Expression Of NLRP3 Inflammasome Research Articles

Pneumocystis murina promotes inflammasome formation and NETosis during Pneumocystis pneumonia.

Pneumocystis jirovecii pneumonia (PjP) poses a serious risk to individuals with compromised immune systems, such as individuals with HIV/AIDS or undergoing immunosuppressive therapies for cancer or solid organ transplants. Severe PjP triggers excessive lung inflammation, resulting in lung function decline and consequential alveolar damage, potentially culminating in acute respiratory distress syndrome. Non-HIV patients face a 30%-60% mortality rate, emphasizing the need for a deeper understanding of inflammatory responses in PjP. Prior research emphasized macrophages in Pneumocystis infections, neglecting neutrophils' role in tissue damage. Consequently, the overemphasis on macrophages led to an incomplete understanding of the role of neutrophils and inflammatory responses. In the current investigation, our RNAseq studies on a murine surrogate model of PjP revealed heightened activation of the NLRP3 inflammasome and NETosis cell death pathways in their lungs. Immunofluorescence staining confirmed neutrophil extracellular trap (NET) presence in the lungs of the P. murina-infected mice, validating our findings. Moreover, isolated neutrophils exhibited NETosis when directly stimulated with P. murina. Isolated NETs compromised P. murina viability in vitro, highlighting the potential role of neutrophils in controlling fungal growth and promoting inflammation during P. murina pneumonia through NLRP3 inflammasome assembly and NETosis. These pathways, essential for inflammation and pathogen elimination, bear the risk of uncontrolled activation leading to excessive tissue damage and persistent inflammation. This pioneering study is the first to identify the formation of NETs and inflammasomes during Pneumocystis infection, paving the way for comprehensive investigations into treatments aimed at mitigating lung damage and augmenting survival rates for individuals with PjP.IMPORTANCEPneumocystis jirovecii pneumonia (PjP) affects individuals with weakened immunity, such as HIV/AIDS, cancer, and organ transplant patients. Severe PjP triggers lung inflammation, impairing function and potentially causing acute respiratory distress syndrome. Non-HIV individuals face a 30%-60% mortality rate, underscoring the need for deeper insight into PjP's inflammatory responses. Past research focused on macrophages in managing Pneumocystis infection and its inflammation, while the role of neutrophils was generally overlooked. In contrast, our findings in P. murina-infected mouse lungs showed neutrophil involvement during inflammation and increased expression of NLRP3 inflammasome and NETosis pathways. Detection of neutrophil extracellular traps further indicated their involvement in the inflammatory process. Although beneficial in combating infection, unregulated neutrophil activation poses a potential threat to lung tissues. Understanding the behavior of neutrophils in Pneumocystis infections is crucial for controlling detrimental reactions and formulating treatments to reduce lung damage, ultimately improving the survival rates of individuals with PjP.

MiR-483-5p-Containing exosomes treatment ameliorated deep vein thrombosis‑induced inflammatory response

Peripheral vascular condition, known as deep vein thrombosis (DVT), is a common ailment that may lead to deadly pulmonary embolism. Inflammation is closely connected to venous thrombosis, which results in blood stasis, leading to ischemia and hypoxia, as indicated by research. The objective of this research was to investigate the mechanism by which exosomes derived from adipose stem cells (ADSCs) prevent deep vein thrombosis.Our data showed that Exo-483 effectively reduced the thrombus weight in DVT rats by intravenous injection. Exo-483 decreased the expression of tissue factor (TF) protein, the influx of inflammatory cells into the thrombosed vein wall, and the levels of cytokines in the serum. Furthermore, Exo-483 suppressed the expression of Mitogen-activated protein kinase 1 (MAPK1) and decreased the expression of NLRP3 inflammasomes. In an oxygen-glucose deprivation (OGD) cell model, the tube-forming and migratory abilities of primary human umbilical vein endothelial cells (HUVEC) and EA.hy926 cells were suppressed by Exo-483 pretreatment.Exo-483 is also linked to regulating Dynamin-related protein 1 (DRP1) production downstream of MAPK1.By decreasing the mitochondrial localization and phosphorylation at the S616 site of DRP1, it diminishes the expression of NLRP3 inflammasomes. Moreover, according to Bioinformatics analysis, miR-483-5p was anticipated to target MAPK1.The research conducted by our team revealed that the miR-483-5p exosome derived from ADSCs exhibited anti-inflammatory properties through the modulation of downstream DRP1-NLRP3 expression by targeting MAPK1.The findings of this research propose that miR-483-5p may be regarded as an innovative treatment target for DVT.

IRGM/Irgm1 increases autophagy to inhibit activation of NLRP3 inflammasome in inflammatory injury induced acute liver failure

Immune-related GTPase M (IRGM) induces autophagy and suppresses inflammation, but its putative role and signaling mechanism remain undefined in the pathogenesis of liver failure. This study aimed to address how IRGM attenuates inflammatory injury by regulating autophagy in liver failure. In this study, a total of 10 patients with hepatitis B virus-related acute-on-chronic liver failure (HBV-ACLF) and 10 healthy controls were prospectively enrolled. Intrahepatic expression of IRGM/Irgm1, NLRP3 inflammasome (NLRP3, ASC, and caspase-1), autophagy-related proteins (LC3II, P62), and inflammatory cytokines (IL-1β, TNF-α) were measured. Autophagy was activated by rapamycin (4 mg/kg) in an acute liver failure (ALF) mouse model, which was used to further study the expression of Irgm1, NLRP3 inflammasome, autophagy-related proteins, and inflammatory cytokines using both qRT-PCR and Western blot analyses. Irgm1 expression was knocked down using Irgm1 short hairpin RNA (shRNA) in lipopolysaccharide (LPS)-induced AML12 cells to investigate the effects of Irgm1 deletion on autophagy and inflammation. We found that the expression of IRGM and autophagy-related proteins was significantly downregulated while the NLRP3 inflammasome was significantly upregulated in the livers of HBV-ACLF patients and the ALF mouse model (all P < 0.05). Rapamycin-induced autophagy ameliorated intrahepatic NLRP3 inflammasome activation and decreased inflammation and necrosis in the ALF mice. Irgm1 knockdown decreased autophagy and significantly upregulated NLRP3 inflammasome activation in AML12 cells (all P < 0.05). Rapamycin-induced autophagy also protected against hepatocyte injury following LPS stimulation in vitro by inhibiting NLRP3 inflammasome activation. Thus, IRGM/Irgm1 alleviates inflammation-mediated hepatocyte injury by regulating autophagy. This study provides new insight into potential molecular targets to treat liver failure.

TRPV4 modulation participates in paraoxon-induced brain injury via NMDA and NLRP3 regulation

ABSTRACT Background Organophosphorus pesticide poisoning can lead to severe brain damage, but the specific mechanisms involved are not fully understood. Our research aims to elucidate the function of the TRPV4 ion channel in the development of brain injury induced by paraoxon (POX). Methods In vivo, we examined the survival rate, behavioral seizures, histopathological alterations, NMDA receptor phosphorylation, as well as the expression of the NLRP3-ASC-caspase-1 complex and downstream inflammatory factors in the POX poisoning model following intervention with the TRPV4 antagonist GSK2193874. In vitro, we investigated the effects of GSK2193874 on NMDA-induced inward current, cell viability, cell death rate, and Ca2+ accumulation in primary hippocampal neurons. Results The treatment with the TRPV4 antagonist increased the survival rate, suppressed the status epilepticus, improved pathological damage, and reduced the phosphorylation level of NMDA receptors after POX exposure. Additionally, it inhibited the upregulation of NLRP3 inflammasome and inflammatory cytokines expression after POX exposure. Moreover, the TRPV4 antagonist corrected the NMDA-induced increase in inward current and cell death rate, decrease in cell viability, and Ca2+ accumulation. Conclusion TRPV4 participates in the mechanisms of brain injury induced by POX exposure through NMDA-mediated excitotoxicity and NLRP3-mediated inflammatory response.

Predicting the potential mechanism of radix chimonanthi pracecocis in treating osteoarthritis by network pharmacology analysis combined with experimental validation

Ethnopharmacological relevanceRadix Chimonanthi Pracecocis (RCP), also known as Tiekuaizi, widely used by the Miao community in Guizhou, exhibits diverse biological activities and holds promise for the treatment of osteoarthritis (OA). However, there is a lack of contemporary pharmacological research in this area. Aims of the studyThis study aims to explore the potential of targets and mechanisms of RCP in the treatment of OA. Materials and methodsThe chemical components of RCP were identified using UPLC-MS/MS, and active components were determined based on the Lipinski rule. RCP and OA-related targets were retrieved from public databases such as TCMSP and GeneCards. Network pharmacology approaches were employed to identify key genes. The limma package (version 3.40.2) in R 4.3.2 was used to screen for differentially expressed genes (DEGs) between OA and healthy individuals in GSE82107. DEGs were analyzed using an independent sample t-test and receiver operating characteristic analysis in GraphPad Prism 9.5.1. Additionally, molecular docking (SYBYL2.1.1) was used to analyze the binding interactions between the active components and target proteins. Finally, we established a papain-induced osteoarthritis (OA) rat model and treated it with RCP aqueous extract by gavage. We validated relevant indicators using real-time fluorescence quantitative polymerase chain reaction, Western blot, immunohistochemistry, and enzyme-linked immunosorbent assays. ResultsSeven active components and 53 targets were identified. The results of GO and KEGG enrichment analyses confirmed the significant role of RCP in the regulation of pyroptosis. Hypoxia-inducible factor-1α (HIF-1α) was identified as a key gene involved in the main biological functions. Molecular docking analysis revealed that Praecoxin, Isofraxidin, Esculin, and Naringenin can bind to the nucleotide-binding domain, leucine-rich repeat, and pyrin domain-containing protein 3 (NLRP3) (T-Score >5). Additionally, Praecoxin can bind to HIF-1α (T-Score >5). In vivo experiments demonstrated that RCP significantly affects the NLRP3 inflammasome, which is regulated by the HIF-1α pathway. RCP inhibited pyroptosis and reduced synovial inflammation. ConclusionsThis study confirmed the efficacy of RCP aqueous extract in the treatment of OA and identified seven active components (esculin, dihydrokaempferol, naringenin, praecoxin, carnosol, hydroxyvalerenic acid, isofraxidin) that may play an anti-pyroptosis role in the treatment of OA by downregulating the expression of HIF-1α and NLRP3 inflammasome.

Sulforaphane decreases oxidative stress and inhibits NLRP3 inflammasome activation in a mouse model of ulcerative colitis

Excessive oxidative stress and NLRP3 inflammasome activation are considered the main drivers of inflammatory bowel disease (IBD), and inhibition of inflammasomes ameliorates clinical symptoms and morphological manifestations of IBD. Herein, we examined the roles of NLRP3 activation in IBD and modulation of NLRP3 by sulforaphane (SFN), a compound with multiple pharmacological activities that is extracted from cruciferous plants. To simulate human IBD, we established a mouse colitis model by administering dextran sodium sulfate in the drinking water. SFN (25, 50 mg·kg−1·d−1, ig) or the positive control sulfasalazine (500 mg/kg, ig) was administered to colitis-affected mice for 7 days. Model mice displayed pathological alterations in colon tissue as well as classic symptoms of colitis beyond substantial tissue inflammation. Expression of NLRP3, ASC, and caspase-1 was significantly elevated in the colonic epithelium. The expression of NLRP3 inflammasomes led to activation of downstream proteins and increases in the cytokines IL-18 and IL-1β. SFN administration either fully or partially reversed these changes, thus restoring IL-18 and IL-1β, substantially inhibiting NLRP3 activation, and decreasing inflammation. SFN alleviated the inflammation induced by LPS and NLRP3 agonists in RAW264.7 cells by decreasing the levels of reactive oxygen species. In summary, our results revealed the pathological roles of oxidative stress and NLRP3 in colitis, and indicated that SFN might serve as a natural NLRP3 inhibitor, thereby providing a new strategy for alternative colitis treatment.

Antigen Presenting Cell activator protein 1 (AP-1) complex contributes to Salt-sensitive Blood Pressure

Salt-sensitivity of blood pressure (SSBP) affects 50% of the hypertensive and 25% of the normotensive population and is a major independent risk factor for cardiovascular morbidity and mortality. We previously found that SSBP is associated with activation of the NLRP3 inflammasome in antigen presenting cells (APCs) via increased oxidated stress and isolevuglandin (IsoLG) formation, but the underlying mechanisms are unknown. The activator protein 1 (AP-1) (FOS/JUN) has been implicated in activation of the NLRP3 inflammasome but its role in SSBP is not known. We hypothesized that AP-1 transcription factor in APCs senses elevated sodium and contributes to SSBP. We further hypothesize that antioxidant flavonoid, diosmetin prevents AP-1 activation and SSBP. Using bulk RNA-sequencing in human monocytes, we found elevated sodium increases expression of the AP-1 gene family when compared to normal sodium concentration, including cFOS (2378.18 ± 480.7 vs 6494.09 ± 945.55, p= 0.0009), FOSB (53.63 ± 17. 55 vs 131.06 ± 10.30, p= 0.397), cJUN (7313.90 ± 984.93 vs 11370.09 ± 1286.35, p= 0.2563) JUNB (4218.4.36 ± 199.17 vs 570.45 ± 200.91, p= 0.0445) and JUND (3309. 63 ± 270.64 vs 8057.90 ± 1043.05, p= 0.00006). In additional experiments, we enrolled people with hypertension and phenotype them for SSBP using an established in-patient protocol of salt-loading/depletion and performed single-cell transcriptomic analyses in vivo on peripheral blood mononuclear cells (PBMCs). We found that expression of the FOSB and JUNB genes are more sensitive in concert with blood pressure in salt-sensitive (SS) but not salt-resistant (SR) humans. Moreover, diosmetin attenuated high salt-induced hypertension, 123.5 ± 4.1 vs 146.8 ± 2.5 mm Hg in controls, **p&lt; 0.01). In addition, diosmetin also reduced high salt-induced inflammation including APC production of IsoLG-protein adducts, IL-1β, and NLRP3 inflammasome expression. These findings reveal a role for immune cell AP-1 (FOS/JUN) signaling in salt-sensitive hypertension and diosmetin may provide a potential therapeutic target for treatment of SSBP. 1R01HL144941-01A1. 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.

The SK4 channel allosteric blocker, BA6b9, reduces atrial fibrillation substrate in rats with reduced ejection fraction.

Atrial fibrillation (AF), the most common cardiac arrhythmia, is strongly associated with several comorbidities including heart failure (HF). AF in general, and specifically in the context of HF, is progressive in nature and associated with poor clinical outcomes. Current therapies for AF are limited in number and efficacy and do not target the underlying causes of atrial remodeling such as inflammation or fibrosis. We previously identified the calcium-activated SK4 K+ channels, which are preferentially expressed in the atria relative to the ventricles in both rat and human hearts, as attractive druggable target for AF treatment. Here, we examined the ability of BA6b9, a novel allosteric inhibitor of SK4 channels that targets the specific calmodulin-PIP2 binding domain, to alter AF susceptibility and atrial remodeling in a systolic HF rat postmyocardial infarction (post-MI) model. Daily BA6b9 injection (20 mg/kg/day) for 3 weeks starting 1-week post-MI prolonged the atrial effective refractory period, reduced AF induction and duration, and dramatically prevented atrial structural remodeling. In the post-MI left atrium (LA), pronounced upregulation of the SK4 K+ channel was observed, with corresponding increases in collagen deposition, α-SMA levels, and NLRP3 inflammasome expression. Strikingly, BA6b9 treatment reversed these changes while also significantly reducing the lateralization of the atrial connexin Cx43 in the LA of post-MI rats. Our findings indicate that the blockade of SK4 K+ channels using BA6b9 not only favors rhythm control but also remarkably reduces atrial structural remodeling, a property that is highly desirable for novel AF therapies, particularly in patients with comorbid HF.

Metformin inhibits activation of NLRP3 inflammasome and inflammatory response in preeclamptic rats

Backgroud: It is widely acknowledged that Metformin (MET), an established medication for managing type 2 diabetes, possesses diverse pharmacological effects. This study aims to investigate the protective effects of MET against Nω-Nitro-L-arginine methyl ester (L-NAME)-induced preeclampsia (PE). MethodsSprague Dawley (SD) rats were exposed to 200 mg/kg L-NAME with or without prior MET treatment. Histopathological analysis was performed using Hematoxylin and Eosin staining. Serum levels of inflammatory, antiangiogenic, and angiogenic factors were quantified using ELISA kits. Immunohistochemistry (IHC) staining was employed to observe NLRP3 and IL-1β expressions in placental tissues. Western blot and Quantitative Real-Time PCR (q-PCR) analyses were conducted to assess protein and mRNA expressions of NLRP3, caspase-1, ASC, and IL-1β. ResultsWe found that MET could mitigate placental histopathological deterioration and improve pregnancy outcomes in L-NAME-induced PE rat models. MET not only suppressed L-NAME-induced elevation of antiangiogenic factors but also stimulated the production of pro-angiogenic factors. Additionally, MET treatment reversed the excessive inflammatory response induced by L-NAME. Furthermore, MET inhibited the activation of the NLRP3 inflammasome triggered by L-NAME, evidenced by the downregulation of NLRP3 expression, caspase-1, and IL-1β. ConclusionsMET demonstrates a protective effect against L-NAME-induced PE rats, potentially mediated through inhibition of the inflammatory response, downregulation of NLRP3 inflammasome expression in the placenta, and regulation of the balance between anti-angiogenic and pro-angiogenic factors.

Atractylodin ameliorates lipopolysaccharide-induced depressive-like behaviors in mice through reducing neuroinflammation and neuronal damage

Depression is a psychiatric disorder associated with multiple factors including microglia-mediated neuroinflammation. Although atractylodin exerted a variety of biological activities, however the effect of atractylodin on neuroinflammation-related depression was still unclear. In this study, the lipopolysaccharide (LPS)-induced mouse model was used to explore the antidepressant effects and molecular mechanisms of atractylodin. The results showed that atractylodin increased sugar preference, also reduced immobility time in FST and TST. Further study showed atractylodin reduced the oxidative stress and the activation of microglia in mouse hippocampus, also inhibited the level of cytokine release, especially IL-1β. The results of western blotting showed that atractylodin significantly inhibited the expression of NLRP3 and pro-IL1β via inhibition of NF-κB pathway. Our studies showed that atractylodin upregulated BDNF/Akt pathway in mouse hippocampus. Therefore, this study firstly indicated that atractylodin can ameliorate lipopolysaccharide-induced depressive-like behaviors in mice through reducing neuroinflammation and neuronal damage, and its molecular mechanism may be associated with the decrease of the expression of NLRP3 inflammasome and upregulation of BDNF/Akt pathway.

Garlic oil supplementation blocks inflammatory pyroptosis-related acute lung injury by suppressing the NF-κB/NLRP3 signaling pathway via H2S generation.

Acute lung injury (ALI) is a major cause of acute respiratory failure with a high morbidity and mortality rate, and effective therapeutic strategies for ALI remain limited. Inflammatory response is considered crucial for the pathogenesis of ALI. Garlic, a globally used cooking spice, reportedly exhibits excellent anti-inflammatory bioactivity. However, protective effects of garlic against ALI have never been reported. This study aimed to investigate the protective effects of garlic oil (GO) supplementation on lipopolysaccharide (LPS)-induced ALI models. Hematoxylin and eosin staining, pathology scores, lung myeloperoxidase (MPO) activity measurement, lung wet/dry (W/D) ratio detection, and bronchoalveolar lavage fluid (BALF) analysis were performed to investigate ALI histopathology. Real-time polymerase chain reaction, western blotting, and enzyme-linked immunosorbent assay were conducted to evaluate the expression levels of inflammatory factors, nuclear factor-κB (NF-κB), NLRP3, pyroptosis-related proteins, and H2S-producing enzymes. GO attenuated LPS-induced pulmonary pathological changes, lung W/D ratio, MPO activity, and inflammatory cytokines in the lungs and BALF. Additionally, GO suppressed LPS-induced NF-κB activation, NLRP3 inflammasome expression, and inflammatory-related pyroptosis. Mechanistically, GO promoted increased H2S production in lung tissues by enhancing the conversion of GO-rich polysulfide compounds or by increasing the expression of H2S-producing enzymes in vivo. Inhibition of endogenous or exogenous H2S production reversed the protective effects of GO on ALI and eliminated the inhibitory effects of GO on NF-κB, NLRP3, and pyroptotic signaling pathways. Overall, these findings indicate that GO has a critical anti-inflammatory effect and protects against LPS-induced ALI by suppressing the NF-κB/NLRP3 signaling pathway via H2S generation.

Effects of electroacupuncture with "intestinal disease prescription" on NLRP3 inflammasome and intestinal mucosal barrier in rats with acute ulcerative colitis.

To observe the effects of electroacupuncture (EA) with "intestinal disease prescription" on the intestinal mucosal barrier and NLRP3 inflammasome in rats with dextran sulfate sodium (DSS)-induced acute ulcerative colitis (UC), and explore the underlying mechanism of EA with "intestinal disease prescription" for the treatment of UC. Thirty-two healthy male SPF-grade SD rats were randomly divided into a blank group, a model group, a medication group, and an EA group, with 8 rats in each group. Except for the blank group, the UC model was established by administering 5% DSS solution for 7 days. After modeling, the rats in the medication group were treated with mesalazine suspension (200 mg/kg) by gavage, while the rats in the EA group were treated with acupuncture at bilateral "Tianshu" (ST 25), "Shangjuxu" (ST 37) and "Zhongwan" (CV 12), with the ipsilateral "Tianshu" (ST 25) and "Shangjuxu" (ST 37) connected to the electrodes of the EA instrument, using disperse-dense wave, with a frequency of 10 Hz/50 Hz, and each intervention lasted for 20 minutes. Both interventions were performed once daily for 3 days. The general conditions of rats were observed daily. After intervention, the disease activity index (DAI) score was calculated; colon tissue morphology was observed using HE staining; serum levels of pro-inflammatory cytokines (interleukin [IL]-18, IL-1β) were measured by ELISA; protein expression of NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), and Caspase-1 in colon tissues was detected by Western blot; positive expression of zonula occludens-1 (ZO-1) and Occludin in colon tissues was examined by immunofluorescence. Compared with the blank group, the rats in the model group exhibited poor general conditions, slow body weight gain, shortened colon length (P<0.01), increased DAI score and spleen index (P<0.01), elevated serum IL-18 and IL-1β levels, and increased protein expression of NLRP3, ASC, and Caspase-1 in colon tissues (P<0.01), along with decreased positive expression of ZO-1 and Occludin in colon tissues (P<0.01). Compared with the model group, the rats in the medication group and the EA group exhibited improved general conditions, accelerated body weight gain, increased colon length (P<0.05), reduced DAI scores and spleen indexes (P<0.05), decreased serum IL-18 and IL-1β levels, and lower protein expression of NLRP3, ASC and Caspase-1 in colon tissues (P<0.05), as well as increased positive expression of ZO-1 and Occludin in colon tissues (P<0.05). There were no significant differences in the above indexes between the medication group and the EA group (P>0.05). Compared with the blank group, the rats in the model group exhibited disrupted colon mucosal morphology, disordered gland arrangement, and atrophy of crypts, along with significant inflammatory cell infiltration. Compared with the model group, the rats in both the medication group and the EA group showed relatively intact colon mucosal morphology, with restored and improved gland and crypt structures, and reduced inflammatory cell infiltration. EA with "intestinal disease prescription" has a significant therapeutic effect on DSS-induced UC, possibly by regulating the expression of NLRP3 inflammasome and proteins related to the intestinal mucosal barrier, thereby alleviating symptoms of ulcerative colitis.

Cardioprotective Potential of Cymbopogon citratus Essential Oil against Isoproterenol-induced Cardiomyocyte Hypertrophy: Possible Involvement of NLRP3 Inflammasome and Oxidative Phosphorylation Complex Subunits.

Cymbopogon citratus (DC.) Stapf is a medicinal and edible herb that is widely used for the treatment of gastric, nervous and hypertensive disorders. In this study, we investigated the cardioprotective effects and mechanisms of the essential oil, the main active ingredient of Cymbopogon citratus, on isoproterenol (ISO)-induced cardiomyocyte hypertrophy. The compositions of Cymbopogon citratus essential oil (CCEO) were determined by gas chromatography-mass spectrometry. Cardiomyocytes were pretreated with 16.9 µg/L CCEO for 1 h followed by 10 µmol/L ISO for 24 h. Cardiac hypertrophy-related indicators and NLRP3 inflammasome expression were evaluated. Subsequently, transcriptome sequencing (RNA-seq) and target verification were used to further explore the underlying mechanism. Our results showed that the CCEO mainly included citronellal (45.66%), geraniol (23.32%), and citronellol (10.37%). CCEO inhibited ISO-induced increases in cell surface area and protein content, as well as the upregulation of fetal gene expression. Moreover, CCEO inhibited ISO-induced NLRP3 inflammasome expression, as evidenced by decreased lactate dehydrogenase content and downregulated mRNA levels of NLRP3, ASC, CASP1, GSDMD, and IL-1β, as well as reduced protein levels of NLRP3, ASC, pro-caspase-1, caspase-1 (p20), GSDMD-FL, GSDMD-N, and pro-IL-1β. The RNA-seq results showed that CCEO inhibited the increase in the mRNA levels of 26 oxidative phosphorylation complex subunits in ISO-treated cardiomyocytes. Our further experiments confirmed that CCEO suppressed ISO-induced upregulation of mt-Nd1, Sdhd, mt-Cytb, Uqcrq, and mt-Atp6 but had no obvious effects on mt-Col expression. CCEO inhibits ISO-induced cardiomyocyte hypertrophy through the suppression of NLRP3 inflammasome expression and the regulation of several oxidative phosphorylation complex subunits.

Metformin Inhibits NLRP3 Inflammasome Expression and Regulates Inflammatory Microenvironment to Delay the Progression of Colorectal Cancer.

Colorectal cancer is a common malignant tumor, with about one million people diagnosed with it worldwide each year. Recent studies have found that metformin can inhibit the production of inflammatory factors and regulate the polarization of immune cells. However, whether metformin can regulate the inflammatory microenvironment and delay the progression of colorectal cancer by inhibiting the inflammatory response has not been deeply studied yet. This study aimed to explore the molecular mechanism by which metformin inhibits the expression of NLRP3 inflammasome, regulates the inflammatory microenvironment, and delays the progression of colorectal cancer through in vitro cell experiments. In this research, NLRP3 was knocked down in human colorectal cancer cells, and metformin was added to them. Cell proliferation ability was detected by CCK8, and cell migration and invasion abilities were assessed by Transwell assay. The apoptosis rate was determined by flow cytometry. In addition, the expression of NLRP3 inflammatory vesicles and inflammatory factors in each group of cells was studied by qRT-PCR and Western blotting. Finally, clinical colorectal cancer samples were analyzed by immunohistochemistry. The results of the study showed that NLRP3 expression was significantly increased in colorectal cancer cell lines and human colorectal cancer tissues. Knockdown of NLRP3 significantly inhibited tumor cell proliferation, migration, and invasion. In addition, the proliferation, migration and invasion of tumor cells were also significantly reduced by the addition of metformin intervention. Furthermore, qRT-PCR and WB results demonstrated that the expression of IL-1β, IL-6, TNF- α, TGF-β, and IL-10 was down-regulated in LS1034 tumor cells after NLRP3 knockdown. In addition, metformin intervention also resulted in different degrees of downregulation of NLRP3 and inflammatory factor expression (p <0.05). Notably, the reduction in inflammatory factors was more pronounced after the combination of NLRP3 knockdown and metformin intervention. Metformin can inhibit the expression of NLRP3 inflammasome, thereby suppressing the expression of inflammation-related factors, reducing the damage of the inflammatory microenvironment to normal cells, and delaying the progression of colorectal cancer.

Holothuria leucospilota polysaccharides (HLP) ameliorate colitis rats via regulation of the metabolic profiling and TLR4/NLRP3 signaling pathways

AbstractRecently, the development of natural polysaccharides for ameliorating immunity and gut metabolism has attracted extensive attention. This study used Holothuria leucospilota polysaccharides (HLP) to explore the improvement mechanism in ulcerative colitis rats on perspectives of immunity and metabolism. The results showed that HLP increased goblet cells’ number and the content of tight junction proteins (zona occludens 1 and occludin) and improved intestinal barrier permeability. The levels of immune cytokines (IL‐4, IL‐6, IL‐10, IL‐18, TNF‐α, and IL‐1β) and the activity of oxidative stress‐related enzymes (superoxide dismutase, catalase, malondialdehyde, and glutathione peroxidase) were regulated. HLP regulated the related genes and proteins expression of immune cytokines, MAPK, and NLRP3 inflammasome. Furthermore, HLP treatment increased the concentration of short‐chain fatty acids (SCFAs) and regulated serum metabolic disorders by regulating amino acid metabolism, SCFA metabolism, and energy metabolism. These results provide a new perspective for developing HLP as a promising functional food for preventing and mitigating colitis.

Long-Term Alcohol Exposure Aggravates Ischemic Stroke-Induced Damage by Promoting Pericyte NLRP3 Inflammasome Activation via Pre-Activating the TLR4/NF-κB Pathway in Rats.

Ischemic stroke (IS) is one of the leading causes of death and disability in the world, and alcohol consumption has been gaining attention as an independent risk factor for IS. Blood-brain barrier (BBB) dysfunction and neuroinflammation are the core of cerebral ischemia/reperfusion (I/R) injury, and pericytes play a crucial role in the structure and function. This study is to explore the effects of long-term alcohol consumption on IS and the potential mechanisms of pericytes. Rat models of long-term alcohol intake followed by transient middle cerebral artery occlusion stroke (EtOH+tMCAO) and cell models of oxygen-glucose deprivation/reoxygenation (OGD/R) with alcohol pre-treatment were constructed. Worsened infarct volume, neurological scores, and BBB disruption were observed in the EtOH+tMCAO group compared with the tMCAO group, and immunofluorescence staining showed increased pericytes NLPR3 inflammasome activation at the ischemic penumbra. In vitro, pericyte mortality and LDH release elevated pre-treated by alcohol after OGD/R, and amplified expression of NLRP3 inflammasome was detected by Western blotting and qPCR. Alcohol pre-treatment activated the TLR4/NF-κB pathway, and transfecting pericytes with TLR4-small interfering RNA (siRNA) to block TLR4 signaling markedly restrained NLRP3 inflammasome over-activation. Injecting TAK-242 in rats alleviated neurological impairment caused by alcohol. Long-term alcohol pre-treatment aggravated ischemic stroke-induced brain damage by activating NLRP3 inflammasome via TLR4/NF-κB signaling pathway in the pericytes.

Coumarin and eugenol ameliorate LPS-induced inflammation in RAW 264.7 cells via modulating the NLRP3 inflammasome pathway

Objective: To investigate the underlying mechanism of anti-inflammatory action of coumarin and eugenol in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. Methods: RAW 264.7 cells were treated with 2.5 μg/mL of LPS, 50 μM of coumarin, and 50 μM eugenol for 24 h. The viability of the cells was assessed using MTT assay. The production of nitric oxide was determined using Griess reagent and DCFH-DA was used to measure the production of reactive oxygen species. The protein expression of NLRP3, IL-1β, NF-κB, and cyclooxygenase 2 was assessed using Western blot analysis. Results: Coumarin and eugenol showed anti-inflammatory effects against LPS-induced inflammatory response by ameliorating the expression of NLRP3 inflammasome and NF-κB, which further led to a subsequent reduction in IL-1β, nitric oxide, and reactive oxygen species. Conclusions: Coumarin and eugenol exert their anti-inflammatory activities by modulating the NLRP3 inflammasome pathway and NF-κB. These compounds may have promising therapeutic applications for the treatment of various inflammatory diseases.

Febuxostat attenuates secondary brain injury caused by cerebral hemorrhage through inhibiting inflammatory pathways.

Neuroinflammation is considered an important step in the progression of secondary brain injury (SBI) induced by cerebral hemorrhage (ICH). The nucleotide-binding and oligomerization structural domain-like receptor family of pyridine structural domain-containing 3 (NLRP3) inflammasomes play an important role in the immune pathophysiology of SBI. Febuxostat (Feb) is a xanthine oxidase inhibitor that is approved for the treatment of gout and has been found to have potent anti-inflammatory effects. However, it has been less studied after ICH and we aimed to explore its protective role in ICH. We established an autologous blood-brain hemorrhage model in C57BL/6 mice. Functions of co-expressed genes were analyzed by trend analysis and bioinformatics analysis. Enzyme-linked immunosorbent assay were used to assess the inflammatory factor levels. Fluoro-Jade B histochemistry and TUNEL staining were used to detect neuronal apoptosis. Immunofluorescence staining and western blotting were used to detect the expression of NLRP3 inflammasomes. Pretreatment with Feb reduced neuronal cell death and degeneration and alleviated neurobehavioral disorders in vivo. Feb was found to modulate inflammation-related pathways by trend analysis and bioinformatics analysis. In addition, Feb inhibited microglia activation and elevated cytokine levels after ICH. Furthermore, double immunofluorescence staining showed that co-localization of NLRP3 with Iba1 positive cells was reduced after treatment with Feb. Finally, we found that Feb inhibited the activation of the NLRP3/ASC/caspase-1 pathway after ICH. By inhibiting the NLRP3 inflammasome, preconditioning Feb attenuates inflammatory injury after ICH. Our findings may provide new insights into the role of Feb in neuroprotection.

NLRP3-mediated autophagy dysfunction links gut microbiota dysbiosis to tau pathology in chronic sleep deprivation.

Emerging evidence indicates that sleep deprivation (SD) can lead to Alzheimer's disease (AD)-related pathological changes and cognitive decline. However, the underlying mechanisms remain obscure. In the present study, we identified the existence of a microbiota-gut-brain axis in cognitive deficits resulting from chronic SD and revealed a potential pathway by which gut microbiota affects cognitive functioning in chronic SD. Our findings demonstrated that chronic SD in mice not only led to cognitive decline but also induced gut microbiota dysbiosis, elevated NLRP3 inflammasome expression, GSK-3β activation, autophagy dysfunction, and tau hyperphosphorylation in the hippocampus. Colonization with the "SD microbiota" replicated the pathological and behavioral abnormalities observed in chronic sleep-deprived mice. Remarkably, both the deletion of NLRP3 in NLRP3 -/- mice and specific knockdown of NLRP3 in the hippocampus restored autophagic flux, suppressed tau hyperphosphorylation, and ameliorated cognitive deficits induced by chronic SD, while GSK-3β activity was not regulated by the NLRP3 inflammasome in chronic SD. Notably, deletion of NLRP3 reversed NLRP3 inflammasome activation, autophagy deficits, and tau hyperphosphorylation induced by GSK-3β activation in primary hippocampal neurons, suggesting that GSK-3β, as a regulator of NLRP3-mediated autophagy dysfunction, plays a significant role in promoting tau hyperphosphorylation. Thus, gut microbiota dysbiosis was identified as a contributor to chronic SD-induced tau pathology via NLRP3-mediated autophagy dysfunction, ultimately leading to cognitive deficits. Overall, these findings highlight GSK-3β as a regulator of NLRP3-mediated autophagy dysfunction, playing a critical role in promoting tau hyperphosphorylation.

Total glucosides of paeony alleviates experimental Sjögren's syndrome through inhibiting NLRP3 inflammasome activation of submandibular gland cells.

The mechanisms by which total glucosides of paeony (TGP) mitigates Sjögren's syndrome (SS) remains elusive. In the present study, we aim to explore the relationship between the therapeutic effects of TGP in the treatment of SS and NLRP3 inflammasome activation in submandibular gland (SG) cells. Female non-obese diabetic (NOD) mice were selected as the model of SS. The mice were divided into PBS and TGP treatment group. For treatment, TGP (400mg·kg-1) was administered intragastrically every day for 4 weeks. The SS-like symptoms and pathological changes of the SG of mice were compared between the PBS and TGP group. The activation of NLRP3 inflammasome in SG was detected by RT-qPCR, immunohistochemistry and western blot. The SG cells stimulated by lipopolysaccharide (LPS) and adenosine triphosphate (ATP) for activation of NLRP3 inflammasome were treated with or without TGP. Then, NLRP3 inflammasome activation was assessed. The IL-1β and IL-18 in homogenate of SG, serum and supernatant were detected by ELISA. Compared with balb/c mice, NOD mice showed SS-like symptoms and lymphocyte infiltration in SG, and the expression of NLRP3 inflammasome in SG was significantly increased. The SS-like symptoms were alleviated, and lymphocyte infiltration in SG was reduced, and the level of NLRP3 inflammasome in SG mice was decreased after TGP treatment. TGP also significantly inhibit the activation of NLRP3 inflammasome of SG cells in vitro. Collectively, our results indicated that TGP alleviates SS through inhibition of the activation of NLRP3 inflammasome of SG. These findings clarified the mechanism underlying the therapeutic effects of TGP on SS, and provided new evidence for the further application of TGP in the treatment of SS.