Activation Of NOD-like Receptor 3 Inflammasome Research Articles

Glucose metabolite methylglyoxal induces vascular endothelial cell pyroptosis via NLRP3 inflammasome activation and oxidative stress in vitro and in vivo

Methylglyoxal (MGO), a reactive dicarbonyl metabolite of glucose, plays a prominent role in the pathogenesis of diabetes and vascular complications. Our previous studies have shown that MGO is associated with increased oxidative stress, inflammatory responses and apoptotic cell death in endothelial cells (ECs). Pyroptosis is a novel form of inflammatory caspase-1-dependent programmed cell death that is closely associated with the activation of the NOD-like receptor 3 (NLRP3) inflammasome. Recent studies have shown that sulforaphane (SFN) can inhibit pyroptosis, but the effects and underlying mechanisms by which SFN affects MGO-induced pyroptosis in endothelial cells have not been determined. Here, we found that SFN prevented MGO-induced pyroptosis by suppressing oxidative stress and inflammation in vitro and in vivo. Our results revealed that SFN dose-dependently prevented MGO-induced HUVEC pyroptosis, inhibited pyroptosis-associated biochemical changes, and attenuated MGO-induced morphological alterations in mitochondria. SFN pretreatment significantly suppressed MGO-induced ROS production and the inflammatory response by inhibiting the NLRP3 inflammasome (NLRP3, ASC, and caspase-1) signaling pathway by activating Nrf2/HO-1 signaling. Similar results were obtained in vivo, and we demonstrated that SFN prevented MGO-induced oxidative damage, inflammation and pyroptosis by reversing the MGO-induced downregulation of the NLRP3 signaling pathway through the upregulation of Nrf2. Additionally, an Nrf2 inhibitor (ML385) noticeably attenuated the protective effects of SFN on MGO-induced pyroptosis and ROS generation by inhibiting the Nrf2/HO-1 signaling pathway, and a ROS scavenger (NAC) and a permeability transition pore inhibitor (CsA) completely reversed these effects. Moreover, NLRP3 inhibitor (MCC950) and caspase-1 inhibitor (VX765) further reduced pyroptosis in endothelial cells that were pretreated with SFN. Collectively, these findings broaden our understanding of the mechanism by which SFN inhibits pyroptosis induced by MGO and suggests important implications for the potential use of SFN in the treatment of vascular diseases.

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.

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.

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.

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.

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.

Anti-Hyperuricemic and Nephroprotective Effects of Dihydroberberine in Potassium Oxonate- and Hypoxanthine-Induced Hyperuricemic Mice

Phellodendri Chinese Cortex has long been used to treat hyperuricemia and gout. Berberine (BBR), its characteristic ingredient, has also been shown to be effective in alleviating monosodium urate crystals-triggered gout inflammation in vitro and in vivo. Dihydroberberine (DHB) is a hydrogenated derivative of BBR that showed improved in vivo efficacy on many metabolic disorders. However, its anti-hyperuricemia effect remains underexplored. In the present work, the hypouricemic and renoprotective effects of DHB on hyperuricemic mice were investigated. The hyperuricemic mice model was induced by intraperitoneal injection of potassium oxonate (PO, 300 mg/kg) combined with intragastric administration of hypoxanthine (HX, 300 mg/kg) for 7 days. Different dosages of DHB (25, 50 mg/kg), BBR (50 mg/kg) or febuxostat (Feb, 5 mg/kg) were orally given to mice 1 h after modeling. The molecular docking results showed that DHB effectively inhibited xanthine oxidase (XOD) by binding with its active site. In vitro, DHB exhibited significant XOD inhibitory activity (IC50 value, 34.37 μM). The in vivo results showed that DHB had obvious hypouricemic and renoprotective effects in hyperuricemic mice. It could not only lower the uric acid and XOD levels in serum, but also suppress the activities of XOD and adenosine deaminase (ADA) in the liver. Furthermore, DHB noticeably down-regulated the renal mRNA and protein expression of XOD. Besides, DHB remarkably and dose-dependently ameliorated renal damage, as evidenced by considerably reducing serum creatinine and blood urea nitrogen (BUN) levels, inflammatory cytokine (TNF-α, IL-1β, IL-6 and IL-18) levels and restoring kidney histological deteriorations. Further mechanistic investigation showed that DHB distinctly down-regulated renal mRNA and protein levels of URAT1, GLUT9, NOD-like receptor 3 (NLRP3), apoptosis-associated speck-like (ASC), caspase-1 and IL-1β. Our study revealed that DHB had outstanding hypouricemic and renoprotective effects via suppressing XOD, URAT1, GLUT9 and NLRP3 inflammasome activation in the kidney.

Lactic Acid Fermentation Is Required for NLRP3 Inflammasome Activation.

Activation of the Nod-like receptor 3 (NLRP3) inflammasome is important for activation of innate immune responses, but improper and excessive activation can cause inflammatory disease. We previously showed that glycolysis, a metabolic pathway that converts glucose into pyruvate, is essential for NLRP3 inflammasome activation in macrophages. Here, we investigated the role of metabolic pathways downstream glycolysis – lactic acid fermentation and pyruvate oxidation—in activation of the NLRP3 inflammasome. Using pharmacological or genetic approaches, we show that decreasing lactic acid fermentation by inhibiting lactate dehydrogenase reduced caspase-1 activation and IL-1β maturation in response to various NLRP3 inflammasome agonists such as nigericin, ATP, monosodium urate (MSU) crystals, or alum, indicating that lactic acid fermentation is required for NLRP3 inflammasome activation. Inhibition of lactate dehydrogenase with GSK2837808A reduced lactate production and activity of the NLRP3 inflammasome regulator, phosphorylated protein kinase R (PKR), but did not reduce the common trigger of NLRP3 inflammasome, potassium efflux, or reactive oxygen species (ROS) production. By contrast, decreasing the activity of pyruvate oxidation by depletion of either mitochondrial pyruvate carrier 2 (MPC2) or pyruvate dehydrogenase E1 subunit alpha 1 (PDHA1) enhanced NLRP3 inflammasome activation, suggesting that inhibition of mitochondrial pyruvate transport enhanced lactic acid fermentation. Moreover, treatment with GSK2837808A reduced MSU-mediated peritonitis in mice, a disease model used for studying the consequences of NLRP3 inflammasome activation. Our results suggest that lactic acid fermentation is important for NLRP3 inflammasome activation, while pyruvate oxidation is not. Thus, reprograming pyruvate metabolism in mitochondria and in the cytoplasm should be considered as a novel strategy for the treatment of NLRP3 inflammasome-associated diseases.

Outer membrane protein A inhibits the degradation of caspase-1 to regulate NLRP3 inflammasome activation and exacerbate the Acinetobacter baumannii pulmonary inflammation

Acinetobacter baumannii (A. baumannii), one of the major pathogens that causes severe nosocomial infections, is characterised by a high prevalence of drug resistance. It has been reported that A. baumannii triggers the NOD-like receptor 3 (NLRP3) inflammasome, but the role of its virulence-related outer membrane protein A (ompA) remains unclear. Therefore, this study aimed to explore the effects of ompA on the NLRP3 inflammasome and its underlying molecular mechanisms. Results showed that ompA enhanced inflammatory damage, which was reduced as a result of knockout of the ompA gene. Additionally, ompA-stimulated expression of NLRP3 inflammasome was significantly blocked by silencing caspase-1, but activation of NLRP3 inflammasome was not altered after silencing ASC; this indicated that ompA was dependent on the caspase-1 pathway to activate the inflammatory response. Simultaneously, the wild-type (WT) strains triggered NLRP3 inflammasome after inhibition of caspase-1 degradation by proteasome inhibitor MG-132, aggravating tissue damage. These findings indicated that ompA may be dependent on the caspase-1 pathway to enhance inflammation and exacerbate tissue damage. Taken together, these results confirmed a novel capsase-1−modulated mechanism underpinning ompA activity, which further reveals the NLRP3 inflammasome pathway as a potential immunomodulatory target against A. baumannii infections.

NIMA-related kinase 7 amplifies NLRP3 inflammasome pro-inflammatory signaling in microglia/macrophages and mice models of spinal cord injury

BackgroundNIMA-related kinase-7 (NEK7) is a serine/threonine kinase that drives cell-cycle dynamics by modulating mitotic spindle formation and cytokinesis. It is also a crucial modulator of the pro-inflammatory effects of NOD-like receptor 3 (NLRP3) inflammasome. However, the role of NEK7 in microglia/macrophages post-spinal cord injury (SCI) is not well defined. MethodsIn this study, we performed both in vivo and in vitro experiments. Using an in vivo mouse SCI model, NEK7 siRNAs were administered intraspinally. For in vitro analysis, BV-2 microglia cells with NEK7-siRNA were stimulated with 1 μg/ml lipopolysaccharide (LPS) and 2 mM Adenosine triphosphate (ATP). ResultsHere, we found that the mRNA and protein levels of NEK7 and NLRP3 inflammasomes were upregulated in spinal cord tissues of injured mice and BV-2 microglia cells exposed to Lipopolysaccharide (LPS) and Adenosine triphosphate (ATP). Further experiments established that NEK7 and NLRP3 interacted in BV-2 microglia cells, an effect that was eliminated following NEK7 ablation. Moreover, NEK7 ablation suppressed the activation of NLRP3 inflammasomes. Although NEK7 inhibition did not significantly improve motor function post-SCI in mice, it was found to attenuate local inflammatory response and inhibit the activation of NLRP3 inflammasome in microglia/macrophages of the injured spinal cord. ConclusionNEK7 amplifies NLRP3 inflammasome pro-inflammatory signaling in BV-2 microglia cells and mice models of SCI. Therefore, agents targeting the NEK7/NLRP3 signaling offers great promise in the treatment of inflammatory response post-SCI.

Cbl Negatively Regulates NLRP3 Inflammasome Activation through GLUT1-Dependent Glycolysis Inhibition.

Activation of the nod-like receptor 3 (NLRP3) inflammasomes is crucial for immune defense, but improper and excessive activation causes inflammatory diseases. We previously reported that Cbl plays a pivotal role in suppressing NLRP3 inflammasome activation by inhibiting Pyk2-mediated apoptosis-associated speck-like protein containing a CARD (ASC) oligomerization. Here, we showed that Cbl dampened NLRP3 inflammasome activation by inhibiting glycolysis, as demonstrated with Cbl knockout cells and treatment with the Cbl inhibitor hydrocotarnine. We revealed that the inhibition of Cbl promoted caspase-1 cleavage and interleukin (IL)-1β secretion through a glycolysis-dependent mechanism. Inhibiting Cbl increased cellular glucose uptake, glycolytic capacity, and mitochondrial oxidative phosphorylation capacity. Upon NLRP3 inflammasome activation, inhibiting Cbl increased glycolysis-dependent activation of mitochondrial respiration and increased the production of reactive oxygen species, which contributes to NLRP3 inflammasome activation and IL-1β secretion. Mechanistically, inhibiting Cbl increased surface expression of glucose transporter 1 (GLUT1) protein through post-transcriptional regulation, which increased cellular glucose uptake and consequently raised glycolytic capacity, and in turn enhanced NLRP3 inflammasome activation. Together, our findings provide new insights into the role of Cbl in NLRP3 inflammasome regulation through GLUT1 downregulation. We also show that a novel Cbl inhibitor, hydrocortanine, increased NLRP3 inflammasome activity via its effect on glycolysis.

Hydroxysafflor yellow A inhibits hypoxia/reoxygenation-induced cardiomyocyte injury via regulating the AMPK/NLRP3 inflammasome pathway

Hydroxysafflor yellow A (HSYA) is an effective therapeutic agent that alleviates myocardial ischaemia/reperfusion injury (MIRI), but the exact mechanisms remain elusive. The aim of this study was to investigate the potential protective effect of HSYA against MIRI through mechanisms related to NLRP3 inflammasome regulation. In this study, hypoxia/reoxygenation (H/R)-induced H9c2 cardiomyocytes were treated with HSYA or the AMPK inhibitor, compound C (CC). Our results showed that HSYA pretreatment improved cardiomyocyte viability, maintained mitochondrial membrane potential, reduced apoptotic cardiomyocytes, decreased caspase-3 activity, and inhibited NOD-like receptor 3 (NLRP3) inflammasome activation during H/R injury. Moreover, the inhibition of AMPK activation by the CC inhibitor partially abolished the effects of HSYA treatment, including suppressing the upregulation of NLRP3 inflammasome components (NLRP3, caspase-1 and interleukin-1β) and promoting autophagy (LC3-II/LC3-I and p62). In conclusion, the protective mechanism of HSYA in H/R-induced cardiomyocyte injury is associated with inhibiting NLRP3 inflammasome activation through the AMPK signalling pathway.

Zika Virus Infection Induces Acute Kidney Injury Through Activating NLRP3 Inflammasome Via Suppressing Bcl-2.

Zika virus (ZIKV) is a newly emerging flavivirus that broadly exhibits in various bodily tissues and fluids, especially in the brain, and ZIKV infection often causes microcephaly. Previous studies have been reported that ZIKV can infect renal cells and can be detected in the urine samples of infected individuals. However, whether ZIKV infection causes renal diseases and its pathogenic mechanisms remains unknown. Here, we identified that ZIKV infection resulted in acute kidney injury (AKI) in both newborn and adult mouse models by increasing the levels of AKI-related biomarkers [e.g., serum creatinine (Scr), kidney injury molecular−1 (Kim-1), and neutrophil gelatinase-associated lipocalin (NGAL)]. ZIKV infection triggered the inflammatory response and renal cell injury by activating Nod-like receptor 3 (NLRP3) inflammasome and secreting interleukin-1β (IL-1β). IL-1β inhibited aquaporins expression and led to water re-absorption disorder. Furthermore, ZIKV infection induced a decreased expression of B-cell lymphoma-2 (Bcl-2) in the kidney. Overexpression of Bcl-2 attenuated ZIKV-induced NLRP3 inflammasome activation in renal cells and down-regulated PARP/caspase-3-mediated renal apoptosis. Overall, our findings demonstrated that ZIKV infection induced AKI by activating NLRP3 inflammasome and apoptosis through suppressing Bcl-2 expression, which provided potential therapeutic targets for ZIKV-associated renal diseases.