FDA-approved cannabidiol [Epidiolex®] alleviates Gulf War Illness-linked cognitive and mood dysfunction, hyperalgesia, neuroinflammatory signaling, and declined neurogenesis.

BACKGROUND: Chronic Gulf War Illness (GWI) is characterized by cognitive and mood impairments, as well as persistent neuroinflammation and oxidative stress. This study aimed to investigate the efficacy of Epidiolex®, a Food and Drug Administration (FDA)-approved cannabidiol (CBD), in improving brain function in a rat model of chronic GWI. METHODS: Six months after exposure to low doses of GWI-related chemicals [pyridostigmine bromide, N,N-diethyl-meta-toluamide (DEET), and permethrin (PER)] along with moderate stress, rats with chronic GWI were administered either vehicle (VEH) or CBD (20 mg/kg, oral) for 16 weeks. Neurobehavioral tests were conducted on 11 weeks after treatment initiation to evaluate the performance of rats in tasks related to associative recognition memory, object location memory, pattern separation, and sucrose preference. The effect of CBD on hyperalgesia was also examined. The brain tissues were processed for immunohistochemical and molecular studies following behavioral tests. RESULTS: GWI rats treated with VEH exhibited impairments in all cognitive tasks and anhedonia, whereas CBD-treated GWI rats showed improvements in all cognitive tasks and no anhedonia. Additionally, CBD treatment alleviated hyperalgesia in GWI rats. Analysis of hippocampal tissues from VEH-treated rats revealed astrocyte hypertrophy and increased percentages of activated microglia presenting NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) complexes as well as elevated levels of proteins involved in NLRP3 inflammasome activation and Janus kinase/signal transducers and activators of the transcription (JAK/STAT) signaling. Furthermore, there were increased concentrations of proinflammatory and oxidative stress markers along with decreased neurogenesis. In contrast, the hippocampus from CBD-treated GWI rats displayed reduced levels of proteins mediating the activation of NLRP3 inflammasomes and JAK/STAT signaling, normalized concentrations of proinflammatory cytokines and oxidative stress markers, and improved neurogenesis. Notably, CBD treatment did not alter the concentration of endogenous cannabinoid anandamide in the hippocampus. CONCLUSIONS: The use of an FDA-approved CBD (Epidiolex®) has been shown to effectively alleviate cognitive and mood impairments as well as hyperalgesia associated with chronic GWI. Importantly, the improvements observed in rats with chronic GWI in this study were attributed to the ability of CBD to significantly suppress signaling pathways that perpetuate chronic neuroinflammation.

Identification of novel NLRP3 inhibitors as therapeutic options for epilepsy by machine learning-based virtual screening, molecular docking and biomolecular simulation studies.

The NOD-Like Receptor Protein-3 (NLRP3) inflammasome is a key therapeutic target for the treatment of epilepsy and has been reported to regulate inflammation in several neurological diseases. In this study, a machine learning-based virtual screening strategy has investigated candidate active compounds that inhibit the NLRP3 inflammasome. As machine learning-based virtual screening has the potential to accurately predict protein-ligand binding and reduce false positives outcomes compared to traditional virtual screening. Briefly, classification models were created using Support Vector Machine (SVM), Random Forest (RF), and K-Nearest Neighbor (KNN) machine learning methods. To determine the most crucial features of a molecule's activity, feature selection was carried out. By utilizing 10-fold cross-validation, the created models were analyzed. Among the generated models, the RF model obtained the best results as compared to others. Therefore, the RF model was used as a screening tool against the large chemical databases. Molecular operating environment (MOE) and PyRx software's were applied for molecular docking. Also, using the Amber Tools program, molecular dynamics (MD) simulation of potent inhibitors was carried out. The results showed that the KNN, SVM, and RF accuracy was 0.911 %, 0.906 %, and 0.946 %, respectively. Moreover, the model has shown sensitivity of 0.82 %, 0.78 %, and 0.86 % and specificity of 0.95 %, 0.96 %, and 0.98 % respectively. By applying the model to the ZINC and South African databases, we identified 98 and 39 compounds, respectively, potentially possessing anti-NLRP3 activity. Also, a molecular docking analysis produced ten ZINC and seven South African compounds that has comparable binding affinities to the reference drug. Moreover, MD analysis of the two complexes revealed that the two compounds (ZINC000009601348 and SANC00225) form stable complexes with varying amounts of binding energy. The in-silico studies indicate that both compounds most likely display their inhibitory effect by inhibiting the NLRP3 protein.

Consecutive palmitoylation and phosphorylation orchestrates NLRP3 membrane trafficking and inflammasome activation.

NLRP3 inflammasome activation, essential for cytokine secretion and pyroptosis in response to diverse stimuli, is closely associated with various diseases. Upon stimulation, NLRP3 undergoes subcellular membrane trafficking and conformational rearrangements, preparing itself for inflammasome assembly at the microtubule-organizing center (MTOC). Here, we elucidate an orchestrated mechanism underlying these ordered processes using human and murine cells. Specifically, NLRP3 undergoes palmitoylation at two sites by palmitoyl transferase zDHHC1, facilitating its trafficking between subcellular membranes, including the mitochondria, trans-Golgi network (TGN), and endosome. This dynamic trafficking culminates in the localization of NLRP3 to the MTOC, where LATS1/2, pre-recruited to MTOC during priming, phosphorylates NLRP3 to further facilitate its interaction with NIMA-related kinase 7 (NEK7), ultimately leading to full NLRP3 activation. Consistently, Zdhhc1-deficiency mitigated LPS-induced inflammation and conferred protection against mortality in mice. Altogether, our findings provide valuable insights into the regulation of NLRP3 membrane trafficking and inflammasome activation, governed by palmitoylation and phosphorylation events.

The new pattern for dual NOTCH pathway involving nuclear transcription and mitochondrial regulation supports therapeutic mechanism of 4-butyl benzophenone derivatives against SIRS.

The systemic inflammatory response syndrome (SIRS) represents a self-amplifying cascade of inflammatory reactions and pathophysiological states triggered by infectious or non-infectious factors. The identification of disease targets and differential proteins in the liver (the unique and important immune organ) of SIRS mice treated with the lead compound D1 was conducted using the Genecards database and proteomic analysis, respectively. Subsequently, NOTCH1 was identified as the potential hub target via an intersection analysis between the aforementioned differentially expressed proteins and disease targets. Based on our previous research on the structure-activity relationship, we designed and synthesized a series of SIRS-related derivatives, wherein butyl, halogen, and ester groups were incorporated into benzophenone, aiming at exploring the anti-inflammatory protective action from the perspective of macrophage polarization. Notably, these derivatives exhibited a direct binding capability to the O-glucosylation site (SER496) or its vicinities (such as SER492, VAL485) of NOTCH1 using docking, SPR, DARTS, and CETSA techniques. Mechanistically, derivative D6 exerted anti-inflammatory effects via the dual NOTCH pathway. Firstly, it could inhibit NOTCH1 nuclear transcriptional activity, attenuate the interaction between NICD and RBPJK, concurrently suppress NF-κB and NLRP3 inflammasome (NLRP3, ASC, and cleaved CASP1) activation, and promote NICD (NOTCH1 active fragments) ubiquitination metabolism (the nuclear transcriptional pathway). Secondly, it might possess the ability to increase PGC1α level, subsequently, enhance ATP and MMP levels, mitigate ROS production, increase mitochondrial numbers, and ameliorate mitochondrial inflammatory damage (the mitochondrial pathway). Importantly, the activator Jagged1 could effectively reverse the aforementioned effects, while the inhibitor DAPT exhibited a synergistic effect, suggesting that the nuclear transcriptional regulation and mitochondrial regulation were both in a NOTCH1-dependent manner. Subsequently, it effectively alleviated the inflammatory response and preserved organ function as evidenced by up-regulating M2-type macrophage-related anti-inflammatory cytokines (IL10, TGFß, CD206, and ARG1) and down-regulating M1-type macrophage-related pro-inflammatory cytokines (NO, IL6, IL18, iNOS, TNFα, CD86, and IL1ß). In a word, derivative D6 modulated macrophage polarization and effectively mitigated SIRS by targeting inhibition of the dual NOTCH pathway.

NLRP3 inflammasome in atherosclerosis: Mechanisms and targeted therapies.

Atherosclerosis (AS) is the primary pathology behind various cardiovascular diseases and the leading cause of death and disability globally. Recent evidence suggests that AS is a chronic vascular inflammatory disease caused by multiple factors. In this context, the NLRP3 inflammasome, acting as a signal transducer of the immune system, plays a critical role in the onset and progression of AS. The NLRP3 inflammasome is involved in endothelial injury, foam cell formation, and pyroptosis in AS. Therefore, targeting the NLRP3 inflammasome offers a new treatment strategy for AS. This review highlights the latest insights into AS pathogenesis and the pharmacological therapies targeting the NLRP3 inflammasome, focusing on optimal targets for small molecule inhibitors. These insights are valuable for rational drug design and the pharmacological assessment of new targeted NLRP3 inflammasome inhibitors in treating AS.

Co-exposure of bisphenol A and selenium deficiency induces pyroptosis via ROS/NLRP3 pathway in chicken spleen.

Bisphenol A (BPA) is widely applied in plastic products, which will produce immunotoxicity to organisms after spilling in the environment, and become a kind of endocrine disruptor. Selenium (Se) is an essential trace element and plays an important role in maintaining redox homeostasis and immune function. BPA exposure and Se deficiency often occur together in livestock and poultry farming, however, studies on the effects of joint exposure on chicken immunotoxins have not been reported. Therefore, this study established a chicken spleen and MDCC-MSB1 cell model under the combined effects of BPA exposure or/and Se deficiency. Transcriptomic analysis showed that BPA exposure and/or Se deficiency induced differential enrichment of positive regulatory pathways such as NLRP3 inflammatory complex assembly, inflammatory response and cellular oxidative stress response. In the -Se+BPA group, pathological damage was significantly increased, Se content decreased, BPA accumulation, oxidative stress and pyroptosis. Meanwhile, the roles and mechanisms of oxidative stress and pyroptosis in BPA exposure or/and Se deficiency-induced splenic tissue injury were investigated by using IF and qRT-PCR methods. The results showed that joint BPA exposure with Se deficiency resulted in more significant changes in the above outcomes than 1 of them. The oxidative stress inhibitor NAC effectually reduced Se deficiency and BPA-induced oxidative stress and pyroptosis, further suggests that oxidative stress mediated Se deficiency or/and BPA-induced pyroptosis. This study revealed that BPA exposure and Se deficiency induced spleen pyroptosis in chickens via the ROS/NLRP3 pathway. These results provide the theoretical basis for the toxicity of BPA in poultry and enrich the toxicological mechanism of combined exposure of Se deficiency and environmental toxins.

Epinephelus coioides NLRP3 inhibits SGIV infection by upregulating Capspase-1 activity.

NLRP3 has an important role in the immune response and viral infection as an essential inflammasome component. However, it is unclear whether the grouper immune system is regulated by NLRP3 inflammasome. In this study, we cloned the NLRP3 gene from Epinephelus coioides. Ec-NLRP3 encodes 893 amino acids and contains two major structural domains, the NACHT domain (69-234aa) and the LRR domain (477-893aa). Tissue distribution analysis showed that Ec-NLRP3 was expressed in all tissues tested, with the spleen exhibiting the highest expression. Additionally, after being infected with SGIV, the expression of the Ec-NLRP3 gene was significantly increased. The results of subcellular localization revealed that Ec-NLRP3 was distributed throughout GS cells. In addition, Ec-NLRP3 co-localized with Ec-ASC and was observed as a cytosolic speck. Ec-NLRP3 overexpression significantly inhibited SGIV infection, which was further inhibited by co-overexpression of Ec-NLRP3 and Ec-ASC. Further studies revealed that overexpression of Ec-NLRP3 significantly upregulated caspase-1 activity, and co-overexpression of Ec-NLRP3 and Ec-ASC further upregulated caspase-1 activity. In addition, inhibition of Caspase-1 activity with VX-765 significantly increased the infection of SGIV. Furthermore, the NLRP3 inflammasome activator Nigericin was able to inhibit the infection of SGIV significantly. The above findings suggest that Ec-NLRP3 inhibits SGIV infection by upregulating caspase-1 activity.

Astrocyte-derived exosomal miR-378a-5p mitigates cerebral ischemic neuroinflammation by modulating NLRP3-mediated pyroptosis.

Objective: This study aimed to investigate the regulatory role of astrocyte-derived exosomes and their microRNAs (miRNAs) in modulating neuronal pyroptosis during cerebral ischemia. Methods: Astrocyte-derived exosomes were studied for treating cerebral ischemia in both in vitro and in vivo models. The effects of astrocyte-derived exosomes on neuroinflammation were investigated by analyzing exosome uptake, nerve damage, and pyroptosis protein expression. High throughput sequencing was used to identify astrocyte-derived exosomal miRNAs linked to pyroptosis, followed by validation via qRT‒PCR. The relationship between these miRNAs and NLRP3 was studied using a dual luciferase reporter assay. This study used miR-378a-5p overexpression and knockdown to manipulate OGD injury in nerve cells. The impact of astrocyte-derived exosomal miR-378a-5p on the regulation of cerebral ischemic neuroinflammation was assessed through analysis of nerve injury and pyroptosis protein expression. Results: Our findings demonstrated that astrocyte-derived exosomes were internalized by neurons both in vitro and in vivo. Additionally, Astrocyte-derived exosomes displayed a neuroprotective effect against OGD-induced neuronal injury and brain injury in the ischemic cortical region of middle cerebral artery occlusion (MCAO) rats while also reducing pyroptosis. Further investigations revealed the involvement of astrocyte-derived exosomal miR-378a-5p in regulating pyroptosis by inhibiting NLRP3. The overexpression of miR-378a-5p mitigated neuronal damage, whereas the knockdown of miR-378a-5p increased NLRP3 expression and exacerbated pyroptosis, thus reversing this neuroprotective effect. Conclusion: Astrocyte-derived exosomal miR-378a-5p has a neuroprotective effect on cerebral ischemia by suppressing neuroinflammation associated with NLRP3-mediated pyroptosis.Further research is required to comprehensively elucidate the signaling pathways by which astrocyte-derived exosomal miR-378a-5p modulates neuronal pyroptosis.

E3 ubiquitin ligase MARCH1 reduces inflammation and pyroptosis in cerebral ischemia-reperfusion injury via PCSK9 downregulation.

Pyroptosis has been regarded as caspase-1-mediated monocyte death that induces inflammation, showing a critical and detrimental role in the development of cerebral ischemia-reperfusion injury (IRI). MARCH1 is an E3 ubiquitin ligase that exerts potential anti-inflammatory functions. Therefore, the study probed into the significance of MARCH1 in inflammation and pyroptosis elicited by cerebral IRI. Middle cerebral artery occlusion/reperfusion (MCAO/R)-treated mice and oxygen glucose deprivation/reoxygenation (OGD/R)-treated hippocampal neurons were established to simulate cerebral IRI in vivo and in vitro. MARCH1 and PCSK9 expression was tested in MCAO/R-operated mice, and their interaction was identified by means of the cycloheximide assay and co-immunoprecipitation. The functional roles of MARCH1 and PCSK9 in cerebral IRI were subsequently determined by examining the neurological function, brain tissue changes, neuronal viability, inflammation, and pyroptosis through ectopic expression and knockdown experiments. PCSK9 expression was increased in the brain tissues of MCAO/R mice, while PCSK9 knockdown reduced brain damage and neurological deficits. Additionally, inflammation and pyroptosis were inhibited in OGD/R-exposed hippocampal neurons upon PCSK9 knockdown, accompanied by LDLR upregulation and NLRP3 inflammasome inactivation. Mechanistic experiments revealed that MARCH1 mediated ubiquitination and degradation of PCSK9, lowering PCSK9 protein expression. Furthermore, it was demonstrated that MARCH1 suppressed inflammation and pyroptosis after cerebral IRI by downregulating PCSK9 both in vivo and in vitro. Taken together, the present study demonstrate the protective effect of MARCH1 against cerebral IRI through PCSK9 downregulation, which might contribute to the discovery of new therapies for improving cerebral IRI.

Therapeutic effect of Sheng Mai San, a traditional Chinese medicine formula, on inflammatory bowel disease via inhibition of NF-κB and NLRP3 inflammasome signaling.

Introduction: Inflammatory bowel disease (IBD) is a globally emergent chronic inflammatory disease which commonly requires lifelong care. To date, there remains a pressing need for the discovery of novel anti-inflammatory therapeutic agents against this disease. Sheng Mai San (SMS) is a traditional Chinese medicine prescription with a long history of use for treating Qi and Yin deficiency and recent studies have shown that SMS exhibits anti-inflammatory potential. However, the effects of SMS on the gastrointestinal system remain poorly studied, and its therapeutic potential and underlying molecular mechanisms in IBD have yet to be discovered. In this study, we examined the therapeutic efficacy of SMS in IBD and its anti-inflammatory activity and underlying molecular mechanism, in vivo and in vitro. Methods: The therapeutic efficacy of SMS in IBD was assessed in the DSS-induced acute colitis mouse model. Body weight, stool consistency, rectal bleeding, colon length, organ coefficient, cytokine levels in colon tissues, infiltration of immune cells, and colon pathology were evaluated. The anti-inflammatory activity of SMS and related molecular mechanisms were further examined in lipopolysaccharide (LPS)-induced macrophages via assessment of pro-inflammatory cytokine secretion and NF-κB, MAPK, STAT3, and NLRP3 signalling. Results: SMS significantly ameliorated the severity of disease in acute colitis mice, as evidenced by an improvement in disease activity index, colon morphology, and histological damage. Additionally, SMS reduced pro-inflammatory cytokine production and infiltration of immune cells in colon tissues. Furthermore, in LPS-induced macrophages, we demonstrated that SMS significantly inhibited the production of cytokines and suppressed the activation of multiple pro-inflammatory signalling pathways, including NF-κB, MAPK, and STAT3. SMS also abolished NLRP3 inflammasome activation and inhibited subsequent caspase-1 activation and IL-1ß secretion, suggesting a new therapeutic target for the treatment of IBD. These mechanistic findings were also confirmed in in vivo assays. Conclusion: This study presents the anti-inflammatory activity and detailed molecular mechanism of SMS, in vitro and in vivo. Importantly, we highlight for the first time the potential of SMS as an effective therapeutic agent against IBD.

NLRP3 and autophagy in retinal ganglion cell inflammation in age-related macular degeneration: potential therapeutic implications.

Retinal degenerative diseases were a large group of diseases characterized by the primary death of retinal ganglion cells (RGCs). Recent studies had shown an interaction between autophagy and nucleotide-binding oligomerization domain-like receptor 3 (NLRP3) inflammasomes, which may affect RGCs in retinal degenerative diseases. The NLRP3 inflammasome was a protein complex that, upon activation, produces caspase-1, mediating the apoptosis of retinal cells and promoting the occurrence and development of retinal degenerative diseases. Upregulated autophagy could inhibit NLRP3 inflammasome activation, while inhibited autophagy can promote NLRP3 inflammasome activation, which leaded to the accelerated emergence of drusen and lipofuscin deposition under the neurosensory retina. The activated NLRP3 inflammasome could further inhibit autophagy, thus forming a vicious cycle that accelerated the damage and death of RGCs. This review discussed the relationship between NLRP3 inflammasome and autophagy and its effects on RGCs in age-related macular degeneration, providing a new perspective and direction for the treatment of retinal diseases.

Magnesium-enriched deep-sea water inhibits NLRP3 inflammasome activation and dampens inflammation.

The NLRP3 inflammasome is an essential component of the innate immune system, but excessive activation can lead to inflammatory diseases. Ion fluxes across the plasma membrane or from intracellular stores are known to regulate NLRP3 inflammasome activation. Deep-sea water (DSW) contains high concentrations of many mineral ions, which could potentially influence NLRP3 inflammasome activation. However, the impact of DSW on NLRP3 inflammasome activation has not been investigated. Here, we demonstrated that DSW with water hardness levels up to 500 mg/L did not affect cell viability or the expression of NLRP3 inflammasome components in macrophages derived from THP-1 cells. However, the DSW significantly inhibited IL-1ß secretion and caspase-1 activation in response to NLRP3 activators such as nigericin, ATP, or monosodium urate (MSU) crystals. Mechanically, it was discovered that the presence of 5 mM magnesium ions (Mg2+), equivalent to the Mg2+ concentration found in the DSW with a water hardness of 500 mg/L, inhibits NLRP3 inflammasome activation. This indicates that Mg2+ contributes to the mechanism by which DSW mitigates NLRP3 inflammasome activation. Moreover, DSW administration effectively lessens MSU-triggered peritonitis in mice, a commonly used model for examining the impacts of NLRP3 inflammasome activation. These results show that DSW enriched with Mg2+ could potentially be beneficial in modulating NLRP3 inflammasome-associated diseases.

UCP2 knockout exacerbates sepsis-induced intestinal injury by promoting NLRP3-mediated pyroptosis.

Sepsis-induced intestinal injury is a common complication that increases the morbidity and mortality associated with sepsis. UCP2, a mitochondrial membrane protein, is involved in numerous cellular processes, including metabolism, inflammation, and pyroptosis. According to our previous studies, UCP2 expression increases in septic intestinal tissue. However, its function in intestinal damage is not known. This work investigated UCP2's role in intestinal injury caused by sepsis. A sepsis mouse model was established in wild-type and UCP2-knockout (UCP2-KO) animals using cecal ligation and puncture (CLP). MCC950, an NLRP3 inflammasome inhibitor, was injected intraperitoneally 3 h before CLP surgery. Overall, significantly higher levels of UCP2 were observed in the intestines of septic mice. UCP2-KO mice subjected to CLP exhibited exacerbated intestinal damage, characterized by enhanced mucosal erosion, inflammatory cell infiltration, and increased intestinal permeability. Furthermore, UCP2 knockout significantly increased oxidative stress, inflammation, and pyroptosis in the CLP mouse intestines. Interestingly, MCC950 not only inhibited pyroptosis but also reversed inflammation, oxidative stress as well as damage to intestinal tissues as a result of UCP2 knockout. Our results highlighted the protective functions of UCP2 in sepsis-associated intestinal injury through modulation of inflammation and oxidative stress via NLRP3 inflammasome-induced pyroptosis.

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

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

ß-Caryophyllene mitigates ischemic stroke-induced white matter lesions by inhibiting pyroptosis.

ß-Caryophyllene (BCP), a selective agonist for cannabinoid receptor 2 (CB2R), has demonstrated promising protective effects in various pathological conditions. However, the neuroprotective effects of BCP on white matter damage induced by ischemic stroke have not been elucidated previously. In this study, we find that BCP not only improves sensorimotor and cognitive function via CB2R but also mitigates white matter lesions in mice following ischemic stroke. Furthermore, BCP enhances the viability of MO3.13 oligodendrocytes after oxygen-glucose deprivation and reoxygenation (OGD/R), attenuating OGD/R-induced cellular damage and pyroptosis. Notably, these protective effects of BCP are partially enhanced by the NLRP3 inhibitor MCC950 and counteracted by the NLRP3 activator nigericin. In addition, nigericin significantly exacerbates neurological outcomes and increases white matter lesions following BCP treatment in middle cerebral artery occlusion (MCAO) mice. These results suggest that BCP may ameliorate neurological deficits and white matter damage induced by cerebral ischemia through inhibiting NLRP3-mediated pyroptosis.

IL-1ß mediates Candida tropicalis-induced immunosuppressive function of MDSCs to foster colorectal cancer.

BACKGROUND: There is increasing evidence that gut fungi dysbiosis plays a crucial role in the development and progression of colorectal cancer (CRC). It has been reported that gut fungi exacerbate the severity of CRC by regulating tumor immunity. Our previous studies have shown that the opportunistic pathogenic fungal pathogen, Candida tropicalis (C. tropicalis) promotes CRC progression by enhancing the immunosuppressive function of MDSCs and activating the NLRP3 inflammasome of MDSCs. However, the relationship between IL-1ß produced by NLRP3 inflammasome activation and the immunosuppressive function of MDSCs enhanced by C. tropicalis in CRC remains unclear. METHODS: The TCGA database was used to analyze the relationship between IL-1ß and genes related to immunosuppressive function of MDSCs in human CRC. The expression of IL-1ß in human CRC tissues was detected by immunofluorescence staining. The proteomic analysis was performed on the culture supernatant of C. tropicalis-stimulated MDSCs. The experiments of supplementing and blocking IL-1ß as well as inhibiting the NLRP3 inflammasome activation were conducted. A mouse colon cancer xenograft model was established by using MC38 colon cancer cell line. RESULTS: Analysis of CRC clinical samples showed that the high expression of IL-1ß was closely related to the immunosuppressive function of tumor-infiltrated MDSCs. The results of in vitro experiments revealed that IL-1ß was the most secreted cytokine of MDSCs stimulated by C. tropicalis. In vitro supplementation of IL-1ß further enhanced the immunosuppressive function of C. tropicalis-stimulated MDSCs and NLRP3-IL-1ß axis mediated the immunosuppressive function of MDSCs enhanced by C. tropicalis. Finally, blockade of IL-1ß secreted by MDSCs augmented antitumor immunity and mitigated C. tropicalis-associated colon cancer. CONCLUSIONS: C. tropicalis promotes excessive secretion of IL-1ß from MDSCs via the NLRP3 inflammasome. IL-1ß further enhances the immunosuppressive function of MDSCs to inhibit antitumor immunity, thus promoting the progression of CRC. Therefore, targeting IL-1ß secreted by MDSCs may be a potential immunotherapeutic strategy for the treatment of CRC.

Safflower Alleviates Pulmonary Arterial Hypertension by Inactivating NLRP3: A Combined Approach of Network Pharmacology and Experimental Verification.

INTRODUCTION: Traditional Chinese medicinal plant, safflower, shows effective for treating pulmonary arterial hypertension (PAH), yet the underlying mechanisms remain largely unexplored. This study is aimed at exploring the potential molecular mechanisms of safflower in the treatment of PAH. METHODS: Network pharmacology approach and molecular docking were applied to identify the core active compounds, therapeutic targets, and potential signaling pathways of safflower against PAH. Meanwhile, high-performance liquid chromatography (HPLC) assay was performed to determine the core compounds from safflower. Further, the mechanism of action of safflower on PAH was verified by in vivo and in vitro experiments. RESULTS: A total of 15 active compounds and 177 targets were screened from safflower against PAH. Enrichment analysis indicated that these therapeutic targets were mainly involved in multiple key pathways, such as TNF signaling pathway and Th17 cell differentiation. Notably, molecular docking revealed that quercetin (core compound in safflower) displayed highest binding capacity with NLRP3. In vivo, safflower exerted therapeutic effects on PAH by inhibiting right ventricular hypertrophy, inflammatory factor release, and pulmonary vascular remodeling. Mechanistically, it significantly reduced the expression of proangiogenesis-related factors (MMP-2, MMP-9, Collagen 1, and Collagen 3) and NLRP3 inflammasome components (NLRP3, ASC, and Caspase-1) in PAH model. Similarly, these results were observed in vitro. Besides, we further confirmed that NLRP3 inhibitor had the same therapeutic effect as safflower in vitro. CONCLUSION: Our findings suggest that safflower mitigates PAH primarily by inhibiting NLRP3 inflammasome activation. This provides novel insights into the potential use of safflower as an alternative therapeutic approach for PAH.

A2 reactive astrocyte-derived exosomes alleviate cerebral ischemia-reperfusion injury by delivering miR-628.

Ischemia and hypoxia activate astrocytes into reactive types A1 and A2, which play roles in damage and protection, respectively. However, the function and mechanism of A1 and A2 astrocyte exosomes are unknown. After astrocyte exosomes were injected into the lateral ventricle, infarct volume, damage to the blood-brain barrier (BBB), apoptosis and the expression of microglia-related proteins were measured. The dual luciferase reporter assay was used to detect the target genes of miR-628, and overexpressing A2-Exos overexpressed and knocked down miR-628 were constructed. qRT-PCR, western blotting and immunofluorescence staining were subsequently performed. A2-Exos obviously reduced the infarct volume, damage to the BBB and apoptosis and promoted M2 microglial polarization. RT-PCR showed that miR-628 was highly expressed in A2-Exos. Dual luciferase reporter assays revealed that NLRP3, S1PR3 and IRF5 are target genes of miR-628. After miR-628 was overexpressed or knocked down, the protective effects of A2-Exos increased or decreased, respectively. A2-Exos reduced pyroptosis and BBB damage and promoted M2 microglial polarization through the inhibition of NLRP3, S1PR3 and IRF5 via the delivery of miR-628. This study explored the mechanism of action of A2-Exos and provided new therapeutic targets and concepts for treating cerebral ischemia.

The protective effect of vinpocetine against Estradiol-benzoate induced cervical hyperkeratosis in female rats via modulation of SIRT1/Nrf2, and NLRP3 inflammasome.

The current study was assigned to determine the putative preventive role of vinpocetine (VIN) in cervical hyperkeratosis (CHK) in female rats. Estradiol Benzoate (EB) was utilized in a dose f (60 µg/100 g, i.m) three times/week for 4 weeks to induce cervical hyperkeratosis. VIN was administered alone in a dose of (10 mg/kg/day, orally) for 4 weeks and in the presence of EB. Levels of malondialdehyde (MDA), total nitrites (NOx), reduced glutathione (GSH), interleukin-18 (IL-18), IL-1ß, tumor necrosis factor-alpha (TNF-α) were measured in cervical tissue. The expression of NLRP3/GSDMD/Caspase-1, and SIRT1/Nrf2 was determined using ELISA. Cervical histopathological examination was also done. EB significantly raised MDA, NOx, TNF-α, IL-18, IL-1ß, and GSDMD and up-regulated NLRP3/Caspase-1 proteins. However, GSH, SIRT1, and Nrf2 levels were reduced in cervical tissue. VIN significantly alleviates all biochemical and histopathological abnormalities. VIN considerably mitigates EB-induced cervical hyperkeratosis via NLRP3-induced pyroptosis and SIRT1/Nrf2 signaling pathway.

NLRP3 inflammasome-induced pyroptosis and serum ASC specks are increased in patients with cardiogenic shock.

BACKGROUND: Cardiogenic shock (CS) is characterized by impaired cardiac function, very high mortality, and limited treatment options. The pro-inflammatory signalling during different phases of CS is incompletely understood. METHODS: We collected serum and plasma (N=44) as well as freshly isolated peripheral blood mononuclear cells (PBMC, N=7) of patients with CS complicating acute myocardial infarction on admission and after revascularization (24h, 48h, 72h) and of healthy controls (serum and plasma N=75; PBMC N=12). RESULTS: PBMC of CS patients had increased gene expression of NLRP3, CASP1, PYCARD, IL1B, and IL18 and showed increased rates of pyroptosis (control: 4.7±0.3% vs. 9.9±1.7% in CS patients, p=0.02). Serum interleukin (IL)-1ß levels were increased after revascularization. IL-18 and IL-6 were higher in patients with CS than in healthy controls but comparable before and after revascularization. Pro-inflammatory apoptosis-associated speck-like proteins containing CARD (ASC) specks were elevated in the serum of CS patients on admission and increased after revascularization (admission: 11.1±4.4 specks/µl, after 24h: 19.0±3.9, p=0.02). ASC specks showed a significant association with 30-day mortality in patients with CS (p<0.05). The estimated regression coefficients and odds ratios indicated a positive relationship between ASC specks and mortality (Odds ratio 1.029, 95% CI, 1.000 to 1.072; p=0.02). CONCLUSIONS: Pyroptosis and circulating ASC specks are increased in patients with CS and are particularly induced after reperfusion This underscores their potential role as a biomarker for poor outcomes in CS patients. ASC specks represent promising new therapeutic targets for CS patients with high inflammatory burden.