A high-throughput gut-on-chip platform to study the epithelial responses to enterotoxins.

Enterotoxins are a type of toxins that primarily affect the intestines. Understanding their harmful effects is essential for food safety and medical research. Current methods lack high-throughput, robust, and translatable models capable of characterizing toxin-specific epithelial damage. Pressing concerns regarding enterotoxin contamination of foods and emerging interest in clinical applications of enterotoxins emphasize the need for new platforms. Here, we demonstrate how Caco-2 tubules can be used to study the effect of enterotoxins on the human intestinal epithelium, reflecting toxins' distinct pathogenic mechanisms. After exposure of the model to toxins nigericin, ochratoxin A, patulin and melittin, we observed dose-dependent reductions in barrier permeability as measured by TEER, which were detected with higher sensitivity than previous studies using conventional models. Combination of LDH release assays and DRAQ7 staining allowed comprehensive evaluation of toxin cytotoxicity, which was only observed after exposure to melittin and ochratoxin A. Furthermore, the study of actin cytoskeleton allowed to assess toxin-induced changes in cell morphology, which were only caused by nigericin. Altogether, our study highlights the potential of our Caco-2 tubular model in becoming a multi-parametric and high-throughput tool to bridge the gap between current enterotoxin research and translatable in vivo models of the human intestinal epithelium.

Geraniin restricts inflammasome activation and macrophage pyroptosis by preventing the interaction between ASC and NLRP3 to exert anti-inflammatory effects.

Geraniin, a chemical component of the traditional Chinese medicine geranii herba, possesses anti-inflammatory and anti-oxidative activities. However, its anti-inflammatory role in managing NLRP3 inflammasome and pyroptosis remains to be elucidated. To investigate the anti-inflammation mechanism of geraniin, LPS-primed macrophages were incubated with classical activators of NLRP3 inflammasome (such as ATP, Nigericin, or MSU crystals), and MSU crystals were injected into the ankle joints of mice to establish an acute gouty arthritis model. The propidium iodide (PI) staining results showed that geraniin could restrain cell death in the ATP- or nigericin-stimulated bone marrow-derived macrophages (BMDMs). Geraniin decreased the release of lactate dehydrogenase (LDH) and interleukin (IL)-1ß from cytoplasm to cell supernatant. Geraniin also inhibited the expression of caspase-1 p20, IL-1ß in cell supernatant and N-terminal of gasdermin D (GSDMD-NT) while blocking the oligomerization of ASC to form speck. The inhibitory effects of geraniin on caspase-1 p20, IL-1ß, GSDMD-NT, and ASC speck were not observed in NLRP3 knockout (NLRP3-/-) BMDMs. Hence, the resistance of geraniin to inflammasome and pyroptosis was contingent upon NLRP3 presence. Geraniin reduced reactive oxygen species (ROS) production and maintained mitochondrial membrane potential while preventing interaction between ASC and NLRP3 protein. Additionally, geraniin diminished MSU crystal-induced mouse ankle joint swelling and IL-1ß expression. Geraniin blocked the recruitment of neutrophils and macrophages to the synovium of joints. Our results demonstrate that geraniin prevents the assembly of ASC and NLRP3 through its antioxidant effect, thereby inhibiting inflammasome activation, pyroptosis, and IL-1ß release to provide potential insights for gouty arthritis targeted therapy.

Puerarin alleviates diabetic nephropathy by inhibiting Caspase-1-mediated pyroptosis.

BACKGROUND AND PURPOSE: Diabetic nephropathy (DN) is an important cause of end-stage renal disease, with podocyte injury as the main feature. Pyroptosis plays a non-negligible role in the process of diabetic nephropathy. Puerarin (PR) treatment of diabetic nephropathy has great potential, but the mechanism is not very clear. This article aims to study the protective effect and mechanism of puerarin on DN. METHODS: Streptozotocin (STZ)-induced C57 BL/6J mouse model of DN was given PR, Necrosulfomide (NSA), Nigericin for 12 weeks; A 60 mM high glucose(HG) induced MPC5 cell injury model was administered to PR, NSA, and Nigericin interventions for 24 h. RESULTS: After 12 weeks of administration, PR reduced fasting blood glucose levels in DN mice, alleviated glomerular lesions, reduced podocyte damage, and protected renal function. Meanwhile, PR also inhibits the expression of pyroptosis-related proteins. In addition, PR alleviated the release of Interleukin 18 (IL-18), Interleukin 1beta (IL-1ß), and lactate dehydrogenase (LDH) in MPC5 cells under HG conditions, downregulated the expression of pyrozozois-related proteins, and improved Caspase-1-mediated pyroptosis in MPC5 cells. CONCLUSION: Our study suggests that the beneficial effects of PR in diabetic nephropathy may be associated with inhibition of Caspase-1-mediated pyroptosis.

Mechanistic basis for potassium efflux-driven activation of the human NLRP1 inflammasome.

Nigericin, an ionophore derived from Streptomyces hygroscopicus, is arguably the most commonly used tool compound to study the NLRP3 inflammasome. Recent findings, however, showed that nigericin also activates the NLRP1 inflammasome in human keratinocytes. In this study, we resolve the mechanistic basis of nigericin-driven NLRP1 inflammasome activation. In multiple nonhematopoietic cell types, nigericin rapidly and specifically inhibits the elongation stage of the ribosome cycle by depleting cytosolic potassium ions. This activates the ribotoxic stress response (RSR) sensor kinase ZAKα, p38, and JNK, as well as the hyperphosphorylation of the NLRP1 linker domain. As a result, nigericin-induced pyroptosis in human keratinocytes is blocked by extracellular potassium supplementation, ZAKα knockout, or pharmacologic inhibitors of ZAKα and p38 kinase activities. By surveying a panel of ionophores, we show that electroneutrality of ion movement is essential to activate ZAKα-driven RSR and a greater extent of K+ depletion is necessary to activate ZAKα-NLRP1 than NLRP3. These findings resolve the mechanism by which nigericin activates NLRP1 in nonhematopoietic cell types and demonstrate an unexpected connection between RSR, perturbations of potassium ion flux, and innate immunity.

Hirudin ameliorates myocardial ischemia-reperfusion injury in a rat model of hemorrhagic shock and resuscitation: roles of NLRP3-signaling pathway.

Severe hemorrhage shock and resuscitation (HSR) has been reported to induce myocardial ischemia-reperfusion injury (MIRI), resulting in a poor prognosis. Hirudin, an effective thrombin inhibitor, can offer protection against MIRI. This study aimed to determine if hirudin administration ameliorates HSR-induced MIRI and the underlying mechanism. A rat model of HSR was established by bleeding rats to a mean arterial blood pressure of 30-35 mmHg for 45 min and then resuscitating them with all the shed blood through the left femoral vein. After HSR, 1 mg/kg of hirudin was administrated immediately. At 24 h after HSR, the cardiac injury was assessed using serum CK-MB, cTnT, hematoxylin-eosin (HE) staining, echocardiography, M1-polarized macrophages, and pyroptosis-associated factors, including cleaved caspase-1, Gasdermin D (GSDMD) N-terminal, IL-1ß, and IL-18 were measured by immunofluorescence and western blot assays. Nigericin, a unique agonist, was utilized to evaluate the responsibilities of NLRP3 signaling. Under the HSR condition, rats exhibited a significant increase in myocardial injury score, an elevation of serum cTnT, CK-MB levels, an aggrandization of M1-polarized macrophages, an upregulation of pyroptosis-associated factors, including cleaved caspase-1, GSDMD N-terminal, IL-1ß, and IL-18, but a significant decrease in left ventricular ejection fraction (EF%) and a reduction of left ventricular fractional shortening (FS%), while hirudin administration partially restored the changes. However, the NLRP3 agonist nigericin reversed the cardioprotective effects of hirudin. We determined the cardioprotective effects of hirudin against HSR-induced MIRI. The mechanism may involve the inhibition of NLRP3-induced pyroptosis.

Potassium ion efflux induces exaggerated mitochondrial damage and non-pyroptotic necrosis when energy metabolism is blocked.

Damage-associated molecular patterns (DAMPs) such as extracellular ATP and nigericin (a bacterial toxin) not only act as potassium ion (K+) efflux inducers to activate NLRP3 inflammasome, leading to pyroptosis, but also induce cell death independently of NLRP3 expression. However, the roles of energy metabolism in determining NLRP3-dependent pyroptosis and -independent necrosis upon K+ efflux are incompletely understood. Here we established cellular models by pharmacological blockade of energy metabolism, followed by stimulation with a K+ efflux inducer (ATP or nigericin). Two energy metabolic inhibitors, namely CPI-613 that targets α-ketoglutarate dehydrogenase and pyruvate dehydrogenase (a rate-limiting enzyme) and 2-deoxy-d-glucose (2-DG) that targets hexokinase, are recruited in this study, and Nlrp3 gene knockout macrophages were used. Our data showed that CPI-613 and 2-DG dose-dependently inhibited NLRP3 inflammasome activation, but profoundly increased cell death in the presence of ATP or nigericin. The cell death was K+ efflux-induced but NLRP3-independent, which was associated with abrupt reactive oxygen species (ROS) production, reduction of mitochondrial membrane potential, and oligomerization of mitochondrial proteins, all indicating mitochondrial damage. Notably, the cell death induced by K+ efflux and blockade of energy metabolism was distinct from pyroptosis, apoptosis, necroptosis or ferroptosis. Furthermore, fructose 1,6-bisphosphate, a high-energy intermediate of glycolysis, significantly suppressed CPI-613+nigericin-induced mitochondrial damage and cell death. Collectively, our data show that energy deficiency diverts NLRP3 inflammasome activation-dependent pyroptosis to Nlrp3-independent necrosis upon K+ efflux inducers, which can be dampened by high-energy intermediate, highlighting a critical role of energy metabolism in cell survival and death under inflammatory conditions.

Characterization and inhibition of inflammasome responses in severe and non-severe asthma.

BACKGROUND: Increased airway NLRP3 inflammasome-mediated IL-1ß responses may underpin severe neutrophilic asthma. However, whether increased inflammasome activation is unique to severe asthma, is a common feature of immune cells in all inflammatory types of severe asthma, and whether inflammasome activation can be therapeutically targeted in patients, remains unknown. OBJECTIVE: To investigate the activation and inhibition of inflammasome-mediated IL-1ß responses in immune cells from patients with asthma. METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from patients with non-severe (n = 59) and severe (n = 36 stable, n = 17 exacerbating) asthma and healthy subjects (n = 39). PBMCs were stimulated with nigericin or lipopolysaccharide (LPS) alone, or in combination (LPS + nigericin), with or without the NLRP3 inhibitor MCC950, and the effects on IL-1ß release were assessed. RESULTS: PBMCs from patients with non-severe or severe asthma produced more IL-1ß in response to nigericin than those from healthy subjects. PBMCs from patients with severe asthma released more IL-1ß in response to LPS + nigericin than those from non-severe asthma. Inflammasome-induced IL-1ß release from PBMCs from patients with severe asthma was not increased during exacerbation compared to when stable. Inflammasome-induced IL-1ß release was not different between male and female, or obese and non-obese patients and correlated with eosinophil and neutrophil numbers in the airways. MCC950 effectively suppressed LPS-, nigericin-, and LPS + nigericin-induced IL-1ß release from PBMCs from all groups. CONCLUSION: An increased ability for inflammasome priming and/or activation is a common feature of systemic immune cells in both severe and non-severe asthma, highlighting inflammasome inhibition as a universal therapy for different subtypes of disease.

Eleutheroside E from pre-treatment of Acanthopanax senticosus (Rupr.etMaxim.) Harms ameliorates high-altitude-induced heart injury by regulating NLRP3 inflammasome-mediated pyroptosis via NLRP3/caspase-1 pathway.

Eleutheroside E, a major natural bioactive compound in Acanthopanax senticosus (Rupr.etMaxim.) Harms, possesses anti-oxidative, anti-fatigue, anti-inflammatory, anti-bacterial and immunoregulatory effects. High-altitude hypobaric hypoxia affects blood flow and oxygen utilisation, resulting in severe heart injury that cannot be reversed, thereby eventually causing or exacerbating high-altitude heart disease and heart failure. The purpose of this study was to determine the cardioprotective effects of eleutheroside E against high-altitude-induced heart injury (HAHI), and to study the mechanisms by which this happens. A hypobaric hypoxia chamber was used in the study to simulate hypobaric hypoxia at the high altitude of 6000 m. 42 male rats were randomly assigned to 6 equal groups and pre-treated with saline, eleutheroside E 100 mg/kg, eleutheroside E 50 mg/kg, or nigericin 4 mg/kg. Eleutheroside E exhibited significant dose-dependent effects on a rat model of HAHI by suppressing inflammation and pyroptosis. Eleutheroside E downregulated the expressions of brain natriuretic peptide (BNP), creatine kinase isoenzymes (CK-MB) and lactic dehydrogenase (LDH). Moreover, The ECG also showed eleutheroside E improved the changes in QT interval, corrected QT interval, QRS interval and heart rate. Eleutheroside E remarkably suppressed the expressions of NLRP3/caspase-1-related proteins and pro-inflammatory factors in heart tissue of the model rats. Nigericin, known as an agonist of NLRP3 inflammasome-mediated pyroptosis, reversed the effects of eleutheroside E. Eleutheroside E prevented HAHI and inhibited inflammation and pyroptosis via the NLRP3/caspase-1 signalling pathway. Taken together, eleutheroside E is a prospective, effective, safe and inexpensive agent that can be used to treat HAHI.

The influence of high glucose conditions on macrophages and its effect on the autophagy pathway.

Introduction: Macrophages are central cells in mediating the inflammatory response. Objective and Methods: We evaluated the effect of high glucose conditions on the inflammatory profile and the autophagy pathway in Bone-Marrow Derived Macrophages (BMDM) from diabetic (D-BMDM) (alloxan: 60mg/kg, i.v.) and non-diabetic (ND-BMDM) C57BL/6 mice. BMDM were cultured in medium with normal glucose (5.5 mM), or high glucose (25 mM) concentration and were primed with Nigericin (20µM) stimulated with LPS (100 ng/mL) at times of 30 minutes; 2; 4; 6 and 24 hours, with the measurement of IL-6, IL-1ß and TNF-α cytokines. Results: We have further identified changes in the secretion of pro-inflammatory cytokines IL-6, IL-1ß and TNF-α, where BMDM showed increased secretion of these cytokines after LPS + Nigericin stimulation. In addition, changes were observed in the autophagy pathway, where the increase in the autophagic protein LC3b and Beclin-1 occurred by macrophages of non-diabetic animals in hyperglycemic medium, without LPS stimulation. D-BMDM showed a reduction on the expression of LC3b and Beclin-1, suggesting an impaired autophagic process in these cells. Conclusion: The results suggest that hyperglycemia alters the inflammatory pathways in macrophages stimulated by LPS, playing an important role in the inflammatory response of diabetic individuals.

Nigericin Induces Apoptosis in Primary Effusion Lymphoma Cells by Mitochondrial Membrane Hyperpolarization and ß-Catenin Destabilization.

BACKGROUND/AIM: Primary effusion lymphoma (PEL) is classified as a rare non-Hodgkin's B-cell lymphoma that is caused by Kaposi's sarcoma-associated herpesvirus (KSHV); PEL cells are latently infected with KSHV. PEL is frequently resistant to conventional chemotherapies. Therefore, the development of novel therapeutic agents is urgently required. Nigericin, a H+ and K+ ionophore, possesses unique pharmacological effects. However, the effects of nigericin on PEL cells remain unknown. MATERIALS AND METHODS: We examined the cytotoxic effects of the K+ ionophores, nigericin, nonactin, and valinomycin, on various B-lymphoma cells including PEL. We also evaluated ionophore-induced changes in signaling pathways involved in KSHV-induced oncogenesis. Moreover, the effects of nigericin on mitochondrial membrane potential and viral reactivation in PEL were analyzed. RESULTS: Although the three tested ionophores inhibited the proliferation of several B-lymphoma cell lines, nigericin inhibited the proliferation of PEL cells compared to KSHV-negative cells. In PEL cells, nigericin disrupted the mitochondrial membrane potential and caused the release of cytochrome c, which triggered caspase-9-mediated apoptosis. Nigericin also induced both an increase in phosphorylated p38 MAPK and proteasomal degradation of ß-catenin. Combination treatment of nigericin with the p38 MAPK inhibitor SB203580 potentiated the cytotoxic effects towards PEL cells, compared to either compound alone. Meanwhile, nigericin did not influence viral replication in PEL cells. CONCLUSION: Nigericin induces apoptosis in PEL cells by mitochondrial dysfunction and down-regulation of Wnt/ß-catenin signaling. Thus, nigericin is a novel drug candidate for treating PEL without the risk of de novo KSHV infection.

The effects of dimethyl fumarate on cytoplasmic LPS-induced noncanonical pyroptosis in periodontal ligament fibroblasts and dental pulp cells.

AIM: Pyroptosis is a type of inflammatory cell death and is related to pulpitis and apical periodontitis. In this study, the aim was to investigate how periodontal ligament fibroblasts (PDLFs) and dental pulp cells (DPCs) respond to pyroptotic stimuli and explore whether dimethyl fumarate (DMF) could block pyroptosis in PDLFs and DPCs. METHODOLOGY: Three methods (stimulation with lipopolysaccharide [LPS] plus nigericin, poly(dA:dT) transfection and LPS transfection) were used to induce pyroptosis in PDLFs and DPCs, two types of fibroblasts related to pulpitis and apical periodontitis. THP-1 cell was used as a positive control. Afterwards, PDLFs and DPCs were treated with or without DMF before inducing pyroptosis to examine the inhibitory effect of DMF. Pyroptotic cell death was measured by lactic dehydrogenase (LDH) release assays, cell viability assays, propidium iodide (PI) staining and flow cytometry. The expression levels of cleaved gasdermin D N-terminal (GSDMD NT), caspase-1 p20, caspase-4 p31 and cleaved PARP were examined by immunoblotting. Immunofluorescence analysis was used to detect the cellular distribution of GSDMD NT. RESULTS: Periodontal ligament fibroblasts and DPCs were more sensitive to cytoplasmic LPS-induced noncanonical pyroptosis than to canonical pyroptosis induced by stimulation with LPS priming plus nigericin or by poly(dA:dT) transfection. In addition, treatment with DMF attenuated cytoplasmic LPS-induced pyroptotic cell death in PDLFs and DPCs. Mechanistically, it was shown that the expression and plasma membrane translocation of GSDMD NT were inhibited in DMF-treated PDLFs and DPCs. CONCLUSIONS: This study indicates that PDLFs and DPCs are more sensitive to cytoplasmic LPS-induced noncanonical pyroptosis and that DMF treatment blocks pyroptosis in LPS-transfected PDLFs and DPCs by targeting GSDMD, suggesting DMF might be a promising drug for the management of pulpitis and apical periodontitis.

Inhibition of sphingosine-1-phosphate receptor 3 suppresses ATP-induced NLRP3 inflammasome activation in macrophages via TWIK2-mediated potassium efflux.

Background: Inhibition of sphingosine kinase 1 (SphK1), which catalyzes bioactive lipid sphingosine-1-phosphate (S1P), attenuates NLRP3 inflammasome activation. S1P exerts most of its function by binding to S1P receptors (S1PR1-5). The roles of S1P receptors in NLRP3 inflammasome activation remain unclear. Materials and methods: The mRNA expressions of S1PRs in bone marrow-derived macrophages (BMDMs) were measured by real-time quantitative polymerase chain reaction (qPCR) assays. BMDMs were primed with LPS and stimulated with NLRP3 activators, including ATP, nigericin, and imiquimod. Interleukin-1ß (IL-1ß) in the cell culture supernatant was detected by enzyme-linked immunosorbent assay (ELISA). Intracellular potassium was labeled with a potassium indicator and was measured by confocal microscopy. Protein expression in whole-cell or plasma membrane fraction was measured by Western blot. Cecal ligation and puncture (CLP) was induced in C57BL/6J mice. Mortality, lung wet/dry ratio, NLRP3 activation, and bacterial loads were measured. Results: Macrophages expressed all five S1PRs in the resting state. The mRNA expression of S1PR3 was upregulated after lipopolysaccharide (LPS) stimulation. Inhibition of S1PR3 suppressed NLRP3 and pro-IL-1ß in macrophages primed with LPS. Inhibition of S1PR3 attenuated ATP-induced NLRP3 inflammasome activation, enhanced nigericin-induced NLRP3 activation, and did not affect imiquimod-induced NLRP3 inflammasome activation. In addition, inhibition of S1PR3 suppressed ATP-induced intracellular potassium efflux. Inhibition of S1PR3 did not affect the mRNA or protein expression of TWIK2 in LPS-primed BMDMs. ATP stimulation induced TWIK2 expression in the plasma membrane of LPS-primed BMDMs, and inhibition of S1PR3 impeded the membrane expression of TWIK2 induced by ATP. Compared with CLP mice treated with vehicle, CLP mice treated with the S1PR3 antagonist, TY52156, had aggravated pulmonary edema, increased bacterial loads in the lung, liver, spleen, and blood, and a higher seven-day mortality rate. Conclusions: Inhibition of S1PR3 suppresses the expression of NLRP3 and pro-IL-1ß during LPS priming, and attenuates ATP-induced NLRP3 inflammasome activation by impeding membrane trafficking of TWIK2 and potassium efflux. Although inhibition of S1PR3 decreases IL-1ß maturation in the lungs, it leads to higher bacterial loads and mortality in CLP mice.

Nigericin treatment activates endoplasmic reticulum apoptosis pathway in goldfish kidney leukocytes.

Nigericin has been reported to induce apoptosis and pyroptosis in mammalian models. However, the effects and mechanism underlying the immune responses of teleost HKLs induced by nigericin remain enigmatic. To decipher the mechanism after nigericin treatment, the transcriptomic profile of goldfish HKLs was analyzed. The results demonstrated that a total of 465 differently expressed genes (DEGs) with 275 up-regulated and 190 down-regulated genes were identified between the control and nigericin treated groups. Among them, the top 20 DEG KEGG enrichment pathways were observed including apoptosis pathways. In addition, the expression level of selected genes (ADP4, ADP5, IRE1, MARCC, ALR1, DDX58) by quantitative real-time PCR showed a significant change after treatment with nigericin, which was generally identical to the expression patterns of the transcriptomic data. Furthermore, the treatment could induce cell death of HKLs, which was confirmed by LDH release and annexin V-FITC/PI assays. Taken together, our results support the idea that nigericin treatment might activate the IRE1-JNK apoptosis pathway in goldfish HKLs, which will provide insights into the mechanisms underlying HKLs immunity towards apoptosis or pyroptosis regulation in teleosts.

Targeting Multiple Homeostasis-Maintaining Systems by Ionophore Nigericin Is a Novel Approach for Senolysis.

Within the present study we proposed a novel approach for senolysis based on the simultaneous disturbance of the several homeostasis-maintaining systems in senescent cells including intracellular ionic balance, energy production and intracellular utilization of damaged products. Of note, we could not induce senolysis by applying ouabain, amiloride, valinomycin or NH4Cl-compounds that modify each of these systems solely. However, we found that ionophore nigericin can disturb plasma membrane potential, intracellular pH, mitochondrial membrane potential and autophagy at once. By affecting all of the tested homeostasis-maintaining systems, nigericin induced senolytic action towards stromal and epithelial senescent cells of different origins. Moreover, the senolytic effect of nigericin was independent of the senescence-inducing stimuli. We uncovered that K+ efflux caused by nigericin initiated pyroptosis in senescent cells. According to our data, the higher sensitivity of senescent cells compared to the control ones towards nigericin-induced death was partially mediated by the lower intracellular K+ content in senescent cells and by their predisposition towards pyroptosis. Finally, we proposed an interval dosing strategy to minimize the negative effects of nigericin on the control cells and to achieve maximal senolytic effect. Hence, our data suggest ionophore nigericin as a new senotherapeutic compound for testing against age-related diseases.

P2X7 Receptor-Induced Bone Cancer Pain by Regulating Microglial Activity via NLRP3/IL-1beta Signaling.

BACKGROUND: Bone cancer pain (BCP) is the most severe and intractable type of cancer pain. Emerging evidence has demonstrated that activated microglia in the spinal cord could release a series of neurotoxic substances to stimulate neurons and form neuronal sensitization. The P2X7 receptor (P2X7R) is a nonselective ATP-gated ion channel predominantly present in microglia in the spinal cord as the key modulator of microglial activity. However, the specific effect and underlying molecular mechanism of P2X7R in BCP have not yet been elucidated. OBJECTIVES: This study aimed at investigating whether P2X7R-induced BCP by regulating microglial activity through NLRP3/IL-1beta signaling involvement in BCP. STUDY DESIGN: Controlled animal study. METHODS: A BCP animal model was established by injecting Walker-256 breast cancer cells into the tibia of female rats. Fifty percent paw withdrawal thresholds (50% PWTs), number of spontaneous flinches (NSF), and limb use scores were used to evaluate behavior in rats. P2X7R inhibitor brilliant blue G (BBG) was used to assess the role of P2X7R in BCP-induced NLRP3 inflammasome activation. Western blot, RT-PCR, and immunofluorescence were used for quantitative comparison. In vitro, BV2 cells were treated with lipopolysaccharide (LPS) and BzATP, in the presence or absence of P2X7 siRNA, with nigericin (an agonist of the NLRP3 inflammasome) to further study the mechanism of P2X7R regulate NLRP3/IL-1beta signaling. RESULTS: The inhibition of spinal P2X7R with BBG could effectively inhibit BCP due to suppressing the expression of NF-kappaB p-p65, NLRP3 inflammasome formation, and downstream pain factors IL-1beta. Furthermore, P2X7 siRNA could reduce microglial activity, the nuclear translocation of NF-kappaB, and the synthesis of NLRP3 and IL-1beta in BV2 cells. In addition, nigericin partially reversed the ameliorating effect of P2X7 siRNA on BV2 cells induced by LPS and BzATP. LIMITATIONS: BBG could relieve BCP but not improve the destruction of bone, which may be related to the specificity of inoculated cells. Further mechanisms should be investigated. CONCLUSION: These findings suggest that targeting the microglial P2X7R activated NLRP3/IL-1beta signaling pathway could serve as a potential strategy for BCP treatment.

Corilagin Restrains NLRP3 Inflammasome Activation and Pyroptosis through the ROS/TXNIP/NLRP3 Pathway to Prevent Inflammation.

Corilagin, a gallotannin, shows excellent antioxidant and anti-inflammatory effects. The NLRP3 inflammasome dysfunction has been implicated in a variety of inflammation diseases. However, it remains unclear how corilagin regulates the NLRP3 inflammasome to relieve gouty arthritis. In this study, bone marrow-derived macrophages (BMDMs) were pretreated with lipopolysaccharide (LPS) and then incubated with NLRP3 inflammasome agonists, such as adenine nucleoside triphosphate (ATP), nigericin, and monosodium urate (MSU) crystals. The MSU crystals were intra-articular injected to induce acute gouty arthritis. Here we showed that corilagin reduced lactate dehydrogenase (LDH) secretion and the proportion of propidium iodide- (PI-)stained cells. Corilagin suppressed the expression of N-terminal of the pyroptosis executive protein gasdermin D (GSDMD-NT). Corilagin restricted caspase-1 p20 and interleukin (IL)-1ß release. Meanwhile, corilagin attenuated ASC oligomerization and speck formation. Our findings confirmed that corilagin diminished NLRP3 inflammasome activation and macrophage pyroptosis. We further discovered that corilagin limited the mitochondrial reactive oxygen species (ROS) production and prevented the interaction between TXNIP and NLRP3, but ROS activator imiquimod could antagonize the inhibitory function of corilagin on NLRP3 inflammasome and macrophage pyroptosis. Additionally, corilagin ameliorated MSU crystals induced joint swelling, inhibited IL-1ß production, and abated macrophage and neutrophil migration into the joint capsule. Collectively, these results demonstrated that corilagin suppressed the ROS/TXNIP/NLRP3 pathway to repress inflammasome activation and pyroptosis and suggest its potential antioxidative role in alleviating NLRP3-dependent gouty arthritis.

NOX4 promotes Kupffer cell inflammatory response via ROS-NLRP3 to aggravate liver inflammatory injury in acute liver injury.

AIM: This work aimed to investigate the mechanism of NOX4 in promoting Kupffer cells (KCs) activation and tissue inflammatory response in acute liver injury. METHODS: Initially, the mouse KCs were cultured in vitro. Thereafter, the NOX4 overexpression plasmid was transfected into KCs to construct the overexpression cell line. Then, KCs inflammatory response was induced by LPS + Nigericin treatment. CCK-8 assay was performed to detect cell viability, flow cytometry (FCM) was conducted to measure cell apoptosis, enzyme-linked immunosorbent assay (ELISA) was performed to detect inflammatory factor levels in the culture medium, NLRP3 and ASC expression in cells was detected by immunofluorescence (IF) staining, and ROS expression was detected by the DCFH-DA probe. Furthermore, the expression levels of NLRP3, ASC and Caspase-1 proteins were detected by Western-Blot (WB) assay. Furthermore, cells were pre-treated with NOX inhibitor or NAC to suppress NOX4 expression or ROS production, aiming to further investigate the effect on KCs inflammatory response. In mouse experiments, the NOX4 knockdown mice and wild-type (WT) mice were adopted for carrying out experiments. The mouse model of ALI was constructed with LPS and D-GalN treatment. Thereafter, the changes in tissue samples were detected by H&E staining, NLRP3 expression was measured by histochemical staining, inflammatory factors in tissues were analyzed by ELISA, and the levels of NLRP3, ASC and Caspase-1 proteins in tissues were detected by WB assay. RESULTS: LPS induced KCs inflammatory response. NOX4 overexpression decreased the mouse viability and increased the apoptosis rate. The levels of inflammatory factors were up-regulated in the culture medium. In addition, ROS were activated, and the positive cell number increased. Moreover, NOX4 promoted NLRP3 activation and significantly increased the expression of NLRP3 and ASC. Pretreatment with NOX4 inhibitor or NAC antagonized the effects of NOX4 and suppressed the KCs inflammatory response. In the mouse model, NOX4 knockdown significantly suppressed the activation and inflammatory response of microglial cells in tissues, reducing the NLRP3 expression in tissues. CONCLUSION: NOX4 activates the NLRP3 inflammasome via ROS to promote inflammatory response in KCs and the release of inflammatory factors, suppressing NOX4 can improve ALI in mice, and NOX4 is promising as a new target for ALI treatment.

Dimethyl fumarate ameliorates autoimmune hepatitis in mice by blocking NLRP3 inflammasome activation.

Dimethyl fumarate (DMF) is a fumaric acid derivative clinically approved for the treatment of some inflammatory diseases, but the underlying mechanism for its therapeutic effects remains incompletely understood. NLR family pyrin domain containing 3 (NLRP3) inflammasome activation has critical roles in innate immune responses to various infections and sterile inflammations. In this study, we aimed to explore whether DMF affects auto-immune hepatitis (AIH) in mice induced by concanavalin A (Con A) by modulating NLRP3 inflammasome activation. The results showed that DMF suppressed the activation of NLRP3 inflammasome activation in lipopolysaccharide-primed murine bone marrow-derived macrophages upon ATP or nigericin treatment, as evidenced by reduced cleavage of pro-caspase-1, release of mature interleukin-1ß (IL-1ß) and generation of gasdermin D N-terminal fragment (GSDMD-NT). DMF also greatly reduced ASC speck formation upon the stimulation of nigericin or ATP, indicating its inhibitory effect on NLRP3 inflammasome assembly. Consistent with reduced generation of GSDMD-NT, ATP or nigericin-induced pyroptosis was markedly suppressed by DMF. Moreover, DMF treatment alleviated mitochondrial damage induced by ATP or nigericin. Interestingly, all these effects were reversed by the protein kinase A (PKA) pathway inhibitors (H89 and MDL-12330A). Mechanistically, DMF enhanced PKA signaling and thus increased NLRP3 phosphorylation at PKA-specific sites to attenuate its activation. Importantly, DMF decreased serum levels of inflammatory cytokines and ameliorated liver injury in Con A-induced AIH of mice, concomitant with reduced the generation of caspase-1p10 and GSDMD-NT and alleviating mitochondrial aggregation in the liver. Collectively, DMF displayed anti-inflammatory effects by inhibiting NLRP3 inflammasome activation likely through regulating PKA signaling, highlighting its potential application in treating AIH.

Astragaloside IV alleviates PM2.5-caused lung toxicity by inhibiting inflammasome-mediated pyroptosis via NLRP3/caspase-1 axis inhibition in mice.

Exposure to particulate matter (PM)2.5 in air pollution is a serious health issue worldwide. At present, effective prevention measures and modalities of treatment for PM2.5-caused lung toxicity are lacking. This study elucidated the protective effect of astragaloside IV (Ast), a natural product from Astragalus membranaceous Bunge, against PM2.5-caused lung toxicity and its possible molecular mechanisms. The mice model of lung toxicity was performed by intratracheal instillation of PM2.5 dust suspension. The investigation was performed with Ast or in combination with nigericin, which is a NOD-like receptor protein 3 (NLRP3) activator. The results revealed that PM2.5 lead significant lung inflammation and promoted the pyroptosis pattern of cell death by upregulating pro-inflammatory cytokines and causing oxidative stress related to the NLRP3 inflammasome-mediated pyroptosis pathway. Ast protected against PM2.5 resulted lung toxicity via suppressing NLRP3 inflammasome-mediated pyroptosis via NLRP3/caspase-1 axis inhibition, thereby protecting the lung against PM2.5-induced lung inflammation and oxidative damage, eventually resulting in prolonged survival in mice. Nigericin partially reversed the protective effects of Ast. The present research provides new insights into the therapeutic potential of Ast, demonstrating that it might be a possible candidate for the prevention of PM2.5-caused respiratory diseases. Targeting the NLRP3 inflammasome might be a novel therapeutic tactic for PM2.5-caused respiratory diseases.

Dehydroepiandrosterone exacerbates nigericin-induced abnormal autophagy and pyroptosis via GPER activation in LPS-primed macrophages.

As a widely acknowledged FDA-approved dietary supplement or over-the-counter medicines, dehydroepiandrosterone (DHEA) exerts anti-inflammatory and immunomodulatory function. Pyroptosis is an important form of programmed cell death (PCD), and which acts a key role in the body's anti-infection and inflammatory responses. But the effects and mechanisms of DHEA on pyroptosis remain unclear. Here, we found that DHEA inhibited the NLRP3 inflammasome components expression by blocking inflammatory signals in lipopolysaccharide (LPS)-primed macrophages, and prevented the bacterial toxin nigericin (Nig)-induced NLRP3 inflammasome assembly. However, DHEA exacerbated NLRP3-independent cell death in Nig-treated inflammatory macrophages. During this process, DHEA induced the abnormal autophagy, which reflected as the blocking of autophagic flux and the accumulation of autophagy receptor p62 (SQSTM1) protein. In addition, DHEA caused a burst of reactive oxygen species (ROS) and activated extracellular signal-regulated kinase (ERK) phosphorylation in LPS plus Nig-stimulated macrophages but not in LPS-treated macrophages. Mechanistically, the present study certified that the activation of G protein-coupled estrogen receptor (GPER) signal mediated the cell death induced by DHEA in Nig-stimulated inflammatory macrophages, as GPER specific inhibitor G15 alleviated the abnormal autophagy and ultimately prevented the gasdermin D (GSDMD)-mediated pyroptosis induced by DHEA. Collectively, DHEA can exacerbate Nig-induced abnormal autophagy and pyroptosis via activation of GPER in LPS-primed macrophages, which prompts us the potential application value of DHEA in anti-infection or anti-tumor immunity.