Caspase-1 Inhibitor Research Articles

Sprayable inflammasome-inhibiting lipid nanorods in a polymeric scaffold for psoriasis therapy.

Localized delivery of inflammasome inhibitors in phagocytic macrophages could be promising for psoriasis treatment. The present work demonstrates the development of non-spherical lipid nanoparticles, mimicking pathogen-like shapes, consisting of an anti-inflammatory inflammasome inhibiting lipid (pyridoxine dipalmitate) as a trojan horse. The nanorods inhibit inflammasome by 3.8- and 4.5-fold compared with nanoellipses and nanospheres, respectively. Nanorods reduce apoptosis-associated speck-like protein and lysosomal rupture, restrain calcium influx, and mitochondrial reactive oxygen species. Dual inflammasome inhibitor (NLRP3/AIM-2-IN-3) loaded nanorods cause synergistic inhibition by 21.5- and 59-folds compared with nanorods and free drug, respectively alongside caspase-1 inhibition. The NLRP3/AIM-2-IN-3 nanorod when transformed into a polymeric scaffold, simultaneously and effectively inhibits RNA levels of NLRP3, AIM2, caspase-1, chemokine ligand-2, gasdermin-D, interleukin-1β, toll-like receptor 7/ 8, and IL-17A by 6.4-, 1.6-, 2.0-, 13.0-, 4.2-, 24.4-, 4.3-, and 1.82-fold, respectively in psoriatic skin in comparison to Imiquimod positive control group in an in-vivo psoriasis-like mice model.

Cinobufotalin inhibits proliferation, migration and invasion in hepatocellular carcinoma by triggering NOX4/NLRP3/GSDMD-dependent pyroptosis

IntroductionPyroptosis is an inflammatory form of programmed cell death that plays a significant role in tumorigenesis. Cinobufotalin (CB), a bufadienolide extracted from toad venom, is associated with antitumor effects in various cancers, including liver cancer. However, the role of CB in pyroptosis and its underlying mechanisms have not been well characterized.MethodsMTT, Colony formation, EdU, Wound healing and Transwell migration and invasion assays were applied to determine the effects of CB on the proliferation, migration, and invasion ability of hepatocellular carcinoma (HCC) cells in vitro. The subcutaneous xenograft mouse model and pulmonary metastasis model were used to evaluate the effect of CB on HCC cells in vivo. PCR, western blot, immunohistochemistry, immunofluorescence, and ELISA were used to verify the expression of proliferation, migration, pyroptosis, and inflammation related molecules after CB treatment. Using si-RNA and inhibitors to interfere with NOX4 and HLRP3 expression to validate the key signaling pathways of pyroptosis induced by CB treatment.ResultsIn vivo experiments using nude mice with xenografted HCC cells and in vitro experiments with HCC cell lines demonstrated that CB treatment significantly inhibited the proliferation, migration, and invasiveness of HCC cells. CB treatment also showed dose-dependent activation of the NLRP3 inflammasome complex in HCC cells, leading to gasdermin D-induced pyroptosis. However, these effects were abrogated via the pretreatment of HCC cells with VX-765, a caspase-1 inhibitor. Additionally, CB increased the production of reactive oxygen species (ROS) and H₂O₂, along with upregulating NOX4 protein expression in HCC cells. Conversely, NOX4 silencing or pretreatment with VAS2870 (an NOX4 inhibitor) or NAC (an ROS scavenger) suppressed the activation of the NLRP3 inflammasome complex and pyroptosis in CB-treated HCC cells.DiscussionOur study demonstrated that CB suppressed the proliferation, migration, and invasiveness of HCC cells by inducing pyroptosis through the activation of the NOX4/NLRP3/GSDMD signaling pathway. Therefore, our results suggest that CB is a promising therapeutic agent for HCC.

Caspase-1-dependent spatiality in triple-negative breast cancer and response to immunotherapy.

Tumor immune microenvironment (TIME) spatial organization predicts outcome and therapy response in triple-negative breast cancer (TNBC). An immunosuppressive TIME containing elevated tumor-associated macrophages (TAM) and scarce CD8+ T cells is associated with poor outcome, but the regulatory mechanisms are poorly understood. Here we show that ETS1-driven caspase-1 expression, required for IL1β processing and TAM recruitment, is negatively regulated by estrogen receptors alpha (ERα) and a defining feature of TNBC. Elevated tumoral caspase-1 is associated with a distinct TIME characterized by increased pro-tumoral TAMs and CD8+ T cell exclusion from tumor nests. Mouse models prove the functional importance of ERα, ETS1, caspase-1 and IL1β in TIME conformation. Caspase-1 inhibition induces an immunoreactive TIME and reverses resistance to immune checkpoint blockade, identifying a therapeutically targetable mechanism that governs TNBC spatial organization.

Role for NLRP3 inflammasome-mediated, Caspase1-dependent response in glaucomatous trabecular meshwork cell death and regulation of aqueous humor outflow

PurposeAcute ocular hypertension (AOH) is the defining feature of acute glaucoma. The mechanical stress and excessive production of reactive oxygen species (ROS) during episodes can directly or indirectly damage the trabecular meshwork (TM). Despite its significance, a clear understanding of its pathogenesis and an effective therapeutic target remain lacking in acute glaucoma. In the present study, we explored the potential molecular mechanisms underlying TM cell death following oxidative damage and AOH. The use of NAC/VX-765 as a potential pharmaceutical intervention for reducing intraocular pressure (IOP) was discussed. MethodsThe levels of NLRP3 and caspase-1 were compared between normal and glaucomatous TM samples. An in vitro oxidative damage model and an AOH rat model were used to investigate the potential molecular mechanism behind TM cell death. The ROS scavenger N-acetyl-L-cysteine (NAC) and caspase-1 inhibitor VX-765 were used to counteract TM damage. ResultsElevated levels of NLRP3 and caspase-1 were observed in patients with acute glaucoma. H2O2 exposure decreased the viability of human trabecular meshwork (HTM) cells and increased intracellular ROS levels. Both Gene and protein expressions of NLRP3, caspase-1, GSDMD-N, and IL-1β were notably upregulated in H2O2-induced HTM cells and the rodent AOH model. Both NAC and VX-765 demonstrated protective effects against TM injury by inhibiting pyroptosis. The IOP-lowering effects of NAC and VX-765 persisted for 7 days. ConclusionsOur findings indicate that the classical pyroptosis pathway, NLRP3/caspase-1/IL-1β, plays a key role in acute glaucomatous TM injury. Targeting pyroptosis provides novel therapeutic avenues for treating AOH-induced irreversible TM injury. This provides not only a promising therapeutic target for glaucoma but also introduces a new approach to intervention.

Glucose metabolite methylglyoxal induces vascular endothelial cell pyroptosis via NLRP3 inflammasome activation and oxidative stress in vitro and in vivo

Methylglyoxal (MGO), a reactive dicarbonyl metabolite of glucose, plays a prominent role in the pathogenesis of diabetes and vascular complications. Our previous studies have shown that MGO is associated with increased oxidative stress, inflammatory responses and apoptotic cell death in endothelial cells (ECs). Pyroptosis is a novel form of inflammatory caspase-1-dependent programmed cell death that is closely associated with the activation of the NOD-like receptor 3 (NLRP3) inflammasome. Recent studies have shown that sulforaphane (SFN) can inhibit pyroptosis, but the effects and underlying mechanisms by which SFN affects MGO-induced pyroptosis in endothelial cells have not been determined. Here, we found that SFN prevented MGO-induced pyroptosis by suppressing oxidative stress and inflammation in vitro and in vivo. Our results revealed that SFN dose-dependently prevented MGO-induced HUVEC pyroptosis, inhibited pyroptosis-associated biochemical changes, and attenuated MGO-induced morphological alterations in mitochondria. SFN pretreatment significantly suppressed MGO-induced ROS production and the inflammatory response by inhibiting the NLRP3 inflammasome (NLRP3, ASC, and caspase-1) signaling pathway by activating Nrf2/HO-1 signaling. Similar results were obtained in vivo, and we demonstrated that SFN prevented MGO-induced oxidative damage, inflammation and pyroptosis by reversing the MGO-induced downregulation of the NLRP3 signaling pathway through the upregulation of Nrf2. Additionally, an Nrf2 inhibitor (ML385) noticeably attenuated the protective effects of SFN on MGO-induced pyroptosis and ROS generation by inhibiting the Nrf2/HO-1 signaling pathway, and a ROS scavenger (NAC) and a permeability transition pore inhibitor (CsA) completely reversed these effects. Moreover, NLRP3 inhibitor (MCC950) and caspase-1 inhibitor (VX765) further reduced pyroptosis in endothelial cells that were pretreated with SFN. Collectively, these findings broaden our understanding of the mechanism by which SFN inhibits pyroptosis induced by MGO and suggests important implications for the potential use of SFN in the treatment of vascular diseases.

Abstract 61: Protective role of caspase-1 inhibition in experimental model of neonatal hyperoxia-induced cardio pulmonary dysfunction in adults born preterm.

Background: Adults born preterm are at an increased risk of systemic hypertension and cardiopulmonary morbidities. However, little is known of the underlying mechanisms connecting neonatal hyperoxia (O 2 ) exposure to these long-term cardiovascular consequences in adults born preterm. Pro-inflammatory caspase-1 mediates the activation of cytokines IL-1β, and pore-forming protein Gasdermin-D. Objective: Test the hypothesis that caspase-1 inhibition prevents long-term cardiopulmonary consequences of preterm birth. Design/Methods: Newborn mice (n=60) exposed to normoxia (RA) or hyperoxia (O 2 ) from postnatal day (P) 1 to 14 were randomly assigned to receive daily intraperitoneal (IP) injections of caspase-1 inhibitor (VX-765, 50 ug/Kg) or DMSO as placebo. The mouse pups recovered in RA into adulthood until 3 months of age. The effect of caspase-1 inhibition on Blood pressure (BP), exercise endurance, cardiac function, systemic vascular stiffness, pulmonary vascular remodeling was assessed at 3 months. Results: Adult rats exposed to neonatal O 2 had significantly increased BP associated with vascular stiffness, reduced exercise capacity, cardiac hypertrophy, and cardiovascular inflammation. Caspase-1 inhibition prevented systemic hypertension, systemic vascular stiffness, improved exercise tolerance and decreased cardiac hypertrophy (Fig 1A-D) in adult mice exposed to neonatal O 2 . Moreover, caspase -1 inhibition improved pulmonary vascular remodeling and pulmonary hypertension in these mice (Fig 2A-E). Additionally, western blot analysis demonstrated that early supplementation of VX-765 in O 2 exposed mice led to reduced cardiac proinflammatory markers GSDMD and IL-1β in adulthood (Fig 3A-B). Conclusion: Neonatal O 2 exposure predisposes to cardiopulmonary morbidities in adults born preterm. Inhibition of caspase-1 reduces cardiovascular inflammation and cardiopulmonary dysfunction in mice exposed to hyperoxia during the neonatal period. The functional and molecular impact of the use of caspase-1 suggests that it can be used as a novel preventive strategy for long-term cardiopulmonary dysfunction due to neonatal O 2 exposure in preterm survivors.

Novel Diclofenac-NO Donor With High Affinity for Human Serum Albumin Induces Endoplasmic Reticulum Stress-mediated Cell Death in Human Pancreatic Cancer Cells.

Nitric oxide (NO) has various physiological activities. In this study, diclofenac (DF) which has a high affinity for human serum albumin (HSA) was nitrosylated to a novel NO donor (NDF). The cytotoxic effects and the mechanism of NDF were investigated. Binding experiments of NDF to HSA were performed by the ultrafiltration method. NO was measured by the Griess method. The number of dead cells were measured using annexin V. Apoptosis and endoplasmic reticulum stress were evaluated by western blotting. NDF competitively inhibits the binding of DF to HSA, suggesting that NDF and DF have equivalent binding characteristics. NDF rapidly released NOx after being dissolved. At 200 μM, NDF induced cell death in human pancreatic cancer cells. Western blotting showed that NDF promoted the cleavage of PARP, caspase-3, and caspase-7. Inhibitors of caspase-1 and caspase-9 significantly suppressed NDF-induced cell death, as did a non-specific caspase inhibitor (Z-VAD). In addition, NDF significantly increased the expression of the endoplasmic reticulum stress marker, CHOP. NDF induces apoptotic cell death by causing endoplasmic reticulum stress. The findings of this study suggest that NDF may become a promising compound for the treatment of pancreatic cancer.

Connexin 43 regulates pyroptosis by influencing intracellular calcium levels in X-ray induced vascular endothelial cell damage.

Our prior research has established that X-ray exposure induces pyroptosis in human umbilical vein endothelial cells (HUVECs), with Cx43 playing a regulatory role in this process. However, the precise mechanism by which Cx43 regulates pyroptosis remains unclear. The objective of this study is to assess the involvement of the calcium signaling pathway in Cx43-mediated regulation of X-ray-induced pyroptosis in HUVECs. HUVECs were exposed to 10 Gy X-ray radiation either alone or combined with Cx43 overexpression or knockdown. Calcium ions (Ca2+) were stained using Fluo-4/AM and analyzed via flow cytometry and confocal microscopy. Pyroptosis was assessed through flow cytometry by staining with FLICA (fluorescent-labeled inhibitor of caspase) and propidium iodide (PI). Calcium signaling was inhibited using BAPTA/AM, 2-APB, or nifedipine. Protein expression levels were assessed by western blotting. X-ray irradiation induced an increase in intracellular calcium levels in HUVECs in a dose- and time-dependent manner. The results demonstrated that regulating calcium release with BAPTA/AM, 2-APB, or nifedipine significantly reduced pyroptosis. Also, the overexpression of Cx43 significantly attenuated the increase in intracellular calcium. Conversely, Cx43 knockdown via siRNA significantly increased the intracellular calcium levels. Also, interfering with calcium signaling using BAPTA/AM, 2-APB, or nifedipine reduced the raised pyroptosis levels induced by Cx43 knockdown. Individual HUVECs exposed to high-dose X-ray irradiation exhibited an increase in intracellular calcium, leading to pyroptosis. Also, upregulating Cx43 expression reduced the pyroptosis levels by inhibiting intracellular calcium concentration. This study introduces new concepts for identifying targets for the prophylaxis and therapy of radiation-induced damage.

Inhibition of Caspase-1-dependent pyroptosis alleviates myocardial ischemia/reperfusion injury during cardiopulmonary bypass (CPB) in type 2 diabetic rats

Cardiovascular complications pose a significant burden in type 2 diabetes mellitus (T2DM), driven by the intricate interplay of chronic hyperglycemia, insulin resistance, and lipid metabolism disturbances. Myocardial ischemia/reperfusion (MI/R) injury during cardiopulmonary bypass (CPB) exacerbates cardiac vulnerability. This study aims to probe the role of Caspase-1-dependent pyroptosis in global ischemia/reperfusion injury among T2DM rats undergoing CPB, elucidating the mechanisms underlying heightened myocardial injury in T2DM. This study established a rat model of T2DM and compared Mean arterial pressure (MAP), heart rate (HR), and hematocrit (Hct) between T2DM and normal rats. Myocardial cell morphology, infarction area, mitochondrial ROS and caspase-1 levels, NLRP3, pro-caspase-1, caspase-1 p10, GSDMD expressions, plasma CK-MB, cTnI, IL-1β, and IL-18 levels were assessed after reperfusion in both T2DM and normal rats. The role of Caspase-1-dependent pyroptosis in myocardial ischemia/reperfusion injury during CPB in T2DM rats was examined using the caspase-1 inhibitor VX-765 and the ROS scavenger NAC. T2DM rats demonstrated impaired glucose tolerance but stable hemodynamics during CPB, while showing heightened vulnerability to MI/R injury. This was marked by substantial lipid deposition, disrupted myocardial fibers, and intensified cellular apoptosis. The activation of caspase-1-mediated pyroptosis and increased reactive oxygen species (ROS) production further contributed to tissue damage and the ensuing inflammatory response. Notably, myocardial injury was mitigated by inhibiting caspase-1 through VX-765, which also attenuated the inflammatory cascade. Likewise, NAC treatment reduced oxidative stress and partially suppressed ROS-mediated caspase-1 activation, resulting in diminished myocardial injury. This study proved that Caspase-1-dependent pyroptosis significantly contributes to the inflammation and injury stemming from global MI/R in T2DM rats under CPB, which correlate with the surplus ROS generated by oxidative stress during reperfusion.

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.

NLRP3 inflammasome activation contributes to the development of the pro-fibrotic phenotype of lung fibroblasts

Idiopathic pulmonary fibrosis (IPF) is an irreversible progressive interstitial lung disease of unknown cause. The poorly understood pathophysiology of IPF poses substantial challenges to the development of effective anti-lung fibrotic drugs. The NLRP3 inflammasome, a key component of the innate immune system, has recently been linked to the pathogenesis of lung fibrosis. However, the specific contributions of NLRP3 inflammasomes to determination of the pro-fibrotic phenotype of lung fibroblasts, which play a central role in the production of extracellular matrix protein, remain to be investigated. Therefore, the present study was performed to elucidate the involvement of NLRP3 inflammasome signalling pathways in modulation of lung fibroblast proliferation and differentiation. We found that activation of NLRP3 inflammasomes increased in lung fibroblasts derived from individuals with pulmonary fibrosis and in normal lung fibroblasts stimulated with transforming growth factor β and platelet-derived growth factor. Importantly, blockage of NLRP3 inflammasome signalling, either by gene silencing of NLRP3 or using pharmacological inhibitors of NLRP3, caspase-1, or IL-1 receptor, inhibited the proliferation, differentiation, and extracellular matrix protein synthesis of activated lung fibroblasts. Moreover, induction of the reactive oxygen species/thioredoxin-interacting protein axis, an upstream signalling pathway of NLRP3 inflammasomes, was essential for maintenance of the pro-fibrotic phenotype of lung fibroblasts. Interestingly, treatments with pharmacological inhibitors of NLRP3 inflammasomes prevented the progression of bleomycin-induced pulmonary fibrosis in mice. Collectively, these findings suggest that aberrant activation of NLRP3 inflammasomes is a critical event in the pathogenesis of IPF and that targeting NLRP3 inflammasomes may serve as a therapeutic strategy for IPF.

Exacerbation of influenza virus induced lung injury by alveolar macrophages and its suppression by pyroptosis blockade in a human lung alveolus chip.

Alveolar macrophages (AMs) are the major sentinel immune cells in human alveoli and play a central role in eliciting host inflammatory responses upon distal lung viral infection. Here, we incorporated peripheral human monocyte-derived macrophages within a microfluidic human Lung Alveolus Chip that recreates the human alveolar-capillary interface under an air-liquid interface along with vascular flow to study how residential AMs contribute to the human pulmonary response to viral infection. When Lung Alveolus Chips that were cultured with macrophages were infected with influenza H3N2, there was a major reduction in viral titers compared to chips without macrophages; however, there was significantly greater inflammation and tissue injury. Pro-inflammatory cytokine levels, recruitment of immune cells circulating through the vascular channel, and expression of genes involved in myelocyte activation were all increased, and this was accompanied by reduced epithelial and endothelial cell viability and compromise of the alveolar tissue barrier. These effects were partially mediated through activation of pyroptosis in macrophages and release of pro-inflammatory mediators, such as interleukin (IL)-1β, and blocking pyroptosis via caspase-1 inhibition suppressed lung inflammation and injury on-chip. These findings demonstrate how integrating tissue resident immune cells within human Lung Alveolus Chip can identify potential new therapeutic targets and uncover cell and molecular mechanisms that contribute to the development of viral pneumonia and acute respiratory distress syndrome (ARDS).

Mesoporous manganese-doped nano-antioxidant suppresses pyroptosis for effective ischemic stroke therapy

Pyroptosis mediates neuroinflammation following ischemic stroke and contributes to its development, indicating that targeting pyroptosis-related pathways could be a potential therapeutic strategy for stroke. In this study, a mesoporous manganese-doped cerium oxide (MMC) nano-antioxidant encapsulated with caspase-1 inhibitor VX765 is rationally designed and engineered to relieve ischemic stroke by integrating the suppression of pyroptosis activity with reactive oxygen species (ROS) scavenging capability. Through the surface modification of Angiopoietin-2 (Ang2), the nano-antioxidant can specifically bind to the low-density lipoprotein receptor-related protein, cross the blood–brain barrier, and ultimately aggregate in ischemic neuronal tissues. In mice subjected to middle cerebral artery occlusion/reperfusion, the nano-antioxidant alleviates oxidative stress, inhibits cell pyroptosis, reduces inflammatory cytokines, and regulates microglial polarization toward M2, ultimately mitigating neuroinflammation and reperfusion injury in brain tissue. Mechanistic studies reveal that the pyroptosis-associated NOD-like receptor signaling pathway, the inflammation-associated TNF signaling pathway, the neutrophil chemotaxis-associated Toll-like receptor signaling pathway, and the oxidative stress-associated NF-kappa B signaling pathway are involved in the antioxidant-mediated stroke therapy. Endowed with pyroptosis-reversing ability and nano-antioxidant activities, this engineered antioxidant presents a novel therapeutic paradigm for minimizing reperfusion injury and enhancing stroke treatment efficacy.

Macrophage-derived exosomal miR-155 regulating hepatocyte pyroptosis in MAFLD

BackgroundPrevious studies have shown that pyroptosis in hepatocyte is essential for the development of MAFLD. Growing evidence has shown that exosomal miRNAs-mediated communication between inflammatory cells and hepatocyte is an important link in MAFLD. In the present study, we aim to elucidate whether macrophage-derived exosomal miRNAs contribute to the hepatocyte pyroptosis in the pathophysiological process of MAFLD. MethodsThe effects of hepatocyte pyroptosis were investigated in an HFD-induced MAFLD mouse model and in the liver tissues from patients with MAFLD using immunohistochemistry, real-time PCR, Western blotting, and luciferase reporter assay, among other techniques. MiR-155 inhibitor tail injections and AAV-FoxO3a-GFP were also administered to respectively inhibit or overexpress its expression in an HFD-induced MAFLD mouse model. ResultsHepatocyte pyroptosis was heightened in the liver tissue of patients with MAFLD or HFD-induced MAFLD mouse. Importantly, treatment with a caspase-1 inhibitor or overexpression of FoxO3a reversed this trend. Our study also demonstrated that miR-155 expression and the number of infiltrated macrophages were increased, and knockdown of miR-155 attenuated hepotocyte pyroptosis and liver fibrosis in HFD-induced mouse. In addition, we demonstrated that macrophage-derived exosomal miR-155 was transferred to hepatocytes, leading to hepatocyte pyroptosis in MAFLD mouse. Furthermore, blockade of exosome secretion improved hepotocyte pyroptosis and liver fibrosis in HFD-induced mouse. On the contrary, macrophage-derived exosomal miR-155 worsened hepotocyte pyroptosis. Moreover, we found that miR-155 promoted hepatocyte pyroptosis in MAFLD by down-regulating FoxO3a. ConclusionsTaken together, our results demonstrated that macrophage-derived exosomal miR-155 promotes hepatocyte pyroptosis and liver fibrosis in MAFLD.

Caspase-1 inhibitor CZL80 protects against acute seizures via amplifying the inhibitory neural transmission

Current anti-seizure medications (ASDs) primarily target ion channels or neurotransmissions; however, their practicability is limited by unwanted side-effects and pharmacoresistance. Cumulative evidence has proposed pro-inflammatory caspase-1 as a potential target for developing ASDs. In this study, we showed that the small-molecular caspase-1 inhibitor CZL80 can prevent seizures in various models including the maximal electroshock (MES), the pentylenetetrazol (PTZ), and the amygdaloid kindled models. Specifically, we discovered that CZL80 prevented death, reduced the duration of generalized seizures, and increased the threshold of generalized seizures in a dose-dependent manner in the MES model. In the PTZ model, CZL80 decreased the seizure stages, prolonged the latency to stage 4 seizures, and decreased the death rate. And in amygdaloid kindled rats, CZL80 inhibited the seizure stages, shortened the durations of both generalized seizures and after-discharges. And the anti-seizure efficacy of CZL80 was diminished in caspase-1 knockout mice. In vitro electrophysiology recordings revealed that CZL80 was able to decreased the excitability of glutamatergic pyramidal neurons, as denoted by reducing the spontaneous neuronal firings and increasing the rheobase injected currents to elicit action potentials. Furthermore, CZL80 was able to increase the amplitudes of inhibitory post-synaptic currents (IPSC), while the excitatory post-synaptic currents (EPSC) were not influenced. Lastly, daily administration of CZL80 for 3 weeks did not influence the normal locomotor functions in mice. In sum, our results highlighted CZL80 as a potential anti-seizure therapy with therapeutic significance.

IPSC-induced neurons with the V337M MAPT mutation are selectively vulnerable to caspase-mediated cleavage of tau and apoptotic cell death

BackgroundTau post-translational modifications (PTMs) result in the gradual build-up of abnormal tau and neuronal degeneration in tauopathies, encompassing variants of frontotemporal lobar degeneration (FTLD) and Alzheimer's disease (AD). Tau proteolytically cleaved by active caspases, including caspase-6, may be neurotoxic and prone to self-aggregation. Also, our recent findings show that caspase-6 truncated tau represents a frequent and understudied aspect of tau pathology in AD in addition to phospho-tau pathology. In AD and Pick's disease, a large percentage of caspase-6 associated cleaved-tau positive neurons lack phospho-tau, suggesting that many vulnerable neurons to tau pathology go undetected when using conventional phospho-tau antibodies and possibly will not respond to phospho-tau based therapies. Therefore, therapeutic strategies against caspase cleaved-tau pathology could be necessary to modulate the extent of tau abnormalities in AD and other tauopathies. MethodsTo understand the timing and progression of caspase activation, tau cleavage, and neuronal death, we created two mAbs targeting caspase-6 tau cleavage sites and probed postmortem brain tissue from an individual with FTLD due to the V337M MAPT mutation. We then assessed tau cleavage and apoptotic stress response in cortical neurons derived from induced pluripotent stem cells (iPSCs) carrying the FTD-related V337M MAPT mutation. Finally, we evaluated the neuroprotective effects of caspase inhibitors in these iPSC-derived neurons. ResultsFTLD V337M MAPT postmortem brain showed positivity for both cleaved tau mAbs and active caspase-6. Relative to isogenic wild-type MAPT controls, V337M MAPT neurons cultured for 3 months post-differentiation showed a time-dependent increase in pathogenic tau in the form of caspase-cleaved tau, phospho-tau, and higher levels of tau oligomers. Accumulation of toxic tau species in V337M MAPT neurons was correlated with increased vulnerability to pro-apoptotic stress. Notably, this mutation-associated cell death was pharmacologically rescued by the inhibition of effector caspases. ConclusionsOur results suggest an upstream, time-dependent accumulation of caspase-6 cleaved tau in V337M MAPT neurons promoting neurotoxicity. These processes can be reversed by caspase inhibition. These results underscore the potential of developing caspase-6 inhibitors as therapeutic agents for FTLD and other tauopathies. Additionally, they highlight the promise of using caspase-cleaved tau as biomarkers for these conditions.

Targeting TANK-binding kinase 1 attenuates painful diabetic neuropathy via inhibiting microglia pyroptosis

BackgroundPainful diabetic neuropathy (PDN) is closely linked to inflammation, which has been demonstrated to be associated with pyroptosis. Emerging evidence has implicated TANK-binding kinase 1 (TBK1) in various inflammatory diseases. However, it remains unknown whether activated TBK1 causes hyperalgesia via pyroptosis.MethodsPDN mice model of type 1 or type 2 diabetic was induced by C57BL/6J or BKS-DB mice with Lepr gene mutation. For type 2 diabetes PDN model, TBK1-siRNA, Caspase-1 inhibitor Ac-YVAD-cmk or TBK1 inhibitor amlexanox (AMX) were delivered by intrathecal injection or intragastric administration. The pain threshold and plantar skin blood perfusion were evaluated through animal experiments. The assessments of spinal cord, dorsal root ganglion, sciatic nerve, plantar skin and serum included western blotting, immunofluorescence, ELISA, and transmission electron microscopy.ResultsIn the PDN mouse model, we found that TBK1 was significantly activated in the spinal dorsal horn (SDH) and mainly located in microglia, and intrathecal injection of chemically modified TBK1-siRNA could improve hyperalgesia. Herein, we described the mechanism that TBK1 could activate the noncanonical nuclear factor κB (NF-κB) pathway, mediate the activation of NLRP3 inflammasome, trigger microglia pyroptosis, and ultimately induce PDN, which could be reversed following TBK1-siRNA injection. We also found that systemic administration of AMX, a TBK1 inhibitor, could effectively improve peripheral nerve injury. These results revealed the key role of TBK1 in PDN and that TBK1 inhibitor AMX could be a potential strategy for treating PDN.ConclusionsOur findings revealed a novel causal role of TBK1 in pathogenesis of PDN, which raises the possibility of applying amlexanox to selectively target TBK1 as a potential therapeutic strategy for PDN.

Caspase-1 Inhibition Ameliorates Photoreceptor Damage Following Retinal Detachment by Inhibiting Microglial Pyroptosis

Retinal detachment (RD) is a sight-threatening condition that occurs in several retinal diseases. Microglia that reside in retina are activated after RD and are involved in the death of photoreceptor cells. The involvement of microglial pyroptosis in the early pathological process of RD is still unclear. It has been shown that VX-765, an inhibitor of Caspase-1, may exert neuroprotective effects by targeting microglial pyroptosis in nervous system disease; however, whether it plays a role in RD is uncertain. This study detected and localized pyroptosis to specific cells by immunofluorescence co-staining and flow cytometry in rat RD models. The majority of gasdermin D N terminal (GSDMD-N) positive cells exhibited IBA1 positive or P2RY12 positive microglia in the early stage of RD, indicating the pyroptosis of microglia. Administration of VX-765 shifted the microglia phenotype from M1 to M2, inhibited microglial migration toward the outer nuclear layer (ONL) post-RD, and most importantly inhibited microglial pyroptosis. The thickness of ONL increased with VX-765 administration and the photoreceptors were more structured and orderly under hematoxylin and eosin staining and transmission electron microscopy, revealing the protective effects of VX-765 on photoreceptors. Overall, this study demonstrates that inflammation induced by pyroptosis of microglia is the early pathological process of RD. VX-765 may serve as a candidate therapeutic approach for the treatment of RD by targeting microglia.

Enhanced Cardiomyocyte NLRP3 Inflammasome-Mediated Pyroptosis Promotes d-Galactose-Induced Cardiac Aging.

Cardiac aging represents an independent risk factor for aging-associated cardiovascular diseases. Although evidence suggests an association between NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome formation and numerous cardiovascular diseases, its role in cardiac aging remains largely unclear. The longevity of mice with wild-type and NLRP3 knockout (NLRP3-/-) genotypes was assessed, with or without d-galactose treatment. Cardiac function was evaluated using echocardiography, and cardiac histopathology was examined through hematoxylin and eosin and Masson's trichrome staining. Senescence-associated β-galactosidase (SA-β-gal) staining was employed to detect cardiac aging. Western blotting was used to assess aging-related proteins (p53, p21) and pyroptosis-related proteins. Additionally, dihydroethidium staining, lactate dehydrogenase release, and interleukin-1β ELISA assays were performed, along with measurements of total superoxide dismutase and malondialdehyde levels. Invitro, H9c2 cells were exposed to d-galactose for 24 hours in the absence or presence of N-acetyl-l-cysteine (reactive oxygen species inhibitor), BAY-117082 (nuclear factor κ-light-chain enhancer of activated B cells inhibitor), MCC950 (NLRP3 inhibitor), and VX-765 (Caspase-1 inhibitor). Immunofluorescence staining was employed to detect p53, gasdermin D, and apoptosis-associated speck-like protein proteins. Intracellular reactive oxygen species levels were assessed using fluorescence microscopy and flow cytometry. Senescence-associated β-galactosidase staining and Western blotting were also employed invitro for the same purpose. The results showed that NLRP3 upregulation was implicated in aging and cardiovascular diseases. Inhibition of NLRP3 extended life span, mitigated the aging phenotype, improved cardiac function and blood pressure, ameliorated lipid metabolism abnormalities, inhibited pyroptosis in cardiomyocytes, and ultimately alleviated cardiac aging. Invitro, the inhibition of reactive oxygen species, nuclear factor κ-light-chain enhancer of activated B cells, NLRP3, or caspase-1 attenuated NLRP3 inflammasome-mediated pyroptosis. The reactive oxygen species/nuclear factor κ-light-chain enhancer of activated B cells/NLRP3 signaling pathway loop contributes to d-galactose-treated cardiomyocyte senescence and cardiac aging.

Structural, computational, docking and biological studies of a triaminopyrimidine caspase-1 inhibitor

In order to more fully characterize the binding properties and activities of a potent, low nanomolar IC50 (half maximal inhibitory concentration) inhibitor of caspase-1, CK-1–41, a series of experiments were designed to probe its structure and function. To accomplish this, larger quantities of the molecule were synthesized in an efficient manner using a microwave method, in only 30 min. The structure of the inhibitor was determined by X-ray crystallography in its neutral and protonated form yielding useful structural information. The protonation site in the X-ray structure was found to be the trialkylamine basic site, but nuclear magnetic resonance (NMR) H/D exchange experiments shows that protonation at the triaminopyrimidine sp2 C is also accessible in solution. Density Functional Theory (DFT) calculations were performed and supported the experimental H/D exchange results. Docking studies with several protonated forms of the inhibitor with caspase-1 revealed the compound binds favorably in an allosteric site, with the strongest binding from the protonated form where the proton is bound to the sp2 C. Due to the involvement of caspase-1 in inflammatory cancers, CK-1–41 was screened against a panel of cancer cell lines displaying modest activity against some of the cell lines tested. These studies further clarify the structure and biological function of the representative member of the triaminopyrimidine inhibitors of caspase-1.