Search Results (56)

Background: Inflammasomes regulate the activation of caspases resulting in inflammation; inflammasome activation is dysregulated in Alzheimer’s disease (AD) and plays a role in the pathogenesis of this condition. Glibenclamide, an anti-inflammatory drug, could be an interesting way to down-modulate neuroinflammation. Methods: In this pilot study we verified with ex vivo experiments whether a glibenclamide-loaded nanovector (GNV) could reduce the NLRP3-inflammasome cascade in cells of AD patients. Monocytes isolated from healthy controls (HC) and AD patients were cultured in medium, alone or stimulated with LPS + nigericin in presence/absence of GNV. ASC-speck positive cells and inflammasome-related genes, proteins, and miRNAs expressions were measured. The polymorphisms of ApoE (Apolipoprotein E), specifically rs7412 and rs429358, as well as those of NLRP3, namely rs35829419, rs10733113, and rs4925663, were also investigated. Results: Results showed that ASC-speck+ cells and Caspase-1, IL-1β, and IL-18 production was significantly reduced (p < 0.005 in all cases) by GNV in LPS + nigericin-stimulated cells of both AD and HC. Notably, the NLRP3 rs10733113 AG genotype was associated with excessive inflammasome-related gene and protein expression. GNV significantly down-regulates inflammasome activation in primary monocytes, at least at protein levels, and its efficacy seems to partially depend on the presence of the NLRP3 rs10733113 genotype. Conclusions: All together, these results showed that GNV is able to dampen inflammation and NLRP-3 inflammasome activation in an ex vivo monocyte model, suggesting a possible role for GNV in controlling AD-associated neuroinflammation. Full article

Background/Objectives: Chronic inflammation and inappropriate NLRP3 inflammasome regulation are related to many brain diseases. Purinergic mediators may play an important role in inflammation regulation and could be targeted for effective therapies for these illnesses. We evaluated resveratrol’s anti-neuroinflammatory potential in BV-2 microglia cells using an innovative in vitro method of NLRP3 inflammasome activation, correlating with the P2X7 purinergic receptor. Methods: In silico analyses were used to estimate resveratrol’s interaction with NLRP3, and its cytotoxicity was measured for 24, 48, and 72 h. Moreover, microglia were exposed to lipopolysaccharide and nigericin to activate the NLRP3 inflammasome and treated with resveratrol between these inflammatory agents. Results: It was found that resveratrol has binding compatible with modulating NLRP3. Specifically, 0.1–25 µM of resveratrol presented a favorable safety profile in BV-2 cells. Microglia exposed to the inflammatory agents had increased levels of oxidative species, the P2X7 receptor, and pro-inflammatory cytokines. However, resveratrol decreased the NLRP3, caspase-1, IL-1β, IL-6, and TNF-α mRNA levels and protein density; on the other hand, IL-10 was increased, acting as a protector, preventing exacerbated inflammation. Under resveratrol exposure, P2X7 was negatively expressed, regulating inflammation to establish homeostasis and microglial proliferation. Additionally, resveratrol activates the A1 adenosine receptor, possibly correlated with neuroprotective effects. Conclusions: We confirmed the anti-neuroinflammatory action of resveratrol via the P2X7 receptor and NLRP3’s combined modulation, regulating the cell cycle and reducing pro-inflammatory and oxidant agents. Considering this pathway, resveratrol could be a candidate for further investigations as a potential treatment against neuroinflammatory diseases. Full article

Cardiac injury after severe trauma is associated with higher mortality in polytrauma patients. Recent evidence suggests that miRNAs play a key role in cardiac pathophysiology and could serve as potential markers of cardiac damage after polytrauma. To explore this hypothesis, plasma miRNA profiles from polytrauma patients (ISS ≥ 16) with and without cardiac injury, stratified by troponin T levels (TnT, > 50 pg/mL vs. < 12 pg/mL), were analysed using NGS and validated via RT-qPCR. Five miRNAs (miR-122-5p, miR-424-5p, miR-885-5p, miR-194-5p, and miR-499a-5p) were found to be significantly upregulated in polytrauma patients with elevated TnT levels. miR-122-5p was associated with markers of right ventricular dysfunction (TAPSE) and left ventricular hypertrophy (IVS/LVPW), while miR-885-5p correlated with left ventricular hypertrophy (IVS/LVPW) and diastolic dysfunction (E/E’ ratio). In vitro, miR-194-5p mimic and miR-499a-5p mimic exhibited more active roles in cardiomyocyte injury by increasing caspase-3/7 activity and/or enhancing caspase-1 activity. Notably, the miR-194-5p mimic significantly enhanced the cytotoxic effects of the polytrauma cocktail, while miR-499a-5p boosted effects of LPS/nigericin stimulation in cardiomyocytes. Our findings identify miR-122-5p and miR-885-5p as potential biomarkers reflecting the cardiomyocyte response to polytrauma-induced inflammation, while miR-499a-5p and miR-194-5p appear to play a direct role in myocardial injury after polytrauma. Full article

Background: Most spheroid models use size measurements as a primary readout parameter; some models extend analysis to T cell infiltration or perform caspase activation assays. However, to our knowledge, T cell motility analysis is not regularly included as an endpoint in imaging studies on cancer spheroids. Methods: Here, we intend to demonstrate that motility analysis of macrophages and T cells is a valuable functional endpoint for studies on molecular interventions in the tumor microenvironment. In particular, T cell migration analysis represents the final step of effector function, as T cells engage with targets cells upon cytotoxic interaction, which is represented by an arrest within the spheroid volume. Therefore, T cell arrest is a novel readout parameter of T cell effector function in spheroids. Results: Here, we demonstrate that incubation of macrophages with nigericin for NLRP3 activation increases T cell velocity, but results in decreased T cellular arrest. This is paralleled by reduced rejection kinetics of pancreatic cancer spheroids in the presence of antigen-dependent T cells and nigericin-treated macrophages. Our model demonstrates consistent changes in T cell motility upon coculturing of T cells and tumors cells with macrophages, including influences of molecular interventions such as NLRP3 activation. Conclusions: Motility analysis using a spheroid model of pancreatic cancer is a more sophisticated alternative to in vitro cytotoxicity assays measuring spheroid size. Ultimately, an optimized spheroid model might replace at least some aspects of animal experiments investigating T cell effector function. Full article

Inflammation is a vital mechanism that defends the organism against infections and restores homeostasis. However, when inflammation becomes uncontrolled, it leads to chronic inflammation. The NLRP3 inflammasome is crucial in chronic inflammatory responses and has become a focal point in research for new anti-inflammatory therapies. Flavonoids like catechin, apigenin, and epicatechin are known for their bioactive properties (antioxidant, anti-inflammatory, etc.), but the mechanisms behind their anti-inflammatory actions remain unclear. This study aimed to explore the ability of various flavonoids (isolated and combined) to modulate the NLRP3 inflammasome using in silico and in vitro models. Computer simulations, such as molecular docking, molecular dynamics, and MM/GBSA calculations examined the interactions between bioactive molecules and NLRP3 PYD. THP1 cells were treated with LPS + nigericin to activate NLRP3, followed by flavonoid treatment at different concentrations. THP1-derived macrophages were also treated following NLRP3 activation protocols. The assays included colorimetric, fluorometric, microscopic, and molecular techniques. The results showed that catechin, apigenin, and epicatechin had high binding affinity to NLRP3 PYD, similar to the known NLRP3 inhibitor MCC950. These flavonoids, particularly at 1 µg/mL, 0.1 µg/mL, and 0.01 µg/mL, respectively, significantly reduced LPS + nigericin effects in both cell types and decreased pro-inflammatory cytokine, caspase-1, and NLRP3 gene expression, suggesting their potential as anti-inflammatory agents through NLRP3 modulation. Full article

The occurrence of inflammation is closely related to the activation of the NLRP3 inflammasome. IL-1β produced during the activation of the NLRP3 inflammasome has strong pro-inflammatory activity and can also promote the release of inflammatory factors by other immune cells, exacerbating inflammatory damage to tissues. Utilizing IL-1β as the detection index to find small-molecule inhibitors targeting NLRP3 from natural products will benefit the search for drugs for inflammation-related diseases. During the exploration of anti-inflammatory active components derived from the flowers of Dolichos lablab L., an ingredient in traditional Chinese medicine with dual applications in both medicinal treatment and dietary consumption, fourteen compounds (1–14), including seven previously unreported ones, named flosdolilabnitrogenousols A–D (1–4) and flosdolilabsaponins A–C (5–7), were found. Their structures were established through extensive NMR spectra determination, HR-ESI-MS analysis, ECD calculations, and chemical reactions. Flosdolilabsaponin A (5) stands out as an exceptionally rare tetracyclic lactone oleane-type saponin. Additionally, the inhibitory activity on IL-1β release of all compounds, without cytotoxicity, was evaluated using BMDMs stimulated with LPS/Nigericin. An Elisa assay revealed that compounds 1, 8, 9, and 11–14 exhibited significant inhibition of IL-1β release at a concentration of 30 μM. Structure–activity relationships were also discussed. This study indicates that D. lablab flowers possess anti-inflammatory activity, which might exert its effect by suppressing the activation of the NLRP3 inflammasome. Full article

The inflammasome is a vital part of the host’s innate immunity activated by cellular infection or stress. Our previous research identified the bovine SP110c isoform (bSP110c) as a novel activator of the inflammasome that promoted the secretion of proinflammatory cytokines IL-1β and IL-18 in macrophages infected with Listeria monocytogenes or stimulated with lipopolysaccharide (LPS). However, the exact molecular mechanism for inhibiting bSP110c-induced inflammasome activation requires further clarification. Here, the researchers identified bovine DDX3X (bDDX3X) as an NLRP3-associated protein and an inhibitor of the bSP110c-induced inflammasome in the human THP1 macrophage cell line. Immunoprecipitation showed that bDDX3X interacted with the bSP110c CARD domain via its helicase domain. The co-expression of bSP110c and bDDX3X in THP1 macrophages significantly prevented the bSP110c-induced activation of inflammasomes. In addition, both bDDX3X and bSP110c interacted with bovine NLRP3 (bNLRP3), and bDDX3X enhanced the interaction between bSP110c and bNLRP3. The expression of bDDX3X in nigericin-stimulated THP1 macrophages significantly suppressed NLRP3 inflammasome activation, ASC speck formation, and pyroptosis. These findings demonstrate that bDDX3X negatively regulates the bSP110c-mediated inflammatory response by restricting the activation of the NLRP3 inflammasome. This discovery unveils a novel regulatory mechanism involving bDDX3X and bSP110c in coordinating inflammasome activation and subsequent cell-fate decisions in LPS-treated macrophages and, in turn, constitutes a step forward toward the implementation of marker-assisted selection in breeding programs aimed at utilizing cattle’s immune defenses. Full article

Hydrolyzed royal jelly peptide (RJP) has garnered attention for its health-promoting functions. However, the potential applications of RJP in skincare have not been fully explored. In this study, we prepared RJP through the enzymatic hydrolysis of royal jelly protein with trypsin and investigated its antioxidant and anti-inflammatory properties on primary human dermal fibroblasts (HDFs). Our results demonstrate that RJP effectively inhibits oxidative damage induced by H₂O₂ and lipid peroxidation triggered by AAPH and t-BuOOH in HDFs. This effect may be attributed to the ability of RJP to enhance the level of glutathione and the activities of catalase and glutathione peroxidase 4, as well as its excellent iron chelating capacity. Furthermore, RJP modulates the NLRP3 inflammasome-mediated inflammatory response in HDFs, suppressing the mRNA expressions of NLRP3 and IL-1β in the primer stage induced by LPS and the release of mature IL-1β induced by ATP, monosodium urate, or nigericin in the activation stage. RJP also represses the expressions of COX2 and iNOS induced by LPS. Finally, we reveal that RJP exhibits superior antioxidant and anti-inflammatory properties over unhydrolyzed royal jelly protein. These findings suggest that RJP exerts protective effects on skin cells through antioxidative and anti-inflammatory mechanisms, indicating its promise for potential therapeutic avenues for managing oxidative stress and inflammation-related skin disorders. Full article

Inflammatory bowel diseases (IBD) are chronic inflammatory disorders affecting the digestive tract, including ulcerative colitis and Crohn’s disease. Ruscogenin, a prominent steroidal sapogenin present in radix ophiopogon japonicus, has shown a protective effect on attenuating the inflammatory response associated with inflammatory diseases, but the efficacy of ruscogenin in IBD remains unclear. The aim of this study is to explore the effect of ruscogenin on intestinal barrier dysfunction and inflammatory responses as well as the underlying mechanism in ulcerative colitis. A dextran sulfate sodium salt (DSS)-induced C57BL/6 mouse colitis model was employed for the in vivo studies, while in vitro experiments were performed in THP-1 cells and human intestinal epithelial cells involved in inducing inflammatory responses and pyroptosis using LPS/nigericin. The results indicated that ruscogenin treatment attenuated the symptoms of ulcerative colitis, reduced the release of inflammatory cytokines and the expression of pyroptosis-associated proteins, and restored the integrity of the intestinal epithelial barrier in colon tissue in mice. Moreover, ruscogenin inhibited LPS/nigericin-induced pyroptosis in THP-1 cells. Mechanically, ruscogenin inhibited NLRP3 inflammasome activation and canonical pyroptosis, at least in part, through the suppression of the TLR4/NF-κB signaling pathway. These findings might provide new insights and a solid foundation for further exploration into the therapeutic potential of ruscogenin in the treatment of IBD. Full article

Inflammasomes are multi-protein complexes, which are formed in response to tissue injury, infections, and metabolic stress. However, aberrant inflammasome activation has been linked to several inflammatory diseases. Anthocyanins have been reported to attenuate NLR family pyrin domain-containing 3 (NLRP3) inflammasome activation, but the influence of grape/blueberry anthocyanins and especially their gut-derived metabolites on NLRP3 inflammasome activation in human monocytes remains unclear. Therefore, human leukemic monocytes (THP-1 cells, Tohoku Hospital Pediatrics-1 cells) were preincubated with different concentrations of grape/blueberry anthocyanins, homovanillyl alcohol, or 2,4,6-trihydroxybenzaldehyde (THBA) before the NLRP3 inflammasome was activated by lipopolysaccharide and/or nigericin. Apoptosis-associated speck-like protein containing a CARD (ASC) speck formation, as well as ASC and NLRP3 protein expression, were determined using flow cytometry. Caspase-1 activity was measured in cultured cells, and pro-inflammatory cytokine secretion was determined using enzyme-linked immunosorbent assays. Anthocyanins and their metabolites had no effect on ASC or NLRP3 protein expression. However, THBA significantly inhibited ASC speck formation in primed and unprimed THP-1 monocytes, while caspase-1 activity was significantly declined by grape/blueberry anthocyanins. Furthermore, reduced inflammasome activation resulted in lower pro-inflammatory cytokine secretion. In conclusion, our results show for the first time that grape/blueberry anthocyanins and their gut-derived metabolites exert anti-inflammatory effects by attenuating NLRP3 inflammasome activation in THP-1 monocytes. Full article

Prostate cancer is the second leading cause of death in men. A challenge in treating prostate cancer is overcoming cell plasticity, which links cell phenotype changes and chemoresistance. In this work, a microfluidic device coupled with electrical impedance spectroscopy (EIS), an electrode-based cell characterization technique, was used to study the electrical characteristics of phenotype changes for (1) prostate cancer cell lines (PC3, DU145, and LNCaP cells), (2) cells grown in 2D monolayer and 3D suspension cell culture conditions, and (3) cells in the presence (or absence) of the anti-cancer drug nigericin. To validate observations of phenotypic change, we measured the gene expression of two epithelial markers, E-cadherin (CDH1) and Tight Junction Protein 1 (ZO-1). Our results showed that PC3, DU145, and LNCaP cells were discernible with EIS. Secondly, moderate phenotype changes based on differences in cell culture conditions were detected with EIS and supported by the gene expression of CDH1. Lastly, we showed that EIS can detect chemoresistant-related cell phenotypes with nigericin drug treatment. EIS is a promising label-free tool for detecting cell phenotype changes associated with chemoresistance. Further development will enable the detection and characterization of many other types of cancer cells. Full article

Background: Inflammasomes recognize endogenous and exogenous danger signals, and subsequently induce the secretion of IL-1β. Studying inflammasomes in the red fox (Vulpes vulpes) is crucial for wildlife veterinary medicine, as it can help control inflammatory diseases in foxes. Methods: We investigated the activation and intracellular mechanisms of three inflammasomes (NLRP3, AIM2, and NLRC4) in fox peripheral blood mononuclear cells (PBMCs), using established triggers and inhibitors derived from humans and mice. Results: Fox PBMCs exhibited normal activation and induction of IL-1β secretion in response to representative inflammasome triggers (ATP and nigericin for NLRP3, dsDNA for AIM2, flagellin for NLRC4). Additionally, PBMCs showed normal IL-1β secretion when inoculated with inflammasome-activating bacteria. In inhibitors of the inflammasome signaling pathway, fox inflammasome activation was compared with mouse inflammasomes. MCC950, a selective NLRP3 inhibitor, suppressed the secretion of dsDNA- and flagellin-mediated IL-1β in foxes, unlike mice. Conclusions: These findings suggest that NLRP3 may have a common role in dsDNA- and flagellin-mediated inflammasome activation in the red fox. It implies that this fox inflammasome biology can be applied to the treatment of inflammasome-mediated diseases in the red fox. Full article

Sepsis is a life-threatening organ dysfunction caused by a dysregulated response to infection. In this context, the aberrant activation of the NLRP3 inflammasome has been documented mostly through the measurement of increased plasmatic concentrations of IL-1β and IL-18. At the cellular level, contradictory results have been published. However, no study has comprehensively monitored NLRP3 inflammasome activation at the basal level and after ex vivo reactivation of whole blood monocytes and neutrophils focusing on ICU patients with bacterial and viral sepsis, including a longitudinal analysis. Thus, we conducted a prospective longitudinal study, examining NLRP3 inflammasome functionality in COVID-19 ICU patients (n = 15) and bacterial septic shock patients (n = 17) during the first week of ICU hospitalization, compared with healthy donors. Using two whole-blood flow cytometry assays, we detected ASC speck-positive monocytes (i.e., monocytes presenting the polymerization of ASC proteins) and activated caspase-1 in polymorphonuclear cells as read-outs, both at baseline and following nigericin stimulation, a drug that forms pores and activates the NLRP3 inflammasome. Our findings showed that, at baseline and regardless of the type of infection, patients exhibited reduced ASC speck-positive monocytes and decreased activated caspase-1 in PMN compared to healthy volunteers. This decrease was prominent at day 0. Following nigericin stimulation, this reduction was also observed and persisted throughout the first week of hospitalization, irrespective of the cellular population or parameter being considered. Notably, at day 0, this diminished activation and response to stimulation of NLRP3 was associated with a higher 28-day mortality rate. Consequently, our observations highlighted a concurrent decline in both basal expression and ex vivo activation of the NLRP3 inflammasome in circulating myeloid cells from patients with bacterial and viral sepsis in association with increased mortality. Full article

The anti-hyperglycemic drug glibenclamide (Glb) might represent an interesting therapeutic option in human neurodegenerative diseases because of its anti-inflammatory activity and its ability to downregulate activation of the NLRP3 inflammasome. Bi-functionalized liposomes that can cross the blood–brain barrier (BBB) may be used to release Glb into the central nervous system (CNS), overcoming its poor solubility and bioavailability. Here, we analyzed in vitro the effect of Glb-loaded nanovectors (GNVs) and Glb itself on NLRP3 inflammasome activation using a lipopolysaccharide- and nigericine-activated THP-1 cell model. Apoptosis-associated speck-like protein containing a CARD (ASC) aggregation and NLRP3-related cytokine (IL-1β, caspase 1, and IL-18) production and gene expression, as well as the concentration of miR-223-3p and miR-7-1-5p, known to modulate the NLRP3 inflammasome, were evaluated in all conditions. Results showed that both GNVs and Glb reduced significantly ASC-speck oligomerization, transcription and translation of NLRP3, as well as the secretion of caspase 1 and IL-1β (p < 0.05 for all). Unexpectedly, GNVs/Glb significantly suppressed miR-223-3p and upregulated miR-7-1-5p expression (p < 0.01). These preliminary results thus suggest that GNVs, similarly to Glb, are able to dampen NLRP3 inflammasome activation, inflammatory cytokine release, and modulate miR-223-3p/miR-7-1-5p. Although the mechanisms underlying the complex relation among these elements remain to be further investigated, these results can open new roads to the use of GNVs as a novel strategy to reduce inflammasome activation in disease and rehabilitation. Full article

Although immune checkpoint inhibitors improved the clinical outcomes of advanced triple negative breast cancer (TBNC) patients, the response rate remains relatively low. Nigericin is an antibiotic derived from Streptomyces hydrophobicus. We found that nigericin caused cell death in TNBC cell lines MDA-MB-231 and 4T1 by inducing concurrent pyroptosis and apoptosis. As nigericin facilitated cellular potassium efflux, we discovered that it caused mitochondrial dysfunction, leading to mitochondrial ROS production, as well as activation of Caspase-1/GSDMD-mediated pyroptosis and Caspase-3-mediated apoptosis in TNBC cells. Notably, nigericin-induced pyroptosis could amplify the anti-tumor immune response by enhancing the infiltration and anti-tumor effect of CD4+ and CD8+ T cells. Moreover, nigericin showed a synergistic therapeutic effect when combined with anti-PD-1 antibody in TNBC treatment. Our study reveals that nigericin may be a promising anti-tumor agent, especially in combination with immune checkpoint inhibitors for advanced TNBC treatment. Full article