Mechanism of Vaginal Epithelial Cell Pyroptosis Induced by the NLRP3 Inflammasome in Vulvovaginal Candidiasis.

PROBLEM: Vulvovaginal candidiasis (VVC) is a common mucosal fungal infection, and Candida albicans is the main causative agent. The NLRP3 inflammasome plays an important role in VVC, but the underlying mechanism is unknown. METHOD OF STUDY: Vaginal epithelial cells were divided into three groups: control, C. albicans strain SC5314 (wild-type, WT), and WT+ Matt Cooper Compound 950 (MCC950, a specific NLRP3 inhibitor). After human vaginal epithelial cells were pretreated with 1 µmol/L MCC950 for 2 h, C. albicans (MOI = 1) was cocultured with the human vaginal epithelial cells for 12 h. The cell supernatants were collected, LDH was detected, and the IL-1ß and IL-18 levels were determined by ELISA. The expression of the pyroptosis-related proteins NLRP3, Caspase-1 p20 and GSDMD was measured by Western blotting analysis. The protein expression of the pyroptosis-related N-terminus of GSDMD (GSDMD-N) was detected by immunofluorescence. RESULTS: In this study, we showed that the WT C. albicans strain induced pyroptosis in vaginal epithelial cells, as indicated by the LDH and proinflammatory cytokine levels and the upregulated levels of the pyroptosis-related proteins NLRP3, Caspase-1 p20, and GSDMD-N. MCC950 reversed the changes in the expression of these proteins and proinflammatory cytokines in vaginal epithelial cells. CONCLUSION: C. albicans activated the NLRP3 inflammasome to induce vaginal epithelial cell pyroptosis. MCC950 inhibited the NLRP3 inflammasome, reduced vaginal epithelial cell pyroptosis, and decreased the release of inflammatory cytokines.

Wogonin ameliorates the proliferation, inflammatory response, and pyroptosis in keratinocytes via NOD-like receptor family pyrin domain containing 3/Caspase-1/Gasdermin-D pathway.

BACKGROUND: Psoriasis refers to a highly prevalent and immunologically mediated dermatosis with considerable deterioration in life quality. Wogonin, a sort of flavonoid, has been mentioned to elicit protective activities in skin diseases. However, whether Wogonin is implicated in the treatment of psoriasis and its specific mechanisms are not fully understood. AIM: The present work attempted to elaborate the role of Wogonin during the process of psoriasis and to concentrate on the associated action mechanism. METHODS: Cell counting kit-8 (CCK-8) method was initially applied to assay the viability of human keratinocyte HaCaT cells treated by varying concentrations of Wogonin. To mimic psoriasis in vitro, HaCaT cells were exposed to M5 cytokines. CCK-8 and 5-Ethynyl-2'-deoxyuridine  assays were adopted for the measurement of cell proliferation. Inflammatory levels were examined with enzyme-linked immunosorbent assay. Immunofluorescence staining tested nucleotide-binding oligomerization domain (NOD)-like receptor family pyrin domain containing 3 (NLRP3) and Caspase-1 expressions. Western blot examined the protein expressions of proliferation-, inflammation-, pyroptosis-associated factors, and NLRP3. RESULTS: Wogonin treatment antagonized the proliferation, inflammatory response, and NLRP3/caspase-1/Gasdermin-D (GSDMD)-mediated pyroptosis in M5-challenged HaCaT cells. Besides, NLRP3 elevation partially abrogated the effects of Wogonin on M5-induced proliferation, inflammatory response, and NLRP3/caspase-1/GSDMD-mediated pyroptosis in HaCaT cells. CONCLUSION: In a word, Wogonin might exert anti-proliferation, anti-inflammatory and anti-pyroptosis activities in M5-induced cell model of psoriasis and the blockade of NLRP3/Caspase-1/GSDMD pathway might be recognized as a potential mechanism underlying the protective mechanism of Wogonin in psoriasis, suggesting Wogonin as a prospective anti-psoriasis drug.

Alveolar hypoxia induces organ-specific inflammasome-related inflammation in male mouse lungs.

Inflammation through activation of caspase-1, seems to play a role in pulmonary hypertension induced by alveolar hypoxia. Whether alveolar hypoxia induces caspase-1-mediated inflammation and influx of leukocytes in other organs than the lungs, is not known. Our aim was to explore sites of caspase-1-related inflammation in alveolar hypoxia. Wild type (WT) mice were exposed to environmental hypoxia or room-air, and organs were analyzed. Right heart catheterization was performed after 14 days of alveolar hypoxia in WT mice and mice transplanted with WT or caspase-1-/- bone marrow. Hypoxia induced leukocyte accumulation and increased caspase-1 protein in the lungs, not in other organs. WT mice transplanted with WT or caspase-1-/- bone marrow showed no difference in pulmonary leukocyte accumulation or development of pulmonary hypertension after alveolar hypoxia. Caspase-1 and IL-18 were detected in bronchial epithelium in WT mice, and hypoxia induced IL-18 secretion from bronchial epithelial cells. IL-18 stimulation generated IL-6 mRNA in monocytes. Phosphorylated STAT3 was increased in hypoxic lungs, not in other organs. Alveolar hypoxia induces caspase-1 activation and leukocyte accumulation specific to the lungs, not in other organs. Caspase-1 activation and IL-18 secretion from bronchial epithelial cells might initiate hypoxia-induced inflammation, leading to pulmonary hypertension.

Dexmedetomidine's protective mechanism against hyperoxic injury in neonatal rats.

Bronchopulmonary dysplasia (BPD) is a common serious complication of premature babies. No effective means control it. Hyperoxia damage is one of the important mechanisms of BPD. The reaserach confirmed pyroptosis existed in BPD. Dexmedetomidine is a new, high-specific α2 receptor agonist. Previous research foundation found that dexmedetomidine has a protective effect on BPD. To investigate how dexmedetomidine improves hyperoxic lung injury in neonatal mice by regulating pyroptosis. Neonatal rats were randomly divided into four groups: normal control group, hyperoxic injury group, air plus dexmedetomidine group, and hyperoxia plus dexmedetomidine group. After seven days the lungs of rats in each group were extracted, and the wet-to-dry weight ratio of the lung was measured. The lung injury in rats was observed using hematoxylin-eosin staining. Additionally, the expression and localization of nucleotide-binding oligomerization domain-like receptor thermal protein domain associated protein 3 (NLRP3), apoptosis-associated speck-like protein (ASC), and gasdermin D (GSDMD) proteins were examined in the lungs of rats using immunofluorescence staining. The mRNA levels of NLRP3, ASC, caspase-1, and interleukin 18 (IL-18) in the lungs of rats were determined using real-time PCR. Moreover, the protein levels of NLRP3, ASC, caspase-1/cleaved caspase-1, interleukin 1beta (IL-1ß), IL-18, and tunor necrosis factor alpha (TNF-α) were detected in lungs of rats using Western blot. The extent of mitochondrial damage in lung tissues of each group was observed by transmission electron microscopy. The lung tissue injury of the neonatal rats was significantly improved in the hyperoxia plus dexmedetomidine group compared to the hyperoxic injury group. Furthermore, the expressions of pyroptosis-related proteins such as NLRP3, ASC, cleaved-caspase-1, and GSDMD were significantly decreased, along with the expressions of inflammatory factors in lung tissues. By inhibiting the NLRP3/caspase-1/GSDMD pyroptosis pathway, dexmedetomidine reduces the activation and release of inflammatory factors and provides a protective effect against hyperoxic lung injury in neonatal mice.

Activation of the NLRP3-CASP-1 inflammasome is restrained by controlling autophagy during Glaesserella parasuis infection.

Infection with Glaesserella parasuis, the primary pathogen behind Glässer's disease, is often associated with diverse clinical symptoms, including serofibrinous polyserositis, arthritis, and meningitis. Autophagy plays a dual role in bacterial infections, exerting either antagonistic or synergistic effects depending on the nature of the pathogen. Our previous studies have demonstrated that autophagy serves as a defense mechanism, combating inflammation and invasion caused by infection of highly virulent G. parasuis. However, the precise mechanisms remain to be elucidated. Pathogens exhibit distinct interactions with inflammasomes and autophagy processes. Herein, we explored the effect of autophagy on inflammasomes during G. parasuis infection. We found that G. parasuis infection triggers NLRP3-dependent pro-CASP-1-IL-18/IL-1ß processing and maturation pathway, resulting in increased release of IL-1ß and IL-18. Inhibition of autophagy enhances NLRP3 inflammasome activity, whereas stimulation of autophagy restricts it during G. parasuis infection. Furthermore, assembled NLRP3 inflammasomes undergo ubiquitination and recruit the autophagic adaptor, p62, facilitating their sequestration into autophagosomes during G. parasuis infection. These results suggest that the induction of autophagy mitigates inflammation by eliminating overactive NLRP3 inflammasomes during G. parasuis infection. Our research uncovers a mechanism whereby G. parasuis infection initiates inflammatory responses by promoting the assembly of the NLRP3 inflammasomes and activating NLRP3-CASP-1, both of which processes are downregulated by autophagy. This suggests that pharmacological manipulation of autophagy could be a promising approach to modulate G. parasuis-induced inflammatory responses.

Elevated NLRP3 Inflammasome Activation Is Associated with Motor Neuron Degeneration in ALS.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by motor neuron degeneration in the central nervous system. Recent research has increasingly linked the activation of nucleotide oligomerization domain-like receptor protein 3 (NLRP3) inflammasome to ALS pathogenesis. NLRP3 activation triggers Caspase 1 (CASP 1) auto-activation, leading to the cleavage of Gasdermin D (GSDMD) and pore formation on the cellular membrane. This process facilitates cytokine secretion and ultimately results in pyroptotic cell death, highlighting the complex interplay of inflammation and neurodegeneration in ALS. This study aimed to characterize the NLRP3 inflammasome components and their colocalization with cellular markers using the wobbler mouse as an ALS animal model. Firstly, we checked the levels of miR-223-3p because of its association with NLRP3 inflammasome activity. The wobbler mice showed an increased expression of miR-223-3p in the ventral horn, spinal cord, and cerebellum tissues. Next, increased levels of NLRP3, pro-CASP 1, cleaved CASP 1 (c-CASP 1), full-length GSDMD, and cleaved GDSMD revealed NLRP3 inflammasome activation in wobbler spinal cords, but not in the cerebellum. Furthermore, we investigated the colocalization of the aforementioned proteins with neurons, microglia, and astrocyte markers in the spinal cord tissue. Evidently, the wobbler mice displayed microgliosis, astrogliosis, and motor neuron degeneration in this tissue. Additionally, we showed the upregulation of protein levels and the colocalization of NLRP3, c-CASP1, and GSDMD in neurons, as well as in microglia and astrocytes. Overall, this study demonstrated the involvement of NLRP3 inflammasome activation and pyroptotic cell death in the spinal cord tissue of wobbler mice, which could further exacerbate the motor neuron degeneration and neuroinflammation in this ALS mouse model.

Hyperglycemia induces microglial pyroptosis by increasing oxygen extraction rate: Implication in neurological impairment during ischemic stroke.

Elevated levels of blood glucose in patients with ischemic stroke are associated with a worse prognosis. The present study aimed to explore whether hyperglycemia promotes microglial pyroptosis by increasing the oxygen extraction rate in an acute ischemic stroke model. C57BL/6 mice that underwent middle cerebral artery occlusion were used for assessment of blood glucose level and neurological function. The cerebral oxygen extraction ratio (CERO2), oxygen consumption rate (OCR) and partial pressure of brain tissue oxygen (PbtO2) were measured. To investigate the significance of the NOD­like receptor protein 3 (NLRP3) inflammasome, NLRP3­/­ mice were used, and the expression levels of NLRP3, caspase­1, full­length gasdermin D (GSDMD­FL), GSDMD­N domain (GSDMD­N), IL­1ß and IL­18 were evaluated. In addition, Z­YVAD­FMK, a caspase­1 inhibitor, was used to treat microglia to determine whether activation of the NLRP3 inflammasome was required for the enhancing effect of hyperglycemia on pyroptosis. It was revealed that hyperglycemia accelerated cerebral injury in the acute ischemic stroke model, as evidenced by decreased latency to fall and the percentage of foot fault. Hyperglycemia aggravated hypoxia by increasing the oxygen extraction rate, as evidenced by increased CERO2 and OCR, and decreased PbtO2 in response to high glucose treatment. Furthermore, hyperglycemia­induced microglial pyroptosis was confirmed by detection of increased levels of caspase­1, GSDMD­N, IL­1ß and IL­18 and a decreased level of GSDMD­FL. However, the knockout of NLRP3 attenuated these effects. Pharmacological inhibition of caspase­1 also reduced the expression levels of GSDMD­N, IL­1ß and IL­18 in microglial cells. These results suggested that hyperglycemia stimulated NLRP3 inflammasome activation by increasing the oxygen extraction rate, thus leading to the aggravation of pyroptosis following ischemic stroke.

Hydrogen sulfide mitigates ox­LDL­induced NLRP3/caspase­1/GSDMD dependent macrophage pyroptosis by S­sulfhydrating caspase­1.

Macrophage pyroptosis mediates vascular inflammation and atherosclerosis (AS). Hydrogen sulfide (H2S) exerts a protective role in preventing inflammation and AS. However, its molecular mechanisms of regulating the pyroptosis signaling pathway and inhibiting macrophage pyroptosis remain unexplored. The present study aimed to determine whether H2S mitigates macrophage pyroptosis by downregulating the pyroptosis signaling pathway and S­sulfhydrating caspase­1 under the stimulation of oxidized low­density lipoprotein (ox­LDL), a pro­atherosclerotic factor. Macrophages derived from THP­1 monocytes were pre­treated using exogenous H2S donors sodium hydrosulfide (NaHS) and D,L­propargylglycine (PAG), a pharmacological inhibitor of endogenous H2S­producing enzymes, alone or in combination. Subsequently, cells were stimulated with ox­LDL or the desulfhydration reagent dithiothreitol (DTT) in the presence or absence of NaHS and/or PAG. Following treatment, the levels of H2S in THP­1 derived macrophages were measured by a methylene blue colorimetric assay. The pyroptotic phenotype of THP­1 cells was observed and evaluated by light microscopy, Hoechst 33342/propidium iodide fluorescent staining and lactate dehydrogenase (LDH) release assay. Caspase­1 activity in THP­1 cells was assayed by caspase­1 activity assay kit. Immunofluorescence staining was used to assess the accumulation of active caspase­1. Western blotting and ELISA were performed to determine the expression of pyroptosis­specific markers (NLRP3, pro­caspase­1, caspase­1, GSDMD and GSDMD­N) in cells and the secretion of pyroptosis­related cytokines [interleukin (IL)­1ß and IL­18] in the cell­free media, respectively. The S­sulfhydration of pro­caspase­1 in cells was assessed using a biotin switch assay. ox­LDL significantly induced macrophage pyroptosis by activating the pyroptosis signaling pathway. Inhibition of endogenous H2S synthesis by PAG augmented the pro­pyroptotic effects of ox­LDL. Conversely, exogenous H2S (NaHS) ameliorated ox­LDL­and ox­LDL + PAG­induced macrophage pyroptosis by suppressing the activation of the pyroptosis signaling pathway. Mechanistically, ox­LDL and the DTT increased caspase­1 activity and downstream events (IL­1ß and IL­18 secretion) of the caspase­1­dependent pyroptosis pathway by reducing S­sulfhydration of pro­caspase­1. Conversely, NaHS increased S­sulfhydration of pro­caspase­1, reducing caspase­1 activity and caspase­1­dependent macrophage pyroptosis. The present study demonstrated the molecular mechanism by which H2S ameliorates macrophage pyroptosis by suppressing the pyroptosis signaling pathway and S­sulfhydration of pro­caspase­1, thereby suppressing the generation of active caspase-1 and activity of caspase-1.

Sex-dependent effects of carbohydrate source and quantity on caspase-1 activity in the mouse central nervous system.

BACKGROUND: Mounting evidence links glucose intolerance and diabetes as aspects of metabolic dysregulation that are associated with an increased risk of developing dementia. Inflammation and inflammasome activation have emerged as a potential link between these disparate pathologies. As diet is a key factor in both the development of metabolic disorders and inflammation, we hypothesize that long term changes in dietary factors can influence nervous system function by regulating inflammasome activity and that this phenotype would be sex-dependent, as sex hormones are known to regulate metabolism and immune processes. METHODS: 5-week-old male and female transgenic mice expressing a caspase-1 bioluminescent reporter underwent cranial window surgeries and were fed control (65% complex carbohydrates, 15% fat), high glycemic index (65% carbohydrates from sucrose, 15% fat), or ketogenic (1% complex carbohydrates, 79% fat) diet from 6 to 26 weeks of age. Glucose regulation was assessed with a glucose tolerance test following a 4-h morning fast. Bioluminescence in the brain was quantified using IVIS in vivo imaging. Blood cytokine levels were measured using cytokine bead array. 16S ribosomal RNA gene amplicon sequencing of mouse feces was performed to assess alterations in the gut microbiome. Behavior associated with these dietary changes was also evaluated. RESULTS: The ketogenic diet caused weight gain and glucose intolerance in both male and female mice. In male mice, the high glycemic diet led to increased caspase-1 biosensor activation over the course of the study, while in females the ketogenic diet drove an increase in biosensor activation compared to their respective controls. These changes correlated with an increase in inflammatory cytokines present in the serum of test mice and the emergence of anxiety-like behavior. The microbiome composition differed significantly between diets; however no significant link between diet, glucose tolerance, or caspase-1 signal was established. CONCLUSIONS: Our findings suggest that diet composition, specifically the source and quantity of carbohydrates, has sex-specific effects on inflammasome activation in the central nervous system and behavior. This phenotype manifested as increased anxiety in male mice, and future studies are needed to determine if this phenotype is linked to alterations in microbiome composition.

Astragaloside IV inhibits inflammation caused by influenza virus via reactive oxygen species/NOD-like receptor thermal protein domain associated protein 3/Caspase-1 signaling pathway.

BACKGROUND: Astragaloside IV (AS-IV) is the most active monomer in the traditional Chinese herbal medicine Radix Astragali, which has a wide range of antiviral, anti-inflammatory, and antifibrosis pharmacological effects, and shows protective effects in acute lung injury. METHODS: This study utilized the immunofluorescence, flow cytometry, enzyme-linked immunosorbent assay, quantitative reverse transcription-polymerase chain reaction, western blot, and hematoxylin and eosin staining methods to investigate the mechanism of AS-IV in reducing viral pneumonia caused by influenza A virus in A549 cells and BALB/c mice. RESULTS: The results showed that AS-IV suppressed reactive oxygen species production in influenza virus-infected A549 cells in a dose-dependent manner, and subsequently inhibited the activation of nucleotide-binding oligomerization domain-like receptor thermal protein domain associated protein 3 inflammasome and Caspase-1, decreased interleukin (IL) -1ß and IL-18 secretion. In BALB/c mice infected with Poly (I:C), oral administration of AS-IV can significantly reduce Poly (I:C)-induced acute pneumonia and lung pathological injury. CONCLUSIONS: AS-IV alleviates the inflammatory response induced by influenza virus in vitro and lung flammation and structural damage caused by poly (I:C) in vivo.

[Coenzyme Q10 alleviates depression-like behaviors in mice with chronic restraint stress by down-regulating the pyroptosis signaling pathway].

OBJECTIVE: To explore the neuroprotective effect of coenzyme Q10 and its possible mechanism in mice with chronic restraint stress (CRS). METHODS: Mouse models of CRS were treated with intraperitoneal injections of coenzyme Q10 at low, moderate and high doses (50, 100 and 200 mg/kg, respectively, n=8), VX765 (a caspase-1 specific inhibitor, 50 mg/kg, n=8), or fluoxetine (10 mg/kg, n=8) on a daily basis for 4 weeks, and the changes in depression-like behaviors of the mice were assessed by sugar water preference test, forced swimming test and tail suspension test. The expression of glial fibrillary acidic protein (GFAP) in the hippocampus of the mice was detected using immunohistochemistry, and the number of synaptic spines was determined with Golgi staining. Western blotting was performed to detect the changes in the expressions of GFAP and pyroptosis-related proteins in the hippocampus, and the colocalization of neurons and caspase-1 p10 was examined with immunofluorescence assay. RESULTS: Compared with the normal control mice, the mouse models of CRS showed significantly reduced sugar water preference and increased immobility time in forced swimming and tail suspension tests (P < 0.05), and these depression-like behaviors were obviously improved by treatment with coenzyme Q10, VX765 or FLX. The mouse models showed a significantly decreased positive rate of GFAP and lowered GFAP protein expression in the hippocampus with obviously decreased synaptic spines, enhanced expressions of GSDMD-N, caspase-1 and IL-1ß, and increased colocalization of neurons and caspase-1 p10 (all P < 0.05). All these changes were significantly ameliorated in the mouse models after treatment with Q10. CONCLUSION: Coenzyme Q10 can alleviate depression-like behaviors in mice with CRS by down-regulating the pyroptosis signaling pathway.

In vitro and in silico analyses reveal the toxicity of metolachlor to grass carp hepatocytes and the antagonism of melatonin.

Due to the widespread use of metolachlor (MET), the accumulation of MET and its metabolites in the environment has brought serious health problems to aquatic organisms. At present, the toxicity of MET on the physiological metabolism of aquatic animals mainly focused on the role of enzymes. There is still a lack of research on the molecular mechanisms of MET hepatotoxicity, especially on antagonizing MET toxicity. Therefore, this study focuses on grass carp hepatocytes (L8824 cells) closely related to toxin accumulation. By establishing a MET exposed L8824 cells model, it is determined that MET exposure induces pyrolytic inflammation of L8824 cells. Subsequent mechanistic studies found that MET exposure induces pyroptosis in L8824 cells through mitochondrial dysfunction, and siCaspase-1 inhibits the MET induced ROS production, suggesting a regulation of ROS-NLRP3- Caspase-1 pyroptotic inflammation cycling center in MET induced injury to L8824 cells. Molecular docking revealed a strong binding energy between melatonin (MT) and Caspase-1. Finally, a model of L8824 cells with MT intervention in MET exposure was established. MT can antagonize the pyroptosis induced by MET exposure in L8824 cells by targeting Caspase-1, thereby restoring mitochondrial function and inhibiting the ROS-pyroptosis cycle. This study discovered targets and mechanisms of MT regulating pyroptosis in MET exposed-L8824 cells, and the results are helpful to provide new targets for the design of MET antidotes.

G protein-coupled estrogen receptor agonist G-1 decreases ADAM10 levels and NLRP3-inflammasome component activation in response to Staphylococcus aureus alpha-hemolysin.

The G protein-coupled estrogen receptor, also known as GPER1 or originally GPR30, is found in various tissues, indicating its diverse functions. It is typically present in immune cells, suggesting its role in regulating immune responses to infectious diseases. Our previous studies have shown that G-1, a selective GPER agonist, can limit the pathogenesis mediated by Staphylococcus aureus alpha-hemolysin (Hla). It aids in clearing bacteria in a mouse skin infection model and restricts the surface display of the Hla receptor, ADAM10 (a disintegrin and metalloprotease 10) in HaCaT keratinocytes. In this report, we delve into the modulation of GPER in human immune cells in relation to the NLRP3 inflammasome. We used macrophage-like differentiated THP-1 cells for our study. We found that treating these cells with G-1 reduces ATP release, decreases the activity of the caspase-1 enzyme, and lessens cell death following Hla intoxication. This is likely due to the reduced levels of ADAM10 and NLRP3 proteins, as well as the decreased display of the ADAM10 receptor in the G-1-treated THP-1 cells. Our studies, along with our previous work, suggest the potential therapeutic use of G-1 in reducing Hla susceptibility in humans. This highlights the importance of GPER in immune regulation and its potential as a therapeutic target.

Ameliorated Effects of Fucoidan on Dextran Sulfate Sodium-Induced Ulcerative Colitis and Accompanying Anxiety and Depressive Behaviors in Aged C57BL/6 Mice.

Fucoidan has shown better effects on the improvement of acute ulcerative colitis (UC). However, the specific mechanisms by which fucoidan improves UC-related behavioral disorders in aged mice, especially its effect on the gut-brain axis, remain to be further explored. C57BL/6 male mice aged 8 months were gavaged with 400 or 100 mg/kg bw day fucoidan for five consecutive weeks, with UC being induced by ad libitum to dextran sulfate sodium (DSS) solution in the fifth week. The results showed that fucoidan ameliorated UC and accompanying anxiety- and depressive-like behaviors with downregulated expressions of (NOD)-like receptor family and pyrin domain-containing 3 (NLRP3), apoptosis-associated speck-like protein (ASC), cysteine aspartate-specific protease-1 (Caspase-1) and interlekin-1ß (IL-1ß), and elevated mRNA levels of brain-derived neurotrophic factor (Bdnf) and postsynaptic-density protein 95 (Psd-95) in cortex and hippocampus. Furthermore, fucoidan improved the permeability of intestinal barrier and blood-brain barrier and restored the abnormal structure of the gut microbiota with a significantly decreased ratio of Firmicutes to Bacteroidota (F/B) and obviously increased abundance of Akkermansia. As a diet-derived bioactive ingredient, fucoidan might be a better alternative for the prevention of UC and accompanying anxiety- and depressive-like behaviors.

Atractylenolide I Alleviates Indomethacin-Induced Gastric Ulcers in Rats by Inhibiting NLRP3 Inflammasome Activation.

Atractylodes macrocephala Koidz, a traditional Chinese medicine, contains atractylenolide I (ATR-I), which has potential anticancer, anti-inflammatory, and immune-modulating properties. This study evaluated the therapeutic potential of ATR-I for indomethacin (IND)-induced gastric mucosal lesions and its underlying mechanisms. Noticeable improvements were observed in the histological morphology and ultrastructures of the rat gastric mucosa after ATR-I treatment. There was improved blood flow, a significant decrease in the expression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), IL-1ß, and IL-18, and a marked increase in prostaglandin E2 (PGE2) expression in ATR-I-treated rats. Furthermore, there was a significant decrease in the mRNA and protein expression levels of NOD-like receptor thermal protein domain associated protein 3 (NLRP3), apoptosis-associated speck-like protein (ASC), cysteinyl aspartate specific proteinase-1 (caspase-1), and nuclear factor-κB (NF-κB) in rats treated with ATR-I. The results show that ATR-I inhibits the NLRP3 inflammasome signaling pathway and effectively alleviates local inflammation, thereby improving the therapeutic outcomes against IND-induced gastric ulcers in rats.

Oxylipins and metabolites from pyroptotic cells act as promoters of tissue repair.

Pyroptosis is a lytic cell death mode that helps limit the spread of infections and is also linked to pathology in sterile inflammatory diseases and autoimmune diseases1-4. During pyroptosis, inflammasome activation and the engagement of caspase-1 lead to cell death, along with the maturation and secretion of the inflammatory cytokine interleukin-1ß (IL-1ß). The dominant effect of IL-1ß in promoting tissue inflammation has clouded the potential influence of other factors released from pyroptotic cells. Here, using a system in which macrophages are induced to undergo pyroptosis without IL-1ß or IL-1α release (denoted Pyro-1), we identify unexpected beneficial effects of the Pyro-1 secretome. First, we noted that the Pyro-1 supernatants upregulated gene signatures linked to migration, cellular proliferation and wound healing. Consistent with this gene signature, Pyro-1 supernatants boosted migration of primary fibroblasts and macrophages, and promoted faster wound closure in vitro and improved tissue repair in vivo. In mechanistic studies, lipidomics and metabolomics of the Pyro-1 supernatants identified the presence of both oxylipins and metabolites, linking them to pro-wound-healing effects. Focusing specifically on the oxylipin prostaglandin E2 (PGE2), we find that its synthesis is induced de novo during pyroptosis, downstream of caspase-1 activation and cyclooxygenase-2 activity; further, PGE2 synthesis occurs late in pyroptosis, with its release dependent on gasdermin D pores opened during pyroptosis. As for the pyroptotic metabolites, they link to immune cell infiltration into the wounds, and polarization to CD301+ macrophages. Collectively, these data advance the concept that the pyroptotic secretome possesses oxylipins and metabolites with tissue repair properties that may be harnessed therapeutically.

Oridonin ameliorates ocular surface inflammatory responses by inhibiting the NLRP3/caspase-1/GSDMD pyroptosis pathway in dry eye.

Chronic inflammation is one of the central drivers in the development of dry eye disease (DED), in which pyroptosis induced by the NLRP3/caspase-1/gasdermin D (GSDMD) pathway plays a key role. This pathway has become a major target for the treatment of a variety of inflammatory disorders. Oridonin (Ori) is a naturally occurring substance with anti-inflammatory properties obtained from Rabdosia rubescens. Whether Ori can exert an anti-inflammatory effect on DED, and its anti-inflammatory mechanism of action, are still unknown. This experiment is intended to investigate the impact of Ori on the hyperosmolarity-induced NLRP3/caspase-1/GSDMD pyroptosis pathway in immortalized human corneal epithelial (HCE-T) cells, as well as its efficacy and mechanism of action on ocular surface injury in DED mice. Our study showed that Ori could inhibit hyperosmotic-induced pyroptosis through the NLRP3/caspase-1/GSDMD pathway in HCE-T cells, and similarly, Ori inhibited the expression of this pathway in DED mice. Moreover, Ori was protective against hyperosmolarity-induced HCE-T cell damage. In addition, we found that the morphology and number of HCE-T cells were altered under culture conditions of various osmolarities. With increasing osmolarity, the proliferation, migration, and healing ability of HCE-T cells decreased significantly, and the expression of N-GSDMD was elevated. In a mouse model of DED, Ori application inhibited the expression of the NLRP3/caspase-1/GSDMD pyroptosis pathway, improved DED signs and injury, decreased corneal sodium fluorescein staining scores, and increased tear volume. Thus, our study suggests that Ori has potential applications for the treatment of DED, provides potential novel therapeutic approaches to treat DED, and provides a theoretical foundation for treating DED using Ori.

NETs contribute to psoriasiform skin inflammation: A novel therapeutic approach targeting IL-36 cytokines by a small molecule tetrahydroxystilbene glucoside.

BACKGROUND: Psoriasis, a chronic immune-mediated skin disease with pathological features such as aberrant differentiation of keratinocytes, dermal-epidermal inflammation, and angiogenesis. 2,3,5,4'-Tetrahydroxy stilbene 2-Ο-ß-d-glucoside (2354Glu) is a natural small molecule polyhydrostilbenes isolated from Polygonum multiglorum Thunb. The regulation of IL-36 subfamily has led to new pharmacologic strategies to reverse psoriasiform dermatitis. PURPOSE: Here we investigated the therapeutic potential of 2354Glu and elucidated the underlying mechanism in psoriasis. METHODS: The effects of 2354Glu on IL-36 signaling were assessed by psoriasiform in vivo, in vitro and ex vivo model. The in vivo mice model of psoriasis-like skin inflammation was established by applying imiquimod (IMQ), and the in vitro and ex vitro models were established by stimulating mouse primary keratinocyte, human keratinocytes cells (HaCaT) and ex vivo skin tissue isolated from the mice back with Polyinosine-polycytidylic acid (Poly(I:C)), IMQ, IL-36γ and Lipopolysaccharide (LPS) respectively. Moreover, NETs formation was inhibited by Cl-amidine to evaluate the effect of NETs in psoriatic mouse model. The effects of 2354Glu on skin inflammation were assessed by western blot, H&E, immunohistochemistry, immunofluorescence, enzyme-linked immunosorbent assay and real-time quantitative PCR. RESULTS: In Poly(I:C)-stimulated keratinocytes, the secretion of IL-36 was inhibited after treatment with 2354Glu, similar to the effects of TLR3, P2X7R and caspase-1 inhibitors. In aldara (imiquimod)-induced mice, 2354Glu (100 and 25 mg/kg) improved immune cell infiltration and hyperkeratosis in psoriasis by directly targeting IL-36 in keratinocytes through P2X7R-caspase-1. When treatment with 2354Glu (25 mg/kg) was insufficient to inhibit IL-36γ, NETs reduced pathological features and IL-36 signaling by interacting with keratinocytes to combat psoriasis like inflammation. CONCLUSION: These results indicated that NETs had a beneficial effect on psoriasiform dermatitis. 2354Glu alleviates psoriasis by directly targeting IL-36/P2X7R axis and NET formation, providing a potential candidate for the treatment of psoriasis.

Tryptanthrin suppresses multiple inflammasome activation to regulate NASH progression by targeting ASC protein.

BACKGROUND: The adaptor protein apoptosis-associated speck-like protein (ASC) containing a caspase recruitment domain (CARD) can be activated through pyrin domain (PYD) interactions between sensors and ASC, and through CARD interactions between caspase-1 and ASC. Although the majority of ternary inflammasome complexes depend on ASC, drugs targeting ASC protein remain scarce. After screening natural compounds from Isatidis Radixin, we found that tryptanthrin (TPR) could inhibit NLRP3-induced IL-1ß and caspase-1 production, but the underlying anti-inflammatory mechanisms remain to be elucidated. PURPOSE: The purpose of this study was to determine the impact of TPR on the NLRP3, NLRC4, and AIM2 inflammasomes and the underlying mechanisms. Additionally, the efficacy of TPR was analysed in the further course of methionine- and choline-deficient (MCD)-induced NASH and lipopolysaccharide (LPS)-induced sepsis models of mice. METHODS: In vitro studies used bone marrow-derived macrophages to assess the anti-inflammatory activity of TPR, and the techniques included western blot, testing of intracellular K+ and Ca2+, immunofluorescence, enzyme-linked immunosorbent assay (ELISA), co-immunoprecipitation, ASC oligomerization assay, surface plasmon resonance (SPR), and molecular docking. We used LPS-induced sepsis models and MCD-induced NASH models in vivo to evaluate the effectiveness of TPR in inhibiting inflammatory diseases. RESULTS: Our observations suggested that TPR could inhibit NLRP3, NLRC4, and AIM2 inflammasome activation. As shown in a mouse model of inflammatory diseases caused by MCD-induced NASH and LPS-induced sepsis, TPR significantly alleviated the progression of diseases. TPR interrupted the interactions between ASC and NLRP3/NLRC4/AIM2 in the co-immunoprecipitation experiment, and stable binding of TPR to ASC was also evident in SPR experiments. The underlying mechanisms of anti-inflammatory activities of TPR might be associated with targeting ASC, in particular, PYD domain of ASC. CONCLUSION: In general, the requirement for ASC in multiple inflammasome complexes makes TPR, as a novel broad-spectrum inflammasome inhibitor, potentially useful for treating a wide range of multifactorial inflammasome-related diseases.

Epimedii Folium decoction ameliorates osteoporosis in mice through NLRP3/caspase-1/IL-1ß signalling pathway and gut-bone axis.

AIM OF THE STUDY: This study aimed to determine the effect of Epimedium brevicornu Maxim. (EF) on osteoporosis (OP) and its underlying molecular mechanisms, and to explore the existence of the "Gut-Bone Axis". MATERIAL AND METHODS: The impact of EF decoction (EFD) on OP was evaluated using istopathological examination and biochemical assays. Targeted metabolomics was employed to identify key molecules and explore their molecular mechanisms. Alterations in the gut microbiota (GM) were evaluated by 16S rRNA gene sequencing. The role of the GM was clarified using an antibiotic cocktail and faecal microbiota transplantation. RESULTS: EFD significantly increased the weight (14.06%), femur length (4.34%), abdominal fat weight (61.14%), uterine weight (69.86%), and insulin-like growth factor 1 (IGF-1) levels (59.48%), while reducing serum type I collagen cross-linked carboxy-terminal peptide (CTX-I) levels (15.02%) in osteoporotic mice. The mechanism of action may involve the regulation of the NLRP3/cleaved caspase-1/IL-1ß signalling pathway in improving intestinal tight junction proteins and bone metabolism. Additionally, EFD modulated the abundance of related GM communities, such as Lactobacillus, Coriobacteriaceae, bacteria of family S24-7, Clostridiales, and Prevotella, and increased propionate and butyrate levels. Antibiotic-induced dysbiosis of gut bacteria disrupted OP regulation of bone metabolism, which was restored by the recovery of GM. CONCLUSIONS: Our study is the first to demonstrate that EFD works in an OP mouse model by utilising GM and butyric acid. Thus, EF shows promise as a potential remedy for OP in the future.