Inhibited Caspase-1 Activation Research Articles

WTAP participates in neuronal damage by protein translation of NLRP3 in an m6A-YTHDF1-dependent manner after traumatic brain injury.

Traumatic brain injury (TBI) is a common complication of acute and severe neurosurgery. Remodeling of N6-methyladenosine (m6A) stabilization may be an attractive treatment option for neurological dysfunction after TBI. In the present study, the authors explored the epigenetic methylation of RNA-mediated NLRP3 inflammasome activation after TBI. Neurological dysfunction, histopathology, and associated molecules were examined in conditional knockout (CKO) WTAP [flox/flox, Camk2a-cre] , WTAP flox/flox , and pAAV-U6-shRNA-YTHDF1-transfected mice. Primary neurons were used in vitro to further explore the molecular mechanisms of action of WTAP/YTHDF1 following neural damage. The authors found that WTAP and m6A levels were upregulated at an early stage after TBI, and conditional deletion of WTAP in neurons did not affect neurological function but promoted functional recovery after TBI. Conditional deletion of WTAP in neurons suppressed neuroinflammation at the TBI early phase: WTAP could directly act on NLRP3 mRNA, regulate NLRP3 mRNA m6A level, and promote NLRP3 expression after neuronal injury. Further investigation found that YTH domain of YTHDF1 could directly bind to NLRP3 mRNA and regulate NLRP3 protein expression. YTHDF1 mutation or silencing improved neuronal injury, inhibited Caspase-1 activation, and decreased IL-1β levels. This effect was mediated via suppression of NLRP3 protein translation, which also reversed the stimulative effect of WTAP overexpression on NLRP3 expression and inflammation. Our results indicate that WTAP participates in neuronal damage by protein translation of NLRP3 in an m6A-YTHDF1-dependent manner after TBI and that WTAP/m6A/YTHDF1 downregulation therapeutics is a viable and promising approach for preserving neuronal function after TBI, which can provide support for targeted drug development.

Discovery of a Covalent Inhibitor of Pro-Caspase-1 Zymogen Blocking NLRP3 Inflammasome Activation and Pyroptosis.

Caspase-1 plays a central role in innate immunity, as its activation by inflammasomes induces the production of proinflammatory cytokines and pyroptosis. However, specific inhibition of the enzymatic activity of this protease is not effective in suppressing inflammation, owing to its enzyme-independent function. Herein, we identified a cyclohexenyl isothiocyanate compound (CIB-1476) that potently inhibited caspase-1 activity and suppressed the assembly and activation of the NLRP3 inflammasome and gasdermin-D-mediated pyroptosis. Mechanistically, CIB-1476 directly targeted pro-caspase-1 as an irreversible covalent inhibitor by binding to Cys285 and Cys397, resulting in more durable anti-inflammatory effects in the suppression of enzyme-dependent IL-1β production and enzyme-independent nuclear factor κB activation. Chemoproteomic profiling demonstrated the engagement of CIB-1476 with caspase-1. CIB-1476 showed potent therapeutic effects by suppressing inflammasome activation in mice, which was abolished in Casp1-/- mice. These results warrant further development of CIB-1476 along with its analogues as a novel strategy for caspase-1 inhibitors.

P159 Inhibition of Caspase-1 signalling subtly affect the gut microbiota and reduces Adherent-invasive Escherichia coli (AIEC)-induced inflammasome activation

Abstract Background Inflammatory bowel disease (IBD), encompassing Crohn’s Disease and ulcerative colitis, is a chronic gastrointestinal disorder with increasing incidence worldwide. Microbial factors such as Adherent-invasive Escherichia coli (AIEC) have been suggested to play a significant role in the pathogenesis of IBD. The inflammasome, a multi-protein complex, senses environmental signals, including bacteria, resulting in the activation of caspase-1 leading to the subsequent maturation and secretion of IL-1β. In this study we examined the impact of AIEC on inflammasome activation and on the inhibition of Caspase-1 signaling in AIEC-colonised mice and on intestinal epithelial cells (IECs). Methods Human IECs (CaCO2Bee1 cells) were cultured for 3hr with AIEC (HM605 and its invasiveness mutant), washed with antibiotics and further cultured in full cell media for 13hrs, followed by analysis on cytokine and inflammasome gene expression and activation by qRT-PCR, ELISA, imaging and flow cytometry. To inhibit caspase-1 activation, cells were pre-treated for 1 hr with the caspase-1 inhibitor zVAD-fmk. To establish AIEC colonisation, C57BL/6 and IL-10-/- mice were treated with streptomycin for 24hrs followed by oral gavage of AIEC-HM605 (10-8 cfu/mL) and faecal and tissue samples were collected at 6 days post AIEC-infection. Caspase-1 inhibitor was orally gavaged every in mice. Faecal samples were prepared for 16S rRNA gene sequencing and colon tissue was analysed for cytokines and inflammasome markers by qRT-PCR and ELISA. Results IECs cultured with AIEC and AIEC-colonised C57BL/6 and IL-10-/- mice resulted in an increased expression and activation of both canonical (Caspase-1) and non-canonical inflammasome (caspase 4). The AIEC-induced inflammasome response was dependent on the invasiveness of AIEC. Treatment with a caspase-1 inhibitor reduced the AIEC-induced inflammasome markers in IECs and in C57BL/6 mice. No major changes in alpha or beta-diversity was detected in AIEC-colonised mice or upon Caspase-1 inhibition. AIEC-colonisation resulted in an increased abundance of Bacteroides and Alistipides and reduced abundance of Lachnospiraceae, alterations reversed by Caspase-1 inhibition. Conclusion We show that the invasion of AIEC in IECs and colonisation of murine intestine results in activation of caspase-1 inflammasome. AIEC colonisation provoke subtle microbiota alterations which are reversed by caspase-1 inhibition. In summary, our data supports a role of AIEC in IBD pathogenesis through the activation of caspase-1 inflammasome.

Resveratrol Alleviates Retinal Ischemia-Reperfusion Injury by Inhibiting the NLRP3/Gasdermin D/Caspase-1/Interleukin-1β Pyroptosis Pathway.

The purpose of this study is to investigate the anti-pyroptotic effect of resveratrol in the context of ischemia-reperfusion (I/R)-induced retinal injury, with a particular focus on Müller glial cells (MGCs) and to elucidate the underlying molecular mechanisms. The retinal I/R model was constructed in mice and pyroptotic markers were measured at six, 12, 24, 48, and 72hours after I/R injury to determine the peak of pyroptotic activity. The effects of resveratrol on pyroptosis, inflammasomes, and the activation of MGCs after I/R injury were observed on the retina of mice. Moreover, induction of pyroptosis in rat Müller glial cells (r-MC) via lipopolysaccharide was used to explore the effects of resveratrol on pyroptosis of r-MC in vitro. After the induction of retinal I/R injury in mice, the intricate involvement of pyroptosis in the progressive degeneration of the retina was observed, reaching its zenith at the onset of 24hours after I/R injury. Resveratrol treatment alleviated I/R injury on the retina, relieved retinal ganglion cells death. In addition, resveratrol inhibited Caspase-1 activation, gasdermin D (GSDMD-N) cleavage, the inflammasome assembly, and the release of inflammatory cytokines, simultaneously relieving the MGCs activation. Furthermore, resveratrol inhibited the pyroptosis-related NLRP3/GSDMD-N/TMS1/ASC/Caspase-1/IL-1β pathway in r-MC cells, and mitigated cells death in vitro. Pyroptosis plays an important role in the pathogenesis of retinal I/R injury. Resveratrol can attenuate pyroptotic-driven damage in the retina and MGC by inhibiting the NLRP3/GSDMD-N/TMS1/ASC/Caspase-1/IL-1β pyroptosis pathway.

Three New Clerodane Diterpenoids and Their NLRP3 Inflammasome Activation Inhibitory Activity from Callicarpa arborea Roxb.

Three new compounds callicarpenoids A-C (1-3), were isolated from the stems of Callicarpa arborea Roxb together with fifteen known compounds (4-18). The structures of these compounds were elucidated using advanced spectroscopic techniques, including 1D and 2D NMR, UV, IR, HR-ESI-MS, ECD, ORD, and quantum chemical calculations. Compound 3, a rare rearranged diterpenoid with a fused 5/6-ring system demonstrated strong potential as an inhibitor of the NLRP3 inflammasome activation with an IC50 value of 3.153 μM. It effectively reduced GSDMD-NT production, inhibited caspase-1 activation, and suppressed IL-1β secretion, thereby mitigating NLRP3 inflammasome-induced pyroptosis in J774A.1 cells. These findings suggest that compound 3 warrants further research and development as a promising NLRP3 inflammasome inhibitor.

Diallyl disulfide attenuates pyroptosis via NLRP3/Caspase-1/IL-1β signaling pathway to exert a protective effect on hypoxic-ischemic brain damage in neonatal rats

Hypoxic-ischemic encephalopathy (HIE) is a perinatal brain disease caused by hypoxia in neonates. It is one of the leading causes of neonatal death in the perinatal period, as well as disability beyond the neonatal period. Due to the lack of a unified and comprehensive treatment strategy for HIE, research into its pathogenesis is essential. Diallyl disulfide (DADS) is an allicin extract, with detoxifying, antibacterial, and cardiovascular disease protective effects. This study aimed to determine whether DADS can alleviate HIE induced brain damage in rats and oxygen-glucose deprivation (OGD)-induced pyroptosis in PC12 cells, as well as whether it can inhibit pyroptosis via the NLRP3/Caspase-1/IL-1β signaling pathway. In vivo, DADS significantly reduced the cerebral infarction volume, alleviated inflammatory reaction, reduced astrocyte activation, promoted tissue structure recovery, improved pyroptosis caused by HIE and improved the prognosis following HI injury. In vitro findings indicated that DADS increased cell activity, decreased LDH activity and reduced the expression of pyroptosis-related proteins, including IL-1β, IL-18, and certain inflammatory factors in PC12 cells caused by OGD. Mechanistically, DADS inhibited pyroptosis and protected against HIE via the NLRP3/Caspase-1/IL-1β pathway. The specific inhibitor of caspase-1, VX-765, inhibited caspase-1 activation, and IL-1β expression was determined. Additionally, the overexpression of NLRP3 reversed the protective effect of allicin against OGD-induced pyroptosis. In conclusion, these findings demonstrated that DADS inhibits the NLRP3/Caspase-1/IL-1β signaling pathway and decreases HI brain damage.

Pyroptosis of chondrocytes activated by synovial inflammation accelerates TMJ osteoarthritis cartilage degeneration via ROS/NLRP3 signaling

ObjectivesSynovial inflammation and chondrocyte death have been widely acknowledged as key contributors to the pathological progression of temporomandibular joint osteoarthritis (TMJ-OA), a degenerative joint disease currently lacking definitive treatments. This study aims to understand the regulatory role of chondrocyte pyroptosis in condylar cartilage degradation during TMJ-OA. MethodsThe levels of cytokines, cartilage degeneration markers, and pyroptotic biomarkers in the synovium and synovial fluid of temporomandibular disorders (TMD) patients were examined. The synovitis, cartilage degradation, and chondrocyte pyroptosis in wild-type and alpha-kinase 1 (ALPK1)-deficient TMJ-OA mice were then compared following monosodium iodoacetate (MIA) induction. Subsequently, we investigated the downstream mechanisms of cytokines- or macrophage supernatants-induced metabolic disorders and pyroptosis in chondrocytes using primary TMJ chondrocytes and ATDC5 chondrocyte cultures. ResultsWe found a positive correlation between pyroptotic biomarkers and cartilage degradation mediators and cytokines in the synovial fluid of TMD patients. MIA-induced TMJ-OA mice demonstrated significant synovitis, cartilage degradation, and chondrocyte pyroptosis, which were mitigated in ALPK1-deficient TMJ-OA mice, inflammation-restrained mice. Ex-vivo study revealed the contribution of reactive oxygen species (ROS) to inflammation-irritated macrophage supernatants-induced pyroptosis and metabolic disorders in chondrocytes. Targeting NOD-like receptor protein 3 (NLRP3) alleviated cytokines- or ROS-induced pyroptosis and metabolic disorders in chondrocytes by inhibiting caspase-1 activation and interleukin-1β (IL-1β) secretion. ConclusionOur findings offer novel insight into the role of synovial inflammation-induced chondrocyte pyroptosis in promoting cartilage degradation during TMJ-OA via the ROS and NLRP3 signaling pathway.

The Role of Interleukin-17A and NLRP3 Inflammasome in the Pathogenesis of Graves' Ophthalmopathy.

The development of Graves' ophthalmopathy (GO) is associated with autoimmune dysfunction. Recent studies have indicated that IL-17A, inflammasomes, and related cytokines may be involved in the etiology of GO. We sought to investigate the pathogenic role of IL-17A and NLRP3 inflammasomes in GO. Orbital fat specimens were collected from 30 patients with GO and 30 non-GO controls. Immunohistochemical staining and orbital fibroblast cultures were conducted for both groups. IL-17A was added to the cell cultures, and cytokine expression, signaling pathways, and inflammasome mechanisms were investigated using reverse transcription polymerase chain reaction, enzyme-linked immunosorbent assay, Western blotting, and small interfering RNA (siRNA) methods. Immunohistochemical staining showed higher NLRP3 expression in GO orbital tissue than in non-GO controls. IL-17A upregulated pro-IL-1β mRNA levels and IL-1β protein levels in the GO group. Furthermore, IL-17A was confirmed to enhance caspase-1 and NLRP3 protein expression in orbital fibroblasts, suggesting NLRP3 inflammasome activation. Inhibiting caspase-1 activity could also decrease IL-1β secretion. In siRNA-transfected orbital fibroblasts, significantly decreased NLRP3 expression was observed, and IL-17A-mediated pro-IL-1β mRNA release was also downregulated. Our observations illustrate that IL-17A promotes IL-1β production from orbital fibroblasts via the NLRP3 inflammasome in GO, and cytokines subsequently released may induce more inflammation and autoimmunity.

Pyrrolidine Dithiocarbamate Suppresses Cutibacterium acnes-Induced Skin Inflammation.

Cutibacterium acnes (C. acnes), a Gram-positive anaerobic bacterium, proliferates in hair follicles and pores and causes inflammation in the skin of young people. The rapid growth of C. acnes triggers macrophages to secrete proinflammatory cytokines. Pyrrolidine dithiocarbamate (PDTC) is a thiol compound that exerts antioxidant and anti-inflammatory effects. Although the anti-inflammatory function of PDTC in several inflammatory disorders has been reported, the effect of PDTC on C. acnes-induced skin inflammation remains unexplored. In the present study, we examined the effect of PDTC on C. acnes-induced inflammatory responses and determined the mechanism by using in vitro and in vivo experimental models. We found that PDTC significantly inhibited the expression of C. acnes-induced proinflammatory mediators, such as interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and NOD-like receptor (NLR) pyrin domain-containing 3 (NLRP3), in mouse-bone-marrow-derived macrophage (BMDM) cells. PDTC suppressed C. acnes-induced activation of nuclear factor-kappa B (NF-κB), which is the major transcription factor for proinflammatory cytokine expression. In addition, we found that PDTC inhibited caspase-1 activation and IL-1β secretion through suppressing NLRP3 and activated the melanoma 2 (AIM2) inflammasome but not the NLR CARD-containing 4 (NLRC4) inflammasome. Moreover, we found that PDTC improved C. acnes-induced inflammation by attenuating C. acnes-induced IL-1β secretion in a mouse acne model. Therefore, our results suggest that PDTC has potential therapeutic value for the amelioration of C. acnes-induced skin inflammation.

Protectin Conjugates in Tissue Regeneration 1 Inhibits Macrophage Pyroptosis by Restricting NLRP3 Inflammasome Assembly to Mitigate Sepsis via the cAMP-PKA Pathway

Protectin conjugates in tissue regeneration 1 (PCTR1) is a novel anti-inflammatory and proresolving lipid mediator biosynthesized from docosahexaenoic acid. Excessive activation of NLR family pyrin domain containing 3 (NLRP3) inflammasome and consequent pyroptosis are involved in diverse inflammatory diseases. However, how PCTR1 affects NLRP3 inflammasome activation and pyroptosis are still unclear. Here, we demonstrated that PCTR1 inhibited NLRP3 inflammasome activation and pyroptosis. These results show that PCTR1 dose-dependently inhibited gasdermin D cleavage in lipopolysaccharide (LPS)-primed murine primary macrophages upon nigericin stimulation. Additionally, PCTR1 treatment after LPS priming inhibited caspase-1 activation and subsequent mature interleukin-1β release independent of the nuclear factor-kappa B pathway. PCTR1 exerted its inhibitory effects by blocking NLRP3-apoptosis-associated speck-like protein containing a CARD (ASC) interaction and ASC oligomerization, thereby restricting NLRP3 inflammasome assembly. However, the inhibitory effect of PCTR1 could be reversed by KH7 and H89, which are the inhibitors of the cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) signaling pathway. Moreover, PCTR1 treatment alleviated lung tissue damage and improved mouse survival in LPS-induced sepsis. Our study unveils the molecular mechanism of negative regulation of NLRP3 inflammasome activation and pyroptosis by a novel lipid mediator and suggests that PCTR1 may serve as a potential treatment option for NLRP3-inflammasome driven diseases.

HSF1 Protects Sepsis-Induced Acute Lung Injury by Inhibiting NLRP3 Inflammasome Activation.

As an important transcription factor, heat shock factor 1 (HSF1) plays an endogenous anti-inflammation role in the body and can alleviate multiple organ dysfunction caused by sepsis, which contributes to an uncontrolled inflammatory response. The NLRP3 inflammasome is a supramolecular complex that plays key roles in immune surveillance. Inflammation is accomplished by NLRP3 inflammasome activation, which leads to the proteolytic maturation of IL-1β and pyroptosis. However, whether HSF1 is involved in the activation of the NLRP3 inflammasome in septic acute lung injury (ALI) has not been reported. Here, we show that HSF1 suppresses NLRP3 inflammasome activation in transcriptional and post-translational modification levels. HSF1 can repress NLRP3 expression via inhibiting NF-κB phosphorylation. HSF1 can inhibit caspase-1 activation and IL-1β maturation via promoting NLRP3 ubiquitination. Our finding not only elucidates a novel mechanism for HSF1-mediated protection of septic ALI but also identifies new therapeutic targets for septic ALI and related diseases.

The toxicity of perfluorodecanoic acid is mainly manifested as a deflected immune function.

Perfluorodecanoic acid (PFDA) is a type of perfluoroalkyl acid (PFAA). PFDA has toxicity similar to dioxin; its effect on the body is not through a single target or a single pathway. However, the mechanism at the global level is still unclear. We treated mice with PFDA and characterized the global changes in gene expression in the liver using microarray analyses. The enriched KEGG pathways and GO analyses revealed that PFDA greatly affected the immune response, which was different from the response of gastric cells previously studied. As a proof of principle, the expressions of IL-1β and IL-18 were both decreased after PFDA treatment, and qRT-PCR and ELISAs verified the reduction of IL-1β and IL-18 in liver tissues. Mechanistic investigations indicated that PFDA inhibited caspase-1 activation, and decreased the mRNA levels of NLRP1, NLRP3, and NLRC4; thus, suggesting that inflammasome assemblies were suppressed. Further microarray data revealed that cIAP2 and its binding proteins, which are critical for regulating inflammasome assembly, were also repressed by PFDA. In addition, flow cytometry results revealed a significant inhibition of Th1 cell differentiation in the livers of PFDA-treated mice. The results of this study suggested that one of the main toxic effects of PFDA on livers was the inhibition of immune response.

Itaconate regulates macrophage function through stressful iron-sulfur cluster disrupting and iron metabolism rebalancing.

Lipopolysaccharide (LPS)-stimulated macrophages express an aconitate decarboxylase (IRG1, also called ACOD1), leading toaccumulationof the endogenous metabolite itaconate. However, the precise mechanisms by which elevated itaconate levels alter macrophage function are not clear. Our hypothesis is itaconate affects macrophage function through some uncertain mechanism. Based on this, we established a transcriptional and proteomic signature of macrophages stimulated by itaconate and identified the pathways of IL-1β secretion and altered iron metabolism. Consistently, the effect of IRG1 deficiency on IL-1β secretion and iron metabolism was confirmed in IRG1 knockout THP-1 cell lines. Several common inhibitors and other compounds were used to examine the molecular mechanisms involved.Only cysteine and antioxidants (catechin hydrate) could inhibit caspase-1 activation and IL-1β secretion in itaconate-stimulated macrophages. We further found that aconitase activity was decreased by itaconate stimulation. Our results demonstrate the counteracting effects of overexpression of mitochondrial aconitase (ACO2,a tricarboxylic acidcycle enzyme) or cytosolic aconitase (ACO1, an iron regulatory protein) on IL-1β secretion and altered iron metabolism. Both enzyme activities were inhibited by itaconate because of iron-sulfur (Fe-S)cluster destruction. Our findings indicate that the immunoregulatory functions of IRG1 and itaconate in macrophages are stressful Fe-S cluster of aconitases disrupting and iron metabolism rebalancing.

Dietary supplementation of camel whey protein attenuates heat stress-induced liver injury by inhibiting NLRP3 inflammasome activation through the HMGB1/RAGE signalling pathway

• The HMGB1/RAGE/NLRP3 signaling pathway is involved in HS-induced liver injury. • CWP inhibited HS-induced liver injury by reducing activity of the NLRP3 inflammasome. • CWP inhibited the activation of NLRP3 inflammasome by restoring HMGB1 nuclear localization. • The combined application of CWP and anti-inflammatory agent completely blocked HS-induced liver injury. Inflammasomes play an important role in promoting heat stress (HS) induced liver injury. Dietary supplementation of camel whey protein (CWP) has been shown to alleviate HS-induced tissue damage. However, whether and how dietary CWP supplementation can reduce HS-induced liver injury remains unclear. Thus, we evaluated the ability of rats supplemented with CWP to resist HS-induced liver injury. To induce liver injury, Sprague-Dawley rats were exposed to HS. The mechanism of action was confirmed using an antagonist of high mobility group box 1 (HMGB1) and the NLR pyrin domain containing 3 (NLRP3) inhibitor before HS. Histological changes in the livers of the HS rats were visualised using haematoxylin and eosin staining. The protein expression of HMG1, receptor for advanced glycation end products (RAGE), NLRP3, Caspase-3, and Bcl-2 were detected using immunohistochemistry or western blotting. The content of IL-1β, the activation of Caspase-1, and the level of alanine transaminase (ALT) were determined using commercial kits. We confirmed that HS activated NLRP3 in the liver, as evidenced by enhanced Caspase-1 activity and increased IL-1β content. Inhibition of NLRP3 activation reversed abnormal expression and nuclear translocation of Caspase-3 and Bcl-2, and alleviated apoptosis, necrosis, and subsequent liver injury. HS-induced hepatocyte NLRP3 activation is dependent on elevated extracellular HMGB1 levels. Glycyrrhizic acid, an antagonist of HMGB1, which is also an anti-inflammatory agent, inhibited HS-induced NLRP3 activation. Interestingly, CWP also reversed HS-induced abnormal expression of HMGB1, RAGE, NLRP3, IL-1β, ALT, Bcl-2, and Caspase-3, inhibited Caspase-1 activity, and alleviated apoptosis and liver histological changes. More importantly, CWP combined with glycyrrhizic acid completely prevented HS-induced hepatocyte apoptosis and injury. These results indicate that CWP ameliorates HS-induced liver injury by inhibiting the HMGB1/RAGE/NLRP3 axis. Dietary supplementation with CWP may be an effective strategy to prevent serious complications from HS.

Depression of Pyroptosis by Inhibiting Caspase-1 Activation Improves Neurological Outcomes of Kernicterus Model Rats.

Kernicterus is a severe complication of extreme neonatal hyperbilirubinemia. Prolonged exposure to high-level unconjugated bilirubin (UCB) directly damages brain tissue. Neuroinflammation is believed to contribute to UCB-induced neurotoxicity. Pyroptosis has been as a highly inflammatory form of programmed cell death. Therefore, this study aimed to explore whether pyroptosis was involved in the pathogenesis of UCB neurotoxicity in kernicterus model rats. VX-765, a specific inhibitor of caspase-1, was intraperitoneally administered to the model rats to observe its effects on the short-term and long-term outcomes of the model animals at the molecular, cellular, morphological, and behavioral levels. The results indicated that UCB significantly induced the activation of caspase-1 and gasdermin D(GSDMD), and VX-765 inhibited caspase-1-GSDMD pathway. Compared with those of the UCB group and the vehicle+UCB group, VX-765-treated rats released lower levels of IL-1β and IL-18. Furthermore, H&E and TUNEL staining showed that nerve cells in the VX-765-treated group were better preserved and had less DNA fragmentation. Most importantly, VX-765 improved both the short-term and long-term neurological functions of kernicterus model rats. This study demonstrated that pyroptosis was involved in the pathogenesis of kernicterus through caspase-1 activation, which could be inhibited by VX-765, exerting a neuroprotective effect in kernicterus model rats.

Seneca Valley Virus 3C Protease Induces Pyroptosis by Directly Cleaving Porcine Gasdermin D.

Seneca Valley virus (SVV), a newly emerging virus belonging to the Picornaviridae family, has caused vesicular disease in the swine industry. However, the molecular mechanism of viral pathogenesis remains poorly understood. This study revealed that SVV infection could induce pyroptosis in SK6 cells in a caspase-dependent and -independent manner. SVV may inhibit caspase-1 activation at late infection because of 3Cpro cleavage of NLRP3, which counteracted pyroptosis activation. Further study showed that 3Cpro targeted porcine gasdermin D (pGSDMD) for cleavage through its protease activity. 3Cpro cleaved porcine GSDMD (pGSDMD) at two sites, glutamine 193 (Q193) and glutamine 277 (Q277), and Q277 was close to the caspase-1-induced pGSDMD cleavage site. pGSDMD1-277 triggered cell death, which was similar to N-terminal fragment produced by caspase-1 cleavage of pGSDMD, and other fragments exhibited no significant inhibitory effects on cellular activity. Ectopic expression of pGSDMD converted 3Cpro-induced apoptosis to pyroptosis in 293T cells. Interestingly, 3Cpro did not cleave mouse GSDMD or human GSDMD. And, both pGSDMD and pGSDMD1-277 exhibited bactericidal activities in vivo. Nevertheless, pGSDMD cannot kill bacteria in vitro. Taken together, our results reveal a novel pyroptosis activation manner produced by viral protease cleavage of pGSDMD, which may provide an important insight into the pathogenesis of SVV and cancer therapy.

1'-Acetoxychavicol acetate inhibits NLRP3-dependent inflammasome activation via mitochondrial ROS suppression.

The nucleotide-binding oligomerization domain-like receptor (NLR) family pyrin domain containing (NLRP) 3 inflammasome is a multiprotein complex that triggers Caspase-1-mediated IL-1β production and pyroptosis, and its dysregulation is associated with the pathogenesis of inflammatory diseases. 1'-Acetoxychavicol acetate (ACA) is a natural compound in the rhizome of tropical ginger Alpinia species with anti-microbial, anti-allergic and anti-cancer properties. In this study, we found that ACA suppressed NLRP3 inflammasome activation in mouse bone marrow-derived macrophages and human THP-1 monocytes. ACA inhibited Caspase-1 activation and IL-1β production by NLRP3 agonists such as nigericin, monosodium urate (MSU) crystals, and ATP. Moreover, it suppressed oligomerization of the adapter molecule, apoptosis-associated speck-like protein containing a CARD (ASC), and Caspase-1-mediated cleavage of pyroptosis executor Gasdermin D. Mechanistically, ACA inhibited generation of mitochondrial reactive oxygen species (ROS) and prevented release of oxidized mitochondrial DNA, which trigger NLRP3 inflammasome activation. ACA also prevented NLRP3 inflammasome activation in vivo, as evidenced in the MSU crystal-induced peritonitis and dextran sodium sulfate-induced colitis mouse models accompanied by decreased Caspase-1 activation. Thus, ACA is a potent inhibitor of the NLRP3 inflammasome for prevention of NLRP3-associated inflammatory diseases.

Epigallocatechin-3-gallate prevents inflammation and diabetes -Induced glucose tolerance through inhibition of NLRP3 inflammasome activation

Epigallocatechin-3-gallate (EGCG), the primary polyphenol component of green tea, has been shown to inhibit both oxidation and inflammation. However, the exact mechanism through which EGCG exhibits anti-inflammatory effects remains unclear. In this study, we assessed the potential pathways by which EGCG regulates NLRP3 inflammasome activity in vitro. We found that EGCG inhibits caspase-1 activation and IL-1β secretion by suppressing NLRP3 inflammasome activation in mouse bone marrow-derived macrophages (BMDMs). EGCG was also observed to block NLRP3-mediated ASC speckle formation and to alleviate pyroptosis in BMDMs. In addition, EGCG treatment could improve high-fat diet (HFD)-induced glucose tolerance and prevent NLRP3 inflammasome-dependent inflammation in a mouse model of HFD-induced type 2 diabetes (T2D). Taken together, our results show that EGCG is a general inhibitor of NLRP3 inflammasome activation and administration of EGCG in T2D mice could improve glucose tolerance in vivo.

Koumine Suppresses IL-1β Secretion and Attenuates Inflammation Associated With Blocking ROS/NF-κB/NLRP3 Axis in Macrophages

Koumine (KM), one of the primary constituents of Gelsemium elegans, has been used for the treatment of inflammatory diseases such as rheumatoid arthritis, but whether KM impacts the activation of the NOD-like receptor protein 3 (NLRP3) inflammasome remains unknown. This study aimed to explore the inhibitory effect of KM on NLRP3 inflammasome activation and the underlying mechanisms both in vitro using macrophages stimulated with LPS plus ATP, nigericin or monosodium urate (MSU) crystals and in vivo using an MSU-induced peritonitis model. We found that KM dose-dependently inhibited IL-1β secretion in macrophages after NLRP3 inflammasome activators stimulation. Furthermore, KM treatment efficiently attenuated the infiltration of neutrophils and suppressed IL-1β production in mice with MSU-induced peritonitis. These results indicated that KM inhibited NLRP3 inflammasome activation, and consistent with this finding, KM effectively inhibited caspase-1 activation, mature IL-1β secretion, NLRP3 formation and pro-IL-1β expression in LPS-primed macrophages treated with ATP, nigericin or MSU. The mechanistic study showed that, KM exerted a potent inhibitory effect on the NLRP3 priming step, which decreased the phosphorylation of IκBα and p65, the nuclear localization of p65, and the secretion of TNF-α and IL-6. Moreover, the assembly of NLRP3 was also interrupted by KM. KM blocked apoptosis-associated speck-like protein containing a CARD (ASC) speck formation and its oligomerization and hampered the NLRP3-ASC interaction. This suppression was attributed to the ability of KM to inhibit the production of reactive oxygen species (ROS). In support of this finding, the inhibitory effect of KM on ROS production was completely counteracted by H2O2, an ROS promoter. Our results provide the first indication that KM exerts an inhibitory effect on NLRP3 inflammasome activation associated with blocking the ROS/NF-κB/NLRP3 signal axis. KM might have potential clinical application in the treatment of NLRP3 inflammasome-related diseases.

VX-765 reduces neuroinflammation after spinal cord injury in mice.

Inflammation is a major cause of neuronal injury after spinal cord injury. We hypothesized that inhibiting caspase-1 activation may reduce neuroinflammation after spinal cord injury, thus producing a protective effect in the injured spinal cord. A mouse model of T9 contusive spinal cord injury was established using an Infinite Horizon Impactor, and VX-765, a selective inhibitor of caspase-1, was administered for 7 successive days after spinal cord injury. The results showed that: (1) VX-765 inhibited spinal cord injury-induced caspase-1 activation and interleukin-1β and interleukin-18 secretion. (2) After spinal cord injury, an increase in M1 cells mainly came from local microglia rather than infiltrating macrophages. (3) Pro-inflammatory Th1Th17 cells were predominant in the Th subsets. VX-765 suppressed total macrophage infiltration, M1 macrophages/microglia, Th1 and Th1Th17 subset differentiation, and cytotoxic T cells activation; increased M2 microglia; and promoted Th2 and Treg differentiation. (4) VX-765 reduced the fibrotic area, promoted white matter myelination, alleviated motor neuron injury, and improved functional recovery. These findings suggest that VX-765 can reduce neuroinflammation and improve nerve function recovery after spinal cord injury by inhibiting caspase-1/interleukin-1β/interleukin-18. This may be a potential strategy for treating spinal cord injury. This study was approved by the Animal Care Ethics Committee of Bengbu Medical College (approval No. 2017-037) on February 23, 2017.