NOD-like Receptor Protein 3 Inflammasome Research Articles

Acute Administration of Edaravone Improves Cognitive Impairment in a Mouse Model of mPFC Ischemia: Crosstalk Between Necroptosis, Neuroinflammation, and Antioxidant Defense.

Edaravone (Eda), a well-known free radical scavenger, has been reported as a possible therapeutic agent for ischemic stroke patients' recovery. This study aimed to investigate the effects of time-dependent treatment with Eda on medial prefrontal cortex (mPFC) ischemia. Mice were randomly allocated into six groups: control, sham, normal saline, Eda-I, Eda-II, and Eda-III. After induction of a photothrombotic ischemia in the mPFC region, Eda-I, Eda-II, and Eda-III groups received 3mg/kg Eda intraperitoneally at the times of 0, 2, and 6h post-surgery. After 1day of recovery, the mice underwent behavioral tests (open field, novel object recognition, and T-maze). Next, necroptosis, NOD-like receptor protein 3 (NLRP3), and nuclear factor erythroid 2-related factor 2 (Nrf2) pathway-related protein levels were measured in the lesioned area using western blot analysis. For double confirmation, IL-1β and IL-18 were also assessed by immunofluorescence in the area. Further, histological evaluations were performed to measure tissue damage. The results showed that mPFC ischemia impaired recognition and spatial working memory without affecting locomotor activity, while immediate Eda administration improved cognitive impairments. Furthermore, acute Eda treatment reduced RIP1, RIP3, and MLKL levels, inhibited NLRP3 inflammasome proteins (NLRP3, ASC, and Cas1), decreased IL-1β and IL-18, upregulated Nrf2 and its targets (NQO-1 and HO-1), and diminished tissue damage. Our results highlighted the effects of acute administration of Eda post-stroke on improving cognitive impairments by suppressing necroptosis and NLRP3 inflammasome pathways and activating the Nrf2 antioxidant defense mechanism.

Copper Overload Promotes β-amyloid Induced NLRP3/Caspase-1/GSDMD-Mediated Pyroptosis in Alzheimer's Disease.

Alzheimer's disease (AD) is characterized by cognitive decline and abnormal protein accumulation. Copper imbalance and pyroptosis play significant roles in the pathogenesis of AD. Recent studies have suggested that dysregulated copper homeostasis contributed to β-amyloid accumulation, which may activate the NOD-like receptor protein 3 (NLRP3)-related pyroptosis pathway, promoting neuronal damages and AD progression. Therefore, the present study aims to investigates whether copper facilitates AD through exacerbating β-amyloid (Aβ) induced activation of NLRP3/Caspase-1/Gasdermin D (GSDMD)-mediated neuronal cell pyroptosis. Mouse hippocampal HT-22 cells were cultured with Aβ1-42 oligomer for 24 h as AD Model group. CuCl2 treatment was administered to the AD cell model, and cell survivability levels were detected by Cell Counting Kit-8 (CCK-8), TdT-mediated dUTP nick end labeling (TUNEL), and other relevant kits. Mitochondrial function was evaluated using Mitochondrial membrane potential dye JC-1 and transmission electron microscopy (TEM). After intervention with the NLRP3 inhibitor MCC950, activation of the NLRP3/Caspase-1/GSDMD pathway by copper ions (Cu2+) was confirmed via Western Blot. Thioredoxin T (ThT) fluorescence assay was performed to observe the aggregation effect of Aβ induced by Cu2+ overload. CuCl2 treatment of the AD cell model resulted in up-regulation of the levels of Lactate Dehydrogenase (LDH), Interleukin-1β (IL-1β), and IL-18 expression, which indicated activation of pyroptosis. We observed a significant decrease in mitochondrial membrane potential, mitochondrial swelling, and loss of mitochondrial cristae by fluorescence microscopy and TEM. ThT fluorescence imaging showed that Cu2+ promoted Aβ aggregation and up-regulated NLRP3, apoptosis-associated speck-like protein containing a CARD (ACS), Caspase-1, Cleaved Caspase-1, GSDMD, and Gasdermin D N-terminal (GSDMD-NT). The NLRP3 inhibitor MCC950 partially reversed Cu2+-mediated pyroptosis in HT-22 cells. Exposure to copper ions disrupt mitochondrial copper homeostasis, promotes Aβ aggregation, and activates NLRP3 inflammasomes, further promoting the Aβ aggregation activated pyroptosis in AD cell models.

N-butanol extract of Broussonetia papyrifera (L.) L′Hér. ex Vent root bark alleviates atopic dermatitis by targeting E3 ubiquitin ligase WWP1 to promote NLRP3 degradation

BackgroundBroussonetia papyrifera (L.) L′Hér. ex Vent (B. papyrifera) is a deciduous tree widely distributed in Asia. Previous studies have revealed that leaves of B. papyrifera ameliorated atopic dermatitis (AD)-like symptoms and inflammatory response. However, the impact and underlying mechanism of other parts of B. papyrifera on AD remain elusive. MethodsThe AD mice induced by 1-Chloro-2,4-dinitrochlorobenzene were used to observe the histopathological alterations in the skin tissues using hematoxylin-eosin staining and toluidine blue staining techniques. Serum levels of inflammatory factors were quantified utilizing ELISA. Pyroptosis was analyzed by lactate dehydrogenase release and flow cytometry in human keratinocytes. The activation of Nod-like receptor protein 3 (NLRP3) inflammasome was analyzed by western blots. Furthermore, the mechanism underlying the inhibition of NLRP3 inflammasome by N-butanol extracts of B. papyrifera root bark (NE-BPRB) was investigated using cellular thermal shift assay and surface plasmon resonance techniques. ResultsTreatment with NE-BPRB significantly ameliorated symptoms of AD mice and reduced serum levels of pro-inflammatory factors. NE-BPRB intervention exhibited inhibitory effects on NLRP3 inflammasome activation and pyroptosis in vitro and in vivo. NE-BPRB and its primary bioactive constituent chlorogenic acid (CA) promote the K48-linked ubiquitination of NLRP3, leading to its proteasomal degradation by binding WW domain containing E3 ubiquitin protein ligase 1 (WWP1). ConclusionsThe NE-BPRB and its primary bioactive constituent, CA, effectively inhibit the formation of the NLRP3 inflammasome and impede cell pyroptosis by promoting K48-linked ubiquitin-dependent proteasomal degradation of NLRP3 through binding to the E3 ubiquitin ligase WWP1, thereby resulting in improved AD.

Breaking the barriers: Overcoming cancer resistance by targeting the NLRP3 inflammasome.

Inflammation has a pivotal role in the initiation and progression of various cancers, contributing to crucial processes such as metastasis, angiogenesis, cell proliferation and invasion. Moreover, the release of cytokines mediated by inflammation within the tumour microenvironment (TME) has a crucial role in orchestrating these events. The activation of inflammatory caspases, facilitated by the recruitment of caspase-1, is initiated by the activation of pattern recognition receptors on the immune cell membrane. This activation results in the production of proinflammatory cytokines, including IL-1β and IL-18, and participates in diverse biological processes with significant implications. The NOD-Like Receptor Protein 3 (NLRP3) inflammasome holds a central role in innate immunity and regulates inflammation through releasing IL-1β and IL-18. Moreover, it interacts with various cellular compartments. Recently, the mechanisms underlying NLRP3 inflammasome activation have garnered considerable attention. Disruption in NLRP3 inflammasome activation has been associated with a spectrum of inflammatory diseases, encompassing diabetes, enteritis, neurodegenerative diseases, obesity and tumours. The NLRP3 impact on tumorigenesis varies across different cancer types, with contrasting roles observed. For example, colorectal cancer associated with colitis can be suppressed by NLRP3, whereas gastric and skin cancers may be promoted by its activity. This review provides comprehensive insights into the structure, biological characteristics and mechanisms of the NLRP3 inflammasome, with a specific focus on the relationship between NLRP3 and tumour-related immune responses, and TME. Furthermore, the review explores potential strategies for targeting cancers via NLRP3 inflammasome modulation. This encompasses innovative approaches, including NLRP3-based nanoparticles, gene-targeted therapy and immune checkpoint inhibitors.

The molecular mechanism of berberine affecting psoriasis skin inflammation by regulating keratinocyte pyroptosis via the p38 MAPK/NF-κB pathway.

Berberine (BBR), a Rhizoma Coptis-sourced isoquinoline alkaloid, is an effective drug for psoriasis treatment with its therapeutic mechanism remaining unclear. We delved into the mechanism of BBR affecting psoriatic skin inflammation by regulating keratinocyte pyroptosis. A psoriasis-like skin inflammation mouse model was induced by imiquimod (IMQ) and treated with BBR and a p38 activator anisomycin. Human epidermal keratinocytes (HEKs) were stimulated with five chemokines (M5) [interleukin (IL)-17A, IL-22A, oncostatin M, tumor necrosis factor-α, IL-1α] to simulate psoriasis immune microenvironment, then treated with BBR and anisomycin. Psoriasis skin lesions, skin tissue damage, cell viability and death, and gasdermin D-N (GSDMD-N) and NOD-like receptor protein 3 (NLRP3) positive cell numbers were assessed. The p38 mitogen-activated protein kinase (MAPK)/nuclear factor-kappa B (NF-κB) pathway and levels of the NLRP3/GSDMD pathway-related proteins and inflammatory factors were determined. BBR alleviated M5-induced HEK pyroptosis by inactivating NLRP3 inflammasomes. BBR inhibited the p38 MAPK/NF-κB pathway, and its effects on HEKs were partly averted by activating the p38 MAPK/NF-κB pathway. BBR repressed NLRP3 inflammasome activation and pyroptosis by inhibiting the p38 MAPK/NF-κB pathway. Collectively, BBR suppressed keratinocyte NLRP3/GSDMD pathway pyroptosis by suppressing the p38 MAPK/NF-κB pathway, thereby affecting psoriasis skin inflammation.

Review on the role of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome pathway in diabetes: mechanistic insights and therapeutic implications.

This review explores the pivotal role of the nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 (NLRP3) inflammasome in the pathogenesis of diabetes and its complications, highlighting the therapeutic potential of various oral hypoglycemic drugs targeting this pathway. NLRP3 inflammasome activation, triggered by metabolic stressors like hyperglycemia, hyperlipidemia, and free fatty acids (FFAs), leads to the release of pro-inflammatory cytokines interleukin-1β and interleukin-18, driving insulin resistance, pancreatic β-cell dysfunction, and systemic inflammation. These processes contribute to diabetic complications such as nephropathy, neuropathy, retinopathy, and cardiovascular diseases (CVD). Here we discuss the various transcriptional, epigenetic, and gut microbiome mediated regulation of NLRP3 activation in diabetes. Different classes of oral hypoglycemic drugs modulate NLRP3 inflammasome activity through various mechanisms: sulfonylureas inhibit NLRP3 activation and reduce inflammatory cytokine levels; sodium-glucose co-transporter 2 inhibitors (SGLT2i) suppress inflammasome activity by reducing oxidative stress and modulating intracellular signaling pathways; dipeptidyl peptidase-4 inhibitors mitigate inflammasome activation, protecting against renal and vascular complications; glucagon-like peptide-1 receptor agonists attenuate NLRP3 activity, reducing inflammation and improving metabolic outcomes; alpha-glucosidase inhibitors and thiazolidinediones exhibit anti-inflammatory properties by directly inhibiting NLRP3 activation. Agents that specifically target NLRP3 and inhibit their activation have been identified recently such as MCC950, Anakinra, CY-09, and many more. Targeting the NLRP3 inflammasome, thus, presents a promising strategy for managing diabetes and its complications, with oral hypoglycemic drugs offering dual benefits of glycemic control and inflammation reduction. Further research into the specific mechanisms and long-term effects of these drugs on NLRP3 inflammasome activity is warranted.

1,25-Dihydroxyvitamin D3 protects against placental inflammation by suppressing NLRP3-mediated IL-1β production via Nrf2 signaling pathway in preeclampsia

BackgroundMaternal vitamin D deficiency is associated with an increased risk of preeclampsia, a potentially life-threatening multi-system disorder specific to human pregnancy. Placental trophoblast dysfunction is a key factor in the development of preeclampsia, and the activation of NOD-like receptor protein 3 (NLRP3) inflammasome may play a crucial role in this process. Previous studies have suggested that vitamin D can exert beneficial effects by suppressing inflammasome activation, but the underlying mechanism has not been fully elucidated. This study aims to explore the protective effects of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on the placenta and to investigate the mechanisms by which 1,25(OH)2D3 attenuates NLRP3 inflammasome activation in a rat model of preeclampsia and hypoxia-cultured placental trophoblast cells. ResultsOur findings demonstrated that supplementation of rats with 1,25(OH)2D3 mitigated placental inflammation and prevented multi-organ dysfunction associated with preeclampsia. Treatment with 1,25(OH)2D3 inhibited inflammasome-mediated inflammation in trophoblast cells via its receptor VDR by reducing the expression of NLRP3, caspase-1, and apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), decreasing IL-1β production, reducing mitochondrial reactive oxygen species generation, and enhancing the expression and enzymatic activity of Cu/Zn-superoxide dismutase (SOD). Mechanistically, 1,25(OH)2D3 upregulated nuclear factor erythroid 2-related factor 2 (Nrf2) signaling, subsequently suppressing NLRP3-mediated IL-1β overproduction in trophoblast cells. ConclusionsOur study indicates that 1,25(OH)2D3 inhibits NLRP3-mediated inflammation in trophoblast cells during preeclampsia by stimulating the Nrf2 signaling pathway and inhibiting oxidative stress.

The hepatoprotective effect of sodium butyrate on hepatic inflammatory injury mediated by the NLRP3 inflammatory pathway in subchronic fluoride-exposed mice.

Excessive subchronic fluoride exposure can cause severe damage to detoxification organs, including the liver. Sodium butyrate has anti-inflammatory, antitumor, antioxidant and immunomodulatory properties. However, relatively few studies have investigated the effects of sodium butyrate on liver injury caused by subchronic fluoride exposure. The purpose of this research was to investigate the effect and mechanism of sodium butyrate on fluoride-induced hepatic inflammatory injury via the expression of nod-like receptor protein 3 (NLRP3). Mice were subjected to randomization into four groups, control group (C), fluorosis group (F), sodium butyrate alone group (S), and treatment group (Y). The mice in groups F and F + S drank 100mg/L sodium fluoride-containing distilled water freely every day. After fluoride exposure lasted for 3 months, the mice in group S and F + S were gavaged with sodium butyrate daily at a concentration of 1000mg/kg. Following the treatment regimen, liver specimens were collected for analysis. The mRNA and protein expression levels of inflammatory factors and NLRP3 and its downstream gene were measured by RT-qPCR and western blotting. The histological hematoxylin and eosin (H&E) staining of liver showed that the subchronic fluoride-exposed group were chronic inflammation. The liver of treatment group were less vacuolar degeneration and inflammatory infiltration. The results of the biochemical assay showed that the subchronic fluoride-exposed group were liver injury. In addition, the detection of oxidative stress indicators showed that chronic subchronic fluoride exposure could lead to an increase in the level of oxidative stress in the liver, and the treatment alleviated this increase. RT-qPCR results showed that compared with those in the control group, the mRNA levels of the inflammatory factors TNF-α, IL-6 and IL-1β, the NLRP3 inflammasome and its downstream factors NLRP3, caspase-1, gasdermin D (GSDMD) and IL-18 increased in the liver tissue of mice in the subchronic fluoride-exposed group. Sodium butyrate released inflammatory factors during subchronic fluoride exposure and inhibited the protein expression of activated NLRP3 to a certain extent. Sodium butyrate may play a protective role by antagonizing the production of activated inflammasomes and their downstream inflammatory factors in the livers of subchronic fluoride-exposed mice.

The role of quercetin in NLRP3-associated inflammation.

Quercetin is a natural flavonoid that is widely found in fruits and vegetables. As an important flavonoid, it exhibits a wide range of biological activities, including antioxidant, anti-inflammatory, antiviral, immunomodulatory, and analgesic activities. Quercetin exerts powerful antioxidant activity by regulating glutathione, enzyme activity, and the production of reactive oxygen species (ROS). Quercetin exerts powerful anti-inflammatory effects by acting on the Nod-likereceptorprotein3 (NLRP3) inflammasome. In diabetes, quercetin has been shown to improve insulin sensitivity and reduce high blood sugar level, while, in neurological diseases, it potentially prevents neuronal degeneration and cognitive decline by regulating neuroinflammation. In addition, in liver diseases, quercetin may improve liver inflammation and fibrosis by regulating the NLRP3 activity. In addition, quercetin may improve inflammation in other diseases based on the NLRP3 inflammasome. With this background, in this review, we have discussed the progress in the study on the mechanism of quercetin toward improving inflammation via NLRP3 inflammasome in the past decade. In addition, from the perspective of quercetin glycoside derivatives, the anti-inflammatory mechanism of hyperoside, rutin, and isoquercetin based on NLRP3 inflammasome has been discussed. Moreover, we have discussed the pharmacokinetics of quercetin and its nanoformulation application, with the aim to provide new ideas for further research on the anti-inflammatory effect of quercetin and its glycoside derivatives based on NLRP3 inflammasome, as well as in drug development and application.

Aerobic Exercise Attenuates Doxorubicin-Induced Cardiomyopathy by Suppressing NLRP3 Inflammasome Activation in a Rat Model.

Doxorubicin (DOX) is a potent chemotherapeutic agent with well-documented dose-dependent cardiotoxicity. Regular exercise is recognized for its cardioprotective effects against DOX-induced cardiac inflammation, although the precise mechanisms remain incompletely understood. The activation of inflammasomes has been implicated in the pathogenesis and treatment of DOX-induced cardiotoxicity, with the nucleotide-binding domain-like receptor protein 3 (NLRP3) inflammasome emerging as a key mediator in cardiovascular inflammation. This study aimed to investigate the role of exercise in modulating the NLRP3 inflammasome to protect against DOX-induced cardiac inflammation. Male Sprague-Dawley rats were randomly assigned to receive a 10-day course of DOX or saline injections, with or without a preceding 10-week treadmill running regimen. Cardiovascular function and histological changes were subsequently evaluated. DOX-induced cardiotoxicity was characterized by cardiac atrophy, systolic dysfunction, and hypotension, alongside activation of the NLRP3 inflammasome. Our findings revealed that regular exercise preserved cardiac mass and hypertrophic indices and prevented DOX-induced cardiac dysfunction, although it did not fully preserve blood pressure. These results underscore the significant cardioprotective effects of exercise against DOX-induced cardiotoxicity. While regular exercise did not entirely prevent DOX-induced hypotension, our findings demonstrate that it confers protection against DOX-induced cardiotoxicity by suppressing NLRP3 inflammasome activation in the heart, underscoring its anti-inflammatory role. Further research should explore the temporal dynamics and interactions among exercise, pyroptosis, and other pathways in DOX-induced cardiotoxicity to enhance translational applications in cardiovascular medicine.

Thioredoxin-Interacting Protein's Role in NLRP3 Activation and Osteoarthritis Pathogenesis by Pyroptosis Pathway: In Vivo Study.

Thioredoxin-interacting protein (TXNIP) has been involved in oxidative stress and activation of the NOD-like receptor protein-3 (NLRP3) inflammasome, directly linking it to the pyroptosis pathway. Furthermore, pyroptosis may contribute to the inflammatory process in osteoarthritis (OA). The purpose of this study was to investigate the role of TXNIP in activating the NLRP3 inflammasome through the pyroptosis pathway in an OA rat model. Destabilization of the medial meniscus (DMM) was induced in the OA model with intra-articular injections of adeno-associated virus (AAV) overexpressing (OE) or knocking down (KD) TXNIP. A total of 48 healthy rats were randomly divided into six groups (N = 8 each). During the experiment, the rats' weights, mechanical pain thresholds, and thermal pain thresholds were measured weekly. Morphology staining, micro-CT, 3D imaging, and immunofluorescence (IF) staining were used to measure the expression level of TXNIP, and ELISA techniques were employed. OE-TXNIP-AAV in DMM rats aggravated cartilage destruction and subchondral bone loss, whereas KD-TXNIP slowed the progression of OA. The histological results showed that DMM modeling and OE-TXNIP-AAV intra-articular injection caused joint structure destruction, decreased anabolic protein expression, and increased catabolic protein expression and pyroptosis markers. Conversely, KD-TXNIP-AAV slowed joint degeneration. OE-TXNIP-AVV worsened OA by accelerating joint degeneration and damage, while KD-TXNIP-AAV treatment had a protective effect.

Corn Peptides Alleviate Nonalcoholic Fatty Liver Fibrosis in Mice by Inhibiting NLRP3 Inflammasome Activation and Regulating Gut Microbiota.

This study aimed to investigate the effects of corn gluten-derived soluble epoxide hydrolase (sEH) inhibitory peptides on nonalcoholic fatty liver fibrosis induced by a high-fat diet and carbon tetrachloride in mice. Mice treated with corn peptides at doses of 500 or 1000 mg/kg/d for 4 weeks exhibited reduced sEH activity in serum and liver, enhanced lipid metabolism, and decreased lipid accumulation and oxidative stress. Corn peptides effectively downregulated the mRNA levels of Pro-IL-1β, Pro-IL-18, NOD-like receptor protein 3 (NLRP3), ASC, Pro-caspase-1, Caspase-1, and GSDMD in the liver. This hepatoprotective effect of corn peptides by inhibiting NLRP3 inflammasome activation was further validated in H2O2-induced HepG2 cells. Moreover, corn peptides restored the composition of the gut microbiota and promoted short-chain fatty acid production. This study provides evidence that corn-derived sEH inhibitory peptides have hepatoprotective activity against nonalcoholic fatty liver fibrosis by suppressing NLRP3 inflammasome activation and modulating gut microbiota.

Daidzein attenuates high glucose-induced inflammatory injury in macrophages by regulating NLRP3 inflammasome signaling pathway

This study aims to explore the inhibitory effect of daidzein on macrophage inflammation induced by high glucose via regulating the NOD-like receptor protein 3(NLRP3) inflammasome signaling pathway. The cell counting kit-8(CCK-8) assay was employed to detect the effects of daidzein at different concentrations on the viability of RAW264.7 cells. Western blot was employed to determine the protein level of tumor necrosis factor(TNF)-α in macrophages exposed to different concentrations of glucose for different time periods as well as the expression levels of proteins involved in the polarization and Toll-like receptor 4(TLR4)-myeloid differentiation factor(MyD88)-NLRP3 inflammasome pathway of the macrophages exposed to high glucose. Enzyme-linked immunosorbent assay was employed to measure the levels of TNF-α, interleukin(IL)-18, and IL-1β secreted by macrophages. The expression level of nuclear factor-kappa B(NF-κB) p65 in macrophages exposed to high glucose was detected by immunofluorescence, and the level of intracellular reactive oxygen species(ROS) was detected by the DCFH-DA fluorescent probe. The mRNA levels of NLRP3, TNF-α, and IL-18 in macrophages were determined by qRT-PCR. The results showed that treatment with 30 mmol·L~(-1) glucose for 48 h was the best condition for the modeling of macrophage injury. Compared with the blank group, the model group showed improved polarization of macrophages, increased secretion of TNF-α, IL-18, and IL-1β, elevated ROS level, and up-regulated expression of NF-κB p65. In addition, the modeling up-regulated the mRNA levels of NLRP3, TNF-α, and IL-18 and the protein levels of TLR4, MyD88, NLRP3, NF-κB p65, p-NF-κB p65, I-κB, p-I-κB, ASC, pro-caspase-1, pro-IL-1β, cleaved IL-1β, and pro-IL-18. Compared with the model group, daidzein(10, 20, and 40 μmol·L~(-1)) lowered the levels of inflammatory cytokines and down-regulated the mRNA levels of NLRP3, TNF-α, and IL-18 as well as the protein levels of TLR4, MyD88, NLRP3, NF-κB p65, p-NF-κB p65, I-κB, p-I-κB, ASC, pro-caspase-1, pro-IL-1β, cleaved IL-1β, and pro-IL-18. In addition, daidzein reduced intracellular ROS. According to the available reports and the experimental results, high glucose can induce the polarization of macrophages and promote the secretion of inflammatory cytokines. Daidzein can inhibit the expression of ROS in macrophages by regulating the NLRP3 inflammasome signaling pathway, thereby reducing the inflammation of macrophages exposed to high glucose.

Inhibition of cGAS attenuates neonatal hypoxic-ischemic encephalopathy via regulating microglia polarization and pyroptosis.

Neonatal hypoxic-ischemic encephalopathy (HIE) is a condition causing brain injury in newborns with unclear pathogenesis. Cyclic GMP-AMP synthase (cGAS)/stimulator of interferon genes (STING) signaling pathway and NOD-like receptor protein 3 (NLRP3) mediated pyroptosis are thought to be involved in the pathological process of HIE, but whether these two mechanisms act independently is still unknown. Therefore, we aim to clarify whether there is any interaction between these two pathways and thus synergistically affects the progression of HIE. The HIE model of neonatal rats was established using the Rice-Vannucci method. The potential therapeutic effect of RU.521 targeting cGAS on HIE was explored through rescue experiment. Twenty-four hours after modeling was selected as observation point, sham + vehicle group, HIE + vehicle group and HIE + RU.521 group were established. A complete medium of BV2 cells was adjusted to a glucose-free medium, and the oxygen-glucose deprivation model was established after continuous hypoxia for 4 hours and reoxygenation for 12 to 24 hours. 2,3,5-triphenyl tetrazolium chloride staining was employed to detect ischemic cerebral infarction in rat brain tissue, and hematoxylin and eosin staining was used to observe tissue injury. Immunofluorescence was applied to monitor the expression of cGAS. Real-time quantitative polymerase chain reaction and western blot were utilized to detect the expression of messenger RNA and protein. cGAS expression was increased in brain tissues of neonatal rats with HIE, and mainly localized in microglia. RU.521 administration reduced infarct size and pathological damage in rat HIE. Moreover, blocking cGAS with RU.521 significantly reduced inflammatory conditions in the brain by down-regulating STING expression, decreasing NLRP3 inflammasome activation and reducing microglial pyroptosis both in vivo and in vitro. Besides, RU.521 promoted the switching of BV2 cells towards the M2 phenotype. This study revealed a link between the cGAS/STING pathway and the NLRP3/GSDMD/pyroptosis pathway in neonatal HIE. Furthermore, the small molecule compound RU.521 can negatively regulate cGAS/STING/NLRP3/pyroptosis axis and promote M2 polarization in microglia, which provides a potential therapeutic strategy for the treatment of neuroinflammation in HIE.

Activation of NLRP3 inflammasome in patients with renal transplantation: relation to allograft dysfunction, inflammation, and renal fibrosis

ABSTRACT Background Chronic allograft dysfunction (CAD) represents a complex process of inflammatory response, fibrosis, and tissue restoration, mediated by a myriad of proinflammatory cytokines, enzymes, and growth factors. NOD-like receptor protein3 (NLRP3) inflammasome is one of the NLR protein families. It drives proteolysis of proinflammatory caspases which induces maturation of pro-interleukin (IL)-1β and pro-IL-18. The aim of this work is to study NLRP3 inflammasome activation in patients with renal transplantation in relation to allograft function, inflammation, and renal fibrosis. Methods Thirty renal transplant recipients (RTR): 15 patients with stable renal function (Group I), 15 patients with CAD (Group II), and 15 healthy subjects (Group III) were included. Serum and urinary NLRP3, caspase-1, and interleukin-1β were measured using an enzyme-linked immunosorbent assay (ELISA) kit. Serum high sensitivity C-reactive protein (hs-CRP) level was measured, and neutrophil-lymphocyte ratio (NLR) was calculated. Renal function was evaluated by serum creatinine (S.Cr) and estimated glomerular filtration rate (eGFR). Resistive index (RI) was calculated with Doppler ultrasonography. In patients with CAD, renal interstitial fibrosis (RIF) was graded in renal biopsies. Results Serum NLRP3, caspase-1, serum and urinary IL-1β, and hs-CRP showed a statistically significant increase in RTR, particularly in those with CAD compared to the controls with a positive correlation with each other, their urinary level, S. Cr, hs-CRP, NLR, RI, and RIF (p < 0.001). The receiver operating characteristic curve showed high diagnostic accuracy of serum NLRP3, caspase-1, and IL-1β in discriminating patients with CAD from patients with stable renal function. Conclusion The NLRP3-caspase1-IL-1β cascade is activated and upregulated in renal transplant recipients, particularly those with advanced renal impairment, and thus may play a role in developing CAD and renal fibrosis. They could be potential biomarkers for post-transplant outcome, follow-up, and early diagnosis. Moreover, their cascade could be a target for future anti-inflammatory therapeutic strategies for allograft dysfunction.

NLRP3 inflammasomes as a target for hesperidin and diosmin flavonoids in varicose vein disease and its complications

Varicose vein disease of the lower extremities is an inflammatory disorder with abnormal structure and functional activity of the venous valves, venous walls and cells, as well as with abnormal activity of the infiltrating leukocytes. The abnormally changed varicose vein are characterized by increased venous pressure, blood accumulation and congestion, ischemia, and metabolic and energy turnover derangement, which result in clinical manifestations of complications, such as pain, edema and formation of trophic ulcers. For many years, flavonoids have been used to treat varicose veins. The most effective flavonoids used in varicose vein disease and its complications, are hesperidin and diosmin, as well as their combinations. The review sets forth the state-of-the-art knowledge on the universal inflammatory processes playing a leading role in the pathophysiology of many cardiovascular disorders, including venous ones. In the recent years, it has been found that one of the main causes of inflammation is the formation of intracellular protein complexes (inflammasomes), which both produce a set of proinflammatory cytokines and are responsible for their excretion from the cells. In addition, inflammasomes control the development of regulated necrosis (pyroptosis) that takes part in the process of ulceration. The inflammasome activity can be modified by various mechanisms, of which the gene independent synthesis of the inflammasomal proteins has been recognized as the leading one. It has been shown that flavonoids inhibit the activation of a key factor NF-kappa B and suppress the synthesis of proteins, including NOD-like receptor protein 3 (NLRP3) inflammasome components, decrease the expression of NLRP3 receptor, protein ASC and caspase 1, as well as diminish interleukin 1 beta, interleukin 6 and tumor necrosis factor-alpha expression. Thus, this is the explanation of positive effects observed with the use of hesperidin, diosmin and their combination in clinical practice.

Hyperglycemia induces microglial pyroptosis by increasing oxygen extraction rate: Implication in neurological impairment during ischemic stroke.

Elevated levels of blood glucose in patients with ischemic stroke are associated with a worse prognosis. The present study aimed to explore whether hyperglycemia promotes microglial pyroptosis by increasing the oxygen extraction rate in an acute ischemic stroke model. C57BL/6 mice that underwent middle cerebral artery occlusion were used for assessment of blood glucose level and neurological function. The cerebral oxygen extraction ratio (CERO2), oxygen consumption rate (OCR) and partial pressure of brain tissue oxygen (PbtO2) were measured. To investigate the significance of the NOD‑like receptor protein 3 (NLRP3) inflammasome, NLRP3‑/‑ mice were used, and the expression levels of NLRP3, caspase‑1, full‑length gasdermin D (GSDMD‑FL), GSDMD‑N domain (GSDMD‑N), IL‑1β and IL‑18 were evaluated. In addition, Z‑YVAD‑FMK, a caspase‑1 inhibitor, was used to treat microglia to determine whether activation of the NLRP3 inflammasome was required for the enhancing effect of hyperglycemia on pyroptosis. It was revealed that hyperglycemia accelerated cerebral injury in the acute ischemic stroke model, as evidenced by decreased latency to fall and the percentage of foot fault. Hyperglycemia aggravated hypoxia by increasing the oxygen extraction rate, as evidenced by increased CERO2 and OCR, and decreased PbtO2 in response to high glucose treatment. Furthermore, hyperglycemia‑induced microglial pyroptosis was confirmed by detection of increased levels of caspase‑1, GSDMD‑N, IL‑1β and IL‑18 and a decreased level of GSDMD‑FL. However, the knockout of NLRP3 attenuated these effects. Pharmacological inhibition of caspase‑1 also reduced the expression levels of GSDMD‑N, IL‑1β and IL‑18 in microglial cells. These results suggested that hyperglycemia stimulated NLRP3 inflammasome activation by increasing the oxygen extraction rate, thus leading to the aggravation of pyroptosis following ischemic stroke.

New applications of Clioquinol in the treatment of inflammation disease by directly targeting arginine 335 of NLRP3

The NOD-like receptor protein 3 (NLRP3) inflammasome is essential in innate immune-mediated inflammation, with its overactivation implicated in various autoinflammatory, metabolic, and neurodegenerative diseases. Pharmacological inhibition of NLRP3 offers a promising treatment strategy for inflammatory conditions, although no medications targeting the NLRP3 inflammasome are currently available. This study demonstrates that clioquinol (CQ), a clinical drug with chelating properties, effectively inhibits NLRP3 activation, resulting in reduced cytokine secretion and cell pyroptosis in both human and mouse macrophages, with a half maximal inhibitory concentration (IC50) of 0.478 μM. Additionally, CQ mitigates experimental acute peritonitis, gouty arthritis, sepsis, and colitis by lowering serum levels of interleukin-1β (IL-1β), IL-6, and tumour necrosis factor-α (TNF-α). Mechanistically, CQ covalently binds to Arginine 335 (R335) in the NACHT domain, inhibiting NLRP3 inflammasome assembly and blocking the interaction between NLRP3 and its component protein. Collectively, this study identifies CQ as an effective natural NLRP3 inhibitor and a potential therapeutic agent for NLRP3-driven diseases.

AMPK Attenuation of β-Adrenergic Receptor-Induced Cardiac Injury via Phosphorylation of β-Arrestin-1-ser330.

β-adrenergic receptor (β-AR) overactivation is a major pathological cue associated with cardiac injury and diseases. AMPK (AMP-activated protein kinase), a conserved energy sensor, regulates energy metabolism and is cardioprotective. However, whether AMPK exerts cardioprotective effects via regulating the signaling pathway downstream of β-AR remains unclear. Using immunoprecipitation, mass spectrometry, site-specific mutation, in vitro kinase assay, and in vivo animal studies, we determined whether AMPK phosphorylates β-arrestin-1 at serine (Ser) 330. Wild-type mice and mice with site-specific mutagenesis (S330A knock-in [KI]/S330D KI) were subcutaneously injected with the β-AR agonist isoproterenol (5 mg/kg) to evaluate the causality between β-adrenergic insult and β-arrestin-1 Ser330 phosphorylation. Cardiac transcriptomics was used to identify changes in gene expression from β-arrestin-1-S330A/S330D mutation and β-adrenergic insult. Metformin could decrease cAMP/PKA (protein kinase A) signaling induced by isoproterenol. AMPK bound to β-arrestin-1 and phosphorylated Ser330 with the highest phosphorylated mass spectrometry score. AMPK activation promoted β-arrestin-1 Ser330 phosphorylation in vitro and in vivo. Neonatal mouse cardiomyocytes overexpressing β-arrestin-1-S330D (active form) inhibited the β-AR/cAMP/PKA axis by increasing PDE (phosphodiesterase) 4 expression and activity. Cardiac transcriptomics revealed that the differentially expressed genes between isoproterenol-treated S330A KI and S330D KI mice were mainly involved in immune processes and inflammatory response. β-arrestin-1 Ser330 phosphorylation inhibited isoproterenol-induced reactive oxygen species production and NLRP3 (NOD-like receptor protein 3) inflammasome activation in neonatal mouse cardiomyocytes. In S330D KI mice, the β-AR-activated cAMP/PKA pathways were attenuated, leading to repressed inflammasome activation, reduced expression of proinflammatory cytokines, and mitigated macrophage infiltration. Compared with S330A KI mice, S330D KI mice showed diminished cardiac fibrosis and improved cardiac function upon isoproterenol exposure. However, the cardiac protection exerted by AMPK was abolished in S330A KI mice. AMPK phosphorylation of β-arrestin-1 Ser330 potentiated PDE4 expression and activity, thereby inhibiting β-AR/cAMP/PKA activation. Subsequently, β-arrestin-1 Ser330 phosphorylation blocks β-AR-induced cardiac inflammasome activation and remodeling.

Changes and clinical significance of NLRP3 inflammasomes and related factors in patients with spinal fracture complicated with acute spinal cord injury

To investigate the changes and clinical significance of NOD like receptor protein 3 (NLRP3) inflammasomes and related factors in patients with spinal fractures complicated with acute spinal cord injury (SCI). Eighty-six spinal fracture patients complicated with acute SCI admitted to hospital from June 2019 to March 2022 were selected as SCI group, There were 48 males and 38 females, with an average age of (43.48±6.58) years old. And 100 healthy volunteers who underwent physical examination during the same time were selected as control group, including 56 males patients and 44 females patients, with an average age of (45.13±6.43) years old. Peripheral blood mononuclear cell (PBMC) were collected, and the mRNA expressions of NLRP3 and Caspase-1 were detected. Serum was collected and the levels of interleukin (IL)- 1β, IL-18 were detected. According to Frankel's grade, the SCI group was divided into complete injury patients and incomplete injury patients, and according to the Japanese Orthopedic Society (JOA) grade, the SCI group was divided into good prognosis group and poor prognosis group. The difference of NLRP3, Caspase-1, IL-1β, IL-18 among groups were compared, the influencing factors for poor prognosis in SCI patients was analyzed by Logistic regression. The mRNA expression levels of NLRP3 (1.41±0.33) and Caspase-1 (1.44±0.35) in PBMC and the levels of IL-1β(45.34±13.22) pg·ml-1, IL-18(40.95±8.77) pg·ml-1 in serum of SCI group were higher than those of the control group[(1.00±0.19), (1.00±0.16), (16.58±4.24) pg·ml-1, (12.57±3.68) pg·ml-1] (P<0.05). The mRNA expression levels of NLRP3(1.63±0.34) and Caspase-1 (1.67±0.27) in PBMC and the levels of IL-1β(51.09±11.10) pg·ml-1, IL-18 (47.65±7.93) pg·ml-1 in serum of patients with complete injury in the SCI group were higher than those of patients with incomplete injury [(1.31±0.27), (1.34±0.33), (42.85±13.36) pg·ml-1, (38.05±7.48) pg·ml-1](P<0.05). The mRNA expression levels of NLRP3 (1.66±0.31) and Caspase-1 (1.72±0.31)in PBMC and the levels of IL-1β(51.21±11.31) pg·ml-1, IL-18 (45.70±7.25) pg·ml-1 in serum, the proportion of complete injury(21 patients), and the proportion of spinal cord edema or bleeding of patients(15 patients) with poor prognosis in the SCI group were higher than those of patients with good prognosis[(1.28±0.26), (1.37±0.36), (42.79±13.25) pg·ml-1、(38.90±8.63) pg·ml-1, 5、20 cases](P<0.05). Complete injury and the mRNA expression of NLRP3 in PBMC were the influencing factors for poor prognosis in the SCI group (P<0.05). The activation of NLRP3 inflammasomes in patients with spinal fractures complicated with acute SCI is associated with worsening injury and poor prognosis, and NLRP3 expression can serve as a marker for evaluating prognosis.