NLR Family Pyrin Domain Containing 3 Inflammasome Activation Research Articles

The absence of IRG1 exacerbates bone loss in a mouse model of ovariectomy-induced osteoporosis by increasing osteoclastogenesis through the potentiation of NLRP3 inflammasome activation.

The immune-responsive gene 1 (IRG1) protein plays a role in various pathological processes by connecting cellular metabolism to a range of cellular activities through the production of itaconate. Recent studies have highlighted the significance of IRG1 and itaconate in bone metabolism and homeostasis. However, the precise role of IRG1 in osteoporosis remains inadequately documented. This study aimed to determine the role of IRG1 in osteoporosis through the utilization of IRG1 knockout (KO) mice and a model of ovariectomy (OVX)-induced osteoporosis. The expression of IRG1 was found to be higher in the bone tissues of postmenopausal osteoporotic mice induced by OVX in comparison to sham control mice. When compared to wild type (WT) mice, OVX-induced bone loss was significantly worse in IRG1 KO mice, and this was accompanied by an increase in osteoclastogenesis and bone resorption. However, the loss of bone and the process of osteoclastogenesis and bone resorption were effectively reversed when the IRG1 KO mice were replenished with itaconate. The osteoclastogenesis induced by receptor activator of nuclear factor kappa-Β ligand (RANKL) in bone marrow-derived macrophages (BMMs) was found to be enhanced by IRG1 deficiency, which could be reversed through the replenishment of itaconate. Further investigation revealed that IRG1 deficiency potentiated the activation of NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome. The inhibition of NLRP3 inflammasome using a targeted inhibitor significantly ameliorated RANKL-induced osteoclastogenesis in IRG1 KO BMMs. Overall, this study highlights the significance of IRG1 in regulating osteoclastogenesis and proposes it as a potential target for osteoporosis treatment.

Gut microbiota: a novel target for exercise-mediated regulation of NLRP3 inflammasome activation.

The NOD-like receptor family pyrin domain-containing 3 (NLRP3) is a key pattern recognition receptor in the innate immune system. Its overactivation leads to the production of pro-inflammatory cytokines, such as IL-1β and IL-18, which contribute to the development and progression of various diseases. In recent years, evidence has shown that gut microbiota plays an important role in regulating the activation of NLRP3 inflammasome. Variations in the function and composition of gut microbiota can directly or indirectly influence NLRP3 inflammasome activation by influencing bacterial components and gut microbiota metabolites. Additionally, exercise has been shown to effectively reduce NLRP3 inflammasome overactivation while promoting beneficial changes in gut microbiota. This suggests that gut microbiota may play a key role in mediating the effects of exercise on NLRP3 inflammasome regulation. This review explores the impact of exercise on gut microbiota and NLRP3 inflammasome activation, and examines the mechanisms through which gut microbiota mediates the anti-inflammatory effects of exercise, providing new avenues for research.

Role of NLRP3 inflammasome in nanoparticle adjuvant-mediated immune response.

The nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family pyrin domain-containing 3 (NLRP3) inflammasome is pivotal in orchestrating the immune response induced by nanoparticle adjuvants. Understanding the intricate mechanisms underlying the activation of NLRP3 inflammasome by these adjuvants is crucial for deciphering their immunomodulatory properties. This review explores the involvement of the NLRP3 inflammasome in mediating immune responses triggered by nanoparticle adjuvants. It delves into the signaling pathways and cellular mechanisms involved in NLRP3 activation, highlighting its significance in modulating the efficacy and safety of nanoparticle-based adjuvants. A comprehensive grasp of the interplay between NLRP3 inflammasome and nanoparticle adjuvants holds promise for optimizing vaccine design and advancing immunotherapeutic strategies.

Aerosol Inhalation of Luteolin-7-O-Glucuronide Exerts Anti-Inflammatory Effects by Inhibiting NLRP3 Inflammasome Activation.

Background: Luteolin-7-O-glucuronide (L7Gn) is a flavonoid isolated from numerous traditional Chinese herbal medicines that exerts anti-inflammatory effects. Previous research has revealed that aerosol inhalation is the most straightforward way of administration for the delivery of respiratory agents. Thus far, the impact of aerosol inhalation of L7Gn on lung inflammation and the underlying mechanisms remain unknown. Methods: The real-time particle size for L7Gn aerosol inhalation was detected by the Spraytec spray droplet size measurement system, including transmission and size diameters. The acute lung injury (ALI) rat model was induced by aerosol inhalation of LPS to evaluate the protective effect of L7Gn. The inhibitory effect of NLRP3 inflammasome activation assays was conducted in LPS-induced MH-S cells. Elisa, Western blotting, and RT-PCR were utilized to investigate the expression of NLRP3 inflammasome-relevant proteins and genes. Results: In this study, we found that inhalation of L7Gn aerosol significantly reduced pulmonary injury by inhibiting inflammatory infiltration and enhancing lung function. Meanwhile, the NLR family pyrin domain containing 3 (NLRP3) inflammasome was activated dramatically, accompanied by upregulated expression of IL-1β and IL-18, both in the ALI rat model and in LPS-induced MH-S cells. Moreover, L7Gn was found to significantly downregulate the expression of NLRP3, ASC, caspase-1, and cleaved caspase-1, which are critical components of the NLRP3 inflammasome, as well as the expression of IL-1β and IL-18. Conclusions: Based on our findings, L7Gn could exert anti-inflammatory effects by inhibiting NLRP3 inflammasome activation, which may emerge as potential therapeutic agents for the treatment of ALI.

NOD3 Reduces Sepsis-Induced Acute Lung Injury by Regulating the Activation of NLRP3 Inflammasome and the Polarization of Alveolar Macrophages.

The pathogenesis of sepsis-induced Acute lung injury (ALI) progresses rapidly, and no effective treatment drugs are known, resulting in a high mortality rate. NLR family pyrin domain containing 3 (NLRP3) inflammasome activation plays an important role in the pathological progression of ALI, and often coincide with the inflammatory activation and polarization of macrophages. NLR family CARD domain-containing protein 3 (NOD3) was reported protecting against sepsis-induced pulmonary pathological injury and inhibiting the inflammatory response in lung tissue. NOD3 can also inhibit NLRP3 inflammasome activation by competitively inhibiting the binding of pro-caspase-1 to apoptosis-related ASC or reducing NLRP3/cryopyrin-induced ASC speckle formation. In this study, we aimed to explore whether NOD3 decrease sepsis-induced lung injury by interfering with NLRP3 inflammasome activation and regulating alveolar macrophages (AMs) polarization. To investigate whether NOD3 reduce sepsis-induced ALI by inhibiting the activation of NLRP3 inflammasome to regulate the polarization of AMs. Sepsis-induced WT (C57BL/6) and NLRC3-/--C57BL/6 mice ALI models were established by intraperitoneal injection of lipopolysaccharide (LPS). In vitro experiments, AMs and bone marrow-derived macrophages (BMDMs) were isolated from WT and NLRC3-/- mice. Using in vivo and in vitro experiments, we found that NOD3 knockout promoted the sepsis-induced inflammatory response in lung tissue. In addition, NOD3 knockout promoted the activation of the TRAF6-NF-κB signaling pathway and the NLRP3 inflammasome in AMs, enhanced the M1-type polarization of AMs and decreased the M2-type polarization of AMs in sepsis-induced lung injury model mice. NOD3 interfered with NLRP3 inflammasome activation by inhibiting NLRP3 inflammasome assembly or negatively regulating the TRAF6-NF-κB signaling pathway, and regulating the polarization of AMs, thereby alleviating sepsis-induced lung injury.

Targeting the ALKBH5-NLRP3 positive feedback loop alleviates cardiomyocyte pyroptosis after myocardial infarction.

Several studies have associated the epitranscriptomic RNA modification of N6-methyladenosine (m6A) with cardiovascular diseases; however, how m6A modification affects cardiomyocyte pyroptosis after myocardial infarction (MI) remains unknown. Here, we showed that AlkB homolog 5 (ALKBH5), an m6A demethylase, is crucial in cardiomyocyte pyroptosis after MI. We used MI rat and mouse models, a cell hypoxia model of rat primary cardiomyocytes (RCMs), and rat embryonic ventricle cell line (H9c2) to explore the functional role of m6A modification and ALKBH5 in the heart and cardiomyocytes. Using plasmids and small interfering RNAs, the expressions of ALKBH5 and NOD-like receptor family pyrin domain-containing 3 (NLRP3) were determined to study their functions in regulating cardiomyocyte m6A and pyroptosis, respectively. We characterized the role of ALKBH5, which exhibited elevated expression in the ischemic heart tissue of rats and mice and hypoxic cardiomyocytes (RCMs and H9c2 cells). ALKBH5 knockdown alleviated hypoxia-induced H9c2 cell pyroptosis by inhibiting NLRP3 inflammasome activation, whereas ALKBH5 overexpression had the opposite effect. NLRP3 knockdown alleviated hypoxia-induced H9c2 cardiomyocyte pyroptosis by inhibiting ALKBH5 expression, whereas NLRP3 overexpression had the opposite effect. Mechanistically, ALKBH5 mediated m6A modification of NLRP3 mRNA in an IGF2BP2-dependent manner, and NLRP3, as a nuclear transcription factor, regulated the ALKBH5 transcription process. Targeting the ALKBH5-NLRP3 loop with the small-molecule inhibitors alleviated cardiomyocyte pyroptosis. Our results highlight that ALKBH5-NLRP3 forms a positive feedback loop that promotes cardiomyocyte pyroptosis after MI. Therefore, inhibiting the ALKBH5-NLRP3 loop is a potential strategy for treating cardiovascular diseases.

Macrophage NLRP3-dependent IL-1β production contributes to aortic fibrosis in heart failure with preserved ejection fraction.

Fibrosis is the main pathological feature of aortic stiffness, which is a common extracardiac comorbidity of heart failure with preserved ejection fraction (HFpEF) and a contributor to left ventricular (LV) diastolic dysfunction. Systemic low-grade inflammation plays a crucial role in the pathogenesis of HFpEF and the development of vascular fibrosis. In this study, we investigate the inflammatory mechanism of aortic fibrosis in HFpEF using a novel mouse model. LV diastolic dysfunction with preserved ejection fraction and aortic fibrosis induced by a high-fat diet (HFD) combined with subcutaneous aldosterone infusion are utilized. The constructed model exhibits augmented macrophage recruitment and NLR family pyrin domain containing 3 (NLRP3)-dependent interleukin (IL)-1β production in fibrotic aortas. In addition, a bone marrow transplant is employed to induce macrophage-specific NLRP3 deficiency in the HFpEF mouse model. These mice show almost completely suppressed cleaved-caspase-1 and mature IL-1β protein expression in the aortas, indicating that macrophage NLRP3 inflammasome activation enhances the IL-1β overproduction in fibrotic aortas. Furthermore, we show that macrophage NLRP3 inflammasome inhibition improves aortic fibrosis and LV diastolic dysfunction. In conclusion, this study demonstrates the pivotal effect of macrophage NLRP3-dependent IL-1β production on aortic fibrosis and cardiac function in HFpEF, suggesting a potential target for HFpEF therapy.

Glutamine metabolism modulates microglial NLRP3 inflammasome activity through mitophagy in Alzheimer’s disease

The NLR family pyrin domain containing 3 (NLRP3) inflammasome in microglia is intimately linked to the pathogenesis of Alzheimer’s disease (AD). Although NLRP3 inflammasome activity is regulated by cellular metabolism, the underlying mechanism remains elusive. Here, we found that under the pathological conditions of AD, the activation of NLRP3 inflammasome in microglia is accompanied by increased glutamine metabolism. Suppression of glutaminase, the rate limiting enzyme in glutamine metabolism, attenuated the NLRP3 inflammasome activation both in the microglia of AD mice and cultured inflammatory microglia. Mechanistically, inhibiting glutaminase blocked the anaplerotic flux of glutamine to the tricarboxylic acid cycle and amino acid synthesis, down-regulated mTORC1 signaling by phosphorylating AMPK, which stimulated mitophagy and limited the accumulation of intracellular reactive oxygen species, ultimately prevented the activation of NLRP3 inflammasomes in activated microglia during AD. Taken together, our findings suggest that glutamine metabolism regulates the activation of NLRP3 inflammasome through mitophagy in microglia, thus providing a potential therapeutic target for AD treatment.

MEK1/2 promote ROS production and deubiquitinate NLRP3 independent of ERK1/2 during NLRP3 inflammasome activation

Inflammasomes are cytosolic supramolecular complexes that play a key role in the innate immune response. Overactivation of NLR family pyrin domain containing 3 (NLRP3) inflammasome leads to multiple diseases. Post-translational modifications (PTMs) are essential modulators of inflammasomes especially in activation phase. Here we found that MEK1/2 kinase activity was indispensable in NLRP3 inflammasome activation both in vitro and in vivo. Inhibition of MEK1/2 resulted in reactive oxygen species (ROS) scavenging and ubiquitination of NLRP3, which further blocked NLRP3 inflammasome activation. These effects were independent of ERK1/2, which were classic downstream of MEK1/2. These investigations proposed a mechanism that MEK1/2 regulated inflammation via non-transcriptional regulation of NLRP3 inflammasome and might help better understanding the effects and side-effects of MEK inhibitors in clinical use.

NLRP3 inflammasome activation and pyroptosis are dispensable for tau pathology.

Neuroinflammation is widely recognized as a key factor in the pathogenesis of Alzheimer's disease (AD), alongside ß-amyloid deposition and the formation of neurofibrillary tangles. The NLR family pyrin domain containing 3 (NLRP3) inflammasome, part of the innate immune system, has been implicated in the neuropathology of both preclinical amyloid and tau transgenic models. Activation of the NLRP3 pathway involves an initial priming step, which increases the expression of Nlrp3 and interleukin (IL)-1β, followed by the assembly of the NLRP3 inflammasome complex, comprising NLRP3, ASC, and caspase-1. This assembly leads to the proteolytic maturation of the pro-inflammatory cytokines IL-1β and IL-18. Additionally, the NLRP3 inflammasome induces Gasdermin D (GSDMD) cleavage, forming membrane pores through which IL-1β and IL-18 are secreted. Inhibition of NLRP3 has been shown to enhance plaque clearance by modulating microglial activation. Furthermore, blocking NLRP3 in tau transgenic mice has been found to reduce tau phosphorylation by affecting the activity of certain tau kinases and phosphatases. In this study, organotypic brain slice cultures from P301S transgenic mice were treated with lipopolysaccharide (LPS) plus nigericin as a positive control or exposed to tau seeds (K18) to evaluate NLRP3 inflammasome activation. The effect of tau seeding on NLRP3 activity was further examined using Meso Scale Discovery (MSD) assays to measure IL1β secretion levels in the presence and absence of NLRP3 inhibitors. The role of NLRP3 activity was investigated in full-body Nlrp3 knockout mice crossbred with the tau transgenic P301S model. Additionally, full-body and microglia-selective Gsdmd knockout mice were crossbred with P301S mice, and tau pathology and neurodegeneration were evaluated at early and late stages of the disease using immunohistochemistry and biochemical assays. Activation of the NLRP3 pathway was observed in the mouse organotypic slice culture (OSC) model following stimulation with LPS and nigericin or exposure to tau seeds. However, Nlrp3 deficiency did not mitigate tauopathy or neurodegeneration in P301S mice in vivo, showing only a minor effect on plasma neurofilament (NF-L) levels. Consistently, Gsdmd deficiency did not alter tau pathology in P301S mice. Furthermore, neither full-body nor microglia-selective Gsdmd deletion had an impact on neuronal pathology or the release of pro-inflammatory cytokines. The absence of key components of the NLRP3 inflammasome pathway did not yield a beneficial effect on tau pathology or neurodegeneration in the preclinical Tau-P301S mouse model of AD. Nonetheless, organotypic slice cultures could serve as a valuable ex vivo mechanistic model for evaluating NLRP3 pathway activation and pharmacological inhibitors.

Galantamine and wedelolactone combined treatment suppresses LPS-induced NLRP3 inflammasome activation in microglial cells

Context Inflammasome NLR family pyrin domain-containing 3 (NLRP3) is associated with neurological disorders. Neuroinflammation can be suppressed by inhibiting NLRP3 inflammasome activation, decreasing neurodegenerative disorder progression. We devised a therapeutic technique that can reduce neuroinflammation induced by microglial activation, avoiding neurodegeneration. We aimed to investigate the mechanisms underlying the pharmacological effects of galantamine and wedelolactone by evaluating the response of the nuclear factor kappa B (NF-κB) signaling pathway and NLRP3 inflammasome in lipopolysaccharide (LPS)-activated N9 microglia. Methods LPS and adenosine triphosphate were used to activate the NLRP3 inflammasome in N9 microglial cells, which were pretreated with galantamine and wedelolactone. Caspase-1, NLRP3, NF-κB, and interleukin (IL)-1β levels were measured using RT-qPCR and immunostaining. Results Combined administration of galantamine and wedelolactone rescued microglial cells from LPS-induced cell death. Furthermore, treatment with galantamine and wedelolactone led to the suppression of NF-κB expression. NLRP3, caspase-1, and IL-1β levels were decreased by the combined treatment. Discussion and Conclusion The concurrent administration of galantamine and wedelolactone effectively suppresses the production of inflammatory cytokines and NLRP3 inflammasome activation in microglia. This inhibitory effect is likely linked to the NF-κB signaling pathway modulation. Therefore, this combined treatment is a potential therapeutic approach for neuroinflammatory diseases.

Bone morphogenetic protein 4 ameliorates bleomycin-induced pulmonary fibrosis in mice by repressing NLRP3 inflammasome activation and epithelial-mesenchymal transition.

Idiopathic pulmonary fibrosis (IPF) is a progressive and deadly lung disease with limited therapeutic options. Bone morphogenetic protein 4 (BMP4), a multifunctional growth factor that belongs to the transforming growth factor-β superfamily, is able to relieve pulmonary fibrosis in mice; nevertheless, the potential mechanism of action remains largely unknown. Growing evidence supports the notion that reiterant damage to the alveolar epithelial cells (AECs) is usually the "prime mover" for pulmonary fibrosis. Here, we examined the effect and mechanisms of BMP4 on bleomycin (BLM)-induced activation of NLR family pyrin domain containing 3 (NLRP3) inflammasome and epithelial-mesenchymal transition (EMT) in vivo and in vitro. The in vivo impact of BMP4 was investigated in a BLM mouse model. Histopathologic changes were analyzed by hematoxylin-eosin (H&E) and Masson's trichrome staining. The NLRP3 inflammasome activation was determined by quantitative real time polymerase chain reaction (qRT-PCR) and immunofluorescence staining. Biomarkers of EMT were measured by qRT-PCR, Western blot and immunofluorescence staining. The in vitro impact of BMP4 on BLM-induced NLRP3 inflammasome activation and EMT was explored in A549 AECs. We also evaluated whether BMP4 inhibited BLM-activated ERK1/2 signaling to address the possible molecular mechanisms. BMP4 was significantly downregulated in the mouse lungs from BLM-induced pulmonary fibrosis. BMP4+/- mice presented with more severe lung fibrosis in response to BLM, and accelerated NLRP3 inflammasome activation and EMT process compared with that in BMP4+/+ mice. Whereas overexpression of BMP4 by injecting adeno-associated virus (AAV) 9 into mice attenuated BLM-induced fibrotic changes, NLRP3 inflammasome activation, and EMT in the mouse lungs, thus exerting protective efficacy against lung fibrosis. In vitro, BMP4 significantly reduced BLM-induced activation of NLRP3 inflammasome and EMT in human alveolar epithelial A549 cells. Mechanically, BMP4 repressed BLM-induced activation of ERK1/2 signaling in vivo and in vitro, suggesting that ERK1/2 inactivation contributes to BMP4-induced effects on BLM-induced activation of NLRP3 inflammasome and EMT. Our findings suggest that BMP4 can suppress NLRP3 inflammasome activation and EMT in AECs via inhibition of ERK1/2 signaling pathway, thus has a potential for the treatment of pulmonary fibrosis.

AdipoRon exerts an antidepressant effect by inhibiting NLRP3 inflammasome activation in microglia via promoting mitophagy

Depression is a serious mental disorder that threatens patients’ physical and mental health worldwide. The activation of the NLR family pyrin domain-containing 3 (NLRP3) inflammasome is essential for microglia-mediated neuroinflammation and neuronal damage in depression. Numerous pathophysiological factors, such as mitochondrial dysfunction and impaired mitophagy, have an essential role in activating the NLRP3 inflammasome. AdipoRon is a potent adiponectin receptor agonist; however, its antidepressant effects have not been thoroughly investigated. In this study, we found that AdipoRon ameliorated depression-like behavior and neuronal damage induced by chronic unpredictable mild stress (CUMS). Further research demonstrated that AdipoRon inhibited the activation of the NLRP3 inflammasome and protected hippocampal neurons from microglial cytotoxicity by promoting mitophagy, increasing the clearance of damaged mitochondria, and reducing mtROS accumulation. Importantly, inhibition of mitophagy attenuated the antidepressant and neuroprotective effects of AdipoRon. Overall, these findings indicate that AdipoRon alleviates depression by inhibiting NLRP3 inflammasome activation in microglia via improving mitophagy.

NLRP3 Inflammasomes: Dual Function in Infectious Diseases.

The Nod-like receptor family pyrin domain containing 3 (NLRP3) inflammasome has been the most distinctive polymer protein complex. After recognizing the endogenous and exogenous danger signals, NLRP3 can cause inflammation by pyroptosis and secretion of mature, bioactive forms of IL-1β and IL-18. The NLRP3 inflammasome is essential in the genesis and progression of infectious illnesses. Herein, we provide a comprehensive review of the NLRP3 inflammasome in infectious diseases, focusing on its two-sided effects. As an essential part of host defense with a protective impact, abnormal NLRP3 inflammasome activation, however, result in a systemic high inflammatory response, leading to subsequent damage. In addition, scientific evidence of small molecules, biologics, and phytochemicals acting on the NLRP3 inflammasome has been reviewed. We believe that the NLRP3 inflammasome helps us understand the pathological mechanism of different stages of infectious diseases and that inhibitors targeting the NLRP3 inflammasome will become a new and valuable research direction for the treatment of infectious diseases.

β2-adrenoceptor agonist formoterol attenuates NLRP3 inflammasome activation and GSDMD-mediated pyroptosis in microglia through enhancing IκBα/NF-κB inhibition, SQSTM1/p62-dependent selective autophagy and ESCRT-III-mediated plasma membrane repair

Microglia are immune cells that play important roles in the formation of the innate immune response within the central nervous system (CNS). The NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome is a multiple protein complex that is crucial for innate immunity, and excessive activation of the inflammasome for various reasons contributes to the pathogenesis of neurodegenerative diseases (NDs). β2-adrenoceptor agonists have become the focus of attention in studies on NDs due to the high synthesis of β2-adrenoceptors in the central nervous system (CNS). Promising results have been obtained from these studies targeting anti-inflammatory and neuroprotective effects. Formoterol is an effective, safe for long-term use, and FDA-approved β2-adrenoceptor agonist with demonstrated anti-inflammatory features in the CNS. In this study, we researched the effects of formoterol on LPS/ATP-stimulated NLRP3 inflammasome activation, pyroptosis, NF-κB, autophagy, and ESCRT-III-mediated plasma membrane repair pathways in the N9 microglia cells. The results showed that formoterol, through the IκBα/NF-κB axis, significantly inhibited NLRP3 inflammasome activation, reduced the level of active caspase-1, secretion of IL-1β and IL-18 proinflammatory cytokine levels, and the levels of pyroptosis. Additionally, we showed that formoterol activates autophagy, autophagosome formation, and ESCRT-III-mediated plasma membrane repair, which are significant pathways in the inhibition of NLRP3 inflammasome activation and pyroptosis. Our study suggests that formoterol efficaciously prevents the NLRP3 inflammasome activation and pyroptosis in microglial cells regulation through IκBα/NF-κB, autophagy, autophagosome formation, and ESCRT-III-mediated plasma membrane repair.

Sodium Tanshinone IIA Sulfonate alleviates vascular senescence in diabetic mice by modulating the A20-NFκB-NLRP3 inflammasome-catalase pathway

Diabetes accelerates vascular senescence, which is the basis for atherosclerosis and stiffness. The activation of NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome and oxidative stress are closely associated with the deteriorative senescence in endothelial cells (ECs) and vascular smooth muscle cells (VSMCs). For decades, Sodium Tanshinone IIA Sulfonate (STS) has been utilized as a cardiovascular medicine with acknowledged anti-inflammatory and anti-oxidative properties. Nevertheless, the impact of STS on vascular senescence remains unexplored in diabetes. Diabetic mice, primary ECs and VSMCs were transfected with the NLRP3 overexpression/knockout plasmid, the tumor necrosis factor alpha-induced protein 3 (TNFAIP3/A20) overexpression/knockout plasmid, and treated with STS to detect senescence-associated markers. In diabetic mice, STS treatment maintained catalase (CAT) level and vascular relaxation, reduced hydrogen peroxide probe (ROSgreen) fluorescence, p21 immunofluorescence, Senescence β-Galactosidase Staining (SA-β-gal) staining area, and collagen deposition in aortas. Mechanistically, STS inhibited NLRP3 phosphorylation (serine 194), NLRP3 dimer formation, NLRP3 expression, and NLRP3-PYCARD (ASC) colocalization. It also suppressed the phosphorylation of IkappaB alpha (IκBα) and NFκB, preserved A20 and CAT levels, reduced ROSgreen density, and decreased the expression of p21 and SA-β-gal staining in ECs and VSMCs under HG culture. Our findings indicate that STS mitigates vascular senescence by modulating the A20-NFκB-NLRP3 inflammasome-CAT pathway in hyperglycemia conditions, offering novel insights into NLRP3 inflammasome activation and ECs and VSMCs senescence under HG culture. This study highlights the potential mechanism of STS in alleviating senescence in diabetic blood vessels, and provides essential evidence for its future clinical application.

Nuclear receptor Rev-erbα alleviates intervertebral disc degeneration by recruiting NCoR-HDAC3 co-repressor and inhibiting NLRP3 inflammasome.

Intervertebral discs (IVDs) are rhythmic tissues that experience daily low-load recovery. Notably, aging and abnormal mechanical stress predispose IVDs to degeneration due to dysrhythmia-induced disordered metabolism. Meanwhile, Rev-erbα acts as a transcriptional repressor in maintaining biorhythms and homeostasis; however, its function in IVD homeostasis and degeneration remains unclear. This study assessed the relationship between low Rev-erbα expression levels and IVD degeneration. Rev-erbα deficiency accelerated needle puncture or aging-induced IVD degeneration, characterized by increased extracellular matrix (ECM) catabolism and nucleus pulposus (NP) cell apoptosis. Mechanistically, Rev-erbα knockdown in NP cells aggravated rhIL1β-induced NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome activation, exacerbating the imbalanced ECM and NP cell apoptosis. Meanwhile, blocking NLRP3 inflammasome activation mitigated Rev-erbα deficiency and needle puncture-induced IVD degeneration. Particularly, Rev-erbα mediated the transcriptional repression of the NLRP3 inflammasome via the ligand heme-binding of nuclear receptor co-repressor (NCoR) and histone deacetylase 3 (HDAC3) complex. Thus, the increased expression of Rev-erbα in NP cells following short-term rhIL1β treatment failed to inhibit NLRP3 transcription in vitro owing to heme depletion. Pharmacological activation of Rev-erbα in vivo and in vitro alleviated IVD degeneration by altering the NLRP3 inflammasome. Taken together, targeting Rev-erbα may be a potential therapeutic strategy for alleviating IVD degeneration and its related diseases.

Fucoidan from Laminaria japonica protects renal tubular epithelial cells from uric acid induced NLRP3-mediated pyroptosis through inhibition of NF-κB pathway

Ethnopharmacological relevanceHyperuricemia is a common metabolic disease with prominent morbidity, it can lead to many adverse effects and complications, such as chronic nephrosis. Fucoidan has been used as natural drug for acute and chronic kidney disease for over 20 years in China, but the precise mechanisms underlying the renal protective function are still indefinable. PurposeThis study is conducted to explore alleviation of fucoidan (FPS) from Laminaria japonica on urate-induced NOD-like receptor family, pyrin domain-containing 3 (NLRP3)-mediated pyroptosis in renal tubular epithelial cells HK-2, as well as the mechanism of nuclear factor κB (NF-κB) signaling pathway involved. Materials and methodsHK-2 cells were treated with FPS, uric acid (UA), and inhibitor of NF-κB signaling pathway. Nitric oxide (NO) content and inducible nitric oxide synthase (iNOS) activity were determined with detection kits. Activation of intercellular NLRP3 inflammasome and NF-κB signaling pathway, gasdermin D (GSDMD) expression level were evaluated with Western blot and quantitative reverse transcription-PCR (qRT-PCR), and immunofluorescent analysis. ResultsData showed that UA induced cellular inflammatory response demonstrated by elevated NO content, iNOS activity and expression level of NLRP3 inflammasome-mediated pyroptosis associated molecules including NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), Caspase-1, interleukin 18 (IL-18) and GSDMD, moreover the NF-κB signaling pathway was activated by UA. However, FPS exposure inhibited efficiently the UA induced adverse effect. ConclusionIt can be concluded that FPS inhibited UA-induced NLRP3-mediated pyroptosis in HK-2 cells through repressing NF-κB signaling pathway.

9-Hydroxy-8-oxypalmatine, a novel liver-mediated oxymetabolite of palmatine, alleviates hyperuricemia and kidney inflammation in hyperuricemic mice

Ethnopharmacological relevancePalmatine is a main bioactive alkaloid of Cortex Phellodendri, which has been commonly prescribed for the treatment of hyperuricemia (HUA) in China. The metabolites of palmatine were crucial to its prominent biological activity. 9-Hydroxy-8-oxypalmatine (9-OPAL) is a novel liver-mediated secondary oxymetabolite of palmatine. Aim of the studyThe current study was to assess the efficacy of 9-OPAL, a novel liver-mediated secondary oxymetabolite of palmatine derived from Cortex Phellodendri, in experimental HUA mouse model and further explore its underlying mechanism. Materials and methodsAn in vitro metabolic experiment with oxypalmatine was carried out using liver samples. We separated and identified a novel liver metabolite, and investigated its anti-HUA effect in mice. HUA mice were induced by potassium oxonate and hypoxanthine daily for one week. After 1 h of modeling, mice were orally administered with different doses of 9-OPAL (5, 10 and 20 mg/kg). The pathological changes of the kidneys were evaluated using hematoxylin-eosin staining (H&E). The acute toxicity of 9-OPAL was assessed. The effects of 9-OPAL on serum levels of uric acid (UA), adenosine deaminase (ADA), xanthine oxidase (XOD), creatinine (CRE), blood urea nitrogen (BUN) and inflammatory cytokines were measured by enzyme-linked immunosorbent assay (ELISA) or biochemical method. Furthermore, Western blot, quantitative real-time PCR (qRT-PCR) and molecular docking were used to investigate the effect of 9-OPAL on the expression of renal urate transporters and NLRP3 signaling pathway in HUA mice. Results9-OPAL had been discovered to be a novel liver-mediated oxymetabolite of palmatine for the first time. Treatment with 9-OPAL significantly reduced the UA, CRE as well as BUN levels, and also effectively attenuated abnormal renal histopathological deterioration with favorable safety profile. Besides, 9-OPAL significantly decreased the serum and hepatic activities of XOD and ADA, dramatically inhibited the up-regulation of UA transporter protein 1 (URAT1) and glucose transporter protein 9 (GLUT9), and reversed the down-regulation of organic anion transporter protein 1 (OAT1). Additionally, 9-OPAL effectively mitigated the renal inflammatory markers (TNF-α, IL-1β, IL-6 and IL-18), and downregulated the transcriptional and translational expressions of renal Nod-like receptor family pyrin domain containing 3 (NLRP3), caspase-1, apoptosis-associated speck-like (ASC) and IL-1β in HUA mice. Molecular docking results revealed 9-OPAL bound firmly with XOD, OAT1, GLUT9, URAT1, NLRP3, caspase-1, ASC and IL-1β. Conclusions9-OPAL was found to be a novel liver-mediated secondary metabolite of palmatine with favorable safety profile. 9-OPAL had eminent anti-hyperuricemic and renal-protective effects, and the mechanisms might be intimately associated with repressing XOD activities, modulating renal urate transporter expression and suppressing the NLRP3 inflammasome activation. Our investigation might also provide further experimental evidence for the traditional application of Cortex Phellodendri in the treatment of HUA.

Sodium tanshinone IIA sulfonate protects vascular relaxation in ApoE-knockout mice by inhibiting the SYK-NLRP3 inflammasome-MMP2/9 pathway

BackgroundHyperlipidemia damages vascular wall and serves as a foundation for diseases such as atherosclerosis, hypertension and stiffness. The NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome is implicated in vascular dysfunction associated with hyperlipidemia-induced vascular injury. Sodium tanshinone IIA sulfonate (STS), a well-established cardiovascular protective drug with recognized anti-inflammatory, antioxidant, and vasodilatory properties, is yet to be thoroughly investigated for its impact on vascular relaxant imbalance induced by hyperlipidemia.MethodsIn this study, we treated ApoE-knockout (ApoE-/-) mouse with STS and assessed the activation of the NLRP3 inflammasome, expression of MMP2/9, integrity of elastic fibers, and vascular constriction and relaxation.ResultsOur findings reveal that STS intervention effectively preserves elastic fibers, significantly restores aortic relaxation function in ApoE-/- mice, and reduces their excessive constriction. Furthermore, STS inhibits the phosphorylation of spleen tyrosine kinase (SYK), suppresses NLRP3 inflammasome activation, and reduces MMP2/9 expression.ConclusionsThese results demonstrate that STS protects vascular relaxation against hyperlipidemia-induced damage through modulation of the SYK-NLRP3 inflammasome-MMP2/9 pathway. This research provides novel insights into the mechanisms underlying vascular relaxation impairment in a hyperlipidemic environment and uncovers a unique mechanism by which STS preserves vascular relaxation, offering valuable foundational research evidence for its clinical application in promoting vascular health.