Mitochondria-Targeting Type-I Photodrug: Harnessing Caspase-3 Activity for Pyroptotic Oncotherapy.

Precise control of cellular signaling events during programmed cell death is crucial yet challenging for cancer therapy. The modulation of signal transduction in cancer cells holds promise but is limited by the lack of efficient, biocompatible, and spatiotemporally controllable approaches. Here we report a photodynamic strategy that modulates both apoptotic and pyroptotic cell death by altering caspase-3 protein activity and the associated signaling crosstalk. This strategy employs a mitochondria-targeting, near-infrared activatable probe (termed M-TOP) that functions via a type-I photochemical mechanism. M-TOP is less dependent on oxygen and more effective in treating drug-resistant cancer cells, even under hypoxic conditions. Our study shows that higher doses of M-TOP induce pyroptotic cell death via the caspase-3/gasdermin-E pathway, whereas lower doses lead to apoptosis. This photodynamic method is effective across diverse gasdermin-E-expressing cancer cells. Moreover, the M-TOP mediated shift from apoptotic to pyroptotic modulation can evoke a controlled inflammatory response, leading to a robust yet balanced immune reaction. This effectively inhibits both distal tumor growth and postsurgical tumor recurrence. This work demonstrates the feasibility of modulating intracellular signaling through the rational design of photodynamic anticancer drugs.

Knockout of Erbin promotes pyroptosis via regulating NLRP3/caspase-1/Gasdermin D pathway in sepsis-induced acute kidney injury.

BACKGROUND: This study aims to investigate the correlation between Erbin and sepsis, and the role of Erbin on the pyroptosis pathway in acute kidney injury caused by sepsis and NLRP3/caspase-1/Gasdermin D pathway. METHODS: In the study, lipopolysaccharide (LPS) treatment or cecal ligation and puncture (CLP) surgery on mice were used to stimulate the in vitro and in vivo sepsis-induced renal injury model. The male C57BL/6 of wild-type mice (WT) and Erbin-knockout mice (Erbin-/-, EKO) were randomly divided into four groups (WT + Sham, WT + CLP, EKO + Sham, EKO + CLP). Inflammatory cytokine, renal function, pyroptotic cell numbers and the levels of protein and mRNA expression of pyroptosis, including the NLRP3 (all P < 0.05), were analyzed and found increase in Erbin-/- mice with CLP and LPS-induced HK-2 cells. RESULTS: The inhibited of Erbin shows a renal damaged effect by promoting NLRP3 inflammasome-mediated pyroptosis in SI-AKI. CONCLUSION: This study demonstrated a novel mechanism by which Erbin regulates NLRP3 inflammasome-mediated pyroptosis in SI-AKI.

TanshinoneIIA inhibits melanocyte pyroptosis by regulating the ROS/NLRP3 signaling axis.

BACKGROUND: Pyroptosis has been implicated in the development of human diseases, including vitiligo. TanshinoneIIA has been confirmed to play anti-vitiligo role. However, whether tanshinoneIIA inhibits vitiligo progression via regulating cell pyroptosis remains unclear. METHODS: Hydrogen peroxide (H2 O2 )-induced melanocytes were used to mimic vitiligo cell model in vitro. Cell viability was assessed by cell counting kit 8 assay, and reactive oxygen species (ROS) production was detected by DCFH-DA staining. Nod-like receptor protein 3 (NLRP3) expression was detected by quantitative real-time PCR, western blot and immunofluorescence staining. Cell pyroptosis was measured using flow cytometry, and the contents of interleukin-1ß and interleukin-18 were determined by ELISA. Besides, the protein levels of apoptosis-associated speck-like protein containing CARD (ASC) and cleaved-Caspase-1 were examined by western blot analysis. RESULTS: H2 O2 could induce ROS production, NLRP3 expression and pyroptosis in melanocytes. TanshinoneIIA inhibited ROS production, pyroptosis, and the expression of NLRP3, ASC and cleaved-caspase-1 in H2 O2 -induced melanocytes. Compared with the function of ROS inhibitor (NAC), tanshinoneIIA acted as a ROS scavenger to relieve melanocyte pyroptosis. In addition, NLRP3 inhibitor (MCC950) also could aggravate the inhibition effect of tanshinoneIIA on melanocyte pyroptosis. CONCLUSION: TanshinoneIIA suppressed melanocyte pyroptosis probably through modulating the ROS/NLRP3 signaling axis, which provides the evidence for therapeutic effect in vitiligo.

Dapagliflozin Attenuates NLRP3/Caspase-1 Signaling Pathway-Mediated Pyroptosis of Vascular Smooth Muscle Cells by Downregulating CTSB.

Background: Atherosclerosis is a chronic inflammatory disease. Pyroptosis triggers and amplifies the inflammatory response and plays an important role in atherosclerosis. Cathepsin B (CTSB) can promote atherosclerosis and activate NOD-like receptor protein 3 (NLRP3) to mediate pyroptosis. Dapagliflozin (DAPA) can inhibit cell pyroptosis to improve atherosclerosis. This study aimed to explore the effect of DAPA on oxidized low-density lipoprotein (ox-LDL)-induced pyroptosis of vascular smooth muscle cells (VSMCs) and its underlying mechanism. Objective: We aimed to investigate the effect of DAPA on ox-LDL-induced pyroptosis of VSMCs in mice and its underlying mechanism. Methods: VSMCs were transfected with CTSB-overexpressing and -silencing lentiviral vectors. VSMCs were treated with different concentrations of ox-LDL (0, 50, 100 and 150 µg/ml ). Then, Hoechst 33342/PI double staining, interleukin (IL)-1ß and lactate dehydrogenase (LDH) release assay were used to detect cell pyroptosis. Western blotting was used to detect pyroptosis indicators protein, based on which the appropriate concentration of ox-LDL was selected. After VSMCs were treated with different concentrations of DAPA (0.1 µM, 1.0 µM, 5.0 µM, 10 µM, 25 µM and 50 µM), the proliferative activity of VSMCs was detected using Cell Counting Kit-8 (CCK8) assay. After VSMCs were pretreated with different DAPA concentrations (0.1 µM, 1.0 µM, 5.0 µM and 10 µM) for 24 hours and then treated with 150 µg/mL ox-LDL for 24 hours, the effects of different concentrations of DAPA on pyroptosis of VSMCs were detected, based on which the appropriate DAPA concentration was selected. After lentivirus transfected VSMCs were treated with 150 µg/mL ox-LDL for 24 hours, the effects of overexpression and silencing of CTSB in pyroptosis were observed. On the basis of DAPA (0.1 µM)- and ox-LDL(150 µg/mL)-treated VSMCs, overexpression and silencing of CTSB were used to observe the effects of DAPA and CTSB on ox-LDL-mediated VSMCs pyroptosis. Results: (1) VSMCs stably transfected with CTSB-overexpressing and -silencing lentiviruses were obtained; 150 µg/mL was the optimal concentration of ox-LDL for inducing pyroptosis of VSMCs, and 0.1 µM was the optimal concentration of DAPA for ameliorating pyroptosis of VSMCs. (2) Ox-LDL-induced pyroptosis of VSMCs was worsened by CTSB overexpression but suppressed by CTSB silencing. (3) DAPA attenuated ox-LDL-induced pyroptosis of VSMCs through downregulating CTSB and NLRP3. (4) Overexpression of CTSB based on DAPA intervention aggravated ox-LDL-induced pyroptosis of VSMCs. Conclusion: DAPA attenuates NLRP3/caspase-1 pathway-mediated pyroptosis of VSMCs through downregulating CTSB.

Inhibitory Effects of Corydalis saxicola Bunting Total Alkaloids on Macrophage Pyroptosis.

This study investigated the effect of Corydalis saxicola Bunting total alkaloids (CSBTA) on pyroptosis in macrophages (Mϕ). In the Mϕ pyroptosis model, an inverted fluorescence microscope was used to assess cell pyroptosis, while a scanning electron microscope was used to observe morphological changes in Mϕ. NLR family pyrin domain-containing 3 (NLRP3), caspase-1, and gasdermin D (GSDMD) expression levels were detected by polymerase chain reaction and western blotting, whereas interleukin-1 (IL-1) and interleukin-18 (IL-18) expression levels were measured by an enzyme-linked immunosorbent assay. After pretreatment with CSBTA or the caspase-1 inhibitor, acetyl-tyrosyl-valyl-alanyl-aspartyl-chloromethylketone (Ac-YVAD-cmk), it was discovered that NLRP3, caspase-1, and GSDMD expressions were significantly reduced at both the mRNA and protein levels, as were IL-1 and IL-18 levels. The inhibitory effects of CSBTA and Ac-YVAD-cmk did not differ significantly. These findings indicate that CSBTA blocks Porphyromonas gingivalis-lipopolysaccharide-induced Mϕ pyroptosis.

Transauricular Vagal Nerve Stimulation at 40 Hz Inhibits Hippocampal P2X7R/NLRP3/Caspase-1 Signaling and Improves Spatial Learning and Memory in 6-Month-Old APP/PS1 Mice.

OBJECTIVES: Transauricular vagal nerve stimulation (taVNS) at 40 Hz attenuates hippocampal amyloid load in 6-month-old amyloid precursor protein/presenilin 1 (APP/PS1) transgenic mice, but it is unclear whether 40-Hz taVNS can improve cognition in these mice. Moreover, the underlying mechanisms are still unclear. MATERIALS AND METHODS: 6-month-old C57BL/6 (wild type [WT]) and APP/PS1 mice were subjected to 40-Hz taVNS. Novel Object Recognition and the Morris Water Maze were used to evaluate cognition. Hippocampal amyloid-ß (Aß)1-40, Aß1-42, pro-interleukin (IL)-1ß, and pro-IL-18 were measured using enzyme-linked immunosorbent assays. Hippocampal Aß42, purinergic 2X7 receptor (P2X7R), nucleotide-binding oligomerization domain-like receptor pyrin domain containing 3 (NLRP3), Caspase-1, IL-1ß, and IL-18 expression were evaluated by western blotting. Histologic assessments including immunofluorescence, immunohistochemistry, Nissl staining, and Congo red staining were used to assess microglial phagocytosis, neuroprotective effects, and Aß plaque load. RESULTS: 40-Hz taVNS improved spatial memory and learning in 6-month-old APP/PS1 mice but did not affect recognition memory. There were no effects on the cognitive behaviors of 6-month-old WT mice. taVNS at 40 Hz modulated microglia; significantly decreased levels of Aß1-40, Aß1-42, pro-IL-1ß, and pro-IL-18; inhibited Aß42, P2X7R, NLRP3, Caspase-1, IL-1ß, and IL-18 expression; reduced Aß deposits; and had neuroprotective effects in the hippocampus of 6-month-old APP/PS1 mice. These changes were not observed in 6-month-old WT mice. CONCLUSION: Our results show that 40-Hz taVNS inhibits the hippocampal P2X7R/NLRP3/Caspase-1 signaling and improves spatial learning and memory in 6-month-old APP/PS1 mice.

Photocatalytic Superoxide Radical Generator that Induces Pyroptosis in Cancer Cells.

Pyroptosis, a newly characterized form of immunogenic cell death, is attracting increasing attention as a promising approach to cancer immunotherapy. However, biocompatible strategies to activate pyroptosis remain rare. Here, we show that a photocatalytic superoxide radical (O2-•) generator, NI-TA, triggers pyroptosis in cancer cells. NI-TA was designed to take advantage of an intramolecular triplet-ground state splitting energy modulation approach. Detailed studies revealed that the pyroptosis triggered by NI-TA under conditions of photoexcitation proceeds through a caspase-3/gasdermin E (GSDME) pathway rather than via canonical processes involving caspase-1/gasdermin-D (GSDMD). NI-TA was found to function via a partial-O2-recycling mode of action and to trigger cell pyroptosis and provide for effective cancer cell ablation even under conditions of hypoxia (≤2% O2). In the case of T47D 3D multicellular spheroids, good antitumor efficiency and stemness inhibition are achieved. This work highlights how photocatalytic chemistry may be leveraged to develop effective pyroptosis-inducing agents.

Paeonol reduces IL-ß production by inhibiting the activation of nucleotide oligomerization domain-like receptor protein-3 inflammasome and nuclear factor-κB in macrophages.

Interleukin-1ß, a key cytokine in gouty inflammation, is precisely regulated by the NLRP3 inflammasome and NF-κB. Our previous study demonstrated that paeonol suppressed IL-1ß production in rats with monosodium urate (MSU)-induced arthritis. Whether NLRP3 inflammasome or NF-κB is responsible for the anti-inflammatory effect of paeonol remains unclear. In this study, J774A.1 cells induced by lipopolysaccharide (LPS) plus MSU, was used to investigate the effect of paeonol on NLRP3 inflammasome activation, and J774A.1 cells induced by LPS alone were used to investigate the effect of paeonol on NF-κB activation. In J774A.1 cells induced by LPS plus MSU, paeonol decreased the levels of IL-1ß and caspase-1 and reduced the MSU-induced interaction of pro-caspase-1 and apoptosis-associated speck-like protein containing caspase recruitment domain (ASC), but did not affect the levels of pro-IL-1ß and pro-caspase-1. In J774A.1 cells induced by LPS alone, paeonol reduced the levels of IL-1ß, NLRP3, p-IKK, p-IκBα, and p-p65, but did not affect ASC levels. Paeonol also promoted the content of IκBα and retained more p65 in the cytoplasm. Furthermore, paeonol reduced the DNA-binding activity of p65 and lowered the levels of p-JNK, p-ERK, and p-p38. These results suggest that paeonol inhibits IL-1ß production by inhibiting the activation of NLRP3 inflammasome, NF-κB, and MAPK signaling pathways.

The p-STAT3/ANXA2 axis promotes caspase-1-mediated hepatocyte pyroptosis in non-alcoholic steatohepatitis.

BACKGROUND: To explore the roles of Annexin A2 (ANXA2) on hepatocyte pyroptosis and hepatic fibrosis in nonalcoholic steatohepatitis (NASH) and underlying molecular mechanism. METHODS: Bioinformatics analyses were performed on transcriptome data of liver tissues from mice and patients with liver fibrosis for screening the hepatocyte pyroptosis-related differential genes. The in vivo NASH mouse model and in vitro NASH cellular model were established. The expression levels of Anxa2/ANXA2 were quantified. Then, the upstream transcription factor of Anxa2 was screened by ChIP-Seq and experimentally verified. The effects of the p-STAT3/ANXA2 axis on Caspase-1 mediated pyroptosis and fibrosis were explored by in vivo and in vitro experiments. RESULTS: Bioinformatics analyses suggested that the expression of Anxa2/ANXA2 was significantly up-regulated in liver tissues of both NASH mice and patients scoring with high pyroptotic activity. Experimental data showed that the ANXA2 expression was positively associated with the development of hepatocyte pyroptosis and fibrosis. As a transcription factor of ANXA2, p-STAT3 can bind to the promoter of Anxa2 and promote its transcription. The inhibition of p-STAT3 can significantly suppress hepatocyte pyroptosis and fibrosis, which was significantly reversed after the over-expression of Anxa2. Caspase-1 was verified as the player of the p-STAT3/ANXA2 axis to promote pyroptosis and fibrosis. By specifically inhibiting Caspase-1, the promotion effect of the p-STAT3/ANXA2 axis on pyroptosis and fibrosis can be significantly weakened. CONCLUSION: The p-STAT3 promoted Anxa2 expression at the transcription level, thus activating the Caspase-1 mediated hepatocyte pyroptosis and fibrosis in NASH.

Mechanism of LSD1 in oxygen-glucose deprivation/reoxygenation-induced pyroptosis of retinal ganglion cells via the miR-21-5p/NLRP12 axis.

BACKGROUND: Retinal ganglion cells (RGCs) are important retinal neurons that connect visual receptors to the brain, and lysine-specific demethylase 1 (LSD1) is implicated in the development of RGCs. This study expounded the mechanism of LSD1 in oxygen-glucose deprivation/reoxygenation (OGD/R)-induced pyroptosis of RGCs. METHODS: Mouse RGCs underwent OGD/R exposure, and then RGC viability was examined using the cell counting kit-8 method. The mRNA levels of Caspase 1, the protein levels of NOD-like receptor family pyrin domain-containing 3 (NLRP3), N-terminal fragment of gasdermin D (GSDMD-N), and cleaved-Caspase1, and the concentrations of interleukin (IL)-1ß and IL-18 were respectively examined. Subsequently, LSD1 expression was intervened to explore the underlying effect of LSD1 on OGD/R-induced pyroptosis of RGCs. Afterwards, the enrichments of LSD1 and histone H3 lysine 4 methylation (H3K4me) 1/2 on the microRNA (miR)-21-5p promoter were determined using chromatin-immunoprecipitation assay. And the binding interaction between miR-21-5p and NLRP12 was detected using dual-luciferase and RNA pull-down assays. Finally, the effects of miR-21-5p/NLRP12 on LSD1-mediated pyroptosis of RGCs were verified through functional rescue experiments. RESULTS: OGD/R treatment increased pyroptosis of RGCs and LSD1 expression. Silencing LSD1 declined levels of Caspase 1 mRNA, NLRP3, GSDMD-N, cleaved-Caspase1, IL-1ß, and IL-18 and limited pyroptosis of OGD/R-treated RGCs. Mechanically, LSD1 suppressed miR-21-5p expression via demethylation of H3K4me2 on the miR-21-5p promoter to hamper the binding of miR-21-5p to NLRP12, and thereby increased NLRP12 expression. Silencing miR-21-5p or overexpressing NLRP12 facilitated OGD/R-induced pyroptosis of RGCs. CONCLUSION: LSD1-mediated demethylation of H3K4me2 decreased miR-21-5p expression to increase NLRP12 expression, promoting pyroptosis of OGD/R-treated RGCs.

Pentraxin-3 Promotes LPS-Induced Pyroptosis in Human Periodontal Ligament Stem Cells.

The aim of this study was to explore the function of Pentraxin-3 (PTX3) in cell viability, pyroptosis, inflammation, osteogenic differentiation, and oxidative stress of lipopolysaccharide (LPS)-stimulated human periodontal ligament stem cells (hPDLSCs). In the study, hPDLSCs were stimulated by LPS from Porphyromonas gingivalis to establish an in vitro inflammatory cellular model. Protein expression was measured using Western blotting. mRNA levels were evaluated by qRT-PCR. Cell viability, inflammatory cytokine production, and caspase-1 activity was measured with commercially available kits. Oxidative stress was assessed by examining reactive oxygen species and nitric oxide production. We found that PTX3 was upregulated in LPS-stimulated hPDLSCs. PTX3 overexpression aggravated LPS-induced cell viability loss, inflammatory cytokine production, and oxidative stress, as well as suppressed the osteogenic differentiation in hPDLSCs, while silencing PTX3 had the opposite effects. Further, PTX3 overexpression promoted NOD-like receptor family, pyrin domain containing protein 3 (NLRP3) inflammasome overactivation and pyroptosis, evidenced by increased protein levels of NLRP3, cleaved-caspase-1, apoptosis-associated speck-like protein (ASC), and N-terminal gasdermin D (GSDMD-N). Inhibition of NLRP3 inflammasome and/or caspase-1 partially attenuated the effects of PTX3 on LPS-stimulated hPDLSCs. This study indicated that PTX3 promotes LPS-induced pyroptosis and inflammation in hPDLSCs through activation of the caspase-1-dependent NLRP3 inflammasome.

The caspase-1 inhibitor VX765 upregulates connexin 43 expression and improves cell-cell communication after myocardial infarction via suppressing the IL-1ß/p38 MAPK pathway.

Connexin 43 (Cx43) is the most important protein in the gap junction channel between cardiomyocytes. Abnormalities of Cx43 change the conduction velocity and direction of cardiomyocytes, leading to reentry and conduction block of the myocardium, thereby causing arrhythmia. It has been shown that IL-1ß reduces the expression of Cx43 in astrocytes and cardiomyocytes in vitro. However, whether caspase-1 and IL-1ß affect connexin 43 after myocardial infarction (MI) is uncertain. In this study we investigated the effects of VX765, a caspase-1 inhibitor, on the expression of Cx43 and cell-to-cell communication after MI. Rats were treated with VX765 (16 mg/kg, i.v.) 1 h before the left anterior descending artery (LAD) ligation, and then once daily for 7 days. The ischemic heart was collected for histochemical analysis and Western blot analysis. We showed that VX765 treatment significantly decreased the infarct area, and alleviated cardiac dysfunction and remodeling by suppressing the NLRP3 inflammasome/caspase-1/IL-1ß expression in the heart after MI. In addition, VX765 treatment markedly raised Cx43 levels in the heart after MI. In vitro experiments were conducted in rat cardiac myocytes (RCMs) stimulated with the supernatant from LPS/ATP-treated rat cardiac fibroblasts (RCFs). Pretreatment of the RCFs with VX765 (25 µM) reversed the downregulation of Cx43 expression in RCMs and significantly improved intercellular communication detected using a scrape-loading/dye transfer assay. We revealed that VX765 suppressed the activation of p38 MAPK signaling in the heart tissue after MI as well as in RCMs stimulated with the supernatant from LPS/ATP-treated RCFs. Taken together, these data show that the caspase-1 inhibitor VX765 upregulates Cx43 expression and improves cell-to-cell communication in rat heart after MI via suppressing the IL-1ß/p38 MAPK pathway.

NLRP3 inflammasome inhibitor ameliorates ischemic stroke by reprogramming the phenotype of microglia/macrophage in a murine model of distal middle cerebral artery occlusion.

Stroke is one of the leading causes of death and disability worldwide. NLRP3 inflammasome has an essential role in the neuropathology of stroke. Recent studies report that shifting the microglial M1 phenotype to the M2 phenotype protects against ischemic stroke. In the present study, the precise effects of Tranilast, a NLPR3 inflammasome inhibitor, on stroke were evaluated. We established a murine model of distal middle cerebral artery occlusion (dMCAO) and administered Tranilast to dMCAO-induced stroke mice. The NLRP3 level, caspase 1 activity, and infarct volume stroke mice were measured. The sensorimotor function, pro-inflammatory cytokine production, and M1/M2 marker expression were measured. The M1 phenotype was induced by treatment of BV2 microglia with lipopolysacharide and interferon γ, and these BV-2 cells were further treated with Tranilast. The expression of CD16 and CD206 was monitored. dMCAO increased the NLRP3 expression and enhanced caspase 1 activity. Tranilast treatment significantly decreased the infarct volume, improved sensorimotor function, and suppressed the production of inflammatory cytokines in stroke mice. Moreover, Tranilast decreased the M1 marker level while promoting the expression of M2 markers. In summary, our findings suggest that Tranilast ameliorates ischemic stroke through stimulating M2 polarization of microglia.

Caspase-1 and Cathepsin B Inhibitors from Marine Invertebrates, Aiming at a Reduction in Neuroinflammation.

Neuroinflammation is a condition associated with several types of dementia, such as Alzheimer's disease (AD), mainly caused by an inflammatory response to amyloid peptides that induce microglial activation, with subsequent cytokine release. Neuronal caspase-1 from inflammasome and cathepsin B are key enzymes mediating neuroinflammation in AD, therefore, revealing new molecules to modulate these enzymes may be an interesting approach to treat neurodegenerative diseases. In this study, we searched for new caspase-1 and cathepsin B inhibitors from five species of Brazilian marine invertebrates (four cnidarians and one echinoderm). The results show that the extract of the box jellyfish Chiropsalmus quadrumanus inhibits caspase-1. This extract was fractionated, and the products monitored for their inhibitory activity, until the obtention of a pure molecule, which was identified as trigonelline by mass spectrometry. Moreover, four extracts inhibit cathepsin B, and Exaiptasia diaphana was selected for subsequent fractionation and characterization, resulting in the identification of betaine as being responsible for the inhibitory action. Both molecules are already found in marine organisms, however, this is the first study showing a potent inhibitory effect on caspase-1 and cathepsin B activities. Therefore, these new prototypes can be considered for the enzyme inhibition and subsequent control of the neuroinflammation.

DDIT3/CHOP promotes LPS/ATP-induced pyroptosis in osteoblasts via mitophagy inhibition.

Inflammatory environments can trigger endoplasmic reticulum (ER) stress and lead to pyroptosis in various tissues and cells, including liver, brain, and immune cells. As a key factor of ER stress, DNA damage-inducible transcript 3 (DDIT3)/CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) is upregulated in osteoblasts during inflammatory stimulation. DDIT3/CHOP may therefore regulate osteoblast pyroptosis in inflammatory conditions. During this investigation, we found that lipopolysaccharides (LPS)/adenosine 5'-triphosphate (ATP) stimulation in vitro induced osteoblasts to undergo pyroptosis, and the expression of DDIT3/CHOP was increased during this process. The overexpression of DDIT3/CHOP further promoted osteoblast pyroptosis as evidenced by the increased expression of the inflammasome NLR family pyrin domain containing 3 (NLRP3) and ratios of caspase-1 p20/caspase-1 and cleaved gasdermin D (GSDMD)/GSDMD. To explore the specific mechanism of this effect, we found through fluorescence imaging and Western blot analysis that LPS/ATP stimulation promoted PTEN-induced kinase 1 (PINK1)/E3 ubiquitin-protein ligase parkin (Parkin)-mediated mitophagy in osteoblasts, and this alteration was suppressed by the DDIT3/CHOP overexpression, resulting in increased ratio of pyroptosis compared with the control groups. The impact of DDIT3/CHOP on pyroptosis in osteoblasts was reversed by the application of carbonyl cyanide 3-chlorophenylhydrazone (CCCP), a specific mitophagy agonist. Therefore, our data demonstrated that DDIT3/CHOP promotes osteoblast pyroptosis by inhibiting PINK1/Parkin-mediated mitophagy in an inflammatory environment.

Paclitaxel may inhibit migration and invasion of gastric cancer cells via nod-like receptor family pyrin domain-containing 3/caspase-1/Gasdermin E mediated pyroptosis pathway.

Gastric cancer (GC) is a gastric epithelium-derived malignancy insensitive to post-surgical radiotherapy. Paclitaxel, an anti-microtubule drug, has been proven to induce apoptosis of GC cells; however, its exact mechanism of action is unclear. Therefore, the molecular mechanism by which paclitaxel inhibits the proliferation, migration and invasion of GC cells was investigated in this study. First off, SNU-719 cells were co-cultured with paclitaxel and/or Caspase1 inhibitor VX765. Then the proliferation ability of the cells was detected by MTT after paclitaxel treatment (0, 10, 20, 40, and 80 nM), the migration ability by scratch assay, and the invasion ability by Transwell assay. Next, the levels of interleukin (IL)-1ß and IL-18 in cell culture supernatant were detected by the enzyme linked immunosorbent assay (ELISA). And the level of lactate dehydrogenase (LDH) in the supernatant was measured by a corresponding kit. Finally, western blot was performed to detect the concentrations of Gasdermin E (GSDME), GSDME-N, nod-like receptor family pyrin domain-containing 3 (NLRP3), caspase-1, cleaved caspase-1 protein in GC cells. As a result, paclitaxel inhibited the proliferation, migration, and invasion of SNU-719 cells in a concentration-dependent manner. Moreover, it induced the pyroptosis of SNU-719 cells. After cell co-culture with VX765 paclitaxel showed decreased inhibitory effect on the migration and invasion of SNU-719 cells. VX765, additionally, suppressed the NLRP3/caspase-1/GSDME mediated pyroptosis pathway activated by paclitaxel. In a nutshell, paclitaxel may inhibit the migration and invasion of GC cells SNU-719 through the NLRP3/caspase-1/GSDME mediated pyroptosis pathway.

Disulfiram accelerates diabetic foot ulcer healing by blocking NET formation via suppressing the NLRP3/Caspase-1/GSDMD pathway.

Diabetic foot ulcer (DFU) is among the most frequent complications of diabetes and is associated with significant morbidity and mortality. Excessive neutrophil extracellular traps (NETs) delay wound healing in diabetic patients. Therefore, interventions targeting NET release need to be developed to effectively prevent NET-based wound healing impairment. Gasdermin D (GSDMD), a pore-forming protein acts as a central executioner of inflammatory cell death and can activate inflammasomes in neutrophils to release NETs. A precise understanding of the mechanism underlying NET-mediated delay in diabetic wound healing may be valuable in identifying potential therapeutic targets to improve clinical outcomes. In this study, we reported that neutrophils were more susceptible to NETosis in diabetic wound environments of patients with DFU. By in vitro experiments and using in vivo mouse models of diabetic wound healing (wide-type, Nlrp3-/-, Casp-1-/-, and Gsdmd-/- mice), we demonstrated that NLRP3/caspase-1/GSDMD pathway on activation controls NET release by neutrophils in diabetic wound tissue. Furthermore, inhibition of GSDMD with disulfiram or genic deletion of Gsdmd abrogated NET formation, thereby accelerating diabetic wound healing. Disulfiram could inhibit NETs-mediated diabetic foot ulcer healing impairment by suppressing the NLRP3/Caspase-1/GSDMD pathway. In summary, our findings uncover a novel therapeutic role of disulfiram in inhibiting NET formation, which is of considerable value in accelerating wound healing in patients with DFU.

Ononin alleviates DSS-induced colitis through inhibiting NLRP3 inflammasome via triggering mitophagy.

BACKGROUND: Ononin, a flavonoid isolated from Astragalus membranaceus root, is the active ingredient of A. membranaceus and has potential anti-inflammatory properties, but its effect on colitis is unclear. AIMS: This study aimed to explore the anticolitis effect of Ononin by establishing a colitis model in mice induced by dextran sulfate sodium (DSS). METHODS: Male C57BL/6 mice were provided DSS, then treated with Ononin (10, 20, 40 mg/kg) or 5-ASA (40 mg/kg). The colitis symptoms were observed, the disease activity index (DAI) score were recorded daily, and colonic inflammation was evaluted by histopathological scoring. The expression of cytokines, inflammatory mediators, and mitophagy/NLRP3 inflammasome-related proteins were measured. RESULTS: Ononin significantly alleviated weight loss and colon shortening in mice with colitis (p < .01). Moreover, Ononin decreased the production of inflammatory cytokines and mediators associated with colitis (p < .05). In addition, Ononin inhibited macrophages infiltration and reduced caspase-1 activation in colitis mice. Caspase-1 activation is closely related to the NLRP3 inflammasome. Therefore, we investigated the effect of Ononin on NLRP3 inflammasome in vitro. The relevant results confirmed that Ononin inhibited NLRP3 inflammasome activation and inhibited mitochondrial damage (p < .05). Further studies revealed that Ononin inhibited mitochondrial damage through triggering mitophagy (p < .05). CONCLUSION: Ononin alleviates DSS-induced colitis by activating mitophagy to inhibit NLRP3 inflammasome.

ASC/Caspase-1-activated endothelial cells pyroptosis is involved in vascular injury induced by arsenic combined with high-fat diet.

Environmental arsenic (As) or high-fat diet (HFD) exposure alone are risk factors for the development of cardiovascular disease (CVDs). However, the effects and mechanisms of co-exposure to As and HFD on the cardiovascular system remain unclear. The current study aimed to investigate the combined effects of As and HFD on vascular injury and shed some light on the underlying mechanisms. The results showed that co-exposure to As and HFD resulted in a significant increase in serum lipid levels and significant lipid accumulation in the aorta of rats compared with exposure to As or HFD alone. Meanwhile, the combined exposure altered blood pressure and disrupted the morphological structure of the abdominal aorta in rats. Furthermore, As combined with HFD exposure upregulated the expression of vascular endothelial cells pyroptosis-related proteins (ASC, Pro-caspase-1, Caspase-1, IL-18, IL-1ß), as well as the expression of vascular endothelial adhesion factors (VCAM-1 and ICAM-1). More importantly, we found that with increasing exposure time, vascular injury-related indicators were significantly higher in the combined exposure group compared with exposure to As or HFD alone, and the vascular injury was more severe in female rats compared with male rats. Taken together, these results suggested that the combination of As and HFD induced vascular endothelial cells pyroptosis through activation of the ASC/Caspase-1 pathway. Therefore, vascular endothelial cells pyroptosis may be a potential molecular mechanism for vascular injury induced by As combined with HFD exposure.

Bisphenol A exacerbates selenium deficiency-induced pyroptosis via the NF-κB/NLRP3/Caspase-1 pathway in chicken trachea.

Selenium deficiency can lead to multiple tissue and organ damage in the body and could coexist with chronic toxic exposures. Contamination from Bisphenol A (BPA) exposure can induce the occurrence of various injuries including pyroptosis. However, it is not clear whether selenium deficiency and BPA exposure affect tracheal tissue pyroptosis in chickens. To investigate whether selenium deficiency and BPA exposure induce chicken tracheal tissue pyroptosis via the NF-κB/NLRP3/Caspase-1 pathway and the effect of their combined exposure on tissue injury, we developed a model of relevant chicken tracheal injury. Sixty broilers were divided into four groups: the control group (C group), selenium-deficient group (SeD group), BPA-exposed group (BPA group) and combined exposure group (SeD + BPA group). The study examined the expression indicators of markers of pyroptosis (NLRP3&GSDMD), NF-κB pathway-related inflammatory factors (NF-κB, iNOS, TNF-α, COX-2), pyroptosis-related factors (ASC, Caspase-1, IL-1ß, IL-18), and some heat shock proteins and interleukins (HSP60, HSP90, IL-6, IL-17) in the samples. The results showed that the expression of the above indicators was significantly upregulated in the different treatment groups (P < 0.05). In addition, the expression levels of the above related indicators were more significantly up-regulated in the combined selenium-deficient and BPA-exposed group compared to the group in which they were individually exposed. It was concluded that selenium deficiency and BPA exposure induced tracheal tissue pyroptosis in chickens through NF-κB/NLRP3/Caspase-1 pathway, and BPA exposure exacerbated selenium deficiency-induced tracheal pyroptosis. The present study provides new ideas into studies related to the co-exposure of organismal micronutrient deficiency and chronic toxicants.