ABSTRACT
Immune checkpoint inhibitor (ICI)-induced myocarditis involves intensive immune/inflammation activation; however, its molecular basis is unclear. Here, we show that gasdermin-E (GSDME), a gasdermin family member, drives ICI-induced myocarditis. Pyroptosis mediated by GSDME, but not the canonical GSDMD, is activated in myocardial tissue of mice and cancer patients with ICI-induced myocarditis. Deficiency of GSDME in male mice alleviates ICI-induced cardiac infiltration of T cells, macrophages, and monocytes, as well as mitochondrial damage and inflammation. Restoration of GSDME expression specifically in cardiomyocytes, rather than myeloid cells, in GSDME-deficient mice reproduces ICI-induced myocarditis. Mechanistically, quantitative proteomics reveal that GSDME-dependent pyroptosis promotes cell death and mitochondrial DNA release, which in turn activates cGAS-STING signaling, triggering a robust interferon response and myocardial immune/inflammation activation. Pharmacological blockade of GSDME attenuates ICI-induced myocarditis and improves long-term survival in mice. Our findings may advance the understanding of ICI-induced myocarditis and suggest that targeting the GSDME-cGAS-STING-interferon axis may help prevent and manage ICI-associated myocarditis.
Subject(s)
Immune Checkpoint Inhibitors , Membrane Proteins , Myocarditis , Nucleotidyltransferases , Pyroptosis , Animals , Myocarditis/immunology , Myocarditis/pathology , Myocarditis/chemically induced , Myocarditis/metabolism , Membrane Proteins/metabolism , Membrane Proteins/genetics , Immune Checkpoint Inhibitors/pharmacology , Immune Checkpoint Inhibitors/adverse effects , Mice , Male , Humans , Nucleotidyltransferases/metabolism , Nucleotidyltransferases/genetics , Signal Transduction , Mice, Inbred C57BL , Mice, Knockout , DNA, Mitochondrial/metabolism , DNA, Mitochondrial/genetics , Female , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/pathology , Myocytes, Cardiac/drug effects , Intracellular Signaling Peptides and Proteins/metabolism , Intracellular Signaling Peptides and Proteins/genetics , Phosphate-Binding Proteins/metabolism , Phosphate-Binding Proteins/genetics , GasderminsABSTRACT
AIM: An analysis of bioinformatics and cell experiments was performed to verify the relationship between gasdermin D (GSDMD), an executive protein of pyroptosis, and Alzheimer's disease (AD). METHODS: The training set GSE33000 was utilized to identify differentially expressed genes (DEGs) in both the AD group and control group, as well as in the GSDMD protein high/low expression group. Subsequently, the weighted gene co-expression network analysis (WGCNA) and the least absolute shrinkage and selection operator (LASSO) regression analysis were conducted, followed by the selection of the key genes for the subsequent Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. The association between GSDMD and AD was assessed and confirmed in the training set GSE33000, as well as in the validation sets GSE5281 and GSE48350. Immunofluorescence (IF) was employed to detect the myelin basic protein (MBP), a distinctive protein found in the rat oligodendrocytes (OLN-93 cells). A range of concentrations (1-15 µmol/L) of ß-amyloid 1-42 (Aß1-42) were exposed to the cells, and the subsequent observations were made regarding cell morphology. Additionally, the assessments were conducted to evaluate the cell viability, the lactate dehydrogenase (LDH) release, the cell membrane permeability, and the GSDMD protein expression. RESULTS: A total of 7,492 DEGs were screened using GSE33000. Subsequently, WGCNA analysis identified 19 genes that exhibited the strongest correlation with clinical traits in AD. Additionally, LASSO regression analysis identified 13 key genes, including GSDMD, AFF1, and ATOH8. Furthermore, the investigation revealed that the key genes were associated with cellular inflammation based on GO and KEGG analyses. Moreover, the area under the curve (AUC) values for the key genes in the training and validation sets were determined to be 0.95 and 0.70, respectively. Significantly, GSDMD demonstrated elevated levels of expression in AD across both datasets. The positivity of MBP expression in cells exceeded 95%. As the concentration of Aß1-42 action gradually escalated, the detrimental effects on cells progressively intensified, resulting in a gradual decline in cell survival rate, accompanied by an increase in lactate dehydrogenase release, cell membrane permeability, and GSDMD protein expression. CONCLUSION: The association between GSDMD and AD has been observed, and it has been found that Aß1-42 can induce a significant upregulation of GSDMD in OLN-93 cells. This suggests that Aß1-42 has the potential to induce cellular pyroptosis and can serve as a valuable cellular pyroptosis model for the study of AD.
Subject(s)
Alzheimer Disease , Phosphate-Binding Proteins , Pyroptosis , Alzheimer Disease/metabolism , Pyroptosis/drug effects , Phosphate-Binding Proteins/metabolism , Phosphate-Binding Proteins/genetics , Humans , Animals , Rats , Intracellular Signaling Peptides and Proteins/metabolism , Intracellular Signaling Peptides and Proteins/genetics , Amyloid beta-Peptides/metabolism , Computational Biology , Peptide Fragments/metabolism , GasderminsABSTRACT
BACKGROUND: Cognitive dysfunction caused by infection frequently emerges as a complication in sepsis survivor patients. However, a comprehensive understanding of its pathogenesis remains elusive. METHODS: In our in vivo experiments, an animal model of endotoxemia was employed, utilizing the Novel Object Recognition Test and Morris Water Maze Test to assess cognitive function. Various techniques, including immunofluorescent staining, Western blotting, bloodâbrain barrier permeability assessment, Limulus Amebocyte Lysate (LAL) assay, and Proximity-ligation assay, were employed to identify brain pathological injury and neuroinflammation. To discern the role of Caspase-11 (Casp11) in hematopoietic or non-hematopoietic cells in endotoxemia-induced cognitive decline, bone marrow chimeras were generated through bone marrow transplantation (BMT) using wild-type (WT) and Casp11-deficient mice. In vitro studies involved treating BV2 cells with E. coli-derived outer membrane vesicles to mimic in vivo conditions. RESULTS: Our findings indicate that the deficiency of Casp11-GSDMD signaling pathways reverses infection-induced cognitive dysfunction. Moreover, cognitive dysfunction can be ameliorated by blocking the IL-1 effect. Mechanistically, the absence of Casp11 signaling significantly mitigated bloodâbrain barrier leakage, microglial activation, and synaptic damage in the hippocampal CA3 region, ultimately leading to improved cognitive function. CONCLUSION: This study unveils the crucial contribution of Casp11 and GSDMD to cognitive impairments and spatial memory loss in a murine sepsis model. Targeting Casp11 signaling emerges as a promising strategy for preventing or treating cognitive dysfunction in patients with severe infections.
Subject(s)
Caspases, Initiator , Caspases , Cognitive Dysfunction , Disease Models, Animal , Signal Transduction , Animals , Cognitive Dysfunction/etiology , Cognitive Dysfunction/metabolism , Mice , Caspases/metabolism , Caspases, Initiator/metabolism , Phosphate-Binding Proteins/metabolism , Phosphate-Binding Proteins/genetics , Blood-Brain Barrier/metabolism , Male , Mice, Inbred C57BL , Mice, Knockout , Endotoxemia/complications , Endotoxemia/metabolism , Endotoxemia/etiology , Hippocampus/metabolism , Hippocampus/pathology , Intracellular Signaling Peptides and Proteins/metabolism , Intracellular Signaling Peptides and Proteins/genetics , Sepsis/complications , Sepsis/metabolism , GasderminsABSTRACT
Anaplastic thyroid cancer (ATC) is among the most aggressive and metastatic malignancies, often resulting in fatal outcomes due to the lack of effective treatments. Prosapogenin A (PA), a bioactive compound prevalent in traditional Chinese herbs, has shown potential as an antineoplastic agent against various human tumors. However, its effects on ATC and the underlying mechanism remain unclear. Here, we demonstrate that PA exhibits significant anti-ATC activity both in vitro and in vivo by inducing GSDME-dependent pyroptosis in ATC cells. Mechanistically, PA promotes lysosomal membrane permeabilization (LMP), leading to the release of cathepsins that activate caspase 8/3 to cleave GSDME. Remarkably, PA significantly upregulates three key functional subunits of V-ATPase-ATP6V1A, ATP6V1B2, and ATP6V0C-resulting in lysosomal over-acidification. This over-acidification exacerbates LMP and subsequent lysosomal damage. Neutralization of lysosomal lumen acidification or inhibition/knockdown of these V-ATPase subunits attenuates PA-induced lysosomal damage, pyroptosis and growth inhibition of ATC cells, highlighting the critical role for lysosomal acidification and LMP in PA's anticancer effects. In summary, our findings uncover a novel link between PA and lysosomal damage-dependent pyroptosis in cancer cells. PA may act as a V-ATPase agonist targeting lysosomal acidification, presenting a new potential therapeutic option for ATC treatment.
Subject(s)
Lysosomes , Pyroptosis , Thyroid Carcinoma, Anaplastic , Vacuolar Proton-Translocating ATPases , Lysosomes/metabolism , Lysosomes/drug effects , Humans , Pyroptosis/drug effects , Vacuolar Proton-Translocating ATPases/metabolism , Thyroid Carcinoma, Anaplastic/metabolism , Thyroid Carcinoma, Anaplastic/pathology , Thyroid Carcinoma, Anaplastic/drug therapy , Animals , Cell Line, Tumor , Sapogenins/pharmacology , Mice , Mice, Nude , Thyroid Neoplasms/pathology , Thyroid Neoplasms/metabolism , Thyroid Neoplasms/drug therapy , Xenograft Model Antitumor Assays , Mice, Inbred BALB C , GasderminsABSTRACT
The septin cytoskeleton is primarily known for roles in cell division and host defense against bacterial infection. Despite recent insights, the full breadth of roles for septins in host defense is poorly understood. In macrophages, Shigella induces pyroptosis, a pro-inflammatory form of cell death dependent upon gasdermin D (GSDMD) pores at the plasma membrane and cell surface protein ninjurin-1 (NINJ1) for membrane rupture. Here, we discover that septins promote macrophage pyroptosis induced by lipopolysaccharide (LPS)/nigericin and Shigella infection, but do not affect cytokine expression or release. We observe that septin filaments assemble at the plasma membrane, and cleavage of GSDMD is impaired in septin-depleted cells. We found that septins regulate mitochondrial dynamics and the expression of NINJ1. Using a Shigella-zebrafish infection model, we show that septin-mediated pyroptosis is an in vivo mechanism of infection control. The discovery of septins as a mediator of pyroptosis may inspire innovative anti-bacterial and anti-inflammatory treatments.
Subject(s)
Cell Adhesion Molecules, Neuronal , Cell Membrane , Intracellular Signaling Peptides and Proteins , Macrophages , Phosphate-Binding Proteins , Pyroptosis , Septins , Pyroptosis/drug effects , Septins/metabolism , Phosphate-Binding Proteins/metabolism , Mice , Animals , Macrophages/metabolism , Cell Membrane/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Intracellular Signaling Peptides and Proteins/genetics , Cell Adhesion Molecules, Neuronal/metabolism , Cell Adhesion Molecules, Neuronal/genetics , Humans , Lipopolysaccharides/pharmacology , Mice, Inbred C57BL , RAW 264.7 Cells , Gasdermins , Nerve Growth FactorsABSTRACT
BACKGROUND: Melanoma, one of the most lethal forms of skin cancer, has the potential to develop in any area where melanocytes are present. Currently, postoperative recurrence due to the emergence of systemic drug resistance represents a significant challenge in the treatment of melanoma. In this study, terphenyllin (TER), a distinctive inhibitory impact on melanoma cells was identified from the natural p-terphenyl metabolite. This study aimed to elucidate the intrinsic mechanism of this inhibitory effect, which may facilitate the discovery of novel chemotherapeutic agents. METHODS: A transcriptome sequencing and metabolomic analysis of TER-treated A375 cells was conducted to identify potential pathways of action. The key proteins were knocked out and backfilled using CRISPR-Cas9 technology and molecular cloning. Subsequently, the results of cytosolic viability, LDH release, immunofluorescence and flow cytometry were employed to demonstrate the cell death status of the drug-treated cells. RESULTS: The p53 signalling pathway was markedly upregulated following TER treatment, leading to the activation of CASP3 via the intrinsic apoptotic pathway. The activated CASP3 initiated apoptosis, while simultaneously continuing to cleave the GSDME, thereby triggering pyroptosis. The knockout of p53, a key protein situated upstream of this pathway, resulted in a significant rescue of TER-induced cell death, as well as an alleviation of the decrease in cell viability. However, the knockout of key proteins situated downstream of the pathway (CASP3 and GSDME) did not result in a rescue of TER-induced cell death, but rather a transformation of the cells from apoptosis and pyroptosis. CONCLUSIONS: The induction of apoptosis and pyroptosis in A375 cells by TER is mediated via the p53-BAX/FAS-CASP3-GSDME signalling pathway. This lays the foundation for TER as a potential anti-melanoma drug in the future. It should be noted that CASP3 and GSDME in this pathway solely regulate the mode of cell death, rather than determine whether cell death occurs. This distinction may prove valuable in future studies of apoptosis and pyroptosis.
Subject(s)
Apoptosis , Caspase 3 , Pyroptosis , Tumor Suppressor Protein p53 , Up-Regulation , Humans , Tumor Suppressor Protein p53/metabolism , Tumor Suppressor Protein p53/genetics , Pyroptosis/drug effects , Pyroptosis/genetics , Apoptosis/drug effects , Up-Regulation/drug effects , Caspase 3/metabolism , Cell Line, Tumor , Melanoma/metabolism , Melanoma/genetics , Melanoma/pathology , Signal Transduction/drug effects , Gene Expression Regulation, Neoplastic/drug effects , GasderminsABSTRACT
Lung cancer causes a significant threat to human health. Despite considerable advancements in the treatment technologies in recent years, the five-year survival rate for lung cancer patients remains low. In this context, the discovery of pyroptosis, a unique cell death mechanism, offers a novel perspective for exploring new pathways of lung cancer treatment. Particularly, the role of gasdermin E (GSDME) in the process of pyroptosis reveals its tremendous potential in lung cancer therapy. Recent studies have made considerable progress in understanding the role of GSDME-mediated pyroptosis in lung cancer growth, the lung cancer microenvironment, and the effect of GSDME methylation on lung cancer treatment. This paper summarizes these research advancements and analyzes the potential and possible side effects of GSDME-mediated pyroptosis in lung cancer therapy, aiming to provide a theoretical foundation for developing more effective strategies for lung cancer treatment.â©.
Subject(s)
Lung Neoplasms , Pyroptosis , Humans , Lung Neoplasms/metabolism , Lung Neoplasms/drug therapy , Lung Neoplasms/pathology , Lung Neoplasms/genetics , Animals , GasderminsABSTRACT
Pyroptosis is an inflammatory programed cell death process that plays a crucial role in cancer therapeutic, while Gasdermin-D is a critical effector protein for pyroptosis execution. This review discusses the intricate interactions between Gasdermin-D and some non-coding RNAs (lncRNA, miRNA, siRNA) and their potential application in the regulation of pyroptosis as an anticancer therapy. Correspondingly, these ncRNAs significantly implicate in Gasdermin-D expression and function regarding the pyroptosis pathway. Functioning as competing endogenous RNAs (ceRNAs), these ncRNAs might regulate Gasdermin-D at the molecular level, underlying fatal cell death caused by cancer and tumor propagation. Therefore, these interactions appeal to therapeutics, offering new avenues for cancer treatment. It address this research gap by discussing the possible roles of ncRNAs as mediators of gasdermin-D regulation. It suggest therapeutic strategies based on the current research findings to ensure the interchange between the ideal pyroptosis and cancer cell death.
Subject(s)
Intracellular Signaling Peptides and Proteins , Neoplasms , Phosphate-Binding Proteins , Pyroptosis , RNA, Untranslated , Pyroptosis/physiology , Humans , Neoplasms/pathology , Neoplasms/genetics , Neoplasms/metabolism , Neoplasms/therapy , Phosphate-Binding Proteins/metabolism , Phosphate-Binding Proteins/genetics , RNA, Untranslated/genetics , RNA, Untranslated/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Intracellular Signaling Peptides and Proteins/genetics , Animals , Gene Expression Regulation, Neoplastic , GasderminsABSTRACT
In recent years, researchers have focused on studying the mechanism of sepsis-induced immunosuppression, but there is still a lack of suitable animal models that accurately reflect the process of sepsis-induced immunosuppression. The aim of this study was to evaluate the immune status at various stages in a model of sepsis-induced secondary pneumonia and to demonstrate whether pyroptosis is one of the modes of immune cell death in sepsis. Firstly, we established a sepsis model in C57BL/6J mice using cecal ligation and puncture (CLP). The surviving mice were treated with a 40 µL suspension of P.aeruginosa (Pa) under anesthesia on day 4 post-CLP to establish a sepsis-induced secondary pneumonia model. Secondly, routine blood tests, serum ALT and PCT levels, gross lung specimens, and H&E staining of the lung and liver tissues were used to assess the successful establishment of this model. Serum levels of TNF-α and IL-6, the CD4+/CD8+ratio in blood, H&E staining of the spleen, and immunohistochemistry of CD4 and CD8 in the spleen were detected to evaluate the immune status of the model mice. Finally, the expression levels of pyroptosis-related proteins in the spleen were detected by Western blot. The expression of GSDMD was assessed using immunohistochemistry, and pyroptosis was directly observed through transmission electron microscopy. The experimental results above confirmed the successful construction of the model for sepsis-induced secondary pneumonia, demonstrating its ability to reflect sepsis-induced immunosuppression. Moreover, the expression of pyroptosis-related proteins, immunohistochemical GSDMD, and transmission electron microscopy of the spleen showed that pyroptosis was one of the modes of immune cell death in sepsis.
Subject(s)
Disease Models, Animal , Mice, Inbred C57BL , Pyroptosis , Sepsis , Spleen , Animals , Sepsis/immunology , Mice , Spleen/immunology , Spleen/pathology , Male , Lung/pathology , Lung/immunology , Tumor Necrosis Factor-alpha/metabolism , Pneumonia/immunology , Pneumonia/pathology , Pneumonia/etiology , Interleukin-6/metabolism , Interleukin-6/blood , Phosphate-Binding Proteins/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , GasderminsABSTRACT
In the contemporary landscape of oncology, immunotherapy, represented by immune checkpoint blockade (ICB) therapy, stands out as a beacon of innovation in cancer treatment. Despite its promise, the therapy's progression is hindered by suboptimal clinical response rates. Addressing this challenge, the modulation of the NLRP3 inflammasome-GSDMD-mediated pyroptosis pathway holds promise as a means to augment the efficacy of immunotherapy. In the pathway, the NLRP3 inflammasome serves as a pivotal molecular sensor that responds to inflammatory stimuli within the organism. Its activation leads to the release of cytokines interleukin 1ß and interleukin 18 through the cleavage of GSDMD, thereby forming membrane pores and potentially resulting in pyroptosis. This cascade of processes exerts a profound impact on tumor development and progression, with its function and expression exhibiting variability across different tumor types and developmental stages. Consequently, understanding the specific roles of the NLRP3 inflammasome and GSDMD-mediated pyroptosis in diverse tumors is imperative for comprehending tumorigenesis and crafting precise therapeutic strategies. This review aims to elucidate the structure and activation mechanisms of the NLRP3 inflammasome, as well as the induction mechanisms of GSDMD-mediated pyroptosis. Additionally, we provide a comprehensive overview of the involvement of this pathway in various cancer types and its applications in tumor immunotherapy, nanotherapy, and other fields. Emphasis is placed on the feasibility of leveraging this approach to enhance ICB therapy within the field of immunotherapy. Furthermore, we discuss the potential applications of this pathway in other immunotherapy methods, such as chimeric antigen receptor T-cell (CAR-T) therapy and tumor vaccines.
Subject(s)
Immunotherapy , Inflammasomes , Intracellular Signaling Peptides and Proteins , NLR Family, Pyrin Domain-Containing 3 Protein , Neoplasms , Phosphate-Binding Proteins , Pyroptosis , Humans , Pyroptosis/immunology , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/immunology , Neoplasms/immunology , Neoplasms/therapy , Immunotherapy/methods , Inflammasomes/metabolism , Inflammasomes/immunology , Phosphate-Binding Proteins/metabolism , Animals , Intracellular Signaling Peptides and Proteins/metabolism , Intracellular Signaling Peptides and Proteins/immunology , GasderminsABSTRACT
Pyroptosis of programmed cell death has been recognized as a more effective way to inhibit the occurrence and development of tumors than the better-studied apoptosis. However, it is still challenging to quickly and effectively trigger pyroptosis of cancer cells for high-efficacy cancer treatment. Here, we report on the first use of mild constant-potential electrostimulation (cp-ES) to quickly trigger cancer cell pyroptosis with a probability up to â¼91.4% and significantly shortened time (within 1 h), â¼3-6 times faster than typical drug stimulation to induce pyroptosis. We find that the ES-induced cancer cell pyroptosis is through the activated caspase-3 (pathway) cleavage of gasdermin E (GSDME) to form an N-terminal fragment (GSDME-N) and observe nuclear shrinkage and reduction of the number of nucleoli as well as down-/up-regulated expression of two important nucleoproteins of nucleolin and nucleophosmin (NPM1). The study enriches the basic understanding of pyroptosis and provides a new avenue for potential effective treatment of cancer.
Subject(s)
Caspase 3 , Nucleophosmin , Pyroptosis , Humans , Caspase 3/metabolism , Nucleolin , Cell Line, Tumor , Cell Nucleus/metabolism , Nuclear Proteins/metabolism , Phosphate-Binding Proteins/metabolism , RNA-Binding Proteins/metabolism , GasderminsABSTRACT
Mitotic catastrophe (MC), which occurs under dysregulated mitosis, represents a fascinating tactic to specifically eradicate tumor cells. Whether pyroptosis can be a death form of MC remains unknown. Proteasome-mediated protein degradation is crucial for M-phase. Bortezomib (BTZ), which inhibits the 20S catalytic particle of proteasome, is approved to treat multiple myeloma and mantle cell lymphoma, but not solid tumors due to primary resistance. To date, whether and how proteasome inhibitor affected the fates of cells in M-phase remains unexplored. Here, we show that BTZ treatment, or silencing of PSMC5, a subunit of 19S regulatory particle of proteasome, causes G2- and M-phase arrest, multi-polar spindle formation, and consequent caspase-3/GSDME-mediated pyroptosis in M-phase (designated as mitotic pyroptosis). Further investigations reveal that inhibitor of WEE1/PKMYT1 (PD0166285), but not inhibitor of ATR, CHK1 or CHK2, abrogates the BTZ-induced G2-phase arrest, thus exacerbates the BTZ-induced mitotic arrest and pyroptosis. Combined BTZ and PD0166285 treatment (named BP-Combo) selectively kills various types of solid tumor cells, and significantly lessens the IC50 of both BTZ and PD0166285 compared to BTZ or PD0166285 monotreatment. Studies using various mouse models show that BP-Combo has much stronger inhibition on tumor growth and metastasis than BTZ or PD0166285 monotreatment, and no obvious toxicity is observed in BP-Combo-treated mice. These findings disclose the effect of proteasome inhibitors in inducing pyroptosis in M-phase, characterize pyroptosis as a new death form of mitotic catastrophe, and identify dual inhibition of proteasome and WEE family kinases as a promising anti-cancer strategy to selectively kill solid tumor cells.
Subject(s)
Bortezomib , Cell Cycle Proteins , Mitosis , Proteasome Endopeptidase Complex , Protein-Tyrosine Kinases , Pyroptosis , Pyroptosis/drug effects , Humans , Mice , Animals , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/metabolism , Mitosis/drug effects , Mitosis/genetics , Proteasome Endopeptidase Complex/metabolism , Proteasome Endopeptidase Complex/genetics , Bortezomib/pharmacology , Cell Line, Tumor , Cell Cycle Proteins/genetics , Cell Cycle Proteins/antagonists & inhibitors , Cell Cycle Proteins/metabolism , Proteasome Inhibitors/pharmacology , Pyrimidines/pharmacology , Pyrazoles/pharmacology , Neoplasms/drug therapy , Neoplasms/genetics , Neoplasms/pathology , Xenograft Model Antitumor Assays , Gasdermins , PyrimidinonesSubject(s)
Phosphate-Binding Proteins , Humans , Phosphate-Binding Proteins/metabolism , Female , Male , GasderminsABSTRACT
Gasdermin E (GSDME), a member of the gasdermin protein family, is associated with post-lingual hearing loss. All GSDME pathogenic mutations lead to skipping exon 8; however, the molecular mechanisms underlying hearing loss caused by GSDME mutants remain unclear. GSDME was recently identified as one of the mediators of programmed cell death, including apoptosis and pyroptosis. Therefore, in this study, we injected mice with GSDME mutant (MT) and examined the expression levels to assess its effect on hearing impairment. We observed loss of hair cells in the organ of Corti and spiral ganglion neurons. Further, the N-terminal release from the GSDME mutant in HEI-OC1 cells caused pyroptosis, characterized by cell swelling and rupture of the plasma membrane, releasing lactate dehydrogenase and cytokines such as interleukin-1ß. We also observed that the N-terminal release from GSDME mutants could permeabilize the mitochondrial membrane, releasing cytochromes and activating the mitochondrial apoptotic pathway, thereby generating possible positive feedback on the cleavage of GSDME. Furthermore, we found that treatment with disulfiram or dimethyl fumarate might inhibit pyroptosis and apoptosis by inhibiting the release of GSDME-N from GSDME mutants. In conclusion, this study elucidated the molecular mechanism associated with hearing loss caused by GSDME gene mutations, offering novel insights for potential treatment strategies.
Subject(s)
Apoptosis , Pyroptosis , Pyroptosis/genetics , Animals , Mice , Gain of Function Mutation , Hearing Loss/genetics , Hearing Loss/pathology , Humans , Spiral Ganglion/metabolism , Spiral Ganglion/pathology , Organ of Corti/metabolism , Organ of Corti/pathology , Hair Cells, Auditory/metabolism , Hair Cells, Auditory/pathology , GasderminsABSTRACT
Bronchopulmonary dysplasia (BPD) is a common serious complication of premature babies. No effective means control it. Hyperoxia damage is one of the important mechanisms of BPD. The reaserach confirmed pyroptosis existed in BPD. Dexmedetomidine is a new, high-specific α2 receptor agonist. Previous research foundation found that dexmedetomidine has a protective effect on BPD. To investigate how dexmedetomidine improves hyperoxic lung injury in neonatal mice by regulating pyroptosis. Neonatal rats were randomly divided into four groups: normal control group, hyperoxic injury group, air plus dexmedetomidine group, and hyperoxia plus dexmedetomidine group. After seven days the lungs of rats in each group were extracted, and the wet-to-dry weight ratio of the lung was measured. The lung injury in rats was observed using hematoxylin-eosin staining. Additionally, the expression and localization of nucleotide-binding oligomerization domain-like receptor thermal protein domain associated protein 3 (NLRP3), apoptosis-associated speck-like protein (ASC), and gasdermin D (GSDMD) proteins were examined in the lungs of rats using immunofluorescence staining. The mRNA levels of NLRP3, ASC, caspase-1, and interleukin 18 (IL-18) in the lungs of rats were determined using real-time PCR. Moreover, the protein levels of NLRP3, ASC, caspase-1/cleaved caspase-1, interleukin 1beta (IL-1ß), IL-18, and tunor necrosis factor alpha (TNF-α) were detected in lungs of rats using Western blot. The extent of mitochondrial damage in lung tissues of each group was observed by transmission electron microscopy. The lung tissue injury of the neonatal rats was significantly improved in the hyperoxia plus dexmedetomidine group compared to the hyperoxic injury group. Furthermore, the expressions of pyroptosis-related proteins such as NLRP3, ASC, cleaved-caspase-1, and GSDMD were significantly decreased, along with the expressions of inflammatory factors in lung tissues. By inhibiting the NLRP3/caspase-1/GSDMD pyroptosis pathway, dexmedetomidine reduces the activation and release of inflammatory factors and provides a protective effect against hyperoxic lung injury in neonatal mice.
Subject(s)
Animals, Newborn , Dexmedetomidine , Hyperoxia , Lung Injury , Lung , NLR Family, Pyrin Domain-Containing 3 Protein , Pyroptosis , Rats, Sprague-Dawley , Animals , Dexmedetomidine/pharmacology , Dexmedetomidine/therapeutic use , Hyperoxia/metabolism , Hyperoxia/complications , Hyperoxia/drug therapy , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Lung/drug effects , Lung/pathology , Lung/metabolism , Pyroptosis/drug effects , Lung Injury/metabolism , Lung Injury/prevention & control , Lung Injury/pathology , Lung Injury/drug therapy , Rats , Phosphate-Binding Proteins/metabolism , CARD Signaling Adaptor Proteins/metabolism , Caspase 1/metabolism , Interleukin-18/metabolism , Adrenergic alpha-2 Receptor Agonists/pharmacology , Adrenergic alpha-2 Receptor Agonists/therapeutic use , Male , GasderminsABSTRACT
Gasdermin D (GSDMD)-mediated pyroptotic cell death drives inflammatory cytokine release and downstream immune responses upon inflammasome activation, which play important roles in host defense and inflammatory disorders. Upon activation by proteases, the GSDMD N-terminal domain (NTD) undergoes oligomerization and membrane translocation in the presence of lipids to assemble pores. Despite intensive studies, the molecular events underlying the transition of GSDMD from an autoinhibited soluble form to an oligomeric pore form inserted into the membrane remain incompletely understood. Previous work characterized S-palmitoylation for gasdermins from bacteria, fungi, invertebrates, as well as mammalian gasdermin E (GSDME). Here, we report that a conserved residue Cys191 in human GSDMD was S-palmitoylated, which promoted GSDMD-mediated pyroptosis and cytokine release. Mutation of Cys191 or treatment with palmitoyltransferase inhibitors cyano-myracrylamide (CMA) or 2-bromopalmitate (2BP) suppressed GSDMD palmitoylation, its localization to the membrane and dampened pyroptosis or IL-1ß secretion. Furthermore, Gsdmd-dependent inflammatory responses were alleviated by inhibition of palmitoylation in vivo. By contrast, coexpression of GSDMD with palmitoyltransferases enhanced pyroptotic cell death, while introduction of exogenous palmitoylation sequences fully restored pyroptotic activities to the C191A mutant, suggesting that palmitoylation-mediated membrane localization may be distinct from other molecular events such as GSDMD conformational change during pore assembly. Collectively, our study suggests that S-palmitoylation may be a shared regulatory mechanism for GSDMD and other gasdermins, which points to potential avenues for therapeutically targeting S-palmitoylation of gasdermins in inflammatory disorders.
Subject(s)
Cysteine , Intracellular Signaling Peptides and Proteins , Lipoylation , Phosphate-Binding Proteins , Pyroptosis , Phosphate-Binding Proteins/metabolism , Phosphate-Binding Proteins/genetics , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Intracellular Signaling Peptides and Proteins/genetics , Cysteine/metabolism , Animals , Mice , Cytokines/metabolism , HEK293 Cells , Inflammasomes/metabolism , GasderminsABSTRACT
Chronic prostatitis is one of the most common urologic diseases that troubles young men, with unclear etiology and ineffective treatment approach. Pyroptosis is a novel model of cell death, and its roles in chronic prostatitis are unknown. In this study, P2X7R, NEK7, and GSDMD-NT expression levels were detected in prostate tissues from benign prostate hyperplasia (BPH) patients and experiment autoimmune prostatitis (EAP) mice. P2X7R agonist, antagonist, NLRP3 inhibitor, and disulfiram were used to explore the roles of the P2X7R-NEK7-NLRP3 axis in prostate epithelial cell pyroptosis and chronic prostatitis development. We found that P2X7R, NEK7, and GSDMD-NT were highly expressed in the prostate epithelial cells of BPH patients with prostatic inflammation and EAP mice. Activation of P2X7R exacerbated prostatic inflammation and increased NLRP3 inflammasome component expressions and T helper 17 (Th17) cell proportion. Moreover, P2X7R-mediated potassium efflux promoted NEK7-NLRP3 interaction, and NLRP3 assembly and activation, which caused GSDMD-NT-mediated prostate epithelial cell pyroptosis to exacerbate EAP development. Disulfiram could effectively improve EAP by inhibiting GSDMD-NT-mediated prostate epithelial cell pyroptosis. In conclusion, the P2X7R-NEK7-NLRP3 axis could promote GSDMD-NT-mediated prostate epithelial cell pyroptosis and chronic prostatitis development, and disulfiram may be an effective drug to treat chronic prostatitis.
Subject(s)
Epithelial Cells , NIMA-Related Kinases , NLR Family, Pyrin Domain-Containing 3 Protein , Phosphate-Binding Proteins , Prostate , Prostatitis , Pyroptosis , Animals , Humans , Male , Mice , Autoimmune Diseases/metabolism , Epithelial Cells/metabolism , Gasdermins , Mice, Inbred C57BL , NIMA-Related Kinases/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Phosphate-Binding Proteins/metabolism , Poly-ADP-Ribose Binding Proteins/metabolism , Prostate/metabolism , Prostatitis/metabolism , Pyroptosis/drug effects , Receptors, Purinergic P2X7/metabolismABSTRACT
Diabetic cardiomyopathy (DCM) is a chronic disease caused by diabetes mellitus, which is recognized as a worldwide challenging disease. This study aimed to investigate the role and the potential mechanism of knocking down the NACHT-, LRR- and PYD domains-containing protein 3 (NLRP3), an inflammasome associated with onset and progression of various diseases, on high glucose or diabetes -induced cardiac cells pyroptosis and ferroptosis, two regulated non-necrosis cell death modalities discovered recent years. In the present study, both in vivo and in vitro studies were conducted simultaneously. Diabetic rats were induced by 55 mg/kg intraperitoneal injection of streptozotocin (STZ). Following the intraperitoneal injection of MCC950 (10 mg/kg), On the other hand, the DCM model in H9C2 cardiac cells was simulated with 35 mmol/L glucose and a short hairpin RNA vector of NLRP3 were transfected to cells. The results showed that in vivo study, myocardial fibers were loosely arranged and showed inflammatory cell infiltration, mitochondrial cristae were broken and the GSDMD-NT expression was found notably increased in the DM group, while the protein expressions of xCT and GPX4 was significantly decreased, both of which were reversed by MCC950. High glucose reduced the cell viability and ATP level in vitro, accompanied by an increase in LDH release. All of the above indicators were reversed after NLRP3 knockdown compared with the HG treated alone. Moreover, the protein expressions of pyroptosis- and ferroptosis-related fators were significantly decreased or increased, consistent with the results shown by immunofluorescence. Furthermore, the protective effects of NLRP3 knockdown against HG were reversed following the mtROS agonist rotenone (ROT) treatment. In conclusion, inhibition of NLRP3 suppressed DM-induced myocardial injury. Promotion of mitochondrial ROS abolished the protective effect of knockdown NLRP3, and induced the happening of pyroptosis and ferroptosis. These findings may present a novel therapeutic underlying mechanism for clinical diabetes-induced myocardial injury treatment.
Subject(s)
Diabetes Mellitus, Experimental , Diabetic Cardiomyopathies , Ferroptosis , Gene Knockdown Techniques , Myocytes, Cardiac , NLR Family, Pyrin Domain-Containing 3 Protein , Pyroptosis , Animals , Ferroptosis/drug effects , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/genetics , Diabetic Cardiomyopathies/metabolism , Diabetic Cardiomyopathies/genetics , Diabetic Cardiomyopathies/pathology , Diabetic Cardiomyopathies/etiology , Diabetic Cardiomyopathies/physiopathology , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/pathology , Male , Diabetes Mellitus, Experimental/complications , Diabetes Mellitus, Experimental/metabolism , Cell Line , Rats, Sprague-Dawley , Rats , Signal Transduction , Reactive Oxygen Species/metabolism , Inflammasomes/metabolism , Sulfonamides/pharmacology , Phosphate-Binding Proteins/metabolism , Phosphate-Binding Proteins/genetics , GasderminsABSTRACT
BACKGROUND: Psoriasis refers to a highly prevalent and immunologically mediated dermatosis with considerable deterioration in life quality. Wogonin, a sort of flavonoid, has been mentioned to elicit protective activities in skin diseases. However, whether Wogonin is implicated in the treatment of psoriasis and its specific mechanisms are not fully understood. AIM: The present work attempted to elaborate the role of Wogonin during the process of psoriasis and to concentrate on the associated action mechanism. METHODS: Cell counting kit-8 (CCK-8) method was initially applied to assay the viability of human keratinocyte HaCaT cells treated by varying concentrations of Wogonin. To mimic psoriasis in vitro, HaCaT cells were exposed to M5 cytokines. CCK-8 and 5-Ethynyl-2'-deoxyuridine assays were adopted for the measurement of cell proliferation. Inflammatory levels were examined with enzyme-linked immunosorbent assay. Immunofluorescence staining tested nucleotide-binding oligomerization domain (NOD)-like receptor family pyrin domain containing 3 (NLRP3) and Caspase-1 expressions. Western blot examined the protein expressions of proliferation-, inflammation-, pyroptosis-associated factors, and NLRP3. RESULTS: Wogonin treatment antagonized the proliferation, inflammatory response, and NLRP3/caspase-1/Gasdermin-D (GSDMD)-mediated pyroptosis in M5-challenged HaCaT cells. Besides, NLRP3 elevation partially abrogated the effects of Wogonin on M5-induced proliferation, inflammatory response, and NLRP3/caspase-1/GSDMD-mediated pyroptosis in HaCaT cells. CONCLUSION: In a word, Wogonin might exert anti-proliferation, anti-inflammatory and anti-pyroptosis activities in M5-induced cell model of psoriasis and the blockade of NLRP3/Caspase-1/GSDMD pathway might be recognized as a potential mechanism underlying the protective mechanism of Wogonin in psoriasis, suggesting Wogonin as a prospective anti-psoriasis drug.
Subject(s)
Caspase 1 , Cell Proliferation , Flavanones , Keratinocytes , NLR Family, Pyrin Domain-Containing 3 Protein , Pyroptosis , Signal Transduction , Humans , Flavanones/pharmacology , Pyroptosis/drug effects , Keratinocytes/drug effects , Keratinocytes/metabolism , Cell Proliferation/drug effects , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Caspase 1/metabolism , Signal Transduction/drug effects , Intracellular Signaling Peptides and Proteins/metabolism , Psoriasis/drug therapy , Psoriasis/metabolism , Psoriasis/pathology , Inflammation/metabolism , Inflammation/drug therapy , HaCaT Cells , Cell Line , Gasdermins , Phosphate-Binding ProteinsABSTRACT
Inflammatory illnesses, such as periodontitis and atherosclerotic coronary heart disease (ASCHD), trigger the production of pro-inflammatory mediators. The aim of this study was to assess the accuracy of using salivary interleukin-1ß (IL-1ß), interleukin-18 (IL-18), and gasdermin D (GSDMD) in discerning patients with periodontitis with and without ASCHD from healthy individuals, and to assess their correlation with clinical periodontal parameters and low-density lipoprotein (LDL) levels. The study involved 120 participants: 30 were healthy subjects (control group, C), 30 had generalized periodontitis (group P), 30 had ASCHD and clinically healthy periodontium (group AS-C), and 30 had ASCHD and generalized periodontitis (group AS-P). Saliva and blood samples were collected, and periodontal characteristics such as plaque index, bleeding on probing, probing pocket depth, and clinical attachment loss were examined. IL-1ß, IL-18, and GSDMD levels from saliva were determined using ELISA. LDL levels were determined from the blood samples. Groups P, AS-C, and AS-P had higher levels of salivary IL-1ß, IL-18, and GSDMD than group C. The receiver operating characteristic (ROC) curves of all biomarkers showed high diagnostic accuracy, with a significant positive correlation with the clinical parameters and LDL levels. The observed correlations between the studied pro-inflammatory mediators and disease severity suggest that these biomarkers could serve as indicators of disease progression in conditions such as periodontitis and ASCHD.