Inflammasome-induced Pyroptosis Research Articles

NKCC1-dependent K+ influx inhibits NLRP3 inflammasome activation and prevents intestinal epithelial cell injury

Background: Loss of function mutations in SLC12A2, the gene encoding the Na+-K+-2Cl− cotransporter (NKCC1) are associated with autoinflammatory and severe chronic inflammation in multiple organs. NLRP(s) are a family of cytosolic sensors that initiate the innate immune response. Once activated, NLRP(s) form inflammasomes, which are multi-protein complex that sense danger signals, damaged and infected cells. Assembly of the inflammasome mediates caspases-1 activation and the production of the proinflammatory cytokines IL-1β and IL-18 and initiates an inflammatory cell death process called pyroptosis. Aberrant activation of NLRP3 and other inflammasomes leads to a variety of autoimmune and chronic inflammatory diseases. Here we ask whether loss of NKCC1 function affects inflammasome activation in intestinal epithelial cells. Methods: Wild-type intestinal epithelial cells (IEC) and CRISPR/Cas9 NKCC1-deleted clones were tested for inflammasome activation. WT and NKCC1-deficient clones were treated with nigericin, ATP or ouabain to activate the inflammasome. Conditioned media, whole cell lysate and fixed cells were collected at different time points and analyzed by immunoblotting, immunofluorescence, and ELISA (to measure cytokine release). Inflammasome-induced pyroptosis was assessed by measuring LDH release in the supernatant and gasdermine D cleavage by immunoblotting. Results: Loss of NKCC1 function induces inflammasome activation by increasing IL-1β secretion in intestinal epithelial cells. Prolong inflammatory cytokine secretion led to increased pyroptosis-mediated cell death. Loss of NKCC1 mediates NLRP3 inflammasome activation by compromising K+ influx. NLRP3 agonists increase the interaction and localization of WNK1 and SPAK kinases with the NLRP3 inflammasome complex. Conclusions: NKCC1 regulates inflammatory cytokines secretion. Loss of NKCC1 inhibits K+ and Cl− inward fluxes, which in turn activate NLRP3 inflammasome. Loss of NKCC1 function is a previously unknown mechanism that causes aberrant inflammasome activation. GM118944 DK093501. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

TXNIP Regulates NLRP3 Inflammasome-Induced Pyroptosis Related to Aging via cAMP/PKA and PI3K/Akt Signaling Pathways.

Aging is an inevitable natural process with time-dependent dysfunction and the occurrence of various diseases, which impose heavy burdens on individuals, families, and society. It has been reported that NLRP3 inflammasome-induced pyroptosis contributes significantly to age-related diseases and aging, while TXNIP is suggested to be involved in regulating pyroptosis mediated by NLRP3. However, the mechanism between TXNIP and NLRP3 inflammasome is still unclear. In this study, we used HT-22 cells to explore the effect of TXNIP on pyroptosis and its potential association with the aging. Also, we delved into the underlying mechanisms. Our findings revealed that TXNIP significantly augmented pyroptosis in HT-22 cells, primarily by enhancing the activation of the NLRP3 inflammasome and promoting the release of proinflammatory cytokines. Remarkably, as TXNIP levels increased, we observed a corresponding rise in the number of p16-positive cells, which is indicative of aging. Furthermore, we conducted experiments to modulate the improvement of TXNIP on NLRP3 inflammasome-induced pyroptosis, that is, the PI3K activator 740 Y-P and the PKA activator DC2797 inhibited the effect, while the PI3K inhibitor LY294002 and the PKA inhibitor H89 enhanced the effect. In conclusion, our study demonstrated that TXNIP regulates NLRP3 inflammasome-induced pyroptosis in HT-22 cells related to aging via the PI3K/Akt and cAMP/PKA pathways.

4-Octyl itaconate alleviates experimental autoimmune prostatitis by inhibiting the NLRP3 inflammasome-induced pyroptosis through activating Nrf2/HO-1 pathway.

Chronic prostatitis demonstrates a prevalence rate of nearly 5%-10% among young and middle-aged individuals, significantly affecting their daily lives. Researchers have obtained significant outcomes investigating the anti-inflammatory properties of itaconic acid (IA) and its derivative, 4-Octyl itaconate (4-OI), against diverse chronic inflammatory disorders, such as osteoarthritis and airway inflammation. Nevertheless, whether IA can also exert anti-inflammatory effects in chronic prostatitis requires extensive research and validation. Human prostate tissues obtained through transurethral prostate resection (TURP) from individuals were divided into three groups based on different levels of inflammation using hematoxylin and eosin staining (H&E). Subsequently, immunohistochemistry (IHC) was employed to detect the expression of immune-responsive gene 1 (IRG-1) in these different groups. The animal experiment of this study induced experimental autoimmune prostatitis (EAP) in nonobese diabetic mice through intradermal prostate antigen injection and complete Freund's adjuvant. Then, the experimental group received intraperitoneal injections of different doses of 4-OI, while the control group received injections of saline. Western blot (WB), H&E staining, and TUNEL staining helped analyze the prostate tissues, while enzyme-linked immunosorbent assay (ELISA) helped evaluate serum inflammatory factors. Reactive oxygen species, superoxide dismutase (SOD), and malondialdehyde (MDA) were assessed for oxidative stress across experimental groups. IHC analysis of human prostate tissue depicts that IRG-1 expression enhances as prostate inflammation worsens, highlighting the critical role of IA in human prostatitis. The application of 4-OI increased Nrf2/HO-1 expression while inhibited NLRP3 expression following the WB results, and its application resulted in a decrease in cell pyroptosis in prostate tissue, demonstrated by the results of TUNEL staining. Administering a Nrf2 inhibitor ML385 1 h before intraperitoneal injection of 50 mg/kg 4-OI reversed the previous conclusion, further confirming the above conclusion from another perspective. Meanwhile, the ELISA results of serum inflammatory factors (IL-1β, IL-6, and TNF-α), as well as the measurements of oxidative stress markers MDA and SOD, further confirmed the specific anti-inflammatory effects of 4-OI in EAP. The present study indicates that 4-OI can alleviates EAP by inhibiting the NLRP3 inflammasome-induced pyroptosis through activating Nrf2/HO-1 pathway, which may facilitate a novel approach toward prostatitis treatment.

Three New Clerodane Diterpenoids and Their NLRP3 Inflammasome Activation Inhibitory Activity from Callicarpa arborea Roxb.

Three new compounds callicarpenoids A-C (1-3), were isolated from the stems of Callicarpa arborea Roxb together with fifteen known compounds (4-18). The structures of these compounds were elucidated using advanced spectroscopic techniques, including 1D and 2D NMR, UV, IR, HR-ESI-MS, ECD, ORD, and quantum chemical calculations. Compound 3, a rare rearranged diterpenoid with a fused 5/6-ring system demonstrated strong potential as an inhibitor of the NLRP3 inflammasome activation with an IC50 value of 3.153 μM. It effectively reduced GSDMD-NT production, inhibited caspase-1 activation, and suppressed IL-1β secretion, thereby mitigating NLRP3 inflammasome-induced pyroptosis in J774A.1 cells. These findings suggest that compound 3 warrants further research and development as a promising NLRP3 inflammasome inhibitor.

ChemR23 activation attenuates cognitive impairment in chronic cerebral hypoperfusion by inhibiting NLRP3 inflammasome-induced neuronal pyroptosis

Neuroinflammation plays critical roles in vascular dementia (VaD), the second leading cause of dementia, which can be induced by chronic cerebral hypoperfusion (CCH). NLRP3 inflammasome-induced pyroptosis, the inflammatory programmed cell death, has been reported to contribute to the development of VaD. ChemR23 is a G protein-coupled receptor that has emerging roles in regulating inflammation. However, the role of ChemR23 signalling in NLRP3 inflammasome-induced pyroptosis in CCH remains elusive. In this study, a CCH rat model was established by permanent bilateral common carotid artery occlusion (BCCAO) surgery. Eight weeks after the surgery, the rats were intraperitoneally injected with the ChemR23 agonist Resolvin E1 (RvE1) or chemerin-9 (C-9). Additionally, primary rat hippocampal neurons and SH-SY5Y cells were adopted to mimic CCH injury in vitro. Our results showed that the levels of ChemR23 expression were decreased from the 8th week after BCCAO, accompanied by significant cognitive impairment. Further analysis revealed that CCH induced neuronal damage, synaptic injury and NLRP3-related pyroptosis activation in hippocampal neurons. However, pharmacologic activation of ChemR23 with RvE1 or C-9 counteracted these changes. In vitro experiments also showed that ChemR23 activation prevented primary neuron pyroptosis induced by chronic hypoxia. In addition, manipulating ChemR23 expression markedly regulated NLRP3 inflammasome-induced neuronal pyroptosis through PI3K/AKT/Nrf2 signalling in SH-SY5Y cells under hypoglycaemic and hypoxic conditions. Collectively, our data demonstrated that ChemR23 activation inhibits NLRP3 inflammasome-induced neuronal pyroptosis and improves cognitive function via the PI3K/AKT/Nrf2 signalling pathway in CCH models. ChemR23 may serve as a potential novel therapeutic target to treat CCH-induced cognitive impairment.

MiR-223-3p attenuates M1 macrophage polarization via suppressing the Notch signaling pathway and NLRP3-mediated pyroptosis in experimental autoimmune uveitis

Autoimmune uveitis is an intraocular inflammatory disease with a high blindness rate in developed countries such as the United States. It is pressing to comprehend the pathogenesis of autoimmune uveitis and develop novel schemes for its treatment. In the present research, we demonstrated that the Notch signaling pathway was activated, and the level of miR-223-3p was significantly reduced in rats with experimental autoimmune uveitis (EAU) compared with the level of normal rats. To investigate the relationship between miR-223-3p and Notch signaling, EAU rats received miR-223-3p-carrying lentivirus, miR-223-3p vector-carrying lentivirus (miR-223-3p-N), and γ-secretase inhibitor (DAPT), respectively. The results of Q-PCR, immunological experiments, and flow cytometry analysis all support the hypothesis that both miR-223-3p and DAPT, a Notch signaling pathway inhibitor, had similar inhibitory effects on the EAU pathological process. That is to say, they could both inhibit the activation of the Notch signaling pathway via modulating recombination signal binding protein-Jκ (RBPJ) to restore the polarization imbalance of M/M2 macrophages in EAU rats. In addition, miR-223-3p could also inhibit NLRP3 inflammasome activation and inflammasome-induced pyroptosis in ocular tissues. Taken together, our findings indicate that miR-223-3p serves as an important regulator in M1 macrophage polarization and pyroptosis, thereby alleviating the inflammatory response in uveitis.

Piceatannol alleviates liver ischaemia/reperfusion injury by inhibiting TLR4/NF-κB/NLRP3 in hepatic macrophages

BackgroundMacrophages present strong immunomodulatory ability and are considered to be core immune cells in the process of hepatic ischaemia‒reperfusion (I/R). The NLRP3 inflammasome is a kind of intracellular multimolecular complex that actively participates in innate immune responses and proinflammatory signalling pathways. Piceatannol (PIC) is a derivative of the natural phenolic compound resveratrol and has antioxidant and anti-inflammatory effects. The purpose of this study was to examine whether pretreatment with PIC can alleviate hepatic I/R injury by targeting NLRP3 inflammasome-induced macrophage pyroptosis. MethodsPIC-pretreated primary hepatic macrophages were subjected to hypoxia/reoxygenation, and liver ischaemia/reperfusion was performed in mice. ResultsPIC pretreatment ameliorated histopathological changes, oxidative stress and inflammation while enhancing antioxidant and anti-inflammasome markers through downregulation of Toll-like receptor 4 (TLR4), p-IκBα (S32), p–NF–κBp65 (S536), NLRP3, caspase-1 (p20), IL-1β, IL-18 and GSDMD-N expression during liver ischaemia‒reperfusion. Moreover, PIC inhibited the translocation of NF-κB p65 after stimulation with hypoxia/reoxygenation in primary hepatic macrophages. ConclusionsThe results indicated that PIC protected the liver against hepatic I/R injury, which was mediated by targeting TLR4-NF-κB-NLRP3-mediated hepatic macrophage pyroptosis.

The role of melatonin in amyloid beta-induced inflammation mediated by inflammasome signaling in neuronal cell lines

Alzheimer's disease (AD) is the most prevalent neurodegenerative disorder. In addition to amyloid beta (Aβ) and tau, neuroinflammation is a crucial element in the etiology of this disease. However, the relevance of inflammasome-induced pyroptosis to AD is unknown. We aimed to clarify whether the anti-inflammatory effects of melatonin could prevent Aβ-mediated activation of the inflammasome. We demonstrated that Aβ upregulated NOD-like receptor family pyrin domain-containing 3 (NLRP3), apoptosis-associated speck-like protein containing a CARD, and cysteinyl aspartate-specific proteinase caspase (caspase 1) expression in SH-SY5Y neuroblastoma cells, resulting in the release of proinflammatory cytokines, including interleukin-1β (IL-1β), interleukin-18 (IL-18) and tumor necrosis factor (TNF-α). Melatonin prevented inflammasome signaling and excessive cytokine release caused by Aβ. We found that ethyl 2[(2-chlorophenyl)(hydroxy) methyl]acrylate (INF-4E, NLRP3 and caspase 1 inhibitor) significantly abolished Aβ-induced proinflammatory cytokine expression. The increase in cleaved-caspase 1, pro-IL18, and cleaved-IL18 caused by Aβ suggested the occurrence of pyroptosis, which was further confirmed by the increased expression of N-terminal gasdermin D (N-GSDMD). Melatonin plays a protective role against Aβ-induced inflammation via an inflammasome-associated mechanism that is essential in inducing the active forms of cytokines and pyroptosis. The ability of melatonin to inhibit inflammasome may represent a turning point in the treatment of AD progression.

Pterostilbene Attenuates High-Intensity Swimming Exercise-Induced Glucose Absorption Dysfunction Associated with the Inhibition of NLRP3 Inflammasome-Induced IECs Pyroptosis

The study investigated the effect of pterostilbene (PTE) on intestinal glucose absorption and its underlying mechanisms in high-intensity swimming exercise (HISE)-treated mice. Male C57BL/6 mice were treated with PTE for 4 weeks and performed high-intensity swimming training in the last week. Intestinal epithelial cells (IECs) were pretreated with 0.5 and 1.0 μM PTE for 24 h before being incubated in hypoxia/reoxygenation condition. Intestinal glucose absorption was detected by using an oral glucose tolerance test and d-xylose absorption assay, and the levels of factors related to mitochondrial function and pyroptosis were measured via western blot analyses, cell mito stress test, and quantitative real-time polymerase chain reaction. In vivo and in vitro, the results showed that PTE attenuated HISE-induced intestinal glucose absorption dysfunction and pyroptosis in mice intestine. Moreover, PTE inhibited NLRP3 inflammasome and the mitochondrial homeostasis as well as the ROS accumulation in IEC in vitro. Additionally, knockdown of SIRT3, a major regulator of mitochondria function, by siRNA or inhibiting its activity by 3-TYP abolished the effects of PTE on pyroptosis, mitochondrial homeostasis, and ROS generation of IEC in vitro. Our results revealed that PTE could alleviate HISE-induced intestinal glucose absorption dysfunction associated with the inhibition of NLRP3 inflammasome-induced IECs pyroptosis.

Intercellular mitochondrial transfer alleviates pyroptosis in dental pulp damage.

Mitochondrial transfer is emerging as a promising therapeutic strategy for tissue repair, but whether it protects against pulpitis remains unclear. Here, we show that hyperactivated nucleotide-binding domain and leucine-rich repeat protein3(NLRP3) inflammasomes with pyroptotic cell death was present in pulpitis tissues, especially in the odontoblast layer, and mitochondrial oxidative stress (OS) was involved in driving this NLRP3 inflammasome-induced pathology. Using bone marrow mesenchymal stem cells (BMSCs) as mitochondrial donor cells, we demonstrated that BMSCs could donate their mitochondria to odontoblasts via tunnelling nanotubes (TNTs) and, thus, reduce mitochondrial OS and the consequent NLRP3 inflammasome-induced pyroptosis in odontoblasts. These protective effects of BMSCs were mostly blocked by inhibitors of the mitochondrial function or TNT formation. In terms of the mechanism of action, TNF-α secreted from pyroptotic odontoblasts activates NF-κB signalling in BMSCs via the paracrine pathway, thereby promoting the TNT formation in BMSCs and enhancing mitochondrial transfer efficiency. Inhibitions of NF-κB signalling and TNF-α secretion in BMSCs suppressed their mitochondrial donation capacity and TNT formation. Collectively, these findings demonstrated that TNT-mediated mitochondrial transfer is a potential protective mechanism of BMSCs under stress conditions, suggesting a new therapeutic strategy of mitochondrial transfer for dental pulp repair.

NLRP3 inflammasome-induced pyroptosis in digestive system tumors.

Programmed cell death (PCD) refers to cell death in a manner that depends on specific genes encoding signals or activities. PCD includes apoptosis, pyroptosis, autophagy and necrosis (programmed necrosis). Among these mechanisms, pyroptosis is mediated by the gasdermin family and is accompanied by inflammatory and immune responses. When pathogens or other danger signals are detected, cytokine action and inflammasomes (cytoplasmic multiprotein complexes) lead to pyroptosis. The relationship between pyroptosis and cancer is complex and the effect of pyroptosis on cancer varies in different tissue and genetic backgrounds. On the one hand, pyroptosis can inhibit tumorigenesis and progression; on the other hand, pyroptosis, as a pro-inflammatory death, can promote tumor growth by creating a microenvironment suitable for tumor cell growth. Indeed, the NLRP3 inflammasome is known to mediate pyroptosis in digestive system tumors, such as gastric cancer, pancreatic ductal adenocarcinoma, gallbladder cancer, oral squamous cell carcinoma, esophageal squamous cell carcinoma, in which a pyroptosis-induced cellular inflammatory response inhibits tumor development. The same process occurs in hepatocellular carcinoma and some colorectal cancers. The current review summarizes mechanisms and pathways of pyroptosis, outlining the involvement of NLRP3 inflammasome-mediated pyroptosis in digestive system tumors.

Protective effect of quercetin on pulmonary dysfunction in streptozotocin-induced diabetic rats via inhibition of NLRP3 signaling pathway

Diabetes mellitus (DM) is a dysmetabolic disease characterized by chronic hyperglycemia. In the developed countries, DM is the commonest life style disease that affects both old and young age. Nod-like receptor protein-3 (NLRP3)-mediated pyroptosis may in fact aid in the development of diabetic complications. Quercetin is a natural flavonoid, can be present in natural foods and plants. Many studies have reported the antioxidant role of quercetin on different tissues, but its effects on NLRP3-mediated pyroptosis in diabetic lung are unclear. The current study aimed to assess quercetin’s protective effects on lung function, oxidative stress, and NLRP3-mediated pyroptosis in Wister rats exposed to streptozotocin (STZ)-induced DM. Forty male Wister rats were randomly allocated into four equal groups. The groups of rats were as follows: group 1 (G1) was kept under normal control conditions; G2 was injected I/P quercetin at a dose of 30 mg/kg b.wt., daily for 30 days; G3 and G4 were injected with a single dose of streptozotocin (STZ) 50 mg/kg b.wt. I/P to induce DM. After 72-h post diabetes induction, the rats of G4 were treated with quercetin as a manner in the second group. The results showed that quercetin ameliorates the pulmonary dysfunctions caused by DM through restoring the levels of glucose, insulin, and arterial blood gases, as well as the oxidative markers. Also, NLRP3-pyroptosis-mediated IL1β was inhibited. Quercetin also reduces the effect of DM on the lung by decreasing the pathological changes in the lung. In conclusion, NLRP3 inflammasome-induced pyroptosis may aggravate lung injury in diabetic rats. Quercetin has the potential to ameliorate diabetes induced pulmonary dysfunction by targeting NLRP3.

GSDMD deficiency ameliorates hyperoxia-induced BPD and ROP in neonatal mice

Bronchopulmonary dysplasia (BPD) and retinopathy of prematurity (ROP) are among the most common morbidities affecting extremely premature infants who receive oxygen therapy. Many clinical studies indicate that BPD is associated with advanced ROP. However, the mechanistic link between hyperoxia, BPD, and ROP remains to be explored. Gasdermin D (GSDMD) is a key executor of inflammasome-induced pyroptosis and inflammation. Inhibition of GSDMD has been shown to attenuate hyperoxia-induced BPD and brain injury in neonatal mice. The objective of this study was to further define the mechanistic roles of GSDMD in the pathogenesis of hyperoxia-induced BPD and ROP in mouse models. Here we show that global GSDMD knockout (GSDMD-KO) protects against hyperoxia-induced BPD by reducing macrophage infiltration, improving alveolarization and vascular development, and decreasing cell death. In addition, GSDMD deficiency prevented hyperoxia-induced ROP by reducing vasoobliteration and neovascularization, improving thinning of multiple retinal tissue layers, and decreasing microglial activation. RNA sequencing analyses of lungs and retinas showed that similar genes, including those from inflammatory, cell death, tissue remodeling, and tissue and vascular developmental signaling pathways, were induced by hyperoxia and impacted by GSDMD-KO in both models. These data highlight the importance of GSDMD in the pathogenesis of BPD and ROP and suggest that targeting GSDMD may be beneficial in preventing and treating BPD and ROP in premature infants.

Edaravone dexborneol provides neuroprotective benefits by suppressing NLRP3 inflammasome-induced microglial pyroptosis in experimental ischemic stroke

Edaravone dexborneol (EDB) is a traditional prescription that consists of two components, edaravone and (+)-borneol, which have synergistic antioxidant and anti-inflammatory activities in animal models of ischemic stroke. Pyroptosis is a form of cell death that has only recently been discovered. In this study, we investigated the therapeutic effects and potential mechanisms of EDB in acute ischemic stroke. We used an in vivo mouse transient middle cerebral artery occlusion (tMCAO) model along with an in vitro BV2 cell oxygen-glucose deprivation (OGD) model to perform specific experiments. The executive protein of pyroptosis, gasdermin D (GSDMD), was increased after tMCAO. The administration of EDB dramatically reduced sensorimotor deficits and infarct sizes in mice with tMCAO. In addition, EDB inhibited the production of the NLRP3-inflammasome and the activation of the NF-κB signaling pathway. This effect inhibited both the in vitro and in vivo expression of inflammatory factors, including IL-1β and IL-18. Collectively, our data indicate that EDB exerted positive effects after ischemic stroke. EDB inhibited the activation of NLRP3 inflammasome-induced microglial pyroptosis in experimental ischemic stroke. The findings of this research indicate that the NF-κB/NLRP3/GSDMD signaling pathway may serve as a therapeutic target for EDB to promote functional recovery after stroke.

JQ1 attenuates neuroinflammation by inhibiting the inflammasome-dependent canonical pyroptosis pathway in SAE

Sepsis-associated encephalopathy (SAE) manifests clinically in hyperneuroinflammation. Pyroptosis, which can induce an inflammatory cascade response, has been considered to be a causative factor of SAE. Evidence has shown that the bromo- and extraterminal (BET) proteins (including BRD2, BRD3, BRD4 and BRDT) inhibitor JQ1 can inhibit inflammation and suppress pyroptosis in various diseases. Therefore, we examined the effect of JQ1 on inflammasome-induced pyroptosis in the hippocampus in a mouse model of sepsis induced by lipopolysaccharide (LPS) injection. The results showed that JQ1 treatment alleviated sepsis-related symptoms, protected the blood–brain barrier (BBB), as indicated by upregulated expression of the tight junction proteins occludin and ZO-1, and remarkably rescued neuronal damage in SAE mice. Mechanistically, we demonstrated that JQ1 intervention inhibited the expression of BRD proteins and decreased the expression of inflammasomes by blocking phospho-nuclear factor kappa B (p-NF-κB) signalling, attenuating the canonical pyroptosis (mediated by cleaved-Caspase1/11) pathway and the release of proinflammatory factors in the hippocampus of septic mice. Interestingly, we also found that JQ1 selectively suppressed the activation of hippocampal microglia in SAE mice. Thus, JQ1 protected the hippocampal BBB and neuronal damage through the attenuation of neuroinflammation by inhibiting the inflammasome-dependent canonical pyroptosis pathway induced by LPS injection in mice, and JQ1 may be a promising target for the prevention of SAE.

PLGA-microspheres-carried circGMCL1 protects against Crohn’s colitis through alleviating NLRP3 inflammasome-induced pyroptosis by promoting autophagy

This study aimed to at explore exploring the biological functions of dysregulated circRNA in Crohn’s disease (CD) pathogenesis, with the overarching goal of and providing potential novel therapeutic targets. CircRNA microarray and quantitative real time-polymerase chain reaction (qRT-PCR) analyses were performed to investigate and verify the candidate dysregulated circRNA. The Next, clinical, in vivo, and in vitro studies were performed to investigate explore the biological function and mechanisms of the candidate circRNA in CD. The therapeutic effect of poly (lactic-co-glycolic acid)-microspheres (PLGA MSs)-carried oe-circGMCL1 in experimental colitis models of IL-10 knock-out mice was assessed. CircGMCL1 was identified as the candidate circRNA by microarray and qRT-PCR analyses. Results showed that circGMCL1 expression was negatively correlated with CD-associated inflammatory indices, suggesting that it is a CD-associated circRNA. Microarray and bioinformatics analyses identified miR-124-3p and Annexin 7 (ANXA7) as its downstream mechanisms. The in vitro studies revealed that circGMCL1 mediates its effects on autophagy and NLRP3 inflammasome-mediated pyroptosis in epithelial cells through the ceRNA network. Moreover, the in vivo studies identified the therapeutic effect of PLGA MSs-carried oe-circGMCL1 in experimental colitis models. This study suggests that circGMCL1 protects intestinal barrier function against Crohn’s colitis through alleviating NLRP3 inflammasome-mediated epithelial pyroptosis by promoting autophagy through regulating ANXA7 via sponging miR-124-3p. Therefore, circGMCL1 can serve as a potential biological therapeutic target for Crohn’s colitis.

Prenylated notoamide-type alkaloids isolated from the fungus Aspergillus sclerotiorum and their inhibition of NLRP3 inflammasome activation and antibacterial activities

Notoamides are a family of prenylated indole alkaloids with unusual ring systems and possessing a range of significant pharmaceutical activities. Based on LC-MS/MS and genome orientations, ten undescribed notoamide-type alkaloids namely sclerotiamides I-R were isolated from a marine gorgonian-derived fungus Aspergillus sclerotiorum LZDX-33-4. Their structures were determined by extensive spectroscopic data, in association with ECD data and single-crystal X-ray diffraction for configurational assignments. Bioassays resulted in sclerotiamide J along with five analogs possessing inhibitory effects against LDH and IL-1β expression in BV-2 cells. Further investigation revealed that sclerotiamide J significantly inhibited NLRP3 inflammasome activation and blocked NLRP3 inflammasome-induced pyroptosis via amelioration of mitochondria damage. In addition, sclerotiamide L exhibited potent inhibition against pathogenic Staphylococcus aureus ATCC 29213 with MIC value of 4.0 μM and the growth of MRSA T144 and Enterococcus faecalis ATCC 29212. This study extends the chemical diversity of notoamide-type alkaloids, and provides potential anti-inflammasome and antibacterial lead compounds for further structure optimization.

Schisandrol B protects against cholestatic liver injury by inhibiting pyroptosis through pregnane X receptor

Previously, we demonstrated that Schisandrol B (SolB) protected against lithocholic acid (LCA)-induced cholestatic liver injury (CLI) through pregnane X receptor (PXR). Additionally, growing evidence has revealed that pyroptosis is involved in CLI. Whether the hepatoprotective effect of SolB driven by PXR activation is related to pyroptosis in CLI remains unclear. First, the hepatoprotective effect of SolB was confirmed, as evidenced by the decreased mortality, morphological and histopathological changes, and biochemical parameters. The upregulated serum lactic dehydrogenase (LDH) level, increased number of TUNEL-positive cells, and formation of hepatocyte membrane pores induced by LCA were significantly alleviated after SolB pretreatment, indicating that SolB attenuated LCA-induced hepatocyte damage. Further analysis revealed that both NOD-like receptor protein 3 (NLRP3) inflammasome-induced canonical pyroptosis and apoptosis protease activating factor-1 (Apaf-1) pyroptosome-induced noncanonical pyroptosis were significantly inhibited after SolB pretreatment, as illustrated by the decreased expression levels of NLRP3, ASC, caspase-1, and GSDMD and the levels of Apaf-1, caspase-11 p20, caspase-3 p20, and GSDME. Furthermore, the activation of the NF-κB and FoxO1 signaling pathways was inhibited after SolB pretreatment. In addition, the activation of PXR via SolB was proven by luciferase reporter gene assays and the upregulation of PXR targets. The results illustrated that SolB could significantly inhibit NLRP3 inflammasome-induced canonical pyroptosis through the PXR/NF-κB/NLRP3 axis and inhibit Apaf-1 pyroptosome-induced noncanonical pyroptosis through the PXR/FoxO1/Apaf-1 axis. Collectively, this study revealed that SolB protected against CLI by inhibiting pyroptosis through PXR, providing new insights for understanding the molecular mechanism of SolB as a promising anti-cholestatic agent.

Puerarin ameliorates acute lung injury by modulating NLRP3 inflammasome-induced pyroptosis

We commenced to analyze putative anti-pyroptosis effects of puerarin (PU) as mediated by the PP2A-HDAC1-NLRP3 pathway in acute lung injury (ALI). ALI animal and cell models were constructed, followed by treatment of PU. Then, the effect of HDAC1, PP2A, and NLRP3 on cell inflammation and pyroptosis was explored. The interaction between HDAC1 and PP2A as well as between PP2A and NLRP3 was analyzed. Our findings suggested that PU downregulated HDAC1 expression to alleviate symptoms of ALI. HDAC1 overexpression promoted inflammation induced by LPS, which reversed the inhibitory effect of PU on ALI. HDAC1 overexpression also decreased PP2A expression, suggesting that PP2A was involved in the effects of HDAC1 on LPS-induced inflammation. PP2A exerted inhibitory effects on NLRP3. Meanwhile, PU hindered the progression of ALI by silencing HDAC1 or overexpressing PP2A both in vivo and in vitro. Taken together, PU restrained pyroptosis of cells induced by NLRP3 inflammasome to abate ALI.

Inhibition of IL1R1 or CASP4 attenuates spinal cord injury through ameliorating NLRP3 inflammasome-induced pyroptosis.

Spinal cord injury (SCI) is a devastating trauma characterized by serious neuroinflammation and permanent neurological dysfunction. However, the molecular mechanism of SCI remains unclear, and few effective medical therapies are available at present. In this study, multiple bioinformatics methods were used to screen out novel targets for SCI, and the mechanism of these candidates during the progression of neuroinflammation as well as the therapeutic effects were both verified in a rat model of traumatic SCI. As a result, CASP4, IGSF6 and IL1R1 were identified as the potential diagnostic and therapeutic targets for SCI by computational analysis, which were enriched in NF-κB and IL6-JAK-STATA3 signaling pathways. In the injured spinal cord, these three signatures were up-regulated and closely correlated with NLRP3 inflammasome formation and gasdermin D (GSDMD) -induced pyroptosis. Intrathecal injection of inhibitors of IL1R1 or CASP4 improved the functional recovery of SCI rats and decreased the expression of these targets and inflammasome component proteins, such as NLRP3 and GSDMD. This treatment also inhibited the pp65 activation into the nucleus and apoptosis progression. In conclusion, our findings of the three targets shed new light on the pathogenesis of SCI, and the use of immunosuppressive agents targeting these proteins exerted anti-inflammatory effects against spinal cord inflammation by inhibiting NF-kB and NLRP3 inflammasome activation, thus blocking GSDMD -induced pyroptosis and immune activation.