NOD-like Receptor Pyrin Domain Containing 3 Research Articles

Corilagin alleviates atherosclerosis by inhibiting NLRP3 inflammasome activation via the Olfr2 signaling pathway in vitro and in vivo

IntroductionAtherosclerosis, a leading cause of global cardiovascular mortality, is characterized by chronic inflammation. Central to this process is the NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasome, which significantly influences atherosclerotic progression. Recent research has identified that the olfactory receptor 2 (Olfr2) in vascular macrophages is instrumental in driving atherosclerosis through NLRP3- dependent IL-1 production.MethodsTo investigate the effects of Corilagin, noted for its anti-inflammatory attributes, on atherosclerotic development and the Olfr2 signaling pathway, our study employed an atherosclerosis model in ApoE−/− mice, fed a high-fat, high-cholesterol diet, alongside cellular models in Ana-1 cells and mouse bone marrow-derived macrophages, stimulated with lipopolysaccharides and oxidized low-density lipoprotein.ResultsThe vivo and vitro experiments indicated that Corilagin could effectively reduce serum lipid levels, alleviate aortic pathological changes, and decrease intimal lipid deposition. Additionally, as results showed, Corilagin was able to cut down expressions of molecules associated with the Olfr2 signaling pathway.DiscussionOur findings indicated that Corilagin effectively inhibited NLRP3 inflammasome activation, consequently diminishing inflammation, macrophage polarization, and pyroptosis in the mouse aorta and cellular models via the Olfr2 pathway. This suggests a novel therapeutic mechanism of Corilagin in the treatment of atherosclerosis.

Modulation of NLRP3 inflammasome-related-inflammation via RIPK1/RIPK3-DRP1 or HIF-1α signaling by phenothiazine in hypothermic and normothermic neuroprotection after acute ischemic stroke

BackgroundInflammation and subsequent mitochondrial dysfunction and cell death worsen outcomes after revascularization in ischemic stroke. Receptor-interacting protein kinase 1 (RIPK1) activated dynamin-related protein 1 (DRP1) in a NLRPyrin domain containing 3 (NLRP3) inflammasome-dependent fashion and Hypoxia-Inducible Factor (HIF)-1α play key roles in the process. This study determined how phenothiazine drugs (chlorpromazine and promethazine (C + P)) with the hypothermic and normothermic modality impacts the RIPK1/RIPK3-DRP1 and HIF-1α pathways in providing neuroprotection. MethodsA total of 150 adult male Sprague-Dawley rats were subjected to 2 h middle cerebral artery occlusion (MCAO) followed by 24 h reperfusion. 8 mg/kg of C + P was administered at onset of reperfusion. Infarct volumes, mRNA and protein expressions of HIF-1α, RIPK1, RIPK3, DRP-1, NLRP3-inflammation and cytochrome c-apoptosis were assessed. Apoptotic cell death, infiltration of neutrophils and macrophages, and mitochondrial function were evaluated. Interaction between RIPK1/RIPK3 and HIF-1α/NLRP3 were determined. In SH-SY5Y cells subjected to oxygen/glucose deprivation (OGD), the normothermic effect of C + P on inflammation and apoptosis were examined. ResultsC + P significantly reduced infarct volumes, mitochondrial dysfunction (ATP and ROS concentration, citrate synthase and ATPase activity), inflammation and apoptosis with and without induced hypothermia. Overexpression of RIPK1, RIPK3, DRP-1, NLRP3-inflammasome and cytochrome c-apoptosis were all significantly reduced by C + P at 33 °C and the RIPK1 inhibitor (Nec1s), suggesting hypothermic effect of C + P via RIPK1/RIPK3-DRP1pathway. When body temperature was maintained at 37 °C, C + P and HIF-1α inhibitor (YC-1) reduced HIF-1α expression, leading to reduction in mitochondrial dysfunction, NLRP3 inflammasome and cytochrome c-apoptosis, as well as the interaction of HIF-1α and NLRP3. These were also evidenced in vitro, indicating a normothermic effect of C + P via HIF-1α. ConclusionHypothermic and normothermic neuroprotection of C + P involve different pathways. The normothermic effect was mediated by HIF-1α, while hypothermic effect was via RIPK1/RIPK3-DRP1 signaling. This provides a theoretical basis for future precise exploration of hypothermic and normothermic neuroprotection.

Aberrant Enhanced NLRP3 Inflammasomes and Cell Pyroptosis in the Brains of Prion-Infected Rodent Models Are Largely Associated with the Proliferative Astrocytes.

Neuroinflammation is a common pathological feature in a number of neurodegenerative diseases, which is mediated primarily by the activated glial cells. Nucleotide-binding oligomerization domain-like receptor pyrin domain-containing-3 (NLRP3) inflammasome-associated neuroinflammatory response is mostly considered. To investigate the situation of the NLRP3-related inflammation in prion disease, we assessed the levels of the main components of NLRP3 inflammasome and its downstream biomarkers in the scrapie-infected rodent brain tissues. The results showed that the transcriptional and expressional levels of NLRP3, caspase-1, and apoptosis-associated speck-like protein (ASC) in the brains of scrapie-infected rodents were significantly increased at terminal stage. The increased NLPR3 overlapped morphologically well with the proliferated GFAP-positive astrocytes, but little with microglia and neurons. Using the brain samples collected at the different time-points after infection, we found the NLRP3 signals increased in a time-dependent manner, which were coincidental with the increase of GFAP. Two main downstream cytokines, IL-1β and IL-18, were also upregulated in the brains of prion-infected mice. Moreover, the gasdermin D (GSDMD) levels, particularly the levels of GSDMD-NT, in the prion-infected brain tissues were remarkably increased, indicating activation of cell pyroptosis. The GSDMD not only co-localized well with the astrocytes but also with neurons at terminal stage, also showing a time-dependent increase after infection. Those data indicate that NLRP3 inflammasomes were remarkably activated in the infected brains, which is largely mediated by the proliferated astrocytes. Both astrocytes and neurons probably undergo a pyroptosis process, which may help the astrocytes to release inflammatory factors and contribute to neuron death during prion infection.

Design and Synthesis of 3-(2H-Chromen-3-yl)-5-aryl-1,2,4-oxadiazole Derivatives as Novel Toll-like Receptor 2/1 Agonists That Inhibit Lung Cancer In Vitro and In Vivo.

Toll-like receptor (TLR) 2 is a transmembrane receptor that participates in the innate immune response by forming a heterodimer with TLR1 or TLR6. TLR2 agonists play an important role in tumor therapy. Herein, we synthesized a series of 3-(2H-chromen-3-yl)-5-aryl-1,2,4-oxadiazole derivatives and identified WYJ-2 as a potent small and selective molecule agonist of TLR2/1, with an EC50 of 18.57 ± 0.98 nM in human TLR2 and TLR1 transient-cotransfected HEK 293T cells. WYJ-2 promoted the formation of TLR2/1 heterodimers and activated the nuclear factor kappa B (NF-κB) signaling pathway. Moreover, our study indicated that WYJ-2 could induce pyroptosis in cancer cells, mediated by activating the NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasome. WYJ-2 exhibited effective anti-non-small cell lung cancer (NSCLC) activity in vitro and in vivo. The discovery that activating TLR2/1 induces pyroptosis in cancer cells may highlight the prospects of TLR2/1 agonists in cancer treatment in the future.

Americanin B inhibits pyroptosis in lipopolysaccharide-induced septic encephalopathy mice through targeting NLRP3 protein

BackgroundSepsis is considered as a severe illness due to its high mortality. Sepsis can cause septic encephalopathy, thus leading to brain injury, behavioral and cognitive dysfunction. Pyroptosis is a type of regulated cell death (RCD) and takes a crucial part in occurrence and development of sepsis. Americanin B (AMEB) is a lignan compounds, which is extracted from Vernicia fordii. In our previous study, AMEB could inhibit microglial activation in inflammatory cell model. However, the function of AMEB in septic encephalopathy mice is uncertain. It would be worthwhile to ascertain the role and mechanism of AMEB in sepsis. PurposeCurrent study designs to certify the relationship between pyroptosis and septic encephalopathy, and investigate whether AMEB can restrain NOD-like receptor pyrin domain-containing 3 (NLRP3) inflammasome activation and restrict pyroptosis by targeting NLRP3 in septic mice model. Study designC57BL/6 mice were utilized to perform sepsis model in vivo experiments. BV-2 cell lines were used for in vitro experiments. MethodsIn vivo sepsis model was established by lipopolysaccharide (LPS) intraperitoneal injection in male C57BL/6 J mice and in vitro model was exposed by LPS plus ATP in BV-2 cells. The survival rate was monitored on the corresponding days. NLRP3, apoptosis associated Speck-like protein (ASC), caspase-1, GasderminD (GSDMD), interleukin-1β (IL-1β) and interleukin-18 (IL-18) level were detected by western blotting and immunofluorescence analysis. Molecular docking, cellular thermal shift assay (CETSA), drug affinity responsive target stability (DARTS) experiments, RNAi transfection and quantitative real-time PCR were applied to confirm the potential target of AMEB. ResultsThe results suggested that AMEB could rise survival percentage and lighten brain injury in LPS-induced sepsis mice. In addition, AMEB could inhibit pyroptosis and the activiation of NLRP3 inflammasome. The inhibiting function of AMEB on the activiation of NLRP3 inflammasome is weakened following si-NLRP3 transfection. Moreover, AMEB exerted anti-pyroptosis effect via targeting NLRP3 protein. ConclusionsOur findings first indicate NLRP3 is an effective druggable target for septic encephalopathy related brain injury, and also provide a candidate-AMEB for the treatment of septic encephalopathy. These emerging findings on AMEB in models of sepsis suggest an innovative approach that may be beneficial in the prevention of septic encephalopathy.

Role of pattern recognition receptors and microbiota-derived ligands in obesity

Obesity is associated with activation of low-grade inflammation in tissues metabolically relevant for the regulation glucose homeostasis. The gut microbiota has been extensively linked to the inflammatory responses observed during obesity emphasizing the interconnection between host immunity and metabolism during obesity. Gut microbiota together with alteration of the gut barrier functions provide a myriad of circulating ligands for the pattern recognition receptors (PRRs) expressed in innate immune cells and nonimmune cells. PRR-dependent signalling drives the expression of a wide range of genes beyond the inflammatory response depending on the specific functions of the targeted cells and on the physiological context. PRRs activation can have opposite effects on host metabolic inflammation. Nucleotide-binding oligomerization domain 1 (NOD1) or NOD-like Receptor pyrin domain containing 3 (NLRP3) activation promote metabolic inflammation and insulin resistance while NOD2 activation improves insulin sensitivity and glucose homeostasis during obesity. Toll-like receptors (TLRs) 2, 4 and 5 also display specific effects on metabolic tissues. TLR5 deficient mice are prone to obesity and inflammation in response to high fat diet, while injection of TLR5 ligand, flagellin, has a protective effect toward diet-induced obesity. To the opposite TLR2 and 4 activations are associated with deleterious metabolic outcome during obesity. TLR4 activation enhances metabolic inflammation and insulin resistance and TLR2 via its activation by molecules derived from the gut microbiota favours the onset of obesity. It is now clear that activation of PRRs by bacterial derived molecules plays a key role in the host metabolic regulation. PRRs are expressed in various cell types complicating the understanding of the mechanisms underlying the relationship between PRRs activation/silencing and metabolic inflammation in obesity context. This review presents an overview of the current understanding of the interrelationship between the gut microbiota and PRRs, with a focus on its consequences for obesity and related metabolic diseases.

Mechanism of Shenling Baizhu Powder on treatment of alcoholic liver disease by regulating TLR4/NLRP3 pathway

This study aims to investigate the regulatory effects of Shenling Baizhu Powder(SBP) on cellular autophagy in alcoholic liver disease(ALD) and its intervention effect through the TLR4/NLRP3 pathway. A rat model of chronic ALD was established by gavage of spirits. An ALD cell model was established by stimulating BRL3A cells with alcohol. High-performance liquid chromatography(HPLC) was utilized for the compositional analysis of SBP. Liver tissue from ALD rats underwent hematoxylin-eosin(HE) and oil red O staining for pathological evaluation. Enzyme-linked immunosorbent assay(ELISA) was applied to quantify lipopolysaccharides(LPS), tumor necrosis factor-alpha(TNF-α), interleukin-1 beta(IL-1β), and interleukin-18(IL-18) levels. Quantitative reverse transcription polymerase chain reaction(qRT-PCR) was conducted to evaluate the mRNA expression of myeloid differentiation factor 88(MyD88) and Toll-like receptor 4(TLR4). The effect of different drugs on BRL3A cell proliferation activity was assessed through CCK-8 analysis. Western blot analysis was performed to examine the protein expression of NOD-like receptor pyrin domain-containing 3(NLRP3), nuclear factor-kappa B P65(NF-κB P65), phosphorylated nuclear factor-kappa B P65(p-P65), caspase-1, P62, Beclin1, and microtubule-associated protein 1 light chain 3(LC3Ⅱ). The results showed that SBP effectively ameliorated hepatic lipid accumulation, reduced liver function, mitigated hepatic tissue inflammation, and reduced levels of LPS, TNF-α, IL-1β, and IL-18. Moreover, SBP exhibited the capacity to modulate hepatic autophagy induced by prolonged alcohol intake through the TLR4/NLRP3 signaling pathway. This modulation resulted in decreased expression of LC3Ⅱ and Beclin1, an elevation in P62 expression, and the promotion of autolysosome formation. These research findings imply that SBP can substantially enhance liver function and mitigate lipid irregularities in the context of chronic ALD. It achieves this by regulating excessive autophagic responses caused by prolonged spirit consumption, primarily through the inhibition of the TLR4/NLRP3 pathway.

FoxO6-Mediated TXNIP Induces Lipid Accumulation in the Liver through NLRP3 Inflammasome Activation.

Hepatic steatosis, which involves the excessive accumulation of lipid droplets in hepatocytes, presents a significant global health concern due to its association with obesity and metabolic disorders. Inflammation plays a crucial role in the progression of hepatic steatosis; however, the precise molecular mechanisms responsible for this process remain unknown. This study investigated the involvement of the nucleotide-binding oligomerization domain-like receptor pyrin domain-containing-3 (NLRP3) inflammasome and the forkhead box O6 (FoxO6) transcription factor in the pathogenesis of hepatic steatosis. We monitored the NLRP3 inflammasome and lipogenesis in mice overexpressing the constitutively active (CA)-FoxO6 allele and FoxO6-null mice. In an in vitro study, we administered palmitate to liver cells overexpressing CA-FoxO6 and measured changes in lipid metabolism. We administered palmitate treatment to clarify the mechanisms through which FoxO6 activates cytokine interleukin (IL)-1β through the NLRP3 inflammasome. The initial experiments revealed that dephosphorylation led to palmitate-induced FoxO6 transcriptional activity. Further palmitate experiments showed increased expression of IL-1β and the hepatic NLRP3 inflammasome complex, including adaptor protein apoptotic speck protein containing a caspase recruitment domain (ASC) and pro-caspase-1. Furthermore, thioredoxin-interacting protein (TXNIP), a key regulator of cellular redox conditions upstream of the NLRP3 inflammasome, was induced by FoxO6 in the liver and HepG2 cells. The findings of this study shed light on the molecular mechanisms underpinning the FoxO6-NLRP3 inflammasome axis in promoting inflammation and lipid accumulation in the liver.

Abstract TP26: Hypothermic and Non-Hypothermic Neuroprotection by Phenothiazine in Acute Ischemic Stroke: Modulation of NLRP3 Inflammasome-Related-Inflammation via Different Pathways of RIPK1/RIPK3-DRP1 or HIF-1α

Background: Inflammation after successful revascularization worsened outcomes in ischemic stroke. NLRPyrin domain containing 3 (NLRP3) inflammasome mediated inflammatory responses, in which receptor-interacting protein kinase (RIPK1) activated dynamin-related protein 1 (DRP1) in a RIPK3-dependent fashion. Hypoxia-inducible factor 1α (HIF-1α) regulated ischemic inflammation through NLRP3 inflammasome complex, thus apoptosis after stroke. Phenothiazine, including chlorpromazine and promethazine (C+P), induced hypothermic and non-hypothermic neuroprotection. We determined effects of RIPK1/RIPK3-DRP1 or HIF-1α pathways underlying the dual neuroprotection by C+P. Methods: 144 adult male Sprague-Dawley rats were subjected to 2 h of middle cerebral artery occlusion (MCAO), followed by 24 h reperfusion. 8mg/kg of C+P was injected at the onset of reperfusion. Infarct volumes, mRNA and protein expressions of HIF-1α, RIPK1, RIPK3, DRP-1, NLRP3-inflammation (NLRP3, ASC, Caspase-1, IL-1β and IL-18) and cytochrome C-apoptosis (cytochrome C, APAF-1, Caspase-9 and Caspase-3) were assessed. Apoptotic cell death, and the infiltration of neutrophils and macrophages were evaluated. Co-localization of RIPK1/RIPK3 and HIF-1α/NLRP3 were determined. Results: C+P significantly reduced brain inflammation, neuronal apoptosis and immune cell infiltration, with or without hypothermia. C+P with induced-hypothermia and RIPK1 inhibitor significantly reduced infarct volumes, the expression of RIPK1, RIPK3, DRP-1, NLRP3-inflammation process and cytochrome C-apoptosis, as well as the co-localization of RIPK1/RIPK3, suggesting the hypothermic effect underlying the RIPK1/RIPK3-DRP1 pathway. In non-hypothermia effect, HIF-1α expression was reduced by C+P or HIF-1α inhibitor, in concomitant with reduced NLRP3 inflammasome, cytochrome C-apoptosis, and decreased interaction of HIF-1α and NLRP3. Conclusion: Hypothermic and non-hypothermic neuroprotection of C+P involve different pathways. The hypothermic effect was via RIPK1/RIPK3-DRP1 signaling, while non-hypothermic effect was mediated by HIF-1α. This provides a theoretical basis for future precise exploration of hypothermic and non-hypothermic neuroprotection.

Cilostazol protects against degenerative cervical myelopathy injury and cell pyroptosis via TXNIP-NLRP3 pathway

Degenerative cervical myelopathy (DCM) is one of the most common and serious neurological diseases. Cilostazol has protective effects of anterior horn motor neurons and prevented the cell apoptosis. However, there was no literatures of Cilostazol on DCM. In this study, we established the DCM rat model to detect the effects of Cilostazol. Meanwhile, the neurobehavioral assessments, histopathology changes, inflammatory cytokines, Thioredoxin-interacting protein (TXNIP), NOD‑like receptor pyrin domain containing 3 (NLRP3) and pro-caspase-1 expressions were detected by Basso, Beattie, and Bresnahan score assessment, Hematoxylin and Eosin Staining, Enzyme-linked immunosorbent assay, immunofluorescence and Western blotting, respectively. After treated with Cilostazol, the Basso, Beattie, and Bresnahan (BBB) score, inclined plane test and forelimb grip strength in DCM rats were significantly increased meanwhile the histopathology injury and inflammatory cytokines were decreased. Additionally, TXNIP, NLRP3 and pro-caspase-1 expressions levels were decreased in Cilostazol treated DCM rats. Interestingly, the using of siTXNIP significantly changed inflammatory cytokines, TXNIP, NLRP3 and pro-caspase-1 expressions, however there was no significance between siTXNIP and Cilostazol + siTXNIP group. These observations showed that Cilostazol rescues DCM injury and ameliorates neuronal destruction mediated by TXNIP/NLRP3/caspase-1 and pro-inflammatory cytokines. As a result of our study, these findings provide further evidence that Cilostazol may represent promising therapeutic candidates for DCM.

Increased expression of NLRP3 associated with elevated levels of HMGB1 in children with febrile seizures: a case–control study

BackgroundHigh mobility group box-1 (HMGB1) is an endogenous danger signal that mediates activation of the innate immune response including NLR pyrin domain containing 3 (NLRP3) inflammasome activation and proinflammatory cytokine release. Although HMGB1 and NLRP3 have been implicated in the pathophysiology of seizures, the correlation between HMGB1 and NLRP3 expression has not been determined in children with febrile seizures (FS). To explore the relationship between extra-cellular HMGB1 and NLRP3 in children with FS, we analyzed serum HMGB1, NLRP3, caspase-1, and proinflammatory cytokines in patients with FS.MethodsThirty children with FS and thirty age-matched febrile controls were included in this study. Blood was obtained from the children with FS within 1 h of the time of the seizure; subsequently, the serum contents of HMGB1, NLRP3, caspase-1, interleukin (IL)-1β, interleukin (IL)-6, and tumour necrosis factor-α (TNF-α) were determined by enzyme-linked immunosorbent assay. The Mann‒Whitney U test was used to compare serum cytokine levels between FS patients and controls. Spearman’s rank correlation coefficient was calculated to detect significant correlations between cytokine levels.ResultsSerum levels of HMGB1, NLRP3, caspase-1, IL-1β, IL-6, and TNF-α were significantly higher in FS patients than in febrile controls (p < 0.05). Serum levels of HMGB1 were significantly correlated with levels of NLRP3 and caspase-1 (both, p < 0.05). Serum levels of caspase-1 were significantly correlated with levels of IL-1β (p < 0.05). Serum levels of IL-1β were significantly correlated with levels of IL-6 and TNF-α (p < 0.05).ConclusionsHMGB1 is up-regulated in the peripheral serum of FS patients, which may be responsible, at least in part, for the increased expression of NLRP3 and Caspase-1. Increased expression of caspase-1 was significantly associated with elevated serum levels of IL-1β. Given that activated Caspase-1 directly regulates the expression of mature IL-1β and positively correlates with activation of the NLRP3 inflammasome, our data suggest that increased levels of peripheral HMGB1 possibly mediate IL-1β secretion through the activation of the NLRP3 inflammasome in children with FS. Thus, both HMGB1 and NLRP3 might be potential targets for preventing or limiting FS.

Binge ethanol exposure in advanced age elevates neuroinflammation and early indicators of neurodegeneration and cognitive impairment in female mice

Binge drinking is rising among aged adults (>65 years of age), however the contribution of alcohol misuse to neurodegenerative disease development is not well understood. Both advanced age and repeated binge ethanol exposure increase neuroinflammation, which is an important component of neurodegeneration and cognitive dysfunction. Surprisingly, the distinct effects of binge ethanol exposure on neuroinflammation and associated degeneration in the aged brain have not been well characterized. Here, we establish a model of intermittent binge ethanol exposure in young and aged female mice to investigate the effects of advanced age and binge ethanol on these outcomes. Following intermittent binge ethanol exposure, expression of pro-inflammatory mediators (tnf-α, il-1β, ccl2) was distinctly increased in isolated hippocampal tissue by the combination of advanced age and ethanol. Binge ethanol exposure also increased measures of senescence, the nod like receptor pyrin domain containing 3 (NLRP3) inflammasome, and microglia reactivity in the brains of aged mice compared to young. Binge ethanol exposure also promoted neuropathology in the hippocampus of aged mice, including tau hyperphosphorylation and neuronal death. We further identified advanced age-related deficits in contextual memory that were further negatively impacted by ethanol exposure. These data suggest binge drinking superimposed with advanced age promotes early markers of neurodegenerative disease development and cognitive decline, which may be driven by heightened neuroinflammatory responses to ethanol. Taken together, we propose this novel exposure model of intermittent binge ethanol can be used to identify therapeutic targets to prevent advanced age- and ethanol-related neurodegeneration.

Abstract B035: The contribution of inflammasomes and cGAS-STING in RT-induced cell fate

Abstract Radiotherapy (RT) is a curative treatment modality for many solid cancers. Our group has previously demonstrated that activation of the cGAS-STING pathway by RT induces inflammatory cytokine production and subsequent stimulation of anti-tumour immunity in concert with immune checkpoint blockade treatment. We have identified three other proteins that contribute to inflammatory signalling after RT: AIM2 (Absent in Melanoma 2), NLRP3 (NOD-Like Receptor Pyrin Domain-Containing 3), and IFI16 (Interferon Gamma Inducible Protein 16). Each of these proteins contribute to form distinct protein complexes called inflammasomes that, like cGAS-STING, sense cytoplasmic DNA. Little is known about how inflammasome signalling may change after RT, or how inflammasomes may influence RT-ICB induced anti-tumour immunity. We hypothesize that RT induces inflammasome signalling to influence specific gene expression and cytokine profiles, in tandem with alterations in cellular fate with implications for systemic anti-tumour immunity triggered by radiotherapy. We will present ongoing work in which we show that pharmacological or genetic disruption of inflammasomes alters cytokine production post-RT. We also demonstrate that manipulation of cell fate through senolytic therapy or manipulation of caspase activity alters the balance of cytokine production post-RT. Together our data strongly implicates the role of several inflammasome complexes in the response of epithelial cells to RT. Our work will also highlight the possible crosstalk between the cGAS-STING pathway and inflammasome complex pathway, both triggered by RT. Understanding and subsequent manipulation of cell fate, in part through inflammasome and cGAS-STING mediated activity, may be a therapeutic strategy to influence treatment responses, such as anti-tumour immunity, in cancer. Citation Format: Cindy T Ha, Shirony Nicholson-Puthenveedu, Shane M Harding. The contribution of inflammasomes and cGAS-STING in RT-induced cell fate [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr B035.

Inhibition of HDAC6 alleviates cancer‑induced bone pain by reducing the activation of NLRP3inflammasome.

Cancer‑induced bone pain (CIBP) is characterized as moderate to severe pain that negatively affects the daily functional status and quality of life of patients. When cancer cells metastasize and grow in bone marrow, this activates neuroinflammation in the spinal cord, which plays a vital role in the generation and persistence of chronic pain. In the present study, a model of CIBP was constructed by inoculating of MRMT‑1 rat breast carcinoma cells into the medullary cavity of the tibia in male Sprague‑Dawley rats. Following two weeks of surgery, CIBP rats exhibited damaged bone structure, increased pain sensitivity and impaired motor coordination. Neuroinflammation was activated in the spinal cords of CIBP rats, presenting with extensive leukocyte filtration, upregulated cytokine levels and activated astrocytes. Histone deacetylase6 (HDAC6) works as a therapeutic target for chronic pain. The intrathecal injection of the HDAC6 inhibitor tubastatin A (TSA) in the lumbar spinal cord resulted in decreased spinal inflammatory cytokine production, suppressed spinal astrocytes activation and reduced NOD‑like receptor pyrin domain containing 3 (NLRP3) inflammasome activity. Consequently, this effect alleviated spontaneous pain and mechanical hyperalgesia and recovered motor coordination in CIBP rats. It was demonstrated by immunoprecipitation assay that TSA treatment reduced the interaction between HDAC6 and NLRP3. Cell research on C6 rat glioma cells served to verify that TSA treatment reduced HDAC6 and NLRP3 expression. In summary, the findings of present study indicated that TSA treatment alleviated cancer‑induced bone pain through the inhibition of HDAC6/NLRP3inflammasome signaling in the spinal cord.

Glycyrrhizin alleviates varicellovirus bovinealpha 1-induced oxidative stress, inflammation, and apoptosis in MDBK cells by inhibiting NF-κB/NLRP3 axis through the Nrf2 signalling pathway.

Varicellovirus bovinealpha 1 (BoAHV-1) is one of the crucial pathogens of bovine respiratory diseases, and its pathogenic mechanism involves oxidative stress, inflammation response, and apoptosis. Glycyrrhizin (GLY) possesses powerful antiviral, antioxidant, anti-inflammatory, and anti-apoptotic bioactivities. However, the anti-BoAHV-1 activity of GLY and its role in BoAHV-1-induced oxidative stress, inflammation, and apoptosis remain unclear. Therefore, the current study investigated the anti-BoAHV-1 effect of GLY and its ability to alleviate BoAHV-1-induced oxidative stress, inflammation, and apoptosis using an in vitro model (MDBK cells). Our results showed that BoAHV-1 titers significantly increased in MDBK cells after infection, and GLY reduced the BoAHV-1 titers in MDBK cells exposed to it. Furthermore, Interleukin (IL)-1β, IL-8, tumor necrosis factor (TNF)-α, phosphorylated NF-κB p65 (p-NF-κB p65), the NLR pyrin domain containing 3 (NLRP3), Caspase-1, and Cleaved Caspase-3 levels were significantly upregulated when MDBK cells were challenged with BoAHV-1. In BAY 11-7085 (a specific NF-κB inhibitor) treated MDBK cells, IL-1β, IL-8, TNF-α, p-NF-κB p65, NLRP3, Caspase-1, and Cleaved Caspase-3 levels were downregulated. Notably, GLY treatment had the same trend as the BAY 11-7085 treatment. Thus, these results suggested that GLY exerted anti-inflammatory and anti-apoptotic activities by blocking NF-κB/NLRP3 axis. In addition, after BoAHV-1 infection, the levels of reactive oxygen species (ROS), malondialdehyde (MDA), and p-NF-κB p65 and apoptosis rate were increased, and catalase (CAT) and glutathione peroxidase (GSH-Px) enzyme activities, as well as NF-E2-related nuclear factor erythroid-2 (Nrf2) protein expression were repressed. Compared with BoAHV-1-infected MDBK cells, GLY treatment significantly downregulated intracellular ROS, MDA, and p-NF-κB p65 levels and apoptotic rates and significantly increased intracellular CAT and GSH-Px enzyme activities and Nrf2 expression. Additionally, ML385 (a specific Nrf2 inhibitor) abolished the enhancing effect of GLY on Nrf2 and the attenuating effect on ROS, p-NF-κB p65, and apoptosis. These results suggested that GLY had an anti-BoAHV-1 effect and could mitigate BoAHV-1-induced oxidative stress, inflammation, and apoptosis by activating the Nrf2 signalling and restraining NF-κB/NLRP3 axis.

Elevated Expression of Two Pore Potassium Channel THIK-1 in Alzheimer's Disease: An Inflammatory Mechanism.

Tandem pore domain halothane-inhibited K+ channel 1 (THIK-1, coded by KCNK13) provides an upstream regulation of the activation of the NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasome, which has been suggested as one of the key mechanisms of the pathological process in neurodegeneration mainly from in vitro and in vivo model systems studies. However, unequivocal evidence from neurodegenerative disorders has been lacking. To investigate the involvement of the THIK-1/NLRP3 pathway in the pathological process of Alzheimer's disease (AD) and Parkinson's disease (PD). This study investigated gene expression of markers in the THIK-1/NLRP3 pathway in an animal model representing AD as well as in human postmortem brains of AD and PD by quantitative real-time PCR. THIK-1 protein expression was determined using automated capillary electrophoresis immunoblotting. Furthermore, DNA methylation of KCNK13 was analysed in AD cohort by pyrosequencing. A substantial upregulation of KCNK13, glial activation markers, NLRP3 inflammasome components, and IL1B was observed in the animal study. Increased expression of KCNK13 support an inflammatory glial cell activation in both advanced AD and PD. The increase in KCNK13 expression was also supported by downregulation in DNA methylation of KCNK13 in AD. The association between THIK-1 K+ channels expression and pathology changes indicates a THIK-1-induced activation of this glial subtype in AD and PD. Therefore, specific blocks of the microglial THIK-1 K+ channels at the early stage of AD and PD may be beneficial for the patients.

A review of studies on the implication of NLRP3 inflammasome for Parkinson’s disease and related candidate treatment targets

Parkinson's disease (PD) is a neurodegenerative disease for which the prevalence is second only to Alzheimer's disease (AD). This disease primarily affects people of middle and old age, significantly impacting their health and quality of life. The main pathological features include the degenerative nigrostriatal dopaminergic (DA) neuron loss and Lewy body (LB) formation. Currently, available PD medications primarily aim to alleviate clinical symptoms, however, there is no universally recognized therapy worldwide that effectively prevents, clinically treats, stops, or reverses the disease. Consequently, the evaluation and exploration of potential therapeutic targets for PD are of utmost importance. Nevertheless, the pathophysiology of PD remains unknown, and neuroinflammation mediated by inflammatory cytokines that prompts neuron death is fundamental for the progression of PD. The nucleotide-binding oligomerization domain-like receptor pyrin domain-containing 3 (NLRP3) inflammasome is a key complex of proteins linking the neuroinflammatory cascade in PD. Moreover, mounting evidence suggests that traditional Chinese medicine (TCM) alleviates PD by suppressing the NLRP3 inflammasome. This article aims to comprehensively review the available studies on the composition and activating mechanism of the NLRP3 inflammasome, along with its significance in PD pathogenesis and potential treatment targets. We also review natural products or synthetic compounds which reduce neuroinflammation via modulating NLRP3 inflammasome activity, aiming to identify new targets for future PD diagnosis and treatment through the exploration of NLRP3 inhibitors. Additionally, this review offers valuable references for developing new PD treatment methods.

Targeting the NLRP3 inflammasome for the treatment of hypertensive target organ damage: Role of natural products and formulations.

Hypertension is a major global health problem that causes target organ damage (TOD) in the heart, brain, kidney, and blood vessels. The mechanisms of hypertensive TOD are not fully understood, and its treatment is challenging. This review provides an overview of the current knowledge on the role of Nod-like receptor pyrin domain containing 3 (NLRP3) inflammasome in hypertensive TOD and the natural products and formulations that inhibit it. We searched PubMed, Web of Science, Google Scholar, and CNKI for relevant articles using the keywords "hypertension," "target organ damage," "NLRP3 inflammasome," "natural products," and "formulations." We reviewed the effects of the NLRP3 inflammasome on hypertensive TOD in different organs and discussed the natural products and formulations that modulate it. In hypertensive TOD, the NLRP3 inflammasome is activated by various stimuli such as oxidative stress and inflammation. Activation of NLRP3 inflammasome leads to the production of pro-inflammatory cytokines that exacerbate tissue damage and dysfunction. Natural products and formulations, including curcumin, resveratrol, triptolide, and allicin, have shown protective effects against hypertensive TOD by inhibiting the NLRP3 inflammasome. The NLRP3 inflammasome is a promising therapeutic target in hypertensive TOD. Natural products and formulations that inhibit the NLRP3 inflammasome may provide novel drug candidates or therapies for hypertensive TOD. Further studies are needed to elucidate the molecular mechanisms and optimize the dosages of these natural products and formulations and evaluate their clinical efficacy and safety.

Tert-butylhydroquinone attenuates LPS-induced pyroptosis of IPEC-J2 cells via downregulating HMGB1/TLR4/NF-κB axis.

Inflammatory response induced by biological stress usually occurs in weaning piglets, it reduces the production performance of piglets and even causes death. Tert-butylhydroquinone (TBHQ) is a food additive that has the effect of anti-inflammation and anti-oxidation. However, there are few reports related to the protective mechanisms of TBHQ on lipopolysaccharide (LPS) induced injury in intestinal porcine epithelial (IPEC-J2) cells. Quantitative real-time polymerase chain reaction and western blot analysis, respectively, detected the mRNA levels and protein expressions related to pyroptosis, tight junction (TJ) protein and high-mobility group box 1/toll-like receptor 4/nuclear factor kappa-B (HMGB1/TLR4/NF-κB) axis. Localisation and expression of NOD-like receptor pyrin domain containing 3 (NLRP3), HMGB1 and P-NF-κB proteins detected by immunofluorescence. The results showed that TBHQ (12.5 and 25 μM) can increase cell activity and reduce intracellular lactate dehydrogenase (LDH) levels in a dose-dependent manner. LPS significantly decreases cell viability and increases the LDH level. However, pretreatment with TBHQ evidently increases cell viability and decreases the LDH level of IPEC-J2 cells. In addition, treatment with LPS decreased the mRNA level and protein expression of zonula occludens-1, occludin and claudin-1, and increased the mRNA level and protein expression of pyroptosis and HMGB1/TLR4/NF-κB axis. Interestingly, pretreatment with TBHQ increased the TJ protein expressions as well as decreased the mRNA level and protein expressions of pyroptosis and HMGB1/TLR4/NF-κB axis. Moreover, the results of immunofluorescence showed that TBHQ significantly reduced the expression of NLRP3, HMGB1 and P-NF-κB in LPS-induced injury of IPEC-J2 cells. Therefore, we come to the conclusion that TBHQ attenuates LPS-induced pyroptosis in IPEC-J2 cells through downregulation of the HMGB1/TLR4/NF-κB axis, TBHQ may become a potential feed additive for preventing inflammatory diarrhoea in piglets.

Genistein upregulates AHR to protect against environmental toxin-induced NASH by inhibiting NLRP3 inflammasome activation and reconstructing antioxidant defense mechanisms

We have previously proven that the environmental toxin could accelerate the development and progression of nonalcoholic steatohepatitis (NASH). However, the underlying mechanism associated with such excessive inflammation hasn't been fully illustrated. Although Genistein has been well accepted for its capability in anti-inflammation and anti-oxidation, its effect in ameliorating contaminants-induced NASH still needs to be identified. In this study, using chickens and primary chicken hepatocytes as models, we found that NOD-like receptor pyrin domain-containing 3 (NLRP3) inflammasome were over-activated in bromoacetic acid (BAA, one of the typical environmental toxins)-induced NASH, characterized by the infiltration of inflammatory cell, and the increase of NLRP3, Caspase-1 p20, and cytokines (IL-1β, IL-18) expressions. Interestingly, genistein treatment could recover these changes, with the signs of restored activities of anti-oxidases, decreased expressions of NLRP3 inflammasome components, and increased levels of elements in phase I metabolic system. The detailed mechanism was that, via up-regulating aryl hydrocarbon receptor (AHR), genistein lifted mRNA levels of Cyp1-related genes to reconstruct cytochrome P450 (CYP450) systems, and the raised AHR negatively regulated NLRP3 inflammasome activity to relieve inflammation. More important, the interaction and co-localization between AHR and NLRP3 was first proved, and genistein could promote the levels of AHR that interacted with NLRP3, which thereafter blocked the activation of NLRP3 inflammasome. Conclusively, in this research, we confirmed the AHR-dependent protective role of genistein in environmental toxin-linked NASH, which shed light on the potential precautions for contaminants-induced NASH.