Caspase-1 Inhibitor VX-765 Research Articles

Age-dependent aggregation of α-synuclein in the nervous system of gut-brain axis is associated with caspase-1 activation.

α-Synuclein (α-Syn) plays a key role in the development of Parkinson' desease (PD). As aging is acknowledged to be the greatest risk factor for PD, here we investigated α-Syn expression in the ileum, thoracic spinal cord, and midbrain of young (1-month-old), middle-aged (6-, 12-month-old) to old (18-month-old) mice. We demonstrated that both the levels of α-Syn monomers, oligomers and ratios of oligomers to monomers were increased with aging in the ileum, thoracic spinal cord, and midbrain. Whereas, the expression of tyrosine hydroxylase (TH), the rate-limiting enzyme for dopamine synthesis, was decreased with aging in the midbrain. We failed to find corresponding α-Syn mRNA increase with aging. However, we found an increased expression of caspase-1 in the ileum, thoracic spinal cord, and midbrain. A specific caspase-1 inhibitor VX765 significantly reduced levels of both the α-Syn monomers and oligomers triggered by the rotenone in vitro. Taken together, the increase in α-Syn aggregation with aging might not occur first in the gut, but simultaneously in the nervous system of gut-brain axis. The mechanism of the age-dependent aggregation of α-Syn in nervous system is likely triggered by the aging-related caspase-1 activation.

Clozapine Induces an Acute Proinflammatory Response That Is Attenuated by Inhibition of Inflammasome Signaling: Implications for Idiosyncratic Drug-Induced Agranulocytosis.

Although clozapine is a highly efficacious schizophrenia treatment, it is under-prescribed due to the risk of idiosyncratic drug-induced agranulocytosis (IDIAG). Clinical data indicate that most patients starting clozapine experience a transient immune response early in treatment and a similar response has been observed in clozapine-treated rats, but the mechanism by which clozapine triggers this transient inflammation remains unclear. Therefore, the aim of this study was to characterize the role of inflammasome activation during the early immune response to clozapine using in vitro and in vivo models. In both differentiated and nondifferentiated human monocytic THP-1 cells, clozapine, but not its structural analogues fluperlapine and olanzapine, caused inflammasome-dependent caspase-1 activation and IL-1β release that was inhibited using the caspase-1 inhibitor yVAD-cmk. In Sprague Dawley rats, a single dose of clozapine caused an increase in circulating neutrophils and a decrease in lymphocytes within hours of drug administration along with transient spikes in the proinflammatory mediators IL-1β, CXCL1, and TNF-α in the blood, spleen, and bone marrow. Blockade of inflammasome signaling using the caspase-1 inhibitor VX-765 or the IL-1 receptor antagonist anakinra attenuated this inflammatory response. These data indicate that caspase-1-dependent IL-1β production is fundamental for the induction of the early immune response to clozapine and, furthermore, support the general hypothesis that inflammasome activation is a common mechanism by which drugs associated with the risk of idiosyncratic reactions trigger early immune system activation. Ultimately, inhibition of inflammasome signaling may reduce the risk of IDIAG, enabling safer, more frequent use of clozapine in patients.

Bisphenol A induces pyroptotic cell death via ROS/NLRP3/Caspase-1 pathway in osteocytes MLO-Y4

Bisphenol A (BPA), a ubiquitous endocrine-disrupting chemical, is commonly used as a plasticizer to manufacture various food packaging materials. Evidence has demonstrated that BPA disturbed bone health. However, few studies focused on the effect of BPA on osteocytes, making up over 95% of all the bone cells. Here, we reported that BPA inhibited the cell viability of MLO-Y4 cells, and increased apoptosis in a dose-dependent manner. Furthermore, BPA up-regulated protein expressions of speck-like protein containing CARD (ASC), NLRP3, cleaved caspase-1 (Casp-1 p20) and cleaved gasdermin D (GSDMD-N), and increased the ratios of interleukin (IL)-1β/pro-IL-1β and IL-18/pro-IL-18 in MLO-Y4 cells. BPA enhanced levels of lactate dehydrogenase (LDH), IL-1β and IL-18 in culture supernatants. This pyroptotic death and the NLPR3 inflammasome activation were reversed by the caspase-1 inhibitor VX765 or the NLRP3 inflammasome inhibitor MCC950. Furthermore, BPA stimulated the production of intracellular reactive oxygen species (ROS), mitochondrial ROS (mtROS), elevated malondialdehyde (MDA) level and decreased superoxide dismutase (SOD) activity, which led to oxidative damage in MLO-Y4 cells. The ROS scavenger N-acetylcysteine (NAC) or the mitochondrial antioxidant Mito-TEMPO inhibited the NLPR3 inflammasome activation and pyroptotic death induced by BPA. Collectively, our data suggest that BPA causes pyroptotic death of osteocytes via ROS/NLRP3/Caspase-1 pathway.

Blockade of the NLRP3/caspase-1 axis attenuates ketamine-induced hippocampus pyroptosis and cognitive impairment in neonatal rats

BackgroundMultiple studies have revealed that repeated or long-term exposure to ketamine causes neurodegeneration and cognitive dysfunction. Pyroptosis is an inflammatory form of programmed cell death that has been linked to various neurological diseases. However, the role of NLRP3/caspase-1 axis-related pyroptosis in ketamine-induced neurotoxicity and cognitive dysfunction remains uncertain.MethodsTo evaluate whether ketamine caused NLRP3/caspase1-dependent pyroptosis, flow cytometry analysis, western blotting, ELISA test, histopathological analysis, Morris water maze (MWM) test, cell viability assay, and lactate dehydrogenase release (LDH) assay were carried out on PC12 cells, HAPI cells, and 7-day-old rats. In addition, the NLRP3 inhibitor MCC950 or the caspase-1 inhibitor VX-765 was used to investigate the role of the NLRP3/caspase-1 axis in ketamine-induced neurotoxicity and cognitive dysfunction.ResultsOur findings demonstrated that ketamine exposure caused cell damage and increased the levels of pyroptosis in PC12 cells, HAPI cells, and the hippocampus of neonatal rats. After continuous exposure to ketamine, targeting NLRP3 and caspase-1 with MCC950 or VX765 improved pyroptosis, reduced neuropathological damages, and alleviated cognitive dysfunction.ConclusionNLRP3/Caspase-1 axis-dependent pyroptosis is involved in ketamine-induced neuroinflammation and cognitive dysfunction, and it provides a promising strategy to treat ketamine-related neurotoxicity.

Transduction of inflammation from peripheral immune cells to the hippocampus induces neuronal hyperexcitability mediated by Caspase-1 activation

BackgroundRecent studies report infiltration of peripheral blood mononuclear cells (PBMCs) into the central nervous system (CNS) in epileptic disorders, suggestive of a potential contribution of PBMC extravasation to the generation of seizures. Nevertheless, the underlying mechanisms involved in PBMC infiltrates promoting neuronal predisposition to ictogenesis remain unclear. Therefore, we developed an in vitro model mimicking infiltration of activated PBMCs into the brain in order to investigate potential transduction of inflammatory signals from PBMCs to the CNS. MethodsTo establish our model, we first extracted PBMCs from rat spleen, then, immunologically primed PBMCs with lipopolysaccharide (LPS), followed by further activation with nigericin. Thereafter, we co-cultured these activated PBMCs with organotypic cortico-hippocampal brain slice cultures (OCHSCs) derived from the same rat, and compared PBMC-OCHSC co-cultures to OCHSCs exposed to PBMCs in the culture media. We further targeted a potential molecular pathway underlying transduction of peripheral inflammation to OCHSCs by incubating OCHSCs with the Caspase-1 inhibitor VX-765 prior to co-culturing PBMCs with OCHSCs. After 24 h, we analyzed inflammation markers in the cortex and the hippocampus using semiquantitative immunofluorescence. In addition, we analyzed neuronal activity by whole-cell patch-clamp recordings in cortical layer II/III and hippocampal CA1 pyramidal neurons. ResultsIn the cortex, co-culturing immunoreactive PBMCs treated with LPS + nigericin on top of OCHSCs upregulated inflammatory markers and enhanced neuronal excitation. In contrast, no excitability changes were detected after adding primed PBMCs (i.e. treated with LPS only), to OCHSCs. Strikingly, in the hippocampus, both immunoreactive and primed PBMCs elicited similar pro-inflammatory and pro-excitatory effects. However, when immunoreactive and primed PBMCs were cultured in the media separately from OCHSCs, only immunoreactive PBMCs gave rise to neuroinflammation and hyperexcitability in the hippocampus, whereas primed PBMCs failed to produce any significant changes. Finally, VX-765 application to OCHSCs, co-cultured with either immunoreactive or primed PBMCs, prevented neuroinflammation and hippocampal hyperexcitability in OCHSCs. ConclusionsOur study shows a higher susceptibility of the hippocampus to peripheral inflammation as compared to the cortex, mediated via Caspase-1-dependent signaling pathways. Thus, our findings suggest that Caspase-1 inhibition may potentially provide therapeutic benefits during hippocampal neuroinflammation and hyperexcitability secondary to peripheral innate immunity.

NLR family pyrin domain containing 3 (NLRP3) and caspase 1 (CASP1) modulation by intracellular Cl- concentration.

The impairment of the cystic fibrosis transmembrane conductance regulator (CFTR) activity induces intracellular chloride (Cl- ) accumulation. The anion Cl- , acting as a second messenger, stimulates the secretion of interleukin-1β (IL-1β), which starts an autocrine positive feedback loop. Here, we show that NLR family pyrin domain containing 3 (NLRP3) and caspase 1 (CASP1) are indirectly modulated by the intracellular Cl- concentration, showing maximal expression and activity at 75mM Cl- , in the presence of the ionophores nigericin and tributyltin. The expression of PYD and CARD domain containing (PYCARD/ASC) remained constant from 0 to 125mM Cl- . The CASP1 inhibitor VX-765 and the NLRP3 inflammasome inhibitor MCC950 completely blocked the Cl- -stimulated IL-1β mRNA expression and partially the IL-1β secretion. DCF fluorescence (cellular reactive oxygen species, cROS) and MitoSOX fluorescence (mitochondrial ROS, mtROS) also showed maximal ROS levels at 75mM Cl- , a response strongly inhibited by the ROS scavenger N-acetyl-L-cysteine (NAC) or the NADPH oxidase (NOX) inhibitor GKT137831. These inhibitors also affected CASP1 and NLRP3 mRNA and protein expression. More importantly, the serum/glucocorticoid regulated kinase 1 (SGK1) inhibitor GSK650394, or its shRNAs, completely abrogated the IL-1β mRNA response to Cl- and the IL-1β secretion, interrupting the autocrine IL-1β loop. The results suggest that Cl- effects are mediated by SGK1, in which under Cl- modulation stimulates the secretion of mature IL-1β, in turn, responsible for the upregulation of ROS, CASP1, NLRP3 and IL-1β itself, through autocrine signalling.

Kupffer cell-targeting strategy for the protection of hepatic ischemia/reperfusion injury

The aim of this study is to evaluate the effect of rare earth upconversion nanoparticles (UCNs) on hepatic ischemia reperfusion injury (IRI) and explore its possible mechanism. Hepatic IRI seriously affects the prognosis of patients undergoing liver surgery. Liver-resident Kupffer cells have been reported to promote IRI. Nanomedicines are known to be effective in the treatment of liver diseases, however, Kupffer cell-targeting nanomedicines for the treatment of IRI are yet to be developed. As potential bioimaging nanomaterials, UCNs have been found to specifically deplete Kupffer cells, but the underlying mechanism is unknown. In this study, we found that UCNs specifically depleted Kupffer cells by pyroptosis, while the co-administration of the caspase-1 inhibitor VX-765 rescued the UCN-induced Kupffer cell pyroptosis in mice. Furthermore, the pre-depletion of Kupffer cells by the UCNs significantly suppressed the release of inflammatory cytokines and effectively improved hepatic IRI. The rescue of the pyroptosis of the Kupffer cells by VX-765 abrogated the protective effect of UCNs on the liver. These results suggest that UCNs are highly promising for the development of Kupffer cell-targeting nanomedicines for intraoperative liver protection.

Mint3 depletion-mediated glycolytic and oxidative alterations promote pyroptosis and prevent the spread of Listeria monocytogenes infection in macrophages

Listeria monocytogenes (LM) infection induces pyroptosis, a form of regulated necrosis, in host macrophages via inflammasome activation. Here, we examined the role of Mint3 in macrophages, which promotes glycolysis via hypoxia-inducible factor-1 activation, during the initiation of pyroptosis following LM infection. Our results showed that Mint3-deficient mice were more resistant to lethal listeriosis than wild-type (WT) mice. Additionally, the mutant mice showed higher levels of IL-1β/IL-18 in the peritoneal fluid during LM infection than WT mice. Moreover, ablation of Mint3 markedly increased the activation of caspase-1, maturation of gasdermin D, and pyroptosis in macrophages infected with LM in vitro, suggesting that Mint3 depletion promotes pyroptosis. Further analyses revealed that Mint3 depletion upregulates inflammasome assembly preceding pyroptosis via glycolysis reduction and reactive oxygen species production. Pharmacological inhibition of glycolysis conferred resistance to listeriosis in a Mint3-dependent manner. Moreover, Mint3-deficient mice treated with the caspase-1 inhibitor VX-765 were as susceptible to LM infection as WT mice. Taken together, these results suggest that Mint3 depletion promotes pyroptosis in host macrophages, thereby preventing the spread of LM infection. Mint3 may serve as a target for treating severe listeriosis by inducing pyroptosis in LM-infected macrophages.

Airborne particulate matter (PM2.5) triggers ocular hypertension and glaucoma through pyroptosis

BackgroundParticulate matter (PM) is strongly linked to human health and has detrimental effects on the eye. Studies have, however, focused on the ocular surface, with limited research on the impact of PM2.5 on intraocular pressure (IOP).MethodsTo investigate the impact of PM2.5 on IOP and the associated mechanism, C57BL/6 mouse eyes were topically exposed to a PM2.5 suspension for 3 months, and human trabecular meshwork (HTM) cells were subjected to various PM2.5 concentrations in vitro. Cell viability, NLRP3/caspase-1, IL-1β, and GSDMD expression, reactive oxygen species (ROS) production and cell contractility were measured by western blot, ELISA, cell counting kit-8, ROS assay kit or a cell contractility assay. ROS scavenger N-acetyl-L-cysteine (NAC) and caspase-1 inhibitor VX-765 were used to intervene in PM2.5-induced damages.ResultsThe results revealed that the IOP increased gradually after PM2.5 exposure, and upregulations of the NLRP3 inflammasome, caspase-1, IL-1β, and GSDMD protein levels were observed in outflow tissues. PM2.5 exposure decreased HTM cell viability and affected contraction. Furthermore, elevated ROS levels were observed as well as an activation of the NLRP3 inflammasome and downstream inflammatory factors caspase-1 and IL-1β. NAC improved HTM cell viability, inhibited the activation of the NLRP3 inflammasome axis, and HTM cell contraction by scavenging ROS. VX-765 showed similar protection against the PM2.5 induced adverse effects.ConclusionThis study provides novel evidence that PM2.5 has a direct toxic effect on intraocular tissues and may contribute to the initiation and development of ocular hypertension and glaucoma. This occurs as a result of increased oxidative stress and the subsequent induction of NLRP3 inflammasome mediated pyroptosis in trabecular meshwork cells.

Peroxynitrite activates NLRP3 inflammasome and contributes to hemorrhagic transformation and poor outcome in ischemic stroke with hyperglycemia

This study aims to test the hypothesis that peroxynitrite-mediated inflammasome activation could be a crucial player in the blood-brain barrier (BBB) disruption, hemorrhagic transformation (HT) and poor outcome in ischemic stroke with hyperglycemia. We used an experimental rat stroke model subjected to 90 min of middle cerebral artery occlusion plus 24 h or 7 days of reperfusion with or without acute hyperglycemia. We detected the production of peroxynitrite, the expression of NADPH oxidase, iNOS, MMPs and NLRP3 inflammasome in the ischemic brains, and evaluated infarct volume, brain edema, HT, neurological deficit score and survival rates. Our results show that: (1) Hyperglycemia increased the expression of NADPH oxidase subunits p47phox and p67phox, and iNOS, and the production of peroxynitrite. (2) Hyperglycemia increased infarct volume, aggravated the BBB hyperpermeability, induced brain edema and HT, and worsened neurological outcomes. These brain damages and poor outcome were reversed by the treatments of FeTmPyP (a representative peroxynitrite decomposition catalyst, PDC), peroxynitrite scavenger uric acid, and iNOS inhibitor 1400W. Furthermore, the activations of MMPs and NLRP3 inflammasome including pro/active-caspase-1 and IL-1β were inhibited both PDC and 1400W, indicating the roles of peroxynitrite in the inductions of MMPs and NLRP3 inflammasome in the ischemic brains under hyperglycemia. (3) NLRP3 inflammasome inhibitor MCC950, caspase-1 inhibitor VX-765 and IL-1β inhibitor diacerein attenuated brain edema, minimized hemorrhagic transformation and improved neurological outcome, demonstrating the roles of NLRP3 inflammasome in the hyperglycemia-mediated HT and poor outcome in the ischemic stroke rats with acute hyperglycemia. In conclusion, peroxynitrite could mediate activations of MMPs and NLRP3 inflammasome, aggravate the BBB damage and HT, and induce poor outcome in ischemic stroke with hyperglycemia. Therefore, targeting peroxynitrite–mediated NLRP3 inflammasome could be a promising strategy for ischemic stroke with hyperglycemia.

ROS/NLRP3/Caspase-1 Pathway Contributes to TCP Particles-Induced Pyroptosis of Calvaria Osteocytes in the Mouse Osteolysis Model

Wear particles-induced inflammatory osteolysis, a major contributing factor of aseptic loosening, affects long-term survival of orthopedic prostheses. Increasing observations have demonstrated that osteocytes death is involved in wear particles-induced periprosthetic osteolysis, but the underling molecular mechanisms remain unclear. We chose micro-sized tricalcium phosphate (TCP) particles to simulate TCP wear debris from loosened TCP implants, which have been widely used as bone substitute biomaterials in orthopedic and dental surgery for almost 40 years, and investigated the direct biological effects of the particles on calvaria osteocytes in vivo . Results showed that TCP wear particles triggered nod-like receptor protein 3 (NLRP3) inflammasome activation and pyroptosis in calvaria osteocytes, as indicated by increased osteocytes death, up-regulation of NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), cleaved Caspase-1 and interleukin (IL)-1β, elevation of IL-1β, IL-18 and lactic dehydrogenase (LDH), which were all inhibited by the Caspase-1 inhibitor VX765. Moreover, the NLRP3 inhibitor MCC950 reduced TCP particles-induced Caspase-1 cleavage, production of IL-18, IL-1β and LDH, and pyroptotic death in calvaria osteocytes. Further experiments revealed that the NLRP3-mediated pyroptosis pathway was mediated by reactive oxygen species (ROS) in TCP particles-induced cell death of calvaria osteocytes, since a ROS scavenger (N-acetyl-cysteine, NAC) efficiently prevented NLRP3 inflammasome activation and pyroptosis. Taken together, TCP wear particles induce pyroptosis of calvaria osteocytes through activation of ROS/NLRP3/Caspase-1 pathway, which is involved in osteoclastogenesis and periprosthetic osteolysis. Our findings strongly suggest that pyroptosis may play a significant role in wear particles-induced cell death of osteocytes and periprosthetic osteolysis, and will provide a novel evidence for elucidating its pathophysiology.

Autophagic Degradation of Gasdermin D Protects against Nucleus Pulposus Cell Pyroptosis and Retards Intervertebral Disc Degeneration In Vivo.

Intervertebral disc degeneration (IDD) is the primary culprit of low back pain and renders heavy social burden worldwide. Pyroptosis is a newly discovered form of programmed cell death, which is also involved in nucleus pulposus (NP) cells during IDD progression. Moderate autophagy activity is critical for NP cell survival, but its relationship with pyroptosis remains unknown. This study is aimed at investigating the relationship between autophagy and pyroptotic cell death. The pyroptosis executor N-terminal domain of gasdermin D (GSDMD-N) and inflammation-related proteins were measured in lipopolysaccharide- (LPS-) treated human NP cells. Inhibition of autophagy by siRNA transfection and chemical drugs aggravated human NP cell pyroptosis. Importantly, we found that the autophagy-lysosome pathway and not the proteasome pathway mediated the degradation of GSDMD-N as lysosome dysfunction promoted the accumulation of cytoplasmic GSDMD-N. Besides, P62/SQSTM1 colocalized with GSDMD-N and mediated its degradation. The administration of the caspase-1 inhibitor VX-765 could reduce cell pyroptosis as confirmed in a rat disc IDD model in vivo, whereas ATG5 knockdown significantly accelerated the progression of IDD. In conclusion, our study indicated that autophagy protects against LPS-induced human NP cell pyroptosis via a P62/SQSTM1-mediated degradation mechanism and the inhibition of pyroptosis retards IDD progression in vivo. These findings deepen the understanding of IDD pathogenesis and hold implications in unraveling therapeutic targets for IDD treatment.

Neural progenitor cell pyroptosis contributes to Zika virus-induced brain atrophy and represents a therapeutic target

Mounting evidence has associated Zika virus (ZIKV) infection with congenital malformations, including microcephaly, which raises global alarm. Nonetheless, mechanisms by which ZIKV disrupts neurogenesis and causes microcephaly are far from being understood. In this study, we discovered direct effects of ZIKV on neural progenitor cell development by inducing caspase-1- and gasdermin D (GSDMD)-mediated pyroptotic cell death, linking ZIKV infection with the development of microcephaly. Importantly, caspase-1 depletion or its inhibitor VX-765 treatment reduced ZIKV-induced inflammatory responses and pyroptosis, and substantially attenuated neuropathology and brain atrophy in vivo. Collectively, our data identify caspase-1- and GSDMD-mediated pyroptosis in neural progenitor cells as a previously unrecognized mechanism for ZIKV-related pathological effects during neural development, and also provide treatment options for ZIKV-associated diseases.

VX-765 attenuates atherosclerosis in ApoE deficient mice by modulating VSMCs pyroptosis

Background and aimsRecent clinical evidences show that patients with atherosclerotic cardiovascular disease can benefit from a targeting IL-1β treatment. Caspase-1 is an important factor for pyroptosis and is responsible for mature and release of interleukin (IL)-1β. Here we investigated the effect of caspase-1 inhibitor VX-765 on atherosclerosis and vascular smooth muscle cells (VSMCs) pyroptosis. MethodsHuman carotid artery plaques and aortas from ApoE−/− mice which were gavaged with VX-765 or vehicle while fed with western diet were examined for plaque burden using Oil Red O staining and Immunohistochemistry staining. Dedifferentiated primary cultured mice VSMCs treated with oxidized low-density lipoprotein (OxLDL) were applied to examine cell pyroptosis. ResultsThe distribution of a-SMA and active pyroptotic indicators had a lot of overlaps near the necrotic core, at the lesion surface and in the intra-plaque hemorrhage area in human or mice plaque. In vitro studies further demonstrated that OxLDL induced VSMCs pyroptosis through activating NLRP3 inflammasome. What's more, VX-765 significantly inhibited the progression of established atheroma and the development of atherosclerosis, without substantially influence lipoprotein level in plasma. VX-765 also significantly reduced VSMCs pyroptosis and IL-1β processing induced by OxLDL. ConclusionsVX-765 inhibits VSMCs pyroptosis during atherogenesis and targeting caspase-1 activity may be a potential treatment strategy for atherosclerotic diseases.

Caspase-1 inhibitor exerts brain-protective effects against sepsis-associated encephalopathy and cognitive impairments in a mouse model of sepsis

Sepsis-associated encephalopathy (SAE) manifested clinically in acute and long-term cognitive impairments and associated with increased morbidity and mortality worldwide. The potential pathological changes of SAE are complex and remain to be elucidated. Pyroptosis, a novel programmed cell death, is executed by caspase-1-cleaved GSDMD N-terminal (GSDMD-NT) and we investigated it in peripheral blood immunocytes of septic patients previously. Here, a caspase-1 inhibitor VX765 was treated with CLP-induced septic mice. Novel object recognition test indicated that VX765 treatment reversed cognitive dysfunction in septic mice. Elevated plus maze, tail suspension test and open field test revealed that depressive-like behaviors of septic mice were relieved. Inhibited caspase-1 suppressed the expressions of GSDMD and its cleavage form GSDMD-NT, and reduced pyroptosis in brain at day 1 and day 7 after sepsis. Meantime, inhibited caspase-1 mitigated the expressions of IL-1β, MCP-1 and TNF-α in serum and brain, diminished microglia activation in septic mice, and reduced sepsis-induced brain-blood barrier disruption and ultrastructure damages in brain as well. Inhibited caspase-1 protected the synapse plasticity and preserved long-term potential, which may be the possible mechanism of cognitive functions protective effects of septic mice. In conclusion, caspase-1 inhibition exerts brain-protective effects against SAE and cognitive impairments in a mouse model of sepsis.

AIM2 promotes non-small-cell lung cancer cell growth through inflammasome-dependent pathway.

The human absent in melanoma 2 (AIM2) is considered as a DNA recognizer. AIM2 has been described as a tumor suppressor gene in the early years. But recent studies suggested that it functions as an oncogene in several cancers. However, its roles in non-small-cell lung cancer (NSCLC) remain unclear. Here we reported that AIM2 highly expressed in NSCLC cells and exhibited a tumor-promoting property both in vitro and in vivo. Besides, AIM2 short hairpin RNA (shRNA)-mediated suppression of cell proliferation was triggered by the accumulation of cells at the G2/M phase. Knockdown of AIM2 reduced the inflammasome formation, while overexpression of AIM2 or stimulation by poly(dA:dT) induced the inflammasome formation. Interestingly, blockade of the inflammasome by caspase-1 inhibitor VX-765 or ASC small interfering RNA (siRNA) abolished the effects brought by AIM2 shRNA and AIM2 plasmid. In summary, our results revealed that AIM2 functioned as an oncogene in NSCLC in an inflammasome-dependent way.

Caspase-1 inhibition alleviates cognitive impairment and neuropathology in an Alzheimer\u2019s disease mouse model

Alzheimer's disease (AD) is an intractable progressive neurodegenerative disease characterized by cognitive decline and dementia. An inflammatory neurodegenerative pathway, involving Caspase-1 activation, is associated with human age-dependent cognitive impairment and several classical AD brain pathologies. Here, we show that the nontoxic and blood–brain barrier permeable small molecule Caspase-1 inhibitor VX-765 dose-dependently reverses episodic and spatial memory impairment, and hyperactivity in the J20 mouse model of AD. Cessation of VX-765 results in the reappearance of memory deficits in the mice after 1 month and recommencement of treatment re-establishes normal cognition. VX-765 prevents progressive amyloid beta peptide deposition, reverses brain inflammation, and normalizes synaptophysin protein levels in mouse hippocampus. Consistent with these findings, Caspase-1 null J20 mice are protected from episodic and spatial memory deficits, neuroinflammation and Aβ accumulation. These results provide in vivo proof of concept for Caspase-1 inhibition against AD cognitive deficits and pathologies.

Caspase-1 inhibition by VX-765 administered at reperfusion in P2Y12 receptor antagonist-treated rats provides long-term reduction in myocardial infarct size and preservation of ventricular function.

Patients with acute myocardial infarction receive a P2Y12 receptor antagonist prior to reperfusion, a treatment that has reduced, but not eliminated, mortality, or heart failure. We tested whether the caspase-1 inhibitor VX-765 given at reperfusion (a requirement for clinical use) can provide sustained reduction of infarction and long-term preservation of ventricular function in a pre-clinical model of ischemia/reperfusion that had been treated with a P2Y12 receptor antagonist. To address, the hypothesis open-chest rats were subjected to 60-min left coronary artery branch occlusion/120-min reperfusion. Vehicle or inhibitors were administered intravenously immediately before reperfusion. With vehicle only, 60.3 ± 3.8% of the risk zone suffered infarction. Ticagrelor, a P2Y12 antagonist, and VX-765 decreased infarct size to 42.8 ± 3.3 and 29.2 ± 4.9%, respectively. Combining ticagrelor with VX-765 further decreased infarction to 17.5 ± 2.3%. Similar to recent clinical trials, combining ticagrelor and ischemic postconditioning did not result in additional cardioprotection. VX-765 plus another P2Y12 antagonist, cangrelor, also decreased infarction and preserved ventricular function when reperfusion was increased to 3days. In addition, VX-765 reduced infarction in blood-free, isolated rat hearts indicating at least a portion of injurious caspase-1 activation originates in cardiac tissue. While the pro-drug VX-765 only protected isolated hearts when started prior to ischemia, its active derivative VRT-043198 provided the same amount of protection when started at reperfusion, indicating that even in blood-free hearts, caspase-1 appears to exert its injury only at reperfusion. Moreover, VX-765 decreased circulating IL-1β, prevented loss of cardiac glycolytic enzymes, preserved mitochondrial complex I activity, and decreased release of lactate dehydrogenase, a marker of pyroptosis. Our results are the first demonstration of a clinical-grade drug given at reperfusion providing additional, sustained infarct size reduction when added to a P2Y12 receptor antagonist.

Effects of caspase-1 inhibitor VX765 on cold-restraint stress-induced acute gastric ulcer in mice

Objective: To investigate the protective effects of caspase-1 inhibitor VX765 on gastric mucosa of mice with cold-restraint stress-induced acute gastric ulcer. Methods: Twenty-four specific pathogen free male C57BL/6J mice were divided into normal control group (NC), cold restrain group (CR), VX765 pre-treatment+ cold restrain group (VCR), and rabeprazole pre-treatment+ cold restrain group (RCR) according to the random number table, with 6 mice in each group. Mice in group NC were injected intraperitoneally with solution of 10 mL/kg dimethylsulfoxide (DMSO) and phosphate buffer solution (PBS). Mice in group CR were inflicted with acute gastric ulcer induced by cold-restraint stress 30 minutes after intraperitoneal injection of solution of DMSO and PBS. Mice in groups VCR and RCR were inflicted with acute gastric ulcer as above 30 minutes after intraperitoneal injection of solution of DMSO and PBS with dose of 12.5 μmol/kg containing 10 mg VX765 and 40 mg/kg containing 20 mg rabeprazole, respectively. Four hour after cold-restraint stress, serum content of tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6)was determined by enzyme-linked immunosorbent assay. Gross condition of gastric tissue was observed. Ulcer index was evaluated. Pathological change of gastric tissue was observed with HE staining. The relative expression of IL-1β, IL-18, and cleaved-caspase-1 in gastric tissue were detected by Western blotting. Mice in group NC were detected as above at the same time point. Data were processed with one-way analysis of variance and Bonferroni test. Results: The serum content of TNF-α and IL-6 and the relative expression of cleaved-caspase-1, IL-1β, and IL-18 in gastric tissue of mice in group NC were significantly lower than those in group CR (with P values below 0.01). The content of the above-mentioned inflammatory indexes in serum and gastric tissue of mice in group VCR was significantly lower than that in group CR (with P values below 0.01). There were no statistically significant differences in content of the above-mentioned inflammatory indexes in serum and gastric tissue of mice between groups RCR and CR (with P values above 0.05). The content of the above-mentioned inflammatory indexes in serum and gastric tissue of mice in group VCR was significantly lower than that in group RCR (with P values below 0.01). Surface of gastric mucosa was smooth and morphology of mucosal cells was normal with clear structure of mice in group NC. Multiple hemorrhage of gastric mucosa, disorderly arrangement of mucosal cells, and large number of inflammatory cell infiltration around necrotic tissue were observed in mice of group CR. Decreased number of gastric mucosa bleeding, intact mucosal structure, and small amount of inflammatory cell infiltration around necrotic tissue were observed in mice of groups VCR and RCR. The ulcer indexes of mice in groups NC, CR, VCR, and RCR were 0, 18.7±1.1, 6.3±1.5, and 8.2±1.3, respectively. The ulcer index of mice in group NC was significantly lower than that in the other 3 groups (with P values below 0.05). The ulcer indexes of mice in groups VCR and RCR were close (P>0.05), which were significantly lower than ulcer index of mice in group CR (with P values below 0.05). Conclusions: VX765 can effectively inhibit the activation of caspase-1, reduce production of inflammatory factor, and alleviate inflammatory response, which have protective effects on gastric mucosa of mice with cold-restraint stress-induced acute gastric ulcer.

The Highly Selective Caspase-1 Inhibitor VX-765 Provides Additive Protection Against Myocardial Infarction in Rat Hearts When Combined With a Platelet Inhibitor.

Use of ischemic postconditioning and other related cardioprotective interventions to treat patients with acute myocardial infarction (AMI) has failed to improve outcomes in clinical trials. Because P2Y12 inhibitors are themselves postconditioning mimetics, it has been postulated that the loading dose of platelet inhibitors routinely given to patients treated for AMI masks the anti-infarct effect of other intended cardioprotective interventions. To further improve outcomes of patients with AMI, an intervention must be able to provide additive protection in the presence of a P2Y12 platelet inhibitor. Previous studies reported an anti-infarct effect using a peptide inhibitor of the pro-inflammatory caspase-1 in animal models of AMI. Herein we tested whether a pharmacologic caspase-1 inhibitor can further limit infarct size in open-chest, anesthetized rats treated with a P2Y12 inhibitor. One hour occlusion of a coronary branch followed by 2 hours of reperfusion was used to simulate clinical AMI and reflow. One group of rats received an intravenous bolus of 16 mg/kg of the highly selective caspase-1 inhibitor VX-765 30 minutes prior to onset of ischemia. A second group received a 60 µg/kg intravenous bolus of the P2Y12 inhibitor cangrelor 10 minutes prior to reperfusion followed by 6 µg/kg/min continuous infusion. A third group received treatment with both inhibitors as above. Control animals received no treatment. Infarct size was measured by tetrazolium stain and volume of muscle at risk by fluorescent microspheres. In untreated hearts, 73.7% ± 4.1% of the ischemic zone infarcted. Treatment with either cangrelor or VX-765 alone reduced infarct size to 43.8% ± 2.4% and 39.6% ± 3.6% of the ischemic zone, respectively. Combining cangrelor and VX-765 was highly protective, resulting in only 14.0% ± 2.9% infarction. The ability of VX-765 to provide protection beyond that of a platelet inhibitor alone positions it as an attractive candidate therapy to further improve outcomes in today's patients with AMI.