ABSTRACT
RESEARCH QUESTION: Does the NOD-like receptor protein 3 (NLRP3) inflammasome have an effect in adenomyosis? DESIGN: Fresh-frozen endometrial tissues and paraffin specimens were obtained from endometrial tissues from patients with adenomyosis and controls. Western blot, quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC) were applied to assess expression of the NLRP3 inflammasome components. Primary eutopic endometrial stromal cells were isolated from the uteri of patients with adenomyosis. After NLRP3 was knocked down using small interfering RNA, proliferation, invasion and epithelial-mesenchymal transition (EMT) were evaluated using EdU, CCK8, transwell assays and western blot. Importantly, a mouse model of adenomyosis was established to evaluate the effects of the NLRP3 inhibitor MCC950 on the formation of adenomyosis. RESULTS: Expression of the NLRP3 inflammasome components was elevated in the ectopic or eutopic endometrium of patients with adenomyosis. NLRP3 knockdown inhibited migration, invasion and EMT in endometrial cells and primary endometrial cells (P < 0.0001). MCC950, which blocks the NLRP3 inflammasome, reduced migration and invasion of endometrial cells (P < 0.01) and primary endometrial cells (P < 0.0001) considerably. Importantly, in the mouse model of adenomyosis, MCC950 had a mitigating effect on the severity of adenomyosis (P < 0.01). CONCLUSIONS: NLRP3 was found to enhance migration, invasion and EMT of human endometrial cells in adenomyosis. Notably, the NLRP3 inhibitor MCC950 reduced migration and invasion of endometrial cells effectively. Furthermore, in the mouse model of adenomyosis, MCC950 exhibited a therapeutic effect by alleviating the severity of adenomyosis.
ABSTRACT
Parkinson's disease (PD) is characterized by the severe loss of dopaminergic neurons in the substantia nigra pars compacta, leading to motor dysfunction. The onset of PD is often accompanied by neuroinflammation and α-Synuclein aggregation, and extensive research has focused on the activation of microglial NLRP3 inflammasomes in PD, which promotes the death of dopaminergic neurons. In this study, a model of cerebral inflammatory response was constructed in wild-type and Parkinï¼/ï¼ mice through bilateral intraventricular injection of LPS. LPS-induced activation of the NLRP3 inflammasome in wild-type mice promotes the progression of PD. The use of MCC950 in wild mice injected with LPS induces activation of Parkin/PINK and improves autophagy, which in turn improves mitochondrial turnover. It also inhibits LPS-induced inflammatory responses, improves motor function, protects dopaminergic neurons, and inhibits microglia activation. Furthermore, Parkinï¼/ï¼ mice exhibited motor dysfunction, loss of dopaminergic neurons, activation of the NLRP3 inflammasome, and α-Synuclein aggregation beginning at an early age. Parkin ± mice exhibited more pronounced microglia activation, greater NLRP3 inflammasome activation, more severe autophagy dysfunction, and more pronounced motor dysfunction after LPS injection compared to wild-type mice. Notably, the use of MCC950 in Parkin ± mice did not ameliorate NLRP3 inflammasome activation, autophagy dysfunction, or α-synuclein aggregation. Thus, MCC950 can only exert its effects in the presence of Parkin/PINK1, and targeting Parkin-mediated NLRP3 inflammasome activation is expected to be a potential therapeutic strategy for Parkinson's disease.
Subject(s)
Furans , Indenes , Inflammasomes , Lipopolysaccharides , Mice, Inbred C57BL , NLR Family, Pyrin Domain-Containing 3 Protein , Neuroinflammatory Diseases , Protein Kinases , Sulfonamides , Ubiquitin-Protein Ligases , Animals , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Ubiquitin-Protein Ligases/metabolism , Ubiquitin-Protein Ligases/genetics , Mice , Furans/pharmacology , Protein Kinases/metabolism , Inflammasomes/metabolism , Inflammasomes/drug effects , Indenes/pharmacology , Neuroinflammatory Diseases/drug therapy , Neuroinflammatory Diseases/metabolism , Sulfonamides/pharmacology , Male , Microglia/drug effects , Microglia/metabolism , Sulfones/pharmacology , Heterocyclic Compounds, 4 or More Rings/pharmacology , Autophagy/drug effects , Autophagy/physiology , Signal Transduction/drug effects , Dopaminergic Neurons/drug effects , Dopaminergic Neurons/metabolism , Dopaminergic Neurons/pathology , Mice, Knockout , alpha-Synuclein/metabolismABSTRACT
BACKGROUND: To explore the neuroprotective mechanism of artemisinin against ischemic stroke from the perspective of NLRP3-mediated pyroptosis. METHODS: Serum metabolomics technology was used to analyze the serum samples of mice, and KEGG metabolic pathway was analyzed for the different metabolites in the samples. PIT model and OGD/R model were used to simulate ischemic stroke damage in vivo and in vitro. Hoechst 33342 staining, Annexin V-FITC/PI staining and TUNEL staining were used to detect the pyroptosis rate of cells. The contents of IL-1ß and IL-18 in PC12 cells and serum of mice were detected by ELISA. The expressions of NLRP3, ASC-1, Caspase-1 and TXNIP in PC12 cells and mouse brain tissue were detected by Western Blot. RESULTS: Serum metabolic profiles of animal models identified 234 different metabolites and 91 metabolic pathways. Compared with the Sham group and the Stroke+ART group, the KEGG pathway in the Stroke group was concentrated in the Necroptosis pathway associated with cell growth and death, and the NLRP3 inflammasome-mediated pyroptosis pathway was activated in the Necroptosis pathway after ischemic stroke. The results of in vivo and in vitro experiments showed that pretreatment with 10⯵M artemisinin reduced ROS production, decreased Δψm, reduced pyroptosis, maintained neuronal cell morphology, and down-regulated the contents of IL-1ß and IL-18 as well as the expression of key proteins of NLRP3, ASC-1, Caspase-1 and TXNIP(p<0.01). CONCLUSION: Artemisinin can reduce neuronal pyroptosis induced by ischemic stroke by inhibiting ROS/TXNIP/NLRP3/Caspase-1 signaling pathway.
Subject(s)
Artemisinins , Carrier Proteins , Caspase 1 , Ischemic Stroke , Mice, Inbred C57BL , NLR Family, Pyrin Domain-Containing 3 Protein , Pyroptosis , Reactive Oxygen Species , Signal Transduction , Animals , Pyroptosis/drug effects , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Caspase 1/metabolism , Signal Transduction/drug effects , Ischemic Stroke/drug therapy , Ischemic Stroke/metabolism , Ischemic Stroke/pathology , Artemisinins/pharmacology , Mice , Carrier Proteins/metabolism , PC12 Cells , Reactive Oxygen Species/metabolism , Male , Rats , Cell Cycle Proteins/metabolism , Neuroprotective Agents/pharmacology , Inflammasomes/metabolism , Inflammasomes/drug effects , Disease Models, Animal , ThioredoxinsABSTRACT
Anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis is a neurological disorder, characterized by cognitive deficits as one of its vital features. The nucleotide-binding oligomerization domain-like receptor (NLRP3) inflammasome is a key contributor to neuroinflammation and cognitive deficits in neurological diseases. However, the underlying mechanism of anti-NMDAR encephalitis remains unclear, and the biological function of the NLRP3 inflammasome in this condition has not been elucidated. In this study, a mouse model of anti-NMDAR encephalitis was induced by active immunization with the GluN1356-385 peptide (NEA model). The NLRP3 inflammasome in the hippocampus and temporal cortex was investigated using real-time quantitative PCR (RT-qPCR), western blotting, and immunofluorescence staining. The impact of MCC950 on cognitive function and NLRP3 inflammation was assessed. Confocal immunofluorescence staining and Sholl analysis were employed to examine the function and morphology of microglia. In the current study, we discovered overactivation of the NLRP3 inflammasome and an enhanced inflammatory response in the NEA model, particularly in the hippocampus and temporal cortex. Furthermore, significant cognitive dysfunction was observed in the NEA model. While, MCC950, a selective inhibitor of the NLRP3 inflammasome, sharply attenuated the inflammatory response in mice, leading to mitigated cognitive deficits of mice and more regular arrangements of neurons and reduced number of hyperchromatic cells were also observed in the hippocampus area. In addition, we found that the excess elevation of NLRP3 inflammasome was mainly expressed in microglia accompanied with the overactivation of microglia, while MCC950 treatment significantly inhibited the increased number and activated morphological changes of microglia in the NEA model. Altogether, our study reveals the vital role of overactivated NLRP3 signaling pathway in aggravating the inflammatory response and cognitive deficits and the potential protective effect of MCC950 in anti-NMDAR encephalitis. Thus, MCC950 represents a promising strategy for anti-inflammation in anti-NMDAR encephalitis and our study lays a theoretical foundation for it to become a clinically targeted drug.
Subject(s)
Anti-N-Methyl-D-Aspartate Receptor Encephalitis , Cognitive Dysfunction , Disease Models, Animal , Hippocampus , Indenes , Inflammasomes , Microglia , NLR Family, Pyrin Domain-Containing 3 Protein , Sulfonamides , Animals , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/antagonists & inhibitors , Cognitive Dysfunction/drug therapy , Cognitive Dysfunction/immunology , Cognitive Dysfunction/etiology , Inflammasomes/metabolism , Inflammasomes/antagonists & inhibitors , Inflammasomes/immunology , Mice , Hippocampus/drug effects , Hippocampus/pathology , Hippocampus/metabolism , Hippocampus/immunology , Anti-N-Methyl-D-Aspartate Receptor Encephalitis/immunology , Anti-N-Methyl-D-Aspartate Receptor Encephalitis/drug therapy , Indenes/therapeutic use , Sulfonamides/therapeutic use , Sulfonamides/pharmacology , Microglia/drug effects , Microglia/immunology , Furans/therapeutic use , Furans/pharmacology , Sulfones/therapeutic use , Sulfones/pharmacology , Mice, Inbred C57BL , Female , Heterocyclic Compounds, 4 or More Rings/therapeutic use , Heterocyclic Compounds, 4 or More Rings/pharmacology , Humans , Male , Temporal Lobe/pathologyABSTRACT
OBJECTIVE: The NLRP3 inflammasome mediates a range of inflammatory responses that are associated with an increasing number of pathological mechanisms. Over-activation of NLRP3 can exacerbate many diseases. However, NLRP3 antagonists have significant therapeutic potential. Moreover, NLRP3 plays an important role in limiting the growth and spread of some tumors, and NLRP3 agonists also have clinical value. MCC950 and BMS986299 are an antagonist and agonist of NLRP3, respectively. In light of the important clinical applications of NLRP3, especially for NLRP3 inhibitors, a computational method was used to investigate the interaction modes of MCC950 and BMS986299 with NLRP3 in order to design and develop more potent NLRP3 regulators. METHODS: In this study, the conformational behaviors of NLRP3 bound to the antagonist MCC950 in an inactive state and the agonist BMS986299 in an active state were investigated using 200 ns equilibrium all-atom molecular dynamics (MD) simulations, and then the analyses of the MD trajectories (RMSD, Rg, RMSF, SASA, PCA, and DCCM) were carried out to explore the mechanism of the antagonist and agonist on NLRP3 in the two different states. RESULTS: The RMSD, RMSF, Rg, SASA, and PCA analyses indicated that NLRP3 was more dispersive and less energetically stable in the active state than in the inactive state and that MCC950 significantly reduced the fluctuations of the interactive residues while BMS986299 did not. The antagonist MCC950 interacted with residues mainly in the NBD, HD1, WHD, and HD2 domains of NLRP3, whereas the agonist BMS986299 mainly in the NBD and WHD of NLRP3. Additionally, both compounds did not interact with residues located in the FISNA domain. The conformation of the FISNA domain appeared to change significantly when NLRP3 was translated from an inactive state to an active state. CONCLUSION: The antagonist may interact with residues mainly in the NBD, HD1, WHD, and HD2 domains, and the agonist may interact in the NBD and WHD domains. Our study provided new insights into the development of NLRP3 regulators.
ABSTRACT
We previously reported that rosmarinic acid (RA) ameliorated renal fibrosis in a unilateral ureteral obstruction (UUO) murine model of chronic kidney disease. This study aimed to determine whether RA attenuates indoxyl sulfate (IS)-induced renal fibrosis by regulating the activation of the NLRP3 inflammasome/IL-1ß/Smad circuit. We discovered the NLRP3 inflammasome was activated in the IS treatment group and downregulated in the RA-treated group in a dose-dependent manner. Additionally, the downstream effectors of the NLRP3 inflammasome, cleaved-caspase-1 and cleaved-IL-1ß showed similar trends in different groups. Moreover, RA administration significantly decreased the ROS levels of reactive oxygen species in IS-treated cells. Our data showed that RA treatment significantly inhibited Smad-2/3 phosphorylation. Notably, the effects of RA on NLRP3 inflammasome/IL-1ß/Smad and fibrosis signaling were reversed by the siRNA-mediated knockdown of NLRP3 or caspase-1 in NRK-52E cells. In vivo, we demonstrated that expression levels of NLRP3, c-caspase-1, c-IL-1ß, collagen I, fibronectin and α-SMA, and TGF- ß 1 were downregulated after treatment of UUO mice with RA or RA + MCC950. Our findings suggested RA and MCC950 synergistically inhibited UUO-induced NLRP3 signaling activation, revealing their renoprotective properties and the potential for combinatory treatment of renal fibrosis and chronic kidney inflammation.
Subject(s)
Cinnamates , Depsides , Fibrosis , Indican , Inflammasomes , Kidney , Mice, Inbred C57BL , NLR Family, Pyrin Domain-Containing 3 Protein , Rosmarinic Acid , Signal Transduction , Animals , Depsides/pharmacology , Depsides/therapeutic use , Cinnamates/pharmacology , Cinnamates/therapeutic use , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Inflammasomes/metabolism , Signal Transduction/drug effects , Male , Kidney/pathology , Kidney/drug effects , Kidney/metabolism , Cell Line , Mice , Interleukin-1beta/metabolism , Ureteral Obstruction/drug therapy , Ureteral Obstruction/pathology , Reactive Oxygen Species/metabolism , Disease Models, Animal , Smad2 Protein/metabolism , Renal Insufficiency, Chronic/drug therapy , Renal Insufficiency, Chronic/chemically induced , Renal Insufficiency, Chronic/pathology , Renal Insufficiency, Chronic/metabolism , Smad3 Protein/metabolism , Caspase 1/metabolism , Kidney Diseases/drug therapy , Kidney Diseases/chemically induced , Kidney Diseases/pathologyABSTRACT
NLRP3 inflammasome activation has emerged as a critical initiator of inflammatory response in ischemic retinopathy. Here, we identified the effect of a potent, selective NLRP3 inhibitor, MCC950, on autophagy and apoptosis under hypoxia. Neonatal mice were exposed to hyperoxia for 5 days to establish oxygen-induced retinopathy (OIR) model. Intravitreal injection of MCC950 was given, and then autophagy and apoptosis markers were assessed. Retinal autophagy, apoptosis, and related pathways were evaluated by western blot, immunofluorescent labeling, transmission electron microscopy, and TUNEL assay. Autophagic activity in Müller glia after NLRP3 inflammasome inhibition, together with its influence on photoreceptor death, was studied using western blot, immunofluorescence staining, mRFP-GFP-LC3 adenovirus transfection, cell viability, proliferation, and apoptosis assays. Results showed that activation of NLRP3 inflammasome in Müller glia was detected in OIR model. MCC950 could improve impaired retinal autophagic flux and attenuate retinal apoptosis while it regulated the retinal AMPK/mTOR/ULK-1 pathway. Suppressed autophagy and depressed proliferation capacity resulting from hypoxia was promoted after MCC950 treatment in Müller glia. Inhibition of AMPK and ULK-1 pathway significantly interfered with the MCC950-induced autophagy activity, indicating MCC950 positively modulated autophagy through AMPK/mTOR/ULK-1 pathway in Müller cells. Furthermore, blockage of autophagy in Müller glia significantly induced apoptosis in the cocultured 661W photoreceptor cells, whereas MCC950 markedly preserved the density of photoreceptor cells. These findings substantiated the therapeutic potential of MCC950 against impaired autophagy and subsequent apoptosis under hypoxia. Such protective effect might involve the modulation of AMPK/mTOR/ULK-1 pathway. Targeting NLRP3 inflammasome in Müller glia could be beneficial for photoreceptor survival under hypoxic conditions.
Subject(s)
Apoptosis , Autophagy , Inflammasomes , NLR Family, Pyrin Domain-Containing 3 Protein , Photoreceptor Cells, Vertebrate , Sulfonamides , Animals , Mice , Apoptosis/drug effects , Autophagy/drug effects , Cyclic S-Oxides/pharmacology , Ependymoglial Cells/metabolism , Ependymoglial Cells/drug effects , Furans/pharmacology , Hypoxia/metabolism , Indenes/pharmacology , Inflammasomes/metabolism , Mice, Inbred C57BL , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Photoreceptor Cells, Vertebrate/metabolism , Photoreceptor Cells, Vertebrate/drug effects , Photoreceptor Cells, Vertebrate/pathology , Signal Transduction/drug effects , Sulfonamides/pharmacology , Sulfones/pharmacologyABSTRACT
N,N-dimethylformamide (DMF) is a widely utilized chemical solvent with various industrial applications. Previous studies have indicated that the liver is the most susceptible target to DMF exposure, whereas the underlying mechanisms remain to be elucidated. This study aimed to investigate the role of NLRP3 inflammasome in DMF-induced liver injury in mice by using two NLRP3 inflammasome inhibitors, Nlrp3-/- mice, Nfe2l2-/- mice, and a macrophage-depleting agent. RNA sequencing revealed that endoplasmic reticulum (ER) stress and NLRP3 inflammasome-associated pathways were activated in the mouse liver after acute DMF exposure, which was validated by Western blotting. Interestingly, DMF-induced liver injury was effectively suppressed by two inflammasome inhibitors, MCC950 and Dapansutrile. In addition, knockout of Nlrp3 markedly attenuated DMF-induced liver injury without affecting the metabolism of DMF. Furthermore, silencing Nfe2l2 aggravated the liver injury and the NLRP3 inflammasome activation in mouse liver. Finally, the depletion of hepatic macrophages by clodronate liposomes significantly reduced the liver damage caused by DMF. These results suggest that NLRP3 inflammasome activation is the upstream molecular event in the development of acute liver injury induced by DMF.
Subject(s)
Dimethylformamide , Inflammasomes , NLR Family, Pyrin Domain-Containing 3 Protein , Animals , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/genetics , Mice , Inflammasomes/metabolism , Chemical and Drug Induced Liver Injury , Liver/drug effects , Mice, Knockout , Endoplasmic Reticulum Stress/drug effectsABSTRACT
Purinergic signaling is an ancient primordial signaling system regulating tissue development and specification of various types of stem cells. Thus, functional purinergic receptors are present in several types of cells in the body, including multiple populations of stem cells. However, one stem cell type that has not been evaluated for expression of purinergic receptors is very small embryonic stem cells (VSELs) isolated from postnatal tissues. Herein, we report that human umbilical cord blood (UCB) and murine bone marrow (BM) purified VSELs express mRNA for P1 and P2 purinergic receptors and CD39 and CD73 ectonucleotidases converting extracellular ATP (eATP) into its signaling metabolite extracellular adenosine (eAdo), that antagonizes eATP effects. More importantly, we demonstrate that human and murine VSELs respond by chemotaxis to eATP, and eAdo inhibits this migration. These responses to eATP are mediated by activation of Nlrp3 inflammasome, and exposure of VSELs to its specific inhibitor MCC950 abolished the chemotactic response to ATP. We conclude that purinergic signaling plays an essential, underappreciated role in the biology of these cells and their potential role in response to tissue/organ injuries.
Subject(s)
Adenosine Triphosphate , Apyrase , Cell Movement , Embryonic Stem Cells , Humans , Adenosine Triphosphate/metabolism , Animals , Mice , Embryonic Stem Cells/metabolism , Embryonic Stem Cells/cytology , Apyrase/metabolism , Receptors, Purinergic/metabolism , 5'-Nucleotidase/metabolism , 5'-Nucleotidase/genetics , Chemotaxis , Antigens, CD/metabolism , Antigens, CD/genetics , Fetal Blood/cytology , Fetal Blood/metabolism , Adenosine/metabolism , Signal TransductionABSTRACT
Plasma cell mastitis (PCM) is a sterile inflammatory condition primarily characterized by periductal inflammation and ductal ectasia. Currently, there is a lack of non-invasive or minimally invasive treatment option other than surgical intervention. The NLRP3 inflammasome has been implicated in the pathogenesis and progression of various inflammatory diseases, however, its involvement in PCM has not yet been reported. In this study, we initially observed the pronounced upregulation of NLRP3 in both human and mouse PCM tissue and elucidated the mechanism underlying the attenuation of PCM through inhibition of NLRP3. We established the PCM murine model and collected samples on day 14, when inflammation reached its peak, for subsequent research purposes. MCC950, an NLRP3 inhibitor, was utilized to effectively ameliorate PCM by significantly reducing plasma cell infiltration in mammary tissue, as well as attenuate the expression of pro-inflammatory cytokines including IL-1ß, TNF-α, IL-2, and IL-6. Mechanistically, we observed that MCC950 augmented the function of myeloid-derived suppressor cells (MDSCs), which in turn inhibited the infiltration of plasma cells. Furthermore, it was noted that depleting MDSCs greatly compromised the therapeutic efficacy of MCC950. Collectively, our findings suggest that the administration of MCC950 has the potential to impede the progression of PCM by augmenting MDSCs both numerically and functionally, ultimately treating PCM effectively. This study provides valuable insights into the utilization of pharmacological agents for PCM treatment.
Subject(s)
Indenes , Mastitis , Myeloid-Derived Suppressor Cells , Female , Humans , Animals , Mice , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Myeloid-Derived Suppressor Cells/metabolism , Plasma Cells/metabolism , Sulfones/pharmacology , Sulfonamides/therapeutic use , Sulfonamides/pharmacology , Inflammasomes/metabolism , Inflammation/drug therapy , Mastitis/drug therapy , Furans/therapeutic use , Furans/pharmacologyABSTRACT
Systemic lupus erythematosus (SLE) is a complex autoimmune disorder with a pathogenesis that remains incompletely understood, resulting in limited treatment options. MCC950, a highly specific NLRP3 inflammasome inhibitor, effectively suppresses the activation of NLRP3, thus reducing the production of caspase-1, the pro-inflammatory cytokines IL-1ß and IL-18. This review highlights the pivotal role of NLRP3 inflammasome activation pathways in the pathogenesis of SLE and discusses the potential therapeutic application of MCC950 in SLE. Notably, it comprehensively elucidates the mechanism of MCC950 targeting the NLRP3 pathway in SLE treatment, outlining its potential role in regulating autophagy and necroptosis. The insights gained contribute to a deeper understanding of the value of MCC950 in SLE therapy, serving as a robust foundation for further research and potential clinical applications.
Subject(s)
Autoimmune Diseases , Indenes , Lupus Erythematosus, Systemic , Humans , Inflammasomes , NLR Family, Pyrin Domain-Containing 3 Protein , Lupus Erythematosus, Systemic/drug therapy , Furans/therapeutic use , Indenes/therapeutic use , SulfonamidesABSTRACT
Diabetic muscle atrophy is an inflammation-related complication of type-2 diabetes mellitus (T2DM). Even though regular exercise prevents further deterioration of atrophic status, there is no effective mediator available for treatment and the underlying cellular mechanisms are less explored. In this study, we investigated the therapeutic potential of MCC950, a specific, small-molecule inhibitor of NLRP3, to treat pyroptosis and diabetic muscle atrophy in mice. Furthermore, we used MCC950 to intervene in the protective effects of aerobic exercise against muscle atrophy in diabetic mice. Blood and gastrocnemius muscle (GAS) samples were collected after 12 weeks of intervention and the atrophic state was assessed. We initially corroborated a diabetic muscle atrophy phenotype in db/db mice (D) by comparison with control m/m mice (W) by examining parameters such as fasting blood glucose (D vs. W: 24.47 ± 0.45 mmol L-1 vs. 4.26 ± 0.6 mmol L-1, p < 0.05), grip strength (D vs. W: 166.87 ± 15.19 g vs. 191.76 ± 14.13 g, p < 0.05), exercise time (D vs. W: 1082.38 ± 104.67 s vs. 1716 ± 168.55 s, p < 0.05) and exercise speed to exhaustion (D vs. W: 24.25 ± 2.12 m min-1 vs. 34.75 ± 2.66 m min-1, p < 0.05), GAS wet weight (D vs. W: 0.07 ± 0.01 g vs. 0.13 ± 0.01 g, p < 0.05), the ratio of GAS wet weight to body weight (D vs. W: 0.18 ± 0.01% vs. 0.54 ± 0.02%, p < 0.05), and muscle fiber cross-sectional area (FCSA) (D vs. W: 1875 ± 368.19 µm2 vs. 2747.83 ± 406.44 µm2, p < 0.05). We found that both MCC950 (10 mg kg-1) treatment and exercise improved the atrophic parameters that had deteriorated in the db/db mice, inhibited serum inflammatory markers and significantly attenuated pyroptosis in atrophic GAS. In addition, a combined MCC950 treatment with exercise (DEI) exhibited a further improvement in glucose uptake capacity and muscle performance. This combined treatment also improved the FCSA of GAS muscle indicated by Laminin immunofluorescence compared to the group with the inhibitor treatment alone (DI) (DEI vs. DI: 2597 ± 310.97 vs. 1974.67 ± 326.15 µm2, p < 0.05) or exercise only (DE) (DEI vs. DE: 2597 ± 310.97 vs. 2006.33 ± 263.468 µm2, p < 0.05). Intriguingly, the combination of MCC950 treatment and exercise significantly reduced NLRP3-mediated inflammatory factors such as cleaved-Caspase-1, GSDMD-N and prevented apoptosis and pyroptosis in atrophic GAS. These findings for the first time demonstrate that targeting NLRP3-mediated pyroptosis with MCC950 improves diabetic muscle homeostasis and muscle function. We also report that inhibiting pyroptosis by MCC950 can enhance the beneficial effects of aerobic exercise on diabetic muscle atrophy. Since T2DM and muscle atrophy are age-related diseases, the young mice used in the current study do not seem to fully reflect the characteristics of diabetic muscle atrophy. Considering the fragile nature of db/db mice and for the complete implementation of the exercise intervention, we used relatively young db/db mice and the atrophic state in the mice was thoroughly confirmed. Taken together, the current study comprehensively investigated the therapeutic effect of NLRP3-mediated pyroptosis inhibited by MCC950 on diabetic muscle mass, strength and exercise performance, as well as the synergistic effects of MCC950 and exercise intervention, therefore providing a novel strategy for the treatment of the disease.
Subject(s)
Diabetes Mellitus, Experimental , Diabetes Mellitus, Type 2 , Diabetic Neuropathies , Mice , Animals , NLR Family, Pyrin Domain-Containing 3 Protein/genetics , Inflammasomes , Diabetes Mellitus, Experimental/complications , Diabetes Mellitus, Experimental/therapy , Pyroptosis , Sulfonamides/pharmacology , Mice, Inbred Strains , Diabetes Mellitus, Type 2/complications , Diabetes Mellitus, Type 2/therapy , Exercise , Muscular Atrophy/drug therapy , Muscular Atrophy/etiologyABSTRACT
Snake bite envenomation causes tissue damage resulting in acute and chronic inflammatory responses. Inflammasome activation is one of the factors involved in tissue damage in a mouse model of snake envenomation. The present study examines the potency of Indian Big Four snake venoms in the activation of inflammasome and its role in local and systemic tissue toxicity. Among Indian Big Four snake venoms, Naja naja venom activated NLRP3 inflammasome in mouse macrophages. Activation of NLRP3 inflammasome was also observed in mouse foot paw and thigh muscle upon administration of N. naja venom. Intraperitoneal administration of N. naja venom cause systemic lung damage showed activation of NLRP3 inflammasome. Treatment with MCC950, a selective NLRP3 inflammasome inhibitor effectively inhibited N. naja venom-induced activation of caspase-1 and liberation of IL-1ß in macrophages. In mice, MCC950 partially inhibited the activation of NLRP3 inflammasome in N. naja venom administered foot paw and thigh muscle. In conclusion, the present data showed that inflammasome is one of the host responses involved in N. naja snake venom-induced toxicities. The inhibition of inflammasome activation will provide new insight into better management of snake bite-induced local tissue damage.
Subject(s)
Inflammasomes , Snake Bites , Mice , Animals , NLR Family, Pyrin Domain-Containing 3 Protein , Naja naja , Elapid Venoms/toxicity , Snake Venoms , SulfonamidesABSTRACT
As a key regulator of the innate immune system, the NLRP3 inflammasome responds to a variety of environmental insults through activation of caspase-1 and release of the proinflammatory cytokines IL-1ß and IL-18. Aberrant NLRP3 inflammasome function is implicated in numerous inflammatory diseases, spurring drug discovery efforts at NLRP3 as a therapeutic target. A diverse array of small molecules is undergoing preclinical/clinical evaluation with a reported mode of action involving direct modulation of the NLRP3 pathway. However, for a subset of these ligands the functional link between live-cell target engagement and pathway inhibition has yet to be fully established. Herein we present a cohort of mechanistic assays to both query direct NLRP3 engagement in cells, and functionally interrogate different nodes of NLRP3 pathway activity. This system enabled the stratification of potency for five confirmed NLRP3 inhibitors, and identification of two reported NLRP3 inhibitors that failed to demonstrate direct pathway antagonism.
Subject(s)
Inflammasomes , NLR Family, Pyrin Domain-Containing 3 Protein , Humans , Inflammasomes/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Cytokines/metabolism , Interleukin-1beta/metabolismABSTRACT
Epilepsy is one of the most common serious chronic brain disorders, affecting up to 70 million people worldwide. Vascular disruption, including blood-brain barrier impairment and pathological angiogenesis, exacerbates its occurrence. However, its underlying mechanisms remain elusive. MCC950 is a specific small-molecule inhibitor that selectively blocks NLRP3 inflammatory vesicle activation across the blood-brain barrier, limits downstream IL-1ß maturation and release, and exerts therapeutic effects across multiple diseases. In the present study, an epilepsy model was established by intraperitoneal administration of Kainic acid to adult male C57BL/6J wild-type mice. The results revealed that the epilepsy susceptibility of MCC950-treated mice was decreased, and neural damage following seizure episodes was reduced. In addition, immunofluorescence staining, RT-qPCR, and Western blot demonstrated that MCC950 inhibited the expression of the NLRP3 inflammasome and its related proteins in microglia, whereas microangiogenesis was found to be increased in the cerebral cortex and hippocampus of epileptic mice, and these effects could be reversed by MCC950. Furthermore, neurobehavioral impairment was observed in the epileptic mouse model, and MCC950 similarly alleviated the aforementioned pathological process. To the best of our knowledge, this is the first study to establish that pathological microangiogenesis is associated with NLRP3/IL-1ß signaling pathway activation in a Kainic acid-induced epilepsy mouse model and that MCC950 administration attenuates the above-mentioned pathological changes and exerts neuroprotective effects. Therefore, MCC950 is a promising therapeutic agent for the treatment of epilepsy.
Subject(s)
Epilepsy , Indenes , Humans , Adult , Male , Animals , Mice , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Sulfones/therapeutic use , Sulfones/pharmacology , Pyroptosis , Angiogenesis , Kainic Acid , Mice, Inbred C57BL , Sulfonamides/therapeutic use , Sulfonamides/pharmacology , Inflammasomes/metabolism , Signal Transduction , Disease Models, Animal , Epilepsy/chemically induced , Epilepsy/drug therapy , Seizures/chemically induced , Seizures/drug therapy , Furans/therapeutic use , Furans/pharmacology , Indenes/therapeutic useABSTRACT
Alzheimer's disease (AD) is the seventh most common cause of mortality and one of the major causes of disability and vulnerability in the elderly. AD is characterized by gradual cognitive deterioration, the buildup of misfolded amyloid beta (Aß) peptide, and the generation of neurofibrillary tangles. Despite enormous scientific progress, there is no effective cure for AD. Thus, exploring new treatment options to stop AD or at least slow down its progress is important. In this study, we investigated the potential therapeutic effects of MCC950 on NLRP3-mediated inflammasome-driven inflammation and autophagy in AD. Rats treated with streptozotocin (STZ) exhibited simultaneous activation of the NLRP3 inflammasome and autophagy, as confirmed by Western blot, immunofluorescence, and co-immunoprecipitation analyses. MCC950, a specific NLRP3 inhibitor, was intraperitoneally administered (50 mg/kg body weight) to rats with AD-like symptoms induced by intracerebroventricular STZ injections (3 mg/kg body weight). MCC950 effectively suppressed STZ-induced cognitive impairment and anxiety by inhibiting NLRP3-dependent neuroinflammation. Moreover, our findings indicate that MCC950 exerts neuroprotective effects by attenuating autophagy in neuronal cells. The inhibiting effects of MCC950 on inflammasome activation and autophagy were reproduced in vitro, provding further mechansistic insights into MCC950 therapeutic action. Our findings suggest that MCC950 impedes the progression of AD and may also improve cognitive function through the mitigation of autophagy and NLRP3 inflammasome inhibition.
Subject(s)
Alzheimer Disease , NLR Family, Pyrin Domain-Containing 3 Protein , Humans , Rats , Animals , Aged , Alzheimer Disease/drug therapy , Inflammasomes , Amyloid beta-Peptides/pharmacology , Neuroinflammatory Diseases , Sulfonamides/pharmacology , Cognition , Autophagy , Body WeightABSTRACT
Bacterial meningitis is considered a life-threatening condition with high mortality rates. In response to the infection, signaling cascades, producing pro-inflammatory mediators trigger an exacerbated host immune response. Another inflammatory pathway occurs through the activation of inflammasomes. Studies highlight the role of the NLR family pyrin domain containing 3 (NLRP3) in central nervous system disorders commonly involved in neuroinflammation. We aimed to investigate the role of NLRP3 and its inhibitor MCC950 on neurochemical, immunological, and behavioral parameters in the early and late stages of experimental pneumococcal meningitis. For this, adult male Wistar rats received an intracisternal injection of Streptococcus pneumoniae or artificial cerebrospinal fluid as a placebo. The animals were divided into control/saline, control/MCC950, meningitis/saline, and meningitis/MCC950. Immediately after the meningitis induction, the animals received 140 ng/kg MCC950 via intracisternal injection. For the acute protocol, 24 h after induction, brain structures were collected to evaluate cytokines, NLRP3, and microglia. In the long-term group, the animals were submitted to open field and recognition of new objects tests at ten days after the meningitis induction. After the behavioral tests, the same markers were evaluated. The animals in the meningitis group at 24 h showed increased levels of cytokines, NLRP3, and IBA-1 expression, and the use of the MCC950 significantly reduced those levels. Although free from infection, ten days after meningitis induction, the animals in the meningitis group had elevated cytokine levels and demonstrated behavioral deficits; however, the single dose of NLRP3 inhibitor rescued the behavior deficits and decreased the brain inflammatory profile.
Subject(s)
Meningitis, Pneumococcal , Animals , Male , Rats , Cytokines/metabolism , Inflammasomes/metabolism , Memory Disorders , Meningitis, Pneumococcal/complications , Meningitis, Pneumococcal/drug therapy , Models, Theoretical , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Rats, Wistar , Sulfonamides/pharmacology , Sulfonamides/therapeutic useABSTRACT
ObjectiveTo investigate the possible mechanism of Shenqi Jianxin Formula (参芪健心方) in the treatment of chronic heart failure (CHF) from the perspective of pyroptosis. MethodsFifty-two rats were randomly divided into sham operation group (n=8) and modeling group (n=44). In the modeling group, the anterior descending branch of the left coronary artery was ligated to construct CHF rat model. Forty successfully-modelled rats were randomly divided into model group, Entresto group, Shenqi Jianxin Formula group, MCC950 group and the combination group (Shenqi Jianxin Formula plus MCC950), with 8 rats in each group. In Shenqi Jianxin Formula group, 7.4 g/(kg·d) of Shenqi Jianxin Formula was given by gavage, while in Entresto group, 68 mg/(kg·d) of Entresto suspension was given by gavage; in MCC950 group, MCC950 was injected intraperitoneally with 10 mg/kg once every other day, and in the combination group, 7.4 g/(kg·d) of Shenqi Jianxin Formula was given by gavage, and MCC950 was injected intraperitoneally with 10 mg/kg once every other day; 10 ml/(kg·d) of saline was given by gavage in the sham operation group and the model group. After 3 weeks of continuous intervention, serum brain B-type natriuretic peptide (BNP), creatine kinase isoenzyme MB (CK-MB), interleukin 1β (IL-1β), and interleukin 18 (IL-18) levels were detected by ELISA; HE staining and MASSON staining were used to observe pathological changes in rat myocardium. Except for the Entresto group, western blot technique was used to detect the expression of NOD-like receptor protein 3 (NLRP3), caspase-1, and apoptosis-associated speck-like protein possessing a caspase-recruiting domain (ASC); RT-PCR was used to detect the expression of NLRP3 and caspase-1 mRNA. ResultsCompared with the sham operation group, HE staining of rats in the model group showed obvious myocardial injury, while MASSON staining showed increased area of collagen fibrosis, and serum BNP, CK-MB, IL-1β, IL-18, myocardial tissue NLRP3, caspase-1, ASC protein expression and NLRP3, caspase-1 mRNA expression were all elevated (P<0.05). Compared with those in the model group, cardiomyocyte injury of rats and collagen fibrosis area were reduced, and serum BNP, CK-MB, IL-1β, and IL-18 contents were all reduced in Shenqi Jianxin Formula group, Entresto group, MCC950 group, and the combination group; except for Entresto group, myocardial tissue NLRP3, caspase-1, ASC protein expression and NLRP3, caspase-1 mRNA expression were reduced in the remaining three medication group (P<0.05). Compared with Shenqi Jianxin Formula group, the MCC950 group and the combination group showed decreased serum IL-1β and IL-18 content, collagen fibrosis area, myocardial tissue NLPR3, caspase-1 protein expression, and caspase-1 mRNA expression, and decreased ASC and NLRP3 mRNA expression was shown in the combination group (P<0.05). Compared with MCC950 group, collagen fibrosis area was reduced, and serum IL-18 content, NLRP3, caspase-1 mRNA expression were reduced in the combination group (P<0.05). ConclusionShenqi Jianxin Formula can effectively improve the myocardial injury and heart failure in rats with CHF, and its mechanism may be related to the inhibition of cardiomyocyte pyroptosis through NLPR3/Caspase-1 pathway to reduce the level of intramyocardial inflammation. The combined use of MCC950 with Shenqi Jianxin Formula could more effectively inhibite myocardial pyroptosis, with better therapeutic result than single use of each part.
ABSTRACT
BACKGROUND: Neuroimmune dysregulation from prenatal alcohol exposure (PAE) may contribute to neurological deficits associated with fetal alcohol spectrum disorders (FASD). PAE is a risk factor for developing peripheral immune and spinal glial sensitization and release of the proinflammatory cytokine IL-1ß, which lead to neuropathic pain (allodynia) from minor nerve injury. Although morphine acts on µ-opioid receptors, it also activates immune receptors, TLR4, and the NLRP3 inflammasome that induces IL-1ß. We hypothesized that PAE induces NLRP3 sensitization by morphine following nerve injury in adult mice. METHODS: We used an established moderate PAE paradigm, in which adult PAE and non-PAE control female mice were exposed to a minor sciatic nerve injury, and subsequent allodynia was measured using the von Frey fiber test. In control mice with standard sciatic damage or PAE mice with minor sciatic damage, the effects of the NLRP3 inhibitor, MCC950, were examined during chronic allodynia. Additionally, minor nerve-injured mice were treated with morphine, with or without MCC950. In vitro studies examined the TLR4-NLRP3-dependent proinflammatory response of peripheral macrophages to morphine and/or lipopolysaccharide, with or without MCC950. RESULTS: Mice with standard sciatic damage or PAE mice with minor sciatic damage developed robust allodynia. Blocking NLRP3 activation fully reversed allodynia in both control and PAE mice. Morphine paradoxically prolonged allodynia in PAE mice, while control mice with minor nerve injury remained stably non-allodynic. Allodynia resolved sooner in nerve-injured PAE mice without morphine treatment than in morphine-treated mice. MCC950 treatment significantly shortened allodynia in morphine-treated PAE mice. Morphine potentiated IL-1ß release from TLR4-activated PAE immune cells, while MCC950 treatment greatly reduced it. CONCLUSIONS: In female mice, PAE prolongs allodynia following morphine treatment through NLRP3 activation. TLR4-activated PAE immune cells showed enhanced IL-1ß release with morphine via NLRP3 actions. Similar studies are needed to examine the adverse impact of morphine in males with PAE. These results are predictive of adverse responses to opioid pain therapeutics in individuals with FASD.
ABSTRACT
The nucleotide-binding oligomerization domain (NOD)-like receptor (NLR), leucine-rich-repeat (LRR), and pyrin domain containing 3 (NLRP3) is one of the key players in neuroinflammation, which is a major pathological hallmark of Alzheimer's Disease (AD). Activated NLRP3 causes release of pro-inflammatory molecules that aggravate neurodegeneration. Thus, pharmacologically inhibiting the NLRP3 inflammasome has the potential to alleviate the inflammatory injury to the neurons. Coumarin is a multifunctional nucleus with potent anti-inflammatory properties and can be utilized to develop novel drugs for the treatment and management of AD. In the present study, we have explored the NLRP3-inhibitory activities of a library of coumarin derivatives through a computational drug discovery approach. Drug-like, PAINS free, and potentially BBB permeable compounds were screened out and subjected to molecular docking and in silico ADMET studies, resulting in three virtual hits, i.e. MolPort-050-872-358, MolPort-050-884-068, and MolPort-051-135-630. The hits exhibited better NLRP3-binding affinity than MCC950, a selective inhibitor of NLRP3. Further, molecular dynamics (MD) simulations, post-MD simulation analyses, and binding free energy calculations of the hits established their potential as promising virtual leads with a common coumarin scaffold for the inhibition of NLRP3 inflammasome.Communicated by Ramaswamy H. Sarma.