of FTF targeting NLRP3 inflammasome, Nrf2, and NF-κB have been determined using western blot, quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR), immunofluorescence assay, nuclear and cytoplasmic extraction, and ubiqutination assay. ResultsFTF-EtOH suppressed CS-induced overproduction of inflammatory factors [e.g., tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β)], and upregulation of the content of intracellular MDA in the lung homogenate of mice. In cell-based models, FTF-EtOH reduced the lipopolysaccharide (LPS)-induced overproduction of inflammatory factors, and attenuated the CS extract-induced overgeneration of reactive oxygen species (ROS). Furthermore, FTF-EtOH up-regulated Nrf2 and its downstream genes through enhancing the stability of Nrf2 protein, and inhibited the activation of NF-κB and NLRP3 inflammasome, which have been confirmed by detecting the protein levels in the mouse model. ConclusionsFTF-EtOH effectively attenuated lung inflammation in vitro and in vivo. The protection of FTF-EtOH against inflammation was produced by activation of Nrf2 and inhibitions of NF-κB and NLRP3 inflammasome. These datas definitely support the ethnopharmacological use of FTF as an anti-inflammatory drug for treating respiratory diseases in TCM.

Internalization of the Membrane Attack Complex Triggers NLRP3 Inflammasome Activation and IL-1β Secretion in Human Macrophages.

Interleukin 1β (IL-1β) plays a major role in inflammation and is secreted by immune cells, such as macrophages, upon recognition of danger signals. Its secretion is regulated by the inflammasome, the assembly of which results in caspase 1 activation leading to gasdermin D (GSDMD) pore formation and IL-1β release. During inflammation, danger signals also activate the complement cascade, resulting in the formation of the membrane attack complex (MAC). Here, we report that stimulation of LPS-primed human macrophages with sub-lytic levels of MAC results in activation of the NOD-like receptor 3 (NLRP3) inflammasome and GSDMD-mediated IL-1β release. The MAC is first internalized into endosomes and then colocalizes with inflammasome components; adapter protein apoptosis associated speck-like protein containing a CARD (ASC) and NLRP3. Pharmacological inhibitors established that MAC-triggered activation of the NLRP3 inflammasome was dependent on MAC endocytosis. Internalization of the MAC also caused dispersion of the trans-Golgi network. Thus, these data uncover a role for the MAC in activating the inflammasome and triggering IL-1β release in human macrophages.

Ulinastatin protects against sepsis‑induced myocardial injury by inhibiting NLRP3 inflammasome activation.

Myocardial injury is the primary manifestation of multiple organ dysfunction during sepsis, however, the mechanisms underlying sepsis-induced myocardial injury remain unclear. Similarly, no effective therapeutics have yet been developed for myocardial injury. In the present study, the role of the NOD-like receptor 3 (NLRP3) inflammasome on cardiac function were characterized and the effects of different ulinastatin (UTI) doses in protecting a septic rat model from myocardial injury were elucidated. To evaluate UTI efficacy on cardiac function, its effects on anti-inflammatory mediators were analyzed and its cardioprotective effects were investigated. It was demonstrated that circulatory levels of tumor necrosis factor-α and interleukin-1β were elevated during sepsis. It was also observed that NLRP3 and caspase-1 expression enhanced post-cecal ligation and puncture (CLP), and that high UTI levels protected against myocardial injury induced by sepsis. To the best of our knowledge, this is the first study to demonstrate that the mechanisms underpinning UTI-mediated myocardial protection were due to the downregulation of the NLRP3/caspase-1/IL-1β signaling pathway. Based on these findings, it is proposed that UTI exerts beneficial effects during sepsis-induced myocardial injury.

Magnoflorine Alleviates "M1" Polarized Macrophage-Induced Intervertebral Disc Degeneration Through Repressing the HMGB1/Myd88/NF-κB Pathway and NLRP3 Inflammasome.

Intervertebral disc degeneration (IDD) is related to the deterioration of nucleus pulposus (NP) cells due to hypertrophic differentiation and calcification. The imbalance of pro-inflammatory (M1 type) and anti-inflammatory (M2 type) macrophages contributes to maintaining tissue integrity. Here, we aimed to probe the effect of Magnoflorine (MAG) on NP cell apoptosis mediated by “M1” polarized macrophages. THP-1 cells were treated with lipopolysaccharide (LPS) to induce “M1” polarized macrophages. Under the treatment with increasing concentrations of MAG, the expression of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α, IL-18), high mobility group box protein 1 (HMGB1), as well as myeloid differentiation factor 88 (MyD88), nuclear factor kappa B (NF-κB) and NOD-like receptor 3 (NLRP3) inflammasomes in THP-1 cells were determined. What’s more, human NP cells were treated with the conditioned medium (CM) from THP-1 cells. The NP cell viability and apoptosis were evaluated. Western blot (WB) was adopted to monitor the expression of apoptosis-related proteins (Bax, Caspase3, and Caspase9), catabolic enzymes (MMP-3, MMP-13, ADAMTS-4, and ADAMTS-5), and extracellular matrix (ECM) compositions (collagen II and aggrecan) in NP cells. As a result, LPS evidently promoted the expression of pro-inflammatory cytokines and HMGB1, the MyD88-NF-κB activation, and the NLRP3 inflammasome profile in THP-1 cells, while MAG obviously inhibited the "M1″ polarization of THP-1 cells. After treatment with “M1” polarized THP-1 cell CM, NP cell viability was decreased, while cell apoptosis, the pro-inflammatory cytokines, apoptosis-related proteins, and catabolic enzymes were distinctly up-regulated, and ECM compositions were reduced. After treatment with MAG, NP cell damages were dramatically eased. Furthermore, MAG dampened the HMGB1 expression and inactivated the MyD88/NF-κB pathway and NLRP3 inflammasome in NP cells. In conclusion, this study confirmed that MAG alleviates “M1” polarized macrophage-mediated NP cell damage by inactivating the HMGB1-MyD88-NF-κB pathway and NLRP3 inflammasome, which provides a new reference for IDD treatment.

NLRP3 Inflammasome Mediates Neurodegeneration in Rats with Chronic Neuropathic Pain.

Patients with chronic neuropathic pain (NP) have a significantly increased risk of central nervous degeneration. Trigeminal neuralgia (TN) is a typical NP, and this manifestation is more obvious. In addition to severe pain, patients with TN are often accompanied by cognitive dysfunction and have a higher risk of central nervous system degeneration, but the mechanism is not clear. The NOD-like receptor 3 (NLRP3) inflammasome assembles inside of microglia on activation, which plays an important role in neurodegeneration such as Alzheimer disease. MCC950 is a specific blocker of NLRP3 inflammasome, which can improve the performance of degenerative diseases. Although NLRP3 inflammasome assembles inside of microglia on activation has been shown to be essential for the development and progression of amyloid pathology, its whether it mediates the neurodegeneration caused by NP is currently unclear. By constructing a rat model of chronic TN, we found that as the course of the disease progresses, TN rats have obvious cognitive and memory deficit. In addition, Tau hyperphosphorylation and Aβ expression increase in the cortex and hippocampus of the brain. At the same time, we found that NLRP3 expression increased significantly in model rats. Interestingly, NLRP3 specific blocker MCC950 can alleviate the neurodegeneration of trigeminal neuralgia rats to a certain extent. It is suggested that our NLRP3 inflammasome plays an important role in the neurodegeneration of trigeminal neuralgia rats. And it is related to the activation of central nervous system inflammation.

Ticagrelor and Dapagliflozin Have Additive Effects in Ameliorating Diabetic Nephropathy in Mice with Type-2 Diabetes Mellitus.

Ticagrelor and dapagliflozin can suppress the activation of the NOD-like receptor 3 (NLRP3)-inflammasome and activate AMP-activated protein kinase (AMPK). The anti-inflammatory effects of dapagliflozin has been shown to depend on AMPK activation. Dapagliflozin and ticagrelor have been shown to have additive effects on the progression of diabetic cardiomyopathy in BTBR ob/ob mice with type-2 diabetes. We assessed whether dapagliflozin and ticagrelor have additive effects on the activation of the NLRP3-inflammasome and the progression of diabetic nephropathy in mice with type-2 diabetes. Eight-week-old BTBR received either no-drug, dapagliflozin (1.5mg/kg/d), ticagrelor (100mg/kg/d), or their combination for 12weeks. Blood was assessed weekly for glucose and urine for glucose and albumin. After 12weeks, blood creatinine, cystatin C, inflammasome activation, and insulin were assessed by ELISA. Renal cortex samples were assessed by hematoxylin and eosin and periodic acid-Schiff staining. RT-PCR and immunoblotting were used to evaluate fibrosis and the activation of Akt, AMPK and the inflammasome. Both ticagrelor and dapagliflozin reduced serum creatinine and cystatin C levels and urinary albumin. Both drugs attenuated the increase in glomerular area and mesangial matrix index. Both drugs decreased collagen-1 and collagen-3 expression and the activation of the NLRP3-inflammasome. Both drugs increased P-AMPK levels, but only dapagliflozin increased P-Akt levels. Overall, the protective effects of dapagliflozin and ticagrelor were additive. Dapagliflozin and ticagrelor attenuated the progression of diabetic nephropathy in BTBR ob/ob mice with additive effects of the combination. This was associated with AMPK activation and reduced activation of the NLRP3 inflammasome, whereas only dapagliflozin increased Akt activation.

MicroRNA-129 overexpression in endothelial cell-derived extracellular vesicle influences inflammatory response caused by myocardial ischemia/reperfusion injury.

Extracellular vesicles (EVs) have the potency to function as modulators in the process of myocardial ischemia/reperfusion (I/R) injury. This investigation was performed to decipher the mechanism of human umbilical vascular endothelial cells (HUVECs)-derived EVs in myocardial I/R injury with the involvement of microRNA-129 (miR-129). HUVECs-secreted EVs were collected and identified. An I/R mouse model was developed, and cardiomyocytes were used for vitro oxygen-glucose deprivation/reperfusion model establishment. Differentially expressed miRNAs in myocardial tissues after EV treatment were assessed using microarray analysis. The target relationship between miR-129 and toll-like receptor 4 (TLR4) was identified using a dual-luciferase assay. Gain- and loss-function studies regarding miR-129 were implemented to figure out its roles in myocardial I/R injury. Meanwhile, the activation of the nuclear factor-kappa-binding (NF-κB) p65 signaling and NOD-like receptor 3 (NLRP3) inflammasome was evaluated. EVs diminished the apoptosis of cardiomyocytes and the secretion of inflammatory factors, and all these trends were reversed by miR-129 reduction. miR-129 bound to the 3'-untranslated region of TLR4 directly. The NF-κB p65 signaling and NLRP3 inflammasome were abnormally activated after I/R injury, whose impairment after EVs was partially restored by miR-129 downregulation. This study illustrated that EVs could carry miR-129 to mitigate myocardial I/R injury via downregulating TLR4 and disrupting the NF-κB signaling and NLRP3 inflammasome.