NOD-like Receptor Protein 3 Inflammasome Activation Research Articles

Interval training suppresses nod-like receptor protein 3 inflammasome activation to improve cardiac function in myocardial infarction rats by hindering the activation of the transforming growth factor-β1 pathway

ObjectiveMyocardial infarction (MI) -induced cardiac dysfunction can be attenuated by aerobic exercises. This study explored the mechanism of interval training (IT) regulating cardiac function in MI rats, providing some theoretical basis for clarifying MI pathogenesis and new ideas for clinically treating MI.MethodsRats were subjected to MI modeling, IT intervention, and treatments of the Transforming growth factor-β1 (TGF-β1) pathway or the nod-like receptor protein 3 (NLRP3) activators. Cardiac function and hemodynamic indicator alterations were observed. Myocardial pathological damage and fibrosis, reactive oxygen species (ROS) level, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) activities, MDA content, inflammasome-associated protein levels, and inflammatory factor levels were assessed. The binding between TGF-β1 and receptor was detected.ResultsMI rats exhibited decreased left ventricle ejection fraction (LVEF), left ventricle fractional shortening (LVFS), left ventricular systolic pressure (LVSP), positive and negative derivates max/min (dP/dt max/min) and increased left ventricular end-systolic pressure (LVEDP), a large number of scar areas in myocardium, disordered cell arrangement and extensive fibrotic lesions, increased TGF-β1 and receptor binding, elevated ROS level and MDA content and weakened SOD, CAT and GSH-Px activities, and up-regulated NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC) and cleaved-caspase-1 levels, while IT intervention caused ameliorated cardiac function. IT inactivated the TGF-β1 pathway to decrease oxidative stress in myocardial tissues of MI rats and inhibit NLRP3 inflammasome activation. Activating NLRP3 partially reversed IT-mediated improvement on cardiac function in MI rats.ConclusionIT diminished oxidative stress in myocardial tissues and suppressed NLRP3 inflammasome activation via inactivating the TGF-β1 pathway, thus improving the cardiac function of MI rats.

Heat stress induces IL-1β and IL-18 overproduction via ROS-activated NLRP3 inflammasome: implication in neuroinflammation in mice with heat stroke.

Heat stroke induced cerebral damage via neuroinflammation. This study aimed to approach whether heat stress would promote NOD-like receptor protein 3 (NLRP3) inflammasome via reactive oxygen species (ROS). The mice were randomly divided into the sham group, the heat stress group, and the heat stress + TEMPOL (ROS scavenger) group. And the NLRP3-/- mice were applied and divided into the NLRP3-/- + sham group and the NLRP3-/- + heat stress group. Furthermore, the BV2 cells were divided into four groups following the intervention measures: the heat stress + TEMPOL group, the heat stress + Z-VAD-FMK (caspase-1 inhibitor) group, the heat stress group, and the control group. ROS levels were examined. The expression levels of NLRP3, caspase-1, IL-1β, and IL-18 were detected by western blotting and double immunofluorescence. We found that heat stress attack induced excessive ROS in microglia and subsequently activated NLRP3 inflammasome in both mice and BV2 cells. When ROS scavenged, the expression level of NLRP3 was downregulated. Furthermore, with NLRP3 inflammasome activation, the expression levels of caspase-1, IL-1β, and IL-18 were increased. In NLRP3-/- mice, however, the caspase-1, IL-1β, and IL-18 were significantly declined. Further experiments showed that pretreatment of caspase-1 inhibitor decreased the expression levels of IL-1β and IL-18. These results suggest that heat stress attack caused neuroinflammation via excessive ROS activating the NLRP3 inflammasome in microglia cells.

Endothelial nucleotide-binding oligomerization domain-like receptor protein 3 inflammasome regulation in atherosclerosis

Abstract Aims The activation of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome in endothelial cells (ECs) contributes to vascular inflammation in atherosclerosis. Considering the high glycolytic rate of ECs, we delineated whether and how glycolysis determines endothelial NLRP3 inflammasome activation in atherosclerosis. Methods and results Our results demonstrated a significant up-regulation of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), a key regulator of glycolysis, in human and mouse atherosclerotic endothelium, which positively correlated with NLRP3 levels. Atherosclerotic stimuli up-regulated endothelial PFKFB3 expression via sterol regulatory element-binding protein 2 (SREBP2) transactivation. EC-selective haplodeficiency of Pfkfb3 in Apoe−/− mice resulted in reduced endothelial NLRP3 inflammasome activation and attenuation of atherogenesis. Mechanistic investigations revealed that PFKFB3-driven glycolysis increased the NADH content and induced oligomerization of C-terminal binding protein 1 (CtBP1), an NADH-sensitive transcriptional co-repressor. The monomer form, but not the oligomer form, of CtBP1 was found to associate with the transcriptional repressor Forkhead box P1 (FOXP1) and acted as a transrepressor of inflammasome components, including NLRP3, caspase-1, and interleukin-1β (IL-1β). Interfering with NADH-induced CtBP1 oligomerization restored its binding to FOXP1 and inhibited the glycolysis-dependent up-regulation of NLRP3, Caspase-1, and IL-1β. Additionally, EC-specific overexpression of NADH-insensitive CtBP1 alleviates atherosclerosis. Conclusion Our findings highlight the existence of a glycolysis-dependent NADH/CtBP/FOXP1-transrepression pathway that regulates endothelial NLRP3 inflammasome activation in atherogenesis. This pathway represents a potential target for selective PFKFB3 inhibitors or strategies aimed at disrupting CtBP1 oligomerization to modulate atherosclerosis.

Teneligliptin mitigates diabetic cardiomyopathy by inhibiting activation of the NLRP3 inflammasome

BACKGROUND Diabetic cardiomyopathy (DCM), which is a complication of diabetes, poses a great threat to public health. Recent studies have confirmed the role of NLRP3 (NOD-like receptor protein 3) activation in DCM development through the inflammatory response. Teneligliptin is an oral hypoglycemic dipeptidyl peptidase-IV inhibitor used to treat diabetes. Teneligliptin has recently been reported to have anti-inflammatory and protective effects on myocardial cells. AIM To examine the therapeutic effects of teneligliptin on DCM in diabetic mice. METHODS Streptozotocin was administered to induce diabetes in mice, followed by treatment with 30 mg/kg teneligliptin. RESULTS Marked increases in cardiomyocyte area and cardiac hypertrophy indicator heart weight/tibia length reductions in fractional shortening, ejection fraction, and heart rate; increases in creatine kinase-MB (CK-MB), aspartate transaminase (AST), and lactate dehydrogenase (LDH) levels; and upregulated NADPH oxidase 4 were observed in diabetic mice, all of which were significantly reversed by teneligliptin. Moreover, NLRP3 inflammasome activation and increased release of interleukin-1β in diabetic mice were inhibited by teneligliptin. Primary mouse cardiomyocytes were treated with high glucose (30 mmol/L) with or without teneligliptin (2.5 or 5 µM) for 24 h. NLRP3 inflammasome activation. Increases in CK-MB, AST, and LDH levels in glucose-stimulated cardiomyocytes were markedly inhibited by teneligliptin, and AMP (p-adenosine 5‘-monophosphate)-p-AMPK (activated protein kinase) levels were increased. Furthermore, the beneficial effects of teneligliptin on hyperglycaemia-induced cardiomyocytes were abolished by the AMPK signaling inhibitor compound C. CONCLUSION Overall, teneligliptin mitigated DCM by mitigating activation of the NLRP3 inflammasome.

Leptin signalling regulates transcriptional differences in granulosa cells from genetically obese mice but not the activation of NLRP3 inflammasome

Obesity is associated with increased ovarian inflammation and the establishment of leptin resistance. We presently investigated the role of impaired leptin signalling on transcriptional regulation in granulosa cells (GCs) collected from genetically obese mice. Furthermore, we characterised the association between ovarian leptin signalling, the activation of the NOD-like receptor protein 3 (NLRP3) inflammasome and macrophage infiltration in obese mice. After phenotype characterisation, ovaries were collected from distinct group of animals for protein and mRNA expression analysis: (i) mice subjected to a diet-induced obesity (DIO) protocol, where one group was fed a high-fat diet (HFD) and another a standard chow diet (CD) for durations of 4 or 16 weeks; (ii) mice genetically deficient in the long isoform of the leptin receptor (ObRb; db/db); (iii) mice genetically deficient in leptin (ob/ob); and (iv) mice rendered pharmacologically hyperleptinemic (LEPT). Next, GCs from antral follicles isolated from db/db and ob/ob mice were subjected to transcriptome analysis. Transcriptional analysis revealed opposing profiles in genes associated with steroidogenesis and prostaglandin action between the genetic models, despite the similarities in body weight. Furthermore, we observed no changes in the mRNA and protein levels of NLRP3 inflammasome components in the ovaries of db/db mice or in markers of M1 and M2 macrophage infiltration. This contrasted with the downregulation of NLRP3 inflammasome components and M1 markers in ob/ob and 16-wk HFD-fed mice. We concluded that leptin signalling regulates NLRP3 inflammasome activation and the expression of M1 markers in the ovaries of obese mice in an ObRb-dependent and ObRb-independent manner. Furthermore, we found no changes in the expression of leptin signalling and NLRP3 inflammasome genes in GCs from db/db and ob/ob mice, which was associated with no effects on macrophage infiltration genes, despite the dysregulation of genes associated with steroidogenesis in homozygous obese db/db. Our results suggest that: (i) the crosstalk between leptin signalling, NLRP3 inflammasome and macrophage infiltration takes place in ovarian components other than the GC compartment; and (ii) transcriptional changes in GCs from homozygous obese ob/ob mice suggest structural rearrangement and organisation, whereas in db/db mice the impairment in steroidogenesis and secretory activity.

Increased Cyclic Guanosine Monophosphate and Interleukin-1Beta Is Activated by Mitochondrial Dysfunction and Associated With Heart Failure in Atrial Fibrillation Patients.

This study aimed to identify the association of cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) synthase-stimulator interferon genes (cGAS-STING) pathway with heart failure (HF) in atrial fibrillation (AF) patients. We prospectively enrolled 106 AF patients without evidence of HF. The serum levels of 2'3'-cyclic GMP-AMP (2'3'-cGAMP) and interleukin (IL)-1β were measured by enzyme-linked immunoassay (ELISA). To determine the underlying mechanism, we supplemented the complex I inhibitor rotenone and the specific cGAS inhibitor RU.521 in neonatal rat ventricular cardiomyocytes. During 18-month follow-up, serum concentrations of 2'3'-cGAMP (baseline 51.82 ± 11.34 pg/mL vs. follow-up 124.50 ± 75.83 pg/mL, Ppaired t < 0.01) and IL-1β (baseline 436.07 ± 165.82 vs. follow-up 632.48 ± 119.25 ng/mL, Ppaired t < 0.01) were substantially upregulated in AF patients with HF as compared with those without HF. Furthermore, serum 2'3'-cGAMP and IL-1β levels at 18-month follow-up were independently associated with the occurrence of HF in AF patients. Inhibition of cGAS by RU.521 effectively reversed the upregulation of 2'3'-cGAMP and STING phosphorylation induced by mitochondrial dysfunction, accompanied with inhibition of nod-like receptor protein 3 (NLRP3) inflammasome, IL-1β and IL-18 secretion. Induction of mitochondrial dysfunction causes an upregulation of 2'3'-cGAMP and activation of NLRP3 inflammasome through cGAS-STING pathway in cardiomyocytes.

Dictyophora indusiata polysaccharide mediates priming of the NLRP3 inflammasome activation via TLR4/ NF-κB signaling pathway to exert immunostimulatory effects.

Dictyophora indusiata, commonly known as bamboo fungus, is a type of edible mushroom that is highly popular worldwide for its rich flavor and nutritional value. It is also recognized for its pharmaceutical efficacy, with medicinal benefits attributed to its consumption. One of the most important components of Dictyophora indusiata is polysaccharide, which has been acknowledged as a promising regulator of biological response due to its immunostimulatory and anti-inflammatory properties. However, the specific roles of polysaccharide in modulating the NOD-like receptor protein 3 (NLRP3) inflammasome activation within macrophages remain relatively under-researched. To investigate this further, the mechanism by which Dictyophora indusiata polysaccharide (DIP) exerts its immunostimulatory activity in RAW 264.7 macrophages was analyzed. Results indicated that DIP has the potential to facilitate the priming of NLRP3 inflammasome activation by enhancing TLR4 expression, phosphorylation of IκB-α, and nuclear translocation of NF-κB p65 subunit. It was noted that DIP was unable to mediate the second step of NLRP3 inflammasome activation. The findings of this study provide compelling evidence that DIP has immunomodulatory effects by modulating the NLRP3 inflammasome in RAW264.7 macrophages.

CXCL13/CXCR5 promote chronic postsurgical pain and astrocyte activation in rats by targeting NLRP3.

Chronic postsurgical pain (CPSP) with high incidence negatively impacts the quality of life. X-C motif chemokine 13 (CXCL13) has been associated with postsurgery inflammation and exacerbates neuropathic pain in patients with CPSP. This study was aimed to illustrate the relationship between CXCL13 and nod-like receptor protein-3 (NLRP3), which is also involved in CPSP. A CPSP model was constructed by skin/muscle incision and retraction (SMIR) in right medial thigh, and the rats were divided into three groups: Sham, SMIR, and SMIR + anti-CXCL13 (intrathecally injected with anti-CXCL13 antibody). Then, the paw withdrawal threshold (PWT) score of rats was recorded. Primary rat astrocytes were isolated and treated with recombinant protein CXCL13 with or without NLRP3 inhibitor INF39. The expressions of CXCL13, CXCR5, IL-1β, IL-18, GFAP, NLRP3, and Caspase-1 p20 were detected by real-time quantitative reverse transcription PCR, western blot, ELISA, immunocytochemistry, and immunofluorescence analyses. The anti-CXCL13 antibody alleviated SMIR-induced decreased PWT and increased expression of GFAP, CXCL13, CXCR5, NLRP3, and Caspase-1 p20 in spinal cord tissues. The production of IL-1β, IL-18, and expression of CXCL13, CXCR5, GFAP, NLRP3, and Caspase-1 p20 were increased in recombinant protein CXCL13-treated primary rat astrocytes in a dose-dependent manner. Treatment with NLRP3 inhibitor INF39 inhibited the function of recombinant protein CXCL13 in primary rat astrocytes. The CXCL13/CXCR5 signaling could promote neuropathic pain, astrocytes activation, and NLRP3 inflammasome activation in CPSP model rats by targeting NLRP3. NLRP3 may be a potential target for the management of CPSP.

Innate and adaptive immune cell interaction drives inflammasome activation and hepatocyte apoptosis in murine liver injury from immune checkpoint inhibitors

Immune checkpoints (CTLA4 & PD-1) are inhibitory pathways that block aberrant immune activity and maintain self-tolerance. Tumors co-opt these checkpoints to avoid immune destruction. Immune checkpoint inhibitors (ICIs) activate immune cells and restore their tumoricidal potential, making them highly efficacious cancer therapies. However, immunotolerant organs such as the liver depend on these tolerogenic mechanisms, and their disruption with ICI use can trigger the unintended side effect of hepatotoxicity termed immune-mediated liver injury from ICIs (ILICI). Learning how to uncouple ILICI from ICI anti-tumor activity is of paramount clinical importance. We developed a murine model to recapitulate human ILICI using CTLA4+/- mice treated with either combined anti-CTLA4 + anti-PDL1 or IgG1 + IgG2. We tested two forms of antisense oligonucleotides to knockdown caspase-3 in a total liver (parenchymal and non-parenchymal cells) or in a hepatocyte-specific manner. We also employed imaging mass cytometry (IMC), a powerful multiplex modality for immunophenotyping and cell interaction analysis in our model. ICI-treated mice had significant evidence of liver injury. We detected cleaved caspase-3 (cC3), indicating apoptosis was occurring, as well as Nod-like receptor protein 3 (NLRP3) inflammasome activation, but no necroptosis. Total liver knockdown of caspase-3 worsened liver injury, and induced further inflammasome activation, and Gasdermin-D-mediated pyroptosis. Hepatocyte-specific knockdown of caspase-3 reduced liver injury and NLRP3 inflammasome activation. IMC-generated single-cell data for 77,692 cells was used to identify 22 unique phenotypic clusters. Spatial analysis revealed that cC3+ hepatocytes had significantly closer interactions with macrophages, Kupffer cells, and NLRP3hi myeloid cells than other cell types. We also observed zones of three-way interaction between cC3+ hepatocytes, CD8 + T-cells, and macrophages. Our work is the first to identify hepatocyte apoptosis and NLRP3 inflammasome activation as drivers of ILICI. Furthermore, we report that the interplay between adaptive and innate immune cells is critical to hepatocyte apoptosis and ILICI.

Panaxydol extracted from Panax ginseng inhibits NLRP3 inflammasome activation to ameliorate NASH-induced liver injury

Activation of NOD-like receptor protein 3 (NLRP3) inflammasome exacerbates liver inflammation and fibrosis in nonalcoholic steatohepatitis (NASH), suggesting that development of inflammasome inhibitor can become leading candidate to ameliorate NASH. Panax ginseng (P. ginseng) contains numerous bioactive natural components to reduce inflammation. This study aims to identify inhibitory components of P. ginseng for NLRP3 inflammasome activation. We separated polar and non-polar fractions of P. ginseng and tested modulation of NLRP3 inflammasome, and then identified pure component for inflammasome inhibitor which ameliorates diet-induced NASH. Non-polar P. ginseng fractions obtained from ethyl acetate solvent attenuated IL-1β secretion and expression of active caspase-1. We revealed that panaxydol (PND) is pure component to inhibit NLRP3 inflammasome activation. PND blocked inflammasome cytokines release, pyroptotic cell death, caspase-1 activation and specking of inflammasome complex. Inhibitory effect of PND was specific to NLRP3-dependent pathway via potential interaction with ATP binding motif of NLRP3. Moreover, in vivo studies showed that PND plays beneficial roles to reduce tissue inflammations through disruption of NLRP3 inflammasome and to ameliorate the development of NASH. These results provide new insight of natural products, panaxydol, for NLRP3 inflammasome inhibitor and could offer potential therapeutic candidate for reliving NASH.

Apolipoprotein C3 (ApoC3) facilitates NLRP3 mediated pyroptosis of macrophages through mitochondrial damage by accelerating of the interaction between SCIMP and SYK pathway in acute lung injury

Respiratory failure caused by severe acute lung injury (ALI) is the main cause of mortality in patients with COVID-19.This study aimed to investigate the effects and underlying biological mechanism of Apolipoprotein C3 (ApoC3) in ALI.To establish an in vivo model, C57BL/6 mice were exposed by lipopolysaccharide (LPS). For the in vitro model, murine bone marrow-derived macrophages (BMDMs) or RAW264.7 cells were stimulated with LPS + adenosine triphosphate (ATP).Serum levels of ApoC3 were found to be upregulated in patients with COVID-19 or pneumonia-induced ALI. Inhibition of ApoC3 reduced lung injury in an ALI model, while overexpression of ApoC3 promoted lung injury. ApoC3 induced mitochondrial damage-mediated pyroptosis in ALI through the activation of the NOD-like receptorprotein 3 (NLRP3) inflammasome. ApoC3 recombinant protein significantly increased SCIMP expression in the lung tissue of mice models with ALI. ApoC3 also facilitated the interaction between the SLP adapter and CSK-interacting membrane protein (SCIMP) protein and Spleen tyrosine kinase (SYK) protein in the ALI model. Moreover, ApoC3 accelerated calcium-dependent reactive oxygen species (ROS) production in the ALI model. The effects of ApoC3 on pyroptosis were mitigated by the use of a pyroptosis inhibitor or an ROS inhibitor in the ALI model. Furthermore, ApoC3 activated the expression of SYK, which in turn induced NLRP3 inflammasome-regulated pyroptosis in the ALI model. METTL3 was found to mediate the m6A mRNA expression of ApoC3.Overall, our study highlights the crucial role of ApoC3 in promoting macrophage pyroptosis in ALI through calcium-dependent ROS production and NLRP3 inflammasome activation via the SCIMP-SYK pathway, providing a potential therapeutic strategy for ALI and other inflammatory diseases.

NLRP3 inflammasome mediates abnormal epithelial regeneration and distal lung remodeling in silica‑induced lung fibrosis.

NOD-like receptor protein 3 (NLRP3) inflammasome is closely related to silica particle‑induced chronic lung inflammation but its role in epithelial remodeling, repair and regeneration in the distal lung during development of silicosis remains to be elucidated. The present study aimed to determine the effects of the NLRP3 inflammasome on epithelial remodeling and cellular regeneration and potential mechanisms in the distal lung of silica‑treated mice at three time points. Pulmonary function assessment, inflammatory cell counting, enzyme‑linked immunosorbent assay, histological and immunological analyses, hydroxyproline assay and western blotting were used in the study. Single intratracheal instillation of a silica suspension caused sustained NLRP3 inflammasome activation in the distal lung. Moreover, a time‑dependent increase in airway resistance and a decrease in lung compliance accompanied progression of pulmonary fibrosis. In the terminal bronchiole, lung remodeling including pyroptosis (membrane‑distributed GSDMD+), excessive proliferation (Ki67+), mucus overproduction (mucin 5 subtype AC and B) and epithelial‑mesenchymal transition (decreased E‑Cadherin+ and increased Vimentin+), was observed by immunofluorescence analysis. Notably, aberrant spatiotemporal expression of the embryonic lung stem/progenitor cell markers SOX2 and SOX9 and ectopic distribution of bronchioalveolar stem cells were observed in the distal lung only on the 7th day after silica instillation (the early inflammatory phase of silicosis). Western blotting revealed that the Sonic hedgehog/Glioma‑associated oncogene (Shh/Gli) and Wnt/β‑catenin pathways were involved in NLRP3 inflammasome activation‑mediated epithelial remodeling and dysregulated regeneration during the inflammatory and fibrotic phases. Overall, sustained NLRP3 inflammasome activation led to epithelial remodeling in the distal lung of mice. Moreover, understanding the spatiotemporal profile of dysregulated epithelial repair and regeneration may provide a novel therapeutic strategy for inhalable particle‑related chronic inflammatory and fibrotic lung disease.

Histone deacetylase 3-specific inhibitor RGFP966 attenuates oxidative stress and inflammation after traumatic brain injury by activating the Nrf2 pathway.

Oxidative stress (OS) and inflammatory reactions play pivotal roles in secondary brain injury after traumatic brain injury (TBI). Histone deacetylase 3 (HDAC3) controls the acetylation of histones and non-histones, which has a significant impact on the central nervous system's reaction to damage. This research determined the implications of RGFP966, a new and specific inhibitor of HDAC3, for the antioxidant (AO) systems mediated by nuclear factor erythroid2-related factor 2 (Nrf2) and the Nod-like receptor protein 3 (NLRP3) inflammasome in TBI. The study also studied the underlying mechanisms of RGFP966's actions. Our objective was to examine the impacts and underlying RGFP966 mechanisms in TBI. In vitro, a rat cortical neuron OS model was induced by H2O2, followed by the addition of RGFP966 to the culture medium. Neurons were collected after 24h for western blot (WB), terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and 2'-7'-dichlorodihydrofluorescein diacetate staining. In vivo, RGFP966 (10mg/kg) was administered post-TBI. Brain tissue water content and modified neurological severity scores were assessed 72h post-injury. Cortical tissues surrounding the focal injury were subjected to western blot, TUNEL staining, Nissl staining and immunofluorescence/immunohistochemistry staining, and malondialdehyde level, hindered glutathione content and superoxide dismutase activity were measured. Serum was collected for the enzyme-linked immunosorbent assay. Nrf2-specific shRNA lentivirus was injected into the lateral ventricle of rats for 7days, and cerebral cortex tissue was analyzed by WB and real-time polymerase chain reaction. During in vitro and in vivo experiments, RGFP966 suppressed HDAC3 expression, promoted Nrf2 nuclear translocation, activated downstream AO enzymes, mitigated excessive reactive oxygen species production and alleviated nerve cell apoptosis. RGFP966 effectively reduced brain edema and histological damage and enhanced neurological and cognitive function in rats with TBI. RGFP966 markedly inhibited NLRP3 inflammasome activation mediated by high-mobility group box 1 (HMGB1)/toll-like receptor 4 (TLR4). Nrf2 knockdown in TBI rats attenuated the AO and anti-inflammatory, neuroprotective impacts of RGFP966. Overall, our findings demonstrate that RGFP966 can mitigate the first brain damage and neurological impairments in TBI. The underlying mechanism involves triggering the Nrf2-mediated AO system and negatively regulating the HMGB1/TLR4-mediated NLRP3 inflammasome pathway.

25-hydroxycholesterol aggravates oxygen-glucose deprivation/reoxygenation-induced pyroptosis through promoting activation of NLRP3 inflammasome in H9C2 cardiomyocytes.

25-hydroxycholesterol (25-HC) plays a role in the regulation of cell survival and immunity. However, the effect of 25-HC on myocardial ischemia/reperfusion (MI/R) injury remains unknown. Our present study aimed to investigate whether 25-HC aggravated MI/R injury through NLRP3 inflammasome-mediated pyroptosis. The overlapping differentially expressed genes (DEGs) in MI/R were identified from the GSE775, GSE45818, GSE58486, and GSE46395 datasets in Gene Expression Omnibus (GEO) database. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were conducted using the database of Annotation, Visualization and Integration Discovery (DAVID). The protein-protein interaction (PPI) network of the overlapping DEGs was established using the Search Tool for the Retrieval of Interacting Genes (STRING) database. These bioinformatics analyses indicated that cholesterol 25-hydroxylase (CH25H) was one of the crucial genes in MI/R injury. The oxygen-glucose deprivation/reoxygenation (OGD/R) cell model was established to simulate MI/R injury. Western blot and RT-qPCR analysis demonstrated that CH25H was significantly upregulated in OGD/R-stimulated H9C2 cardiomyocytes. Moreover, knockdown of CH25H inhibited the OGD/R-induced pyroptosis and nod-like receptor protein 3 (NLRP3) inflammasome activation, as demonstrated by cell counting kit-8 (CCK8), lactate dehydrogenase (LDH), RT-qPCR, and western blotting assays. Conversely, 25-HC, which is synthesized by CH25H, promoted activation of NLRP3 inflammasome in OGD/R-stimulated H9C2 cardiomyocytes. In addition, the NLRP3 inhibitor BAY11-7082 attenuated 25-HC-induced H9C2 cell injury and pyroptosis under OGD/R condition. In conclusion, 25-HC could aggravate OGD/R-induced pyroptosis through promoting activation of NLRP3 inflammasome in H9C2 cells.

Arsenic Exposure Induces Neuro-immune Toxicity in the Cerebral Cortex and the Hippocampus via Neuroglia and NLRP3 Inflammasome Activation in C57BL/6 Mice.

This study aimed to examine the immuntoxic effects of arsenic in the nervous system. Our results showed that arsenic increased corticocerebral and hippocampal weights (p < 0.05). Morris water maze tests revealed that arsenic significantly increased the time spent in latency to platform on the fourth day in 50mg/L arsenic exposure and the fifth day in 25 and 50mg/L arsenic exposure, as well as reduced the path length in target quadrant, time spent in target quadrant, and crossing times of the platform (p < 0.05). Hematoxylin-eosin staining showed that the vacuolated degeneration and pyknosis was found in the cerebral cortex and hippocampus of arsenic-treated mice. The mRNA levels of corticocerebral and hippocampal brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF) were decreased in the 50mg/L arsenic-treated group (p < 0.05). In addition, immunofluorescence staining showed that 25 and 50mg/L arsenic all increased the expression of CD11b and glial fibrillary acidic protein (GFAP) in the cerebral cortex and hippocampus (p < 0.05). Arsenic markedly raised antigen-presenting molecule MHCII and CD40 mRNA levels in the cerebral cortex and hippocampus and upregulated the cell chemokine receptor 5 (CCR5) and CCR7 mRNA levels in the cerebral cortex at the 50mg/L arsenic group, and increased the CCR7 mRNA levels in the hippocampus at the 25 and 50mg/L arsenic groups (p < 0.05). Arsenic activated the nucleotide-binding domain-like receptor protein-3 (NLRP3) inflammasome, and enhanced its upstream promoter NF-κB protein level and downstream regulators IL-18 mRNA levels. Collectively, these results provide new evidences for the neuro-immune toxicity of arsenic.

Anesthesia/surgery-induced learning and memory dysfunction by inhibiting mitophagy-mediated NLRP3 inflammasome inactivation in aged mice

Postoperative cognitive dysfunction (POCD) is a common postoperative complication, not only affects the quality of life of the elderly and increases the mortality rate, but also brings a greater burden to the family and society. Previous studies demonstrated that Nod-like receptor protein 3 (NLRP3) inflammasome participates in various inflammatory and neurodegenerative diseases. However, possible mitophagy mechanism in anesthesia/surgery-elicited NLRP3 inflammasome activation remains to be elucidated. Hence, this study clarified whether mitophagy dysfunction is related to anesthesia/surgery-elicited NLRP3 inflammasome activation. POCD model was established in aged C57BL/6 J mice by tibial fracture fixation under isoflurane anesthesia. Morris Water Maze (MWM) was used to evaluate learning and memory abilities. We found that in vitro experiments, lipopolysaccharide (LPS) significantly facilitated NLRP3 inflammasome activation and mitophagy inhibition in BV2 cells. Rapamycin restored mitophagy and improved mitochondrial function, and inhibited NLRP3 inflammasome activation induced by LPS. In vivo experiments, anesthesia and surgery caused upregulation of hippocampal NLRP3, caspase recruitment domain (ASC) and interleukin-1β (IL-1 β), and downregulation of microtubule-associated protein light chain 3II (LC3II) and Beclin1 in aged mice. Olaparib inhibited anesthesia/surgery-induced NLRP3, ASC, and IL-1β over-expression in the hippocampus, while upregulated the expression of LC3II and Beclin1. Furthermore, Olaparib improved cognitive impairment in older mice. These results revealed that mitophagy was involved in NLRP3 inflammasome-mediated anesthesia/surgery-induced cognitive deficits in aged mice. Overall, our results suggested that mitophagy was related in NLRP3 inflammasome-induced cognitive deficits after anesthesia and surgery in aged mice. Activating mitophagy may have clinical benefits in the prevention of cognitive impairment induced by anesthesia and surgery in elderly patients.

Inhibition of NLRP3 inflammasome-A potential mechanistic therapeutic for treatment of polycystic ovary syndrome?

This review article explores the relationship between the NOD-like receptor protein 3 (NLRP3) inflammasome and the risk of developing polycystic ovary syndrome (PCOS). The NLRP3 inflammasome, a fundamental element of the innate immune system, plays a crucial role in the production of proinflammatory mediators and pyroptosis, a type inflammatory cell death. We conducted a thorough search on scientific databases to gather relevant information on this topic, utilizing relevant keywords. The reviewed studies indicated a correlation between PCOS and a higher incidence of granulosa cell (GC) death and the presence of ovarian tissue fibrosis. NLRP3 inflammasome stimulation and subsequent pyroptosis in GCs play a significant role in the pathophysiology of PCOS. Active NLRP3 inflammasome is involved in the production of inflammatory mediators like interleukin-1β (IL-1β) and IL-18, contributing to the development of PCOS, particularly in overweight patients. Therefore, inhibiting NLRP3 activation and pyroptosis could potentially offer novel therapeutic strategies for PCOS. Some limited studies have explored the use of agents with antioxidant and anti-inflammatory properties, as well as gene therapy approaches, to target the NLRP3 and pyroptosis signaling pathways. This study overview the understanding of the relationship between NLRP3 inflammasome activation, pyroptosis, and PCOS. It highlights the potential of targeting the NLRP3 inflammasome as an approach for treating PCOS. Nonetheless, further research and clinical trials are imperative to validate these results and explore the effectiveness of NLRP3 inflammasome inhibition in the management of PCOS.

[Guiqi Yiyuan Ointment protects rat left lung from bystander effect of right lung injury induced by ~(12)C~(6+) beam].

This study observed the effects of Guiqi Yiyuan Ointment(GQYY) on the left lung subjecting to bystander effect of right lung injury induced by ~(12)C~(6+) beam in rats and decipher the underlying mechanism from NOD-like receptor protein 3(NLRP3)/apoptosis-associated speck-like protein containing a CARD(ASC)/cysteinyl aspartate specific proteinase-1(caspase-1) pathway. Wistar rats were randomized into 7 groups: blank, model, inhibitor [200 mg·kg~(-1), N-acetylcysteine(NAC)], western drug [140 mg·kg~(-1) amifostine(AMI)], and high-, medium-, and low-dose(4.8, 2.4, and 1.2 g·kg~(-1), respectively) GQYY groups. The model of bystander effect damage was established by 4 Gy ~(12)C~(6+) beam irradiation of the right lung(with the other part shielded by a lead plate). The pathological changes in the lung tissue, the level of reactive oxygen species(ROS) in the lung tissue, and the levels of superoxide dismutase(SOD) and malondialdehyde(MDA) in the serum were observed and measured in each group. Furthermore, the mRNA and protein levels of NLRP3, ASC, caspase-1, and phosphorylated nuclear factor-κB p65(p-NF-κB p65)/nuclear factor-κB p65(NF-κB p65) were determined. Compared with the blank group, the model group showed thickened alveolar wall, narrowed alveolar cavity, and presence of massive red blood cells and inflammatory infiltration in the alveolar wall and alveolar cavity. In addition, the model group showed elevated ROS levels in both left and right lungs, elevated MDA level, lowered SOD level, and up-regulated mRNA and protein levels of NLRP3, ASC, caspase-1, and p-NF-κB p65/NF-κB p65. Compared with the model group, the drug administration in all the groups reduced inflammatory cell infiltration in the lung tissue. The inhibitor group and the western drug group showed enlarged alveolar cavity, thinned interstitium, and reduced inflammation. There was a small amount of alveolar wall rupture in the high-and medium-dose GQYY groups and reduced inflammatory cell infiltration in the low dose GQYY group. Compared with the model group, drug administration lowered level of ROS in the left and right lungs, lowered the MDA level, elevated the SOD level, and down-regulated the mRNA and protein levels of NLRP3, ASC, caspase-1, and p-NF-κB p65/NF-κB p65. GQYY can effectively reduce the damage caused by radiation and bystander effect, which may be associated with the ROS-mediated NLRP3 inflammasome activation.

NLRP3 Inflammasome-Mediated Platelet Activation and Thrombus Formation in Sickle Cell Mice Can be Targeted By the BTK Inhibitor Ibrutinib

Introduction: Platelets are abnormally activated in sickle cell disease (SCD) and are involved in promoting vascular occlusions and thrombosis. The NOD like receptor protein 3 (NLRP3) inflammasome is a critical inflammatory mediator that has recently been found to be upregulated in platelets in SCD. Our recent discovery of Bruton Tyrosine Kinase (BTK) as a regulator of the NLRP3 inflammasome has provided an opportunity to suppress NLRP3 inflammasome activation in platelets via the BTK inhibitor ibrutinib. The role of the NLRP3 inflammasome in regulating platelet activation in SCD and its potential targetability through BTK inhibition is poorly understood. Methods: Townes SCD mice, which express human sickle hemoglobin (HbSS), and control mice (HbAA) at age 16 to 28 weeks were used for this study. Where indicated, mice were injected with the NLRP3 inhibitor MCC950, the BTK inhibitor ibrutinib or vehicle control 4 hours prior to blood collection. NLRP3 inflammasome activation was assessed in isolated platelets with fluorescent labeled inhibitor of caspase-1 (FLICA) assay and immunofluorescence staining of intraplatelet NLRP3 and the adaptor apoptosis-associated speck-like protein containing a CARD (ASC) with confocal microscopy. We measured platelet surface expression of P-selectin (CD62P), platelet ATP secretion, platelet spreading, platelet aggregation, and in vitro thrombus formation using flow cytometry, luminescence, scanning electron microscopy, whole blood impedance aggregometry, and a flow chamber with blood perfused at shear rates of 1,700 s -1, respectively. Isolated platelets or blood samples were treated with the NLRP3 activator nigericin where indicated. Collagen was used as platelet agonist. Data are shown as mean ± SD. Results: We found increased platelet P-selectin expression (CD62P%: 74.57 ± 3.03 vs 35.18 ± 0.73, p&amp;lt;0.01), platelet ATP secretion (AUC: 16.24 ± 2.17 vs 3.16 ± 0.94, p&amp;lt;0.01), platelet spreading (spreading area, µm 2: 5.47 ± 1.97 vs 2.35 ± 0.23, p&amp;lt;0.01), platelet aggregation (AUC: 68.18 ± 6.84 vs 36.28 ± 3.29, p&amp;lt;0.01), and thrombus formation (thrombus area %: 64.24 ± 14.64 vs 27.44 ± 5.99, p&amp;lt;0.05) in blood from SCD mice as compared to control mice. NLRP3:ASC speck formation (Pearson coefficient: 0.62 ± 0.03 vs 0.38 ± 0.02, p&amp;lt;0.001) and caspase-1 activation levels (RFUs: 476.6 ± 54.84 vs 237.8 ± 50.09, p&amp;lt;0.001) were elevated in platelets from SCD mice as compared to control mice, indicating platelet NLRP3 inflammasome activation in SCD mice. Next, we investigated the role of the NLRP3 inflammasome in regulating platelet function in SCD mice. Treatment of mice with the NLRP3 inhibitor MCC950 resulted in a reduction of upregulated platelet P-selectin expression (CD62P%: 33.55 ± 17.21 vs 74.23 ± 9.95, p&amp;lt;0.001), platelet ATP secretion (AUC: 5.98 ± 1.2 vs 13 ± 2.91, p&amp;lt;0.001), platelet spreading (spreading area, µm 2: 2.42 ± 0.75 vs 5.47 ± 1.97, p&amp;lt;0.001), platelet aggregation (AUC: 39.85 ± 2.5 vs 64.65 ± 6.5, p&amp;lt;0.001), and thrombus formation (thrombus area %: 41.63 ± 7.78 vs 70.22 ± 5.5, p&amp;lt;0.001) (MCC950 vs vehicle) in SCD mice but had no effect in control mice. We next investigated the effect of the BTK inhibitor ibrutinib. Injection of ibrutinib interfered with elevated platelet NLRP3 inflammasome activation in SCD mice, as shown by quantification of intraplatelet NLRP3:ASC specks (Pearson coefficient: 0.56 ± 0.04 vs 0.72 ± 0.03, p&amp;lt;0.001) and platelet caspase-1 activity (RFUs: 193 ± 55.24 vs 386.8 ± 88.08, p&amp;lt;0.001) (ibrutinib vs vehicle) but had no effect in control mice. Ibrutinib treatment decreased upregulated platelet P-selectin (CD62P%: 37.81 ± 10.65 vs 64.74 ± 17.73, p&amp;lt;0.01), platelet ATP secretion (AUC: 6.78 ± 0.24 vs 13.9 ± 1.12, p&amp;lt;0.001), platelet spreading (spreading area, µm 2: 2.32 ± 0.79 vs 6.45 ± 0.99, p&amp;lt;0.001), platelet aggregation (AUC: 40.08 ± 3.56 vs 62.13 ± 3.99, p&amp;lt;0.001), and thrombus formation (thrombus area, %: 44.42 ± 8.24 vs 66.97 ± 11.83, p&amp;lt;0.001) (ibrutinib vs vehicle) in SCD mice. The NLRP3 activator nigericin reversed these inhibitory effects of ibrutinib in SCD mice, indicating that ibrutinib interferes with platelet activation and thrombus formation in SCD mice through the NLRP3 inflammasome. Conclusion: The NLRP3 inflammasome is a critical mediator of platelet activation and thrombus formation in SCD mice, which is targetable by the BTK inhibitor ibrutinib, making it a potentially useful therapeutic approach for managing the prothrombotic state in SCD.

RhoGDI3 at the trans-Golgi network participates in NLRP3 inflammasome activation, VSMC phenotypic modulation, and neointima formation

Background and aimsThe pathological roles and mechanisms of Rho-specific guanine nucleotide dissociation inhibitor 3 (RhoGDI3) in vascular smooth muscle cell (VSMC) phenotypic modulation and neointima formation are currently unknown. This study aimed to investigate how RhoGDI3 regulates the Nod-like receptor protein 3 (NLRP3) inflammasome in platelet-derived growth factor-BB (PDGF-BB)-induced neointima formation. MethodsFor in vitro assays, human aortic VSMCs (HA-VSMCs) were transfected with pcDNA3.1-GDI3 and RhoGDI3 siRNA to overexpress and knockdown RhoGDI3, respectively. HA-VSMCs were also treated with an NLRP3 inhibitor (CY-09) or agonist (NSS). Protein transcription and expression, cell proliferation and migration, Golgi morphology, and protein binding and colocalization were measured. For the in vivo assays, balloon injury (BI) rats were injected with recombinant adenovirus carrying RhoGDI3 shRNA. Carotid arterial morphology, protein expression and colocalization, and activation of the NLRP3 inflammasome were measured. ResultsPDGF-BB treatment induced transcription and expression of RhoGDI3 through PDGF receptor αβ (PDGFRαβ) rather than PDGFRαα or PDGFRββ in HA-VSMCs. RhoGDI3 suppression blocked PDGF-BB-induced VSMC phenotypic transformation. In contrast, RhoGDI3 overexpression further promoted PDGF-BB-induced VSMC dedifferentiation. The in vivo results also confirmed that RhoGDI3 expressed in VSMCs participated in neointima formation and muscle fiber and collagen deposition caused by balloon injury. In addition, PDGF-BB increased binding of RhoGDI3 to NLRP3 and apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) at the trans-Golgi membrane, which depended on the normal Golgi network. However, recruitment of NLRP3 and ASC to the trans-Golgi network after PDGF-BB treatment was independent of RhoGDI3. Moreover, RhoGDI3 knockdown significantly inhibited ASC expression and NLRP3 inflammasome assembly and activation and reduced NLRP3 protein stability in PDGF-BB-treated HA-VSMCs. Inhibiting NLRP3 effectively prevented PDGF-BB-induced VSMC phenotypic modulation, and an NLRP3 agonist reversed the decline in VSMC phenotypic transformation caused by RhoGDI3 knockdown. Furthermore, RhoGDI3 suppression reduced the protein levels and assembly of NLRP3 and ASC, and the activation of the NLRP3 inflammasome in VSMCs in a rat balloon injury model. ConclusionsThe results of this study reveal a novel mechanism through which RhoGDI3 regulates VSMC phenotypic modulation and neointima formation by activating the NLRP3 inflammasome.