Nod-like Receptor Protein Research Articles

RRx-001 Affects Multiple Toxicities in Patients Receiving Concomitant Chemoradiation for Head and Neck Cancers: Observations of the PREVLAR Phase II Trial

PURPOSE Phase IIa trial results suggested that RRx-001, an nucleotide-binding domain-like receptor protein 3 inhibitor and nuclear factor erythroid 2–related factor 2 activator, attenuated severe oral mucositis (SOM) in patients treated with concomitant chemoradiation (CRT) for oral cancer (OC) and oropharyngeal cancer (OPC). Given the shared pathobiology of CRT injury among other exposed tissues, we initiated a secondary analysis of reported adverse events (AEs) to assess RRx-001's potential benefit on non-SOM regimen-related toxicities. METHODS PREVLAR was an open-label randomized trial that has been completed and published. Patients were treated with CRT for locally advanced OC or OPC. Arms 1-3 received doses of RRx-001 twice a week beginning 2 weeks before the start of CRT. Arm 2 (n = 11) received additional RRx-001 once during weeks 2 and 5. Arm 3 (n = 13) received RRx-001 once a week for the first 6 weeks of CRT. Arm 4 (n = 10) received CRT only. Patients received standard intensity modulated radiation therapy (daily fractions of 2-2.2 Gy/minimum cumulative dose of 55 Gy) plus cisplatin. Comprehensive toxicity assessment was based on reported AEs graded using Common Terminology Criteria for Adverse Events v5.0 and categorized with the Medical Dictionary for Regulatory Activities. A two-sided Fisher's exact test was performed to compare AE incidences between RRx-001 arms and standard of care in a secondary analysis of the data. Overall false-positive rate in Fisher's exact test was controlled via Hochberg's adjustment method. RESULTS Compared with the control cohort, patients receiving RRx-001 demonstrated fewer AEs attributable to their CRT regimen. Statistically significant differences in AE incidences favoring RRx-001 were dependent on the RRx-001 dosing regimen. CONCLUSION This secondary analysis suggests that RRx-001 infusion may reduce multiple, biologically related AEs associated with a standard CRT regimen. More broadly, it suggests that it is possible that agents that effectively target central pathobiology targets may mitigate more than one toxicity.

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.

Coenzyme Q10 ameliorates lipopolysaccharide-induced acute lung injury by attenuating oxidative stress and NLRP3 inflammation through regulating mitochondrial dynamics

Increasing evidence has demonstrated that coenzyme Q10 (CoQ10) exhibits a range of biological properties. Herein, we explored the protective effect and potential molecular mechanism of CoQ10 on lipopolysaccharide (LPS)-induced acute lung injury (ALI). We found that medium (10 mg/kg) and high (50 mg/kg) doses of CoQ10 ameliorated LPS (50 µg/µL)-induced ALI to varying degrees, as demonstrated by reduced lung coefficient, lower wet/dry weight lung tissue ratio, decreased bronchoalveolar lavage fluid protein concentration, less anatomical and histopathological damage to the lung, and increased expression of proteins related to lung epithelial barrier structure. CoQ10 also alleviated LPS-induced oxidative stress and inflammation mediated by NOD-like receptor protein 3 (NLRP3) by reducing the reactive oxygen species (ROS), malondialdehyde, and mitochondrial ROS concentrations, increasing superoxide dismutase, glutathione, and catalase activity, and decreasing NLRP3 expression at the protein and mRNA levels. Moreover, CoQ10 alleviated structural and functional damage to the mitochondria, inhibited mitochondrial fission, and promoted mitochondrial fusion, mainly by inhibiting phosphorylation of dynamin-related protein 1 (Drp1) at Ser616 and Ser637. Correlation analysis revealed that mitochondrial fission (especially Drp1) was positively correlated with oxidative stress, NLRP3-mediated inflammation, and structural damage to the lung epithelial barrier. Molecular docking analysis showed that CoQ10 binds stably to Drp1, with a binding energy of −5.9 kcal/mol. Furthermore, the use of schaftoside (a Drp1 inhibitor) has further elucidated the mechanism of action of CoQ10. Together, these results suggest that CoQ10 alleviates LPS-induced ALI by regulating mitochondrial dynamics, attenuating oxidative stress, and decreasing NLRP3-medated inflammation, thereby promoting lung epithelial barrier structural remodeling.

Sulfiredoxin 1 ameliorates doxorubicin-induced cardiotoxicity by suppressing oxidative stress and inflammation via the Sirt1/NLRP3 pathway

BackgroundDoxorubicin (DOX) is limited in clinical use due to its cardiotoxic side effects. Oxidative stress and inflammation are pivotal mechanisms underlying doxorubicin-induced cardiotoxicity (DIC). Sulfiredoxin 1 (Srxn1) plays a central role in antioxidant effects. However, the role of Srxn1 in DIC has not yet been fully elucidated. This study aims to explore the effects and underlying mechanisms of Srxn1 on DIC. MethodsWe overexpressed Srxn1 in the myocardium using an adeno-associated virus 9 (AAV9) system, delivered through tail vein injection. C57BL/6 mice received intraperitoneal injections of DOX (4 mg/kg) weekly for four consecutive weeks to establish a mouse model of DIC. We used echocardiography, histopathological, and molecular techniques to elucidate the effects and mechanisms. ResultsOur findings demonstrate that overexpression of Srxn1 significantly enhanced cardiac function and mitigated myocardial injury in mice exposed to DOX. Overexpressing Srxn1 attenuated oxidative stress and inflammation induced by DOX. Furthermore, Srxn1 overexpression led to upregulation of sirtuin 1 (Sirt1) expression and inhibited the activation of the NOD-like receptor protein 3 (NLRP3) inflammasome. Notably, the protective effects of Srxn1 were significantly abrogated by the Sirt1 inhibitor EX527. ConclusionThe protective effects of Srxn1 against DOX-induced cardiac oxidative stress and inflammation operate by targeting the Sirt1/NLRP3 signaling pathway to alleviate DIC. Srxn1 could be a potential candidate for the treatment of DOX-induced myocardial injury.

The phosphorylation of Smad3 by CaMKIIγ leads to the hepatocyte pyroptosis under perfluorooctane sulfonate exposure

Perfluorooctane sulfonate (PFOS) is a persistent organic pollutant and accumulated in the liver of mammals. PFOS exposure is closely associated with the development of pyroptosis. Nevertheless, the underlying mechanism is unclear. We found here that PFOS induced pyroptosis in the mice liver and L-02 cells as demonstrated by activation of the NOD-like receptor protein 3 inflammasome, gasdermin D cleavage and increased release of interleukin-1β and interleukin-18. The level of cytoplasmic calcium was accelerated in hepatocytes upon exposure to PFOS. The phosphorylated/activated form of calcium/calmodulin-dependent protein kinase II (CaMKII) was augmented by PFOS in vivo and in vitro. PFOS-induced pyroptosis was relieved by CaMKII inhibitor. Among various CaMKII subtypes, we identified that CaMKIIγ was activated specifically by PFOS. CaMKIIγ interacted with Smad family member 3 (Smad3) under PFOS exposure. PFOS increased the phosphorylation of Smad3, and CaMKII inhibitor or CaMKIIγ siRNA alleviated PFOS-caused phosphorylation of Smad3. Inhibiting Smad3 activity was found to alleviate PFOS-induced hepatocyte pyroptosis. This study puts forward that CaMKIIγ-Smad3 is the linkage between calcium homeostasis disturbance and pyroptosis, providing a mechanistic explanation for PFOS-induced pyroptosis.

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.

LncRNA GHET1 from bone mesenchymal stem cell–derived exosomes improves doxorubicin-induced pyroptosis of cardiomyocytes by mediating NLRP3

Doxorubicin (DOX) is an important chemotherapeutic agent for the treatment of hematologic tumors and breast carcinoma. However, its clinical application is limited owing to severe cardiotoxicity. Pyroptosis is a form of programmed cell death linked to DOX-induced cardiotoxicity. Bone mesenchymal stem cell–derived exosomes (BMSC-Exos) and endothelial progenitor cells-derived exosomes (EPC-Exos) have a protective role in the myocardium. Here we found that BMSC-Exos could improve DOX-induced cardiotoxicity by inhibiting pyroptosis, but EPC-Exos couldn’t. Compared with EPCs-Exo, BMSC-Exo-overexpressing lncRNA GHET1 more effectively suppressed pyroptosis, protecting against DOX-induced cardiotoxicity. Further studies showed that lncRNA GHET1 effectively decreased the expression of Nod-like receptor protein 3 (NLRP3), which plays a vital role in pyroptosis by binding to IGF2 mRNA-binding protein 1 (IGF2BP1), a non-catalytic posttranscriptional enhancer of NLRP3 mRNA. In summary, lncRNA GHET1 released by BMSC-Exo ameliorated DOX-induced pyroptosis by targeting IGF2BP1 to reduce posttranscriptional stabilization of NLRP3.

Unravelling the Significance of NLRP3 and IL-β1 in Oral Squamous Cell Carcinoma and Potentially Malignant Oral Disorders: A Diagnostic and Prognostic Exploration.

Nucleotide-binding domain-like receptor protein 3 (NLRP3), an inflammasome, is reported to be dysregulated or aberrantly expressed in chronic inflammation, leading to a myriad of inflammatory disorders, autoimmune diseases, and cancer. This study aimed to explore the expression and role of NLRP3 protein and the secreted cytokine IL-β1 in oral squamous cell carcinoma (OSCC) and potentially malignant oral disorders (PMOD). Tissue NLRP3 expression was quantified using sandwich ELISA in 30 cases each of OSCC, PMOD, and normal oral mucosa. Serum IL-β1 level was also measured by ELISA to determine their correlation. In surgically treated OSCC cases, pathological parameters such as tumor size, depth of invasion (DOI), pTNM stage, and perineural & lymphovascular invasion were assessed and correlated with NLRP3 & IL-β1 levels to investigate their roles in tumor progression, invasion, and metastasis. Tissue NLRP3 expression was markedly elevated in OSCC, with significant IL-β1 levels observed in the serum of both OSCC and PMOD cases. Both markers showed a pronounced increase with the severity of dysplasia, indicating a strong association (p = 0.003%). The expression levels of tissue NLRP3 and serum IL-β1 were positively correlated with DOI and tumor size. Furthermore, their elevated levels, alongside higher histological grades, indicate roles in the dedifferentiation and progression of tumor cells. The findings indicated that increased expression of NLRP3 and IL-β1 in PMOD correlates with higher transformation rates, along with tumor progression and dedifferentiation in OSCC. Consequently, these markers hold promise as valuable targets for prognostic assessment, diagnostics, and therapeutic strategies in OSCC.

CD8+ T-cell deficiency protects mice from abdominal aortic aneurysm formation in response to calcium chloride2.

Abdominal aortic aneurysm (AAA) is an aneurysm-like dilated and highly fatal cardiovascular disease. CD8+ T cells have been shown to be critical for vascular pathological processes, but the contribution of these lymphocytes to vascular diseases remains elusive. Eight-week-old male wildtype (CD8+/+) and Cd8a knockout (CD8-/-) mice were used in a calcium chloride2 (CaCl2)-induced experimental AAA model. At 6 weeks after surgery, CD8+ T-cell deletion prevented the formation of AAA, accompanied by reductions of the levels of inflammatory (interferon-γ [IFN-γ], interleukin-1β, monocyte chemoattractant protein-1, intracellular adhesion molecule-1, vascular cell adhesion molecule-1, NOD-like receptor protein 3, caspase-1), oxidative stress [NADPH oxidase and gp91phox], and proteolysis (cathepsin S, cathepsin K, matrix metalloproteinase-2 [MMP-2] and MMP-9) proteins and/or genes in plasma and/or AAA tissues. Immunoreactivities of MMP-2 and MMP-9 were observed in macrophages. An injection of IFN-γ and adoptive transfer of CD8+ T cells of IFN-γ+/+ mice diminished CD8-/--mediated vasculoprotective actions in the AAA mice. In vitro, IFN-γ enhanced MMP-2 and MMP-9 gelatinolytic activities in macrophage and/or vascular smooth muscle cells. The vasculoprotective effects of CD8+ T-cell deletion in a mouse CaCl2-induced AAA model were likely attributable to, at least in part, the attenuation of IFN-γ-dependent inflammation action, oxidative stress production, and proteolysis, suggesting a novel therapeutic target for AAA formation by regulating CD8+ T-cell-derived IFN-γ secretion.

Osteoarthritis rat serum-derived extracellular vesicles aggravate osteoarthritis development by inducing NLRP3-mediated pyroptotic cell death and cellular inflammation.

Osteoarthritis (OA), degenerative joint disease, is the most prevalent form of arthritis worldwide. Besides its substantial burden on society, the high OA morbidity greatly diminishes patients' quality of life. According to recent research, patients-derived serum extracellular vesicles (EVs) are critically involved in sustaining the corresponding disease progression. However, limited research has fully explored the specific functions and molecular mechanisms of OA serum-derived EVs in disease progression. Consequently, we aimed to investigate the underlying mechanism of OA rats-derived serum EVs in regulating OA progression. Before constructing the exosome-cell co-culture system, EVs were extracted from OA and control rat serum and co-cultured with bone marrow mesenchymal stem cells (BM-MSCs). Western blotting (WB), RT-qPCR, and enzyme-linked immunosorbent assay (ELISA) results revealed that OA rat serum-derived EVs upregulated cell pyroptosis-related markers, including nod-Like receptor protein-3 (NLRP3), apoptosis-associated speck-like protein (ASC), gasdermin D (GSDMD), and cleaved caspase-1. The OA rat-EVs also induced the release of LDH and inflammatory cytokines, including interleukin (IL)-1β, IL-18, IL-6, and TNF-α. Additional experiments revealed that OA rat-EVs delivered miR-133a-3p to BM-MSCs and upregulated miR-133a-3p to degrade sirtuin 1 (SIRT1), and activating the downstream NF-κB signaling pathway. Furthermore, the rescuing experiments confirmed that silencing SIRT1 abrogated the miR-133a-3p-induced protective effects in OA-EVs-treated BM-MSCs. In conclusion, OA rats-derived miR-133a-3p-containing EVs modulated the downstream SIRT1/NF-κB pathway-mediated pyroptotic cell death and inflammation in OA. In other words, this study confirmed the role and underlying mechanisms by which OA-associated serum EVs regulate pyroptosis and inflammation response in OA development.

Based on SIRT1/NF-κB/NLRP3 Signal Pathway to Explore the Effect of Yiqi Huatan Huoxue Recipe on Inflammatory Injury in AIT Mice by Pyroptosis.

Autoimmune thyroiditis (AIT) is the most common autoimmune thyroid disease. In recent decades, its incidence and prevalence have sharply increased. Yiqi Huatan Huoxue recipe is a traditional Chinese medicine formula we use to treat AIT. Its clinical efficacy is clear, but the specific mechanism remains unclear. This study aims to explore whether pyroptosis mediated by the SIRT1/NF-κB/NLRP3 signaling pathway is one of the therapeutic mechanisms of Yiqi Huatan Huoxue recipe. Forty 8-week-old female NOD.H-2h4 mice were randomly divided into four groups: the normal group (NG), model group (MG), Yiqi Huatan Huoxue recipe group (YG), and western medicine group (selenium yeast tablet, SeG). The normal group was gavaged with distilled water, while the remaining groups were gavaged with 0.05% sodium iodide (NaI) solution for 8 weeks. After the AIT animal model formed naturally, the mice were euthanized by gavage after 8 weeks. Hematoxylin-eosin staining was used to observe thyroid tissue changes, and enzymelinked immunosorbent assay (ELISA) was used to detect serum anti-thyroglobulin antibodies (TGAb) and mouse anti-thyroid peroxidase antibodies (TPOAb). Real-time quantitative PCR (qRT-PCR), Western blot, and immunohistochemistry were used to detect the expression of sirtuin 1 (SIRT1), nuclear factor κB p65 (NF-κB p65), nod-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), caspase- 1, gasdermin D (GSDMD), and interleukin (IL)-1β in thyroid tissue. Compared with the NG group, the thyroid structure of rats in the MG group was severely damaged, with significant lymphocyte infiltration, significantly increased serum TGAb and TPOAb levels, and significantly increased expression levels of SRIT1, NF-κB p65, NLRP3, ASC, Caspase-1, GSDMD, IL-1β mRNA, and protein. Compared with the MG group, the thyroid structure damage and lymphocyte infiltration in rats of each treatment group were improved, and the serum TGAb, TPOAb, SRIT1, NF-κB p65, NLRP3, ASC, Caspase-1, GSDMD, IL-1β mRNA, and protein expression levels were significantly reduced. Yiqi Huatan Huoxue recipe can alleviate thyroid structural damage in AIT mice, and its mechanism may be related to the upregulation of SIRT1, NF-κB deacetylation, and inhibition of NLRP3-mediated pyroptosis.

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.

Activation of pregnane X receptor protects against cholestatic liver injury by inhibiting hepatocyte pyroptosis.

Our previous study shows that activation of pregnane X receptor (PXR) exerts hepatoprotection against lithocholic acid (LCA)-induced cholestatic liver injury. In this study we investigated whether PXR activation could inhibit hepatocyte pyroptosis, as well as the underlying mechanisms. Male mice were treated with mouse PXR agonist pregnenolone 16α-carbonitrile (PCN, 50 mg·kg-1·d-1, i.p.) for 7 days, and received LCA (125 mg/kg, i.p., bid) from D4, then sacrificed 12 h after the last LCA injection. We showed that LCA injection resulted in severe cholestatic liver injury characterized by significant increases in gallbladder size, hepatocellular necrosis, and neutrophil infiltration with a mortality rate of 68%; PCN treatment significantly inhibited hepatocyte pyroptosis during LCA-induced cholestatic liver injury, as evidenced by reduced serum lactic dehydrogenase (LDH) levels, TUNEL-positive cells and hepatocyte membrane damage. Furthermore, PXR activation suppressed both the NOD-like receptor protein 3 (NLRP3) inflammasome-induced canonical pyroptosis and the apoptosis protease activating factor-1 (APAF-1) pyroptosome-induced non-canonical pyroptosis. Inhibition of the nuclear factor kappa B (NF-κB) and forkhead box O1 (FOXO1) signaling pathways was also observed following PXR activation. Notably, dual luciferase reporter assay showed that PXR activation inhibited the transcriptional effects of NF-κB on NLRP3, as well as FOXO1 on APAF-1. Our results demonstrate that PXR activation protects against cholestatic liver injury by inhibiting the canonical pyroptosis through the NF-κB-NLRP3 axis and the non-canonical pyroptosis through the FOXO1-APAF-1 axis, providing new evidence for PXR as a prospective anti-cholestatic target.

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.

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.

The Protective Effects of Ruscogenin Against Lipopolysaccharide-Induced Myocardial Injury in Septic Mice.

Sepsis-induced myocardial dysfunction commonly occurs in individuals with sepsis and is a severe complication with high morbidity and mortality rates. This study aimed to investigate the effects and potential mechanisms of the natural steroidal sapogenin ruscogenin (RUS) against lipopolysaccharide (LPS)-induced myocardial injury in septic mice. We found that RUS effectively alleviated myocardial pathological damage, normalized cardiac function, and increased survival in septic mice. RNA sequencing demonstrated that RUS administration significantly inhibited the activation of the NOD-like receptor signaling pathway in the myocardial tissues of septic mice. Subsequent experiments further confirmed that RUS suppressed myocardial inflammation and pyroptosis during sepsis. In addition, cultured HL-1 cardiomyocytes were challenged with LPS, and we observed that RUS could protect these cells against LPS-induced cytotoxicity by suppressing inflammation and pyroptosis. Notably, both the in vivo and in vitro findings indicated that RUS inhibited NOD-like receptor protein-3 (NLRP3) upregulation in cardiomyocytes stimulated with LPS. As expected, knockdown of NLRP3 blocked the LPS-induced activation of inflammation and pyroptosis in HL-1 cells. Furthermore, the cardioprotective effects of RUS on HL-1 cells under LPS stimulation were abolished by the novel NLRP3 agonist BMS-986299. Taken together, our results suggest that RUS can alleviate myocardial injury during sepsis, at least in part by suppressing NLRP3-mediated inflammation and pyroptosis, highlighting the potential of this molecule as a promising candidate for sepsis-induced myocardial dysfunction therapy.

Novel genetic variants in the NLRP3 inflammasome-related PANX1 and APP genes predict survival of patients with hepatitis B virus-related hepatocellular carcinoma.

The nod-like receptor protein 3 (NLRP3) is one of the most characterized inflammasomes involved in the pathogenesis of several cancers, including hepatocellular carcinoma (HCC). However, the effects of genetic variants in the NLRP3 inflammasome-related genes on survival of hepatitis B virus (HBV)-related HCC patients are unclear. We performed multivariable Cox proportional hazards regression analysis to evaluate associations between 299 single-nucleotide polymorphisms (SNPs) in 16 NLRP3 inflammasome-related genes and overall survival (OS) of 866 patients with HBV-related HCC. We further performed expression quantitative trait loci (eQTL) analysis using the data from the GTEx project and 1000 Genomes projects, and performed differential expression analysis using the TCGA dataset to explore possible molecular mechanisms underlying the observed associations. We found that two functional SNPs (PANX1 rs3020013 A > G and APP rs9976425 C > T) were significantly associated with HBV-related HCC OS with the adjusted hazard ratio (HR) of 0.83 [95% confidence interval (CI) = 0.73-0.95, P = 0.008], and 1.26 (95% CI = 1.02-1.55, P = 0.033), respectively. Moreover, the eQTL analysis revealed that the rs3020013 G allele was correlated with decreased mRNA expression levels of PANX1 in both normal liver tissues (P = 0.044) and whole blood (P < 0.001) in the GTEx dataset, and PANX1 mRNA expression levels were significantly higher in HCC samples and associated with a poorer survival of HCC patients. However, we did not observe such correlations for APP rs9976425. These results indicated that SNPs in the NLRP3 inflammasome-related genes may serve as potential biomarkers for HBV-related HCC survival, once replicated by additional larger studies.

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.

Oncoprotein-induced transcript 3 protein-enriched extracellular vesicles promotes NLRP3 ubiquitination to alleviate acute lung injury after cardiac surgery

Acute lung injury (ALI) including acute respiratory distress syndrome (ARDS) is a major complication and increase the mortality of patients with cardiac surgery. We previously found that the protein cargoes enriched in circulating extracellular vesicles (EVs) are closely associated with cardiopulmonary disease. We aimed to evaluate the implication of EVs on cardiac surgery-associated ALI/ARDS. The correlations between “oncoprotein-induced transcript 3 protein (OIT3) positive” circulating EVs and postoperative ARDS were assessed. The effects of OIT3-overexpressed EVs on the cardiopulmonary bypass (CPB) -induced ALI in vivo and inflammation of human bronchial epithelial cells (BEAS-2B) were detected. OIT3 enriched in circulating EVs is reduced after cardiac surgery with CPB, especially with postoperative ARDS. The “OIT3 positive” EVs negatively correlate with lung edema, hypoxemia and CPB time. The OIT3-overexpressed EVs can be absorbed by pulmonary epithelial cells and OIT3 transferred by EVs triggered K48- and K63-linked polyubiquitination to inactivate NOD-like receptor protein 3 (NLRP3) inflammasome, and restrains pro-inflammatory cytokines releasing and immune cells infiltration in lung tissues, contributing to the alleviation of CPB-induced ALI. Overexpression of OIT3 in human bronchial epithelial cells have similar results. OIT3 promotes the E3 ligase Cbl proto-oncogene B associated with NLRP3 to induce the ubiquitination of NLRP3. Immunofluorescence tests reveal that OIT3 is reduced in the generation from the liver sinusoids endothelial cells (LSECs) and secretion in liver-derived EVs after CPB. In conclusion, OIT3 enriched in EVs is a promising biomarker of postoperative ARDS and a therapeutic target for ALI after cardiac surgery.

Oxycodone attenuates lipopolysaccharide-induced myocardial injury by inhibiting inflammation, oxidation and pyroptosis via Nrf2/HO-1 signalling pathway.

Myocardial injury and cardiovascular dysfunction are the most common complications of sepsis, and effective therapeutic candidate is still lacking. This study aims to investigate the protective effect of oxycodone in myocardial injury of lipopolysaccharide-induced sepsis and its related signalling pathways. Wild-type and nuclear factor erythroid 2-related factor 2 (Nrf2)-knockout mice, as well as H9c2 cardiomyocytes cultures treated with lipopolysaccharide (LPS) were used as models of septic myocardial injury. H9c2 cardiomyocytes culture showed that oxycodone protected cells from pyroptosis induced by LPS. Mice model confirmed that oxycodone pretreatment significantly attenuated myocardial pathological damage and improved cardiac function demonstrated by increased ejection fraction (EF) and fractional shortening (FS), as well as decreased cardiac troponin I (cTnI) and creatine kinase isoenzymes MB (CK-MB). Oxycodone also reduced the levels of inflammatory factors and oxidative stress damage induced by LPS, which involves pyroptosis-related proteins including: Nod-like receptor protein 3 (NLRP3), Caspase 1, Apoptosis-associated speck-like protein contain a CARD (ASC), and Gasdermin D (GSDMD). These changes were mediated by Nrf2 and heme oxygenase-1 (HO-1) because Nrf2-knockout mice or Nrf2 knockdown in H9c2 cells significantly reversed the beneficial effect of oxycodone on oxidative stress, inflammatory responses and NLRP3-mediated pyroptosis. Our findings yielded that oxycodone therapy reduces LPS-induced myocardial injury by suppressing NLRP3-mediated pyroptosis via the Nrf2/HO-1 signalling pathway in vivo and in vitro.