Eclipta prostrata improves alveolar development of bronchopulmonary dysplasia via suppressing the NLRP3 inflammasome in a DLD-dependent manner.

OBJECTIVES: Bronchopulmonary dysplasia (BPD), the most common late morbidity in preterm infants, is characterized by impaired alveolar development caused by persistent lung inflammation. Studies have shown that NOD-, LRR- and pyrin domain-containing 3 (NLRP3) inflammasome-mediated inflammation is critically involved in the development of BPD. As a traditional Chinese medicinal herb, Eclipta prostrata (EAP) exhibits potent anti-inflammatory properties. Our study aims to investigate whether EAP could improve the lung development of BPD by suppressing the lung inflammatory response. METHODS: The BPD rat model was established by intra-amniotic injection of lipopolysaccharide (LPS) and postnatal exposure to hyperoxia. Changes in the NLRP3 inflammasome and pyroptosis were assessed by treatment with EAP. The effect of EAP on the NLRP3 inflammasome was tested in vitro using the THP-1 cell line and primary alveolar macrophages. Proteomics analysis was used to elucidate the mechanism of action of EAP. RESULTS: Histopathological and immunofluorescence results of lung tissues revealed that LPS and hyperoxia induced lung injury and triggered NLRP3 inflammasome activation and pyroptosis in alveolar macrophages. EAP ameliorated BPD lung injury, inhibited NLRP3 inflammasome activation and reduced gasdermin D (GSDMD) expression in alveolar macrophages. EAP downregulated the expression of NLRP3 inflammasome pathway molecules (NLRP3, caspase-1, and IL-1ß) and GSDMD in LPS-stimulated THP-1 macrophages and primary alveolar macrophages. In addition, proteomics analysis identified that dihydrolipoamide dehydrogenase (DLD) interacted with EAP. Inhibition of DLD activity abolished the protective effects of EAP. CONCLUSIONS: Our study suggested that EAP could attenuate arrest of alveolar development via inhibiting NLRP3 inflammasome in a DLD-dependent way, and could be a potential therapeutic method for BPD.

Novel genetic variants in the NLRP3 inflammasome-related PANX1 and APP genes predict survival of patients with hepatitis B virus-related hepatocellular carcinoma.

BACKGROUND: The nod-like receptor protein 3 (NLRP3) is one of the most characterized inflammasomes involved in the pathogenesis of several cancers, including hepatocellular carcinoma (HCC). However, the effects of genetic variants in the NLRP3 inflammasome-related genes on survival of hepatitis B virus (HBV)-related HCC patients are unclear. METHODS: We performed multivariable Cox proportional hazards regression analysis to evaluate associations between 299 single-nucleotide polymorphisms (SNPs) in 16 NLRP3 inflammasome-related genes and overall survival (OS) of 866 patients with HBV-related HCC. We further performed expression quantitative trait loci (eQTL) analysis using the data from the GTEx project and 1000 Genomes projects, and performed differential expression analysis using the TCGA dataset to explore possible molecular mechanisms underlying the observed associations. RESULTS: We found that two functional SNPs (PANX1 rs3020013 A > G and APP rs9976425 C > T) were significantly associated with HBV-related HCC OS with the adjusted hazard ratio (HR) of 0.83 [95% confidence interval (CI) = 0.73-0.95, P = 0.008], and 1.26 (95% CI = 1.02-1.55, P = 0.033), respectively. Moreover, the eQTL analysis revealed that the rs3020013 G allele was correlated with decreased mRNA expression levels of PANX1 in both normal liver tissues (P = 0.044) and whole blood (P < 0.001) in the GTEx dataset, and PANX1 mRNA expression levels were significantly higher in HCC samples and associated with a poorer survival of HCC patients. However, we did not observe such correlations for APP rs9976425. CONCLUSIONS: These results indicated that SNPs in the NLRP3 inflammasome-related genes may serve as potential biomarkers for HBV-related HCC survival, once replicated by additional larger studies.

An integrated network pharmacology and molecular docking approach to reveal the role of Arctigenin against Cutibacterium acnes-induced skin inflammation by targeting the CYP19A1.

Acne caused by inflammation of hair follicles and sebaceous glands is a common chronic skin disease. Arctigenin (ATG) is an extract of Arctium lappa L., which has significant anti-inflammatory effects. However, the effect and mechanism of ATG in cutaneous inflammation mediated by Cutibacterium acnes (C. acnes) has not been fully evaluated. The purpose of this study was to explore the effect and potential mechanism of ATG in the treatment of acne through network pharmacology and experimental confirmation. An acne model was established by injected live C. acnes into living mice and treated with ATG. Our data showed that ATG effectively improved acne induced by live C. acnes, which was confirmed by determining ear swelling rate, estradiol concentration and hematoxylin and eosin (H&E) staining. In addition, ATG inhibited the NLRP3 inflammasome signaling pathway in mice ear tissues and reduced the secretion of pro-inflammatory cytokines IL-1ß to relieve inflammation. The results of network pharmacology and molecular docking confirmed that ATG can regulate 17ß-Estradiol (E2) levels through targeted to CYP19A1, and finally inhibited skin inflammation. Taken together, our results confirmed that ATG regulated E2 secretion by targeting CYP19A1, thereby inhibiting the NLRP3 inflammasome signaling pathway and improving inflammation levels in acne mice. This study provides a basis for the feasibility of ATG in treating acne in clinical practice.

GDF11 protects against mitochondrial-dysfunction-dependent NLRP3 inflammasome activation to attenuate osteoarthritis.

INTRODUCTION: Osteoarthritis (OA) is a highly prevalent degenerative disease worldwide, and tumor necrosis factor (TNF-α) is closely associated with its development. Growth differentiation factor 11 (GDF11) has demonstrated anti-injury and anti-aging abilities in certain tissues; however, its regulatory role in OA remains unclear and requires further investigation. OBJECTIVES: To identify whether GDF11 can attenuate osteoarthritis. To exploring the the potential mechanism of GDF11 in alleviating osteoarthritis. METHODS: In this study, we cultured and stimulated mouse primary chondrocytes with or without TNF-α, analyzing the resulting damage phenotype through microarray analysis. Additionally, we employed GDF11 conditional knockout mice OA model to examine the relationship between GDF11 and OA. To investigate the target of GDF11's function, we utilized NLRP3 knockout mice and its inhibitor to verify the potential involvement of the NLRP3 inflammasome. RESULTS: Our in vitro experiments demonstrated that endogenous overexpression of GDF11 significantly inhibited TNF-α-induced cartilage matrix degradation and inflammatory expression in chondrocytes. Furthermore, loss of GDF11 led to NLRP3 inflammasome activation, inflammation, and metabolic dysfunction. In an in vivo surgically induced mouse model, intraarticular administration of recombinant human GDF11 alleviated OA pathogenesis, whereas GDF11 conditional knockout reversed this effect. Additionally, findings from the NLRP3-knockout DMM mouse model revealed that GDF11 exerted its protective effect by inhibiting NLRP3. CONCLUSION: These findings demonstrate the ability of GDF11 to suppress TNF-α-induced inflammation and cartilage degeneration by preventing mitochondrial dysfunction and inhibiting NLRP3 inflammasome activation, suggesting its potential as a promising therapeutic drug for osteoarthritis.

Graphene oxide-decorated microporous sulfonated polyetheretherketone for guiding osteoporotic bone regeneration.

Recent studies have indicated that the nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome is an ideal therapeutic target for osteoporosis because it affects the differentiation of osteoblasts and osteoclasts. RNA sequencing utilizing multifunctional graphene oxide (GO) nanosheets revealed a correlation between GO nanomaterials and the NLRP3 inflammasome, as well as osteogenic genes in macrophages. This study aimed to construct a bone microenvironment-responsive multifunctional two-dimensional GO coating on the surface of microporous sulfonated polyetheretherketone (SPEEK) via polydopamine modification (SPEEK@PDA-GO). In vitro analysis showed that the SPEEK@PDA-GO implants weakened the STAT3-mediated NLRP3/caspase-1/IL-1ß signaling pathway in macrophages and subsequently prevented the formation of an extracellular inflammatory microenvironment, which is crucial for osteoclastogenesis. SPEEK@PDA-GO displayed significantly higher expression of M2 macrophage markers and osteogenic genes, indicating that the multifunctional GO nanosheets could facilitate bone regeneration via their immunomodulatory properties. The ability of SPEEK@PDA-GO to stimulate new bone formation and block bone loss caused by estrogen loss due to ovariectomy was also analyzed. The findings of this study offer valuable information on the possible involvement of the NLRP3 inflammasome in the interaction between the immune system and bone health in patients with osteoporosis.

Constituents from leaves of Macaranga hemsleyana.

Objective: To study constituents of the leaves of Macaranga hemsleyana, and evaluate their inhibitory effects against NOD-like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome activation, and antiproliferative activity. Methods: The constituents were isolated and purified by column chromatography on MCI gel CHP20P/P120, silica gel, Sephadex LH-20, and HPLC. The structures of compounds were determined by 1D, 2D NMR, and HR-ESI-MS data. The inhibitory effect of compounds on inflammasome activation was determined by lactate dehydrogenase (LDH) procedure. The antiproliferative activity was evaluated using MTT assay. Results: The study led to the isolation of 23 compounds, including one new compound, identified as (2Z)-3-[4-(ß-D-glucopyranosyloxy)-2'-hydroxy-5'-methoxyphenyl]-2-propenoic acid (1), together with 22 known compounds recognized as 1,4-dihydro-4-oxo-3-pyridinecarbonitrile (2), methyl 4-methoxynicotinate (3), 4-methoxynicotinonitrile (4), 1-(3-O-ß-D-glucopyranosyl-4,5-dihydroxyphenyl)-ethanone (5), neoisoastilbin (6), isoastilbin (7), aromadendrin (8), neoastilbin (9), astilbin (10), quercitrin (11), neoschaftoside (12), apigenin 6,8-bis-C-α-L-arabinoside (13), vitexin (14), bergenin (15), scopoletin (16), glucopyranoside salicyl (17), koaburside (18), benzyl ß-D-glucoside (19), icariside B5 (20), roseoside (21), loliolide (22), and adenosine (23). The tested compounds did not show LDH inhibition nor antiproliferative activity. Conclusion: Compound 1 was a new glycoside. Compounds 2 and 3 were obtained for the first time from natural source. The 22 known compounds constituted of alkaloids (2-4, 23), phenolics (5, 15, 17, 18), flavonoids (6-14), coumarin (16), benzyl glycoside (19), and norsesquiterpenes (20-22). All the compounds, 1-23, were revealed from M. hemsleyana for the first time. This is the initial uncovering of molecules 1-10, 12, 13, 17-19, and 23 from the genus Macaranga. The isolated compounds, 11, 14-16, and 20-22 established taxonomic classification of M. hemsleyana in Euphorbiaceae family. Flavonoids were outstanding as chemosystematic markers of Macaranga genus.

9-Hydroxy-8-oxypalmatine, a novel liver-mediated oxymetabolite of palmatine, alleviates hyperuricemia and kidney inflammation in hyperuricemic mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Palmatine is a main bioactive alkaloid of Cortex Phellodendri, which has been commonly prescribed for the treatment of hyperuricemia (HUA) in China. The metabolites of palmatine were crucial to its prominent biological activity. 9-Hydroxy-8-oxypalmatine (9-OPAL) is a novel liver-mediated secondary oxymetabolite of palmatine. AIM OF THE STUDY: The current study was to assess the efficacy of 9-OPAL, a novel liver-mediated secondary oxymetabolite of palmatine derived from Cortex Phellodendri, in experimental HUA mouse model and further explore its underlying mechanism. MATERIALS AND METHODS: An in vitro metabolic experiment with oxypalmatine was carried out using liver samples. We separated and identified a novel liver metabolite, and investigated its anti-HUA effect in mice. HUA mice were induced by potassium oxonate and hypoxanthine daily for one week. After 1 h of modeling, mice were orally administered with different doses of 9-OPAL (5, 10 and 20 mg/kg). The pathological changes of the kidneys were evaluated using hematoxylin-eosin staining (H&E). The acute toxicity of 9-OPAL was assessed. The effects of 9-OPAL on serum levels of uric acid (UA), adenosine deaminase (ADA), xanthine oxidase (XOD), creatinine (CRE), blood urea nitrogen (BUN) and inflammatory cytokines were measured by enzyme-linked immunosorbent assay (ELISA) or biochemical method. Furthermore, Western blot, quantitative real-time PCR (qRT-PCR) and molecular docking were used to investigate the effect of 9-OPAL on the expression of renal urate transporters and NLRP3 signaling pathway in HUA mice. RESULTS: 9-OPAL had been discovered to be a novel liver-mediated oxymetabolite of palmatine for the first time. Treatment with 9-OPAL significantly reduced the UA, CRE as well as BUN levels, and also effectively attenuated abnormal renal histopathological deterioration with favorable safety profile. Besides, 9-OPAL significantly decreased the serum and hepatic activities of XOD and ADA, dramatically inhibited the up-regulation of UA transporter protein 1 (URAT1) and glucose transporter protein 9 (GLUT9), and reversed the down-regulation of organic anion transporter protein 1 (OAT1). Additionally, 9-OPAL effectively mitigated the renal inflammatory markers (TNF-α, IL-1ß, IL-6 and IL-18), and downregulated the transcriptional and translational expressions of renal Nod-like receptor family pyrin domain containing 3 (NLRP3), caspase-1, apoptosis-associated speck-like (ASC) and IL-1ß in HUA mice. Molecular docking results revealed 9-OPAL bound firmly with XOD, OAT1, GLUT9, URAT1, NLRP3, caspase-1, ASC and IL-1ß. CONCLUSIONS: 9-OPAL was found to be a novel liver-mediated secondary metabolite of palmatine with favorable safety profile. 9-OPAL had eminent anti-hyperuricemic and renal-protective effects, and the mechanisms might be intimately associated with repressing XOD activities, modulating renal urate transporter expression and suppressing the NLRP3 inflammasome activation. Our investigation might also provide further experimental evidence for the traditional application of Cortex Phellodendri in the treatment of HUA.

The role of Pim-1 kinases in inflammatory signaling pathways.

OBJECTIVE AND DESIGN: This observational study investigated the regulatory mechanism of Pim-1 in inflammatory signaling pathways. MATERIALS: THP-1, RAW 264.7, BV2, and Jurkat human T cell lines were used. TREATMENT: None. METHODS: Lipopolysaccharide (LPS) was used to induce inflammation, followed by PIM1 knockdown. Western blot, immunoprecipitation, immunofluorescence, and RT-PCR assays were used to assess the effect of PIM1 knockdown on LPS-induced inflammation. RESULTS: PIM1 knockdown in macrophage-like THP-1 cells suppressed LPS-induced upregulation of pro-inflammatory cytokines, inducible nitric oxide synthase, cyclooxygenase-2, phosphorylated Janus kinase, signal transducer and activator of transcription 3, extracellular signal-regulated kinase, c-Jun N-terminal kinase, p38, and nuclear factor kappa B p65 (NF-κB p65). It also suppressed upregulation of inhibitor of NF-κB kinase α/ß and enhanced the nuclear translocation of NF-κB p65. Moreover, it inhibited the upregulation of Nod-like receptor family pyrin domain-containing 3 (NLRP3) and cleavage of caspase-1 induced by co-treatment of LPS with adenosine triphosphate. Additionally, p-transforming growth factor-ß-activated kinase 1 (TAK1) interacted with Pim-1. All three members of Pim kinases (Pim-1, Pim-2, and Pim-3) were required for LPS-mediated inflammation in macrophages; however, unlike Pim-1 and Pim-3, Pim-2 functioned as a negative regulator of T cell activity. CONCLUSIONS: Pim-1 interacts with TAK1 in LPS-induced inflammatory responses and is involved in MAPK/NF-κB/NLRP3 signaling pathways. Additionally, considering the negative regulatory role of Pim-2 in T cells, further in-depth studies on their respective functions are needed.

GDF15 regulated by HDAC2 exerts suppressive effects on oxygen-glucose deprivation/reoxygenation-induced neuronal cell pyroptosis via the NLRP3 inflammasome.

Background: Pyroptosis, inflammation-related programed cell death mediated by NLRP3 inflammasome, is involved in the pathogenesis of cerebral hypoxic-ischemic injury. Our study aims to explore the biological role of growth differentiation factor (GDF)15 in oxygen-glucose deprivation/reoxygenation (OGD/R)-induced neuronal pyroptosis. Methods: HT22 neurons were subjected to OGD/R to simulate cerebral hypoxic-ischemic injury. Cells were transfected with plasmids to overexpress GDF15, or lentiviral-based shRNAs constructs to silence GDF15. ELISA assay was used to detect GDF15, IL-1ß, IL-18, and neuron specific enolase (NSE) levels. Cell pyroptosis was measured by flow cytometery. Chromatin immunoprecipitation assay was used to detect interaction of H3K27ac with GDF15 promoter. GDF15, NLRP3, Caspase-1 p20 and GSDMD-N expressions were measured by Western blotting. Results: Patients with malignant middle cerebral artery infarction showed decreased GDF15, but increased IL-1ß, IL-18, and NSE levels in serum compared to healthy controls. OGD/R treatment caused significant increases in the levels of IL-1ß, IL-18 and NSE, percentages of pyroptotic cells, and expressions of NLRP3, Caspase-1 p20, and GSDMD in HT22 cells, which were markedly reversed by GDF15 overexpression. However, GDF15 knockdown resulted in neuronal injury similar to those observed in OGD/R treatment. The GDF15 knockdown-induced effects were counteracted by treatment with NLRP3 inhibitor. OGD/R decreased the enrichment of H3K27ac in the promoter of GDF15 to down-regulate GDF15, but was compromised by co-treatment with HDAC2 inhibitor. Conclusion: Our data demonstrates that GDF15 attenuates OGD/R-induced pyroptosis through NLRP3 inflammasome. HDAC2 is involved in mediating OGD-induced GDF15 down-regulation via H3K27ac modification. GDF15 overexpression and HDAC2 inhibition hold potential as useful therapeutic strategies for neuroprotection.

Huajuxiaoji Formula Alleviates Phenyl Sulfate-Induced Diabetic Kidney Disease by Inhibiting NLRP3 Inflammasome Activation and Pyroptosis.

Background: One of the most common microvascular complications of diabetes is diabetic kidney disease (DKD). The Huajuxiaoji formula (HJXJ) has shown clinical efficacy for DKD; however, its regulatory mechanisms against DKD remain elusive. We investigated NLRP3 inflammasome and the mechanisms of HJXJ by which HJXJ alleviates DKD. Methods: Phenyl sulfate (PS) was used to establish DKD models. HJXJ was administered to mice through intragastric or made into a pharmaceutical serum for the cell cultures. Biological indicator levels in mouse blood and urine were analyzed, and kidney tissues were used for HE, Masson, and PAS staining. ELISA and western blotting were used to detect inflammatory cytokines and protein levels, respectively. Reactive oxygen species (ROS) production and pyroptosis were evaluated using flow cytometry. Lentiviral vector-mediated overexpression of NLRP3 was performed to determine whether NLRP3 participates in the antipyroptotic effect of HJXJ. Results: HJXJ significantly reduced the severity of the injury and, in a dose-dependent manner, decreased the levels of biological markers including creatinine, blood urea nitrogen, urine protein, and endotoxin, as well as inflammatory cytokines such as interleukin (IL)-1ß, IL-18, tumor necrosis factor-α, and IL-6 in DKD mice. Treatment with HJXJ reversed the downregulation of podocin, nephrin, ZO-1, and occludin and upregulated ROS, NLRP3, Caspase-1 P20, and GSDMD-N induced by PS. Moreover, the upregulation of NLRP3 expression increased the number of cells positive for pyroptosis. HJXJ suppressed pyroptosis and inflammasome activation by inhibiting NLRP3 expression. Conclusions: Generally, HJXJ has the potential to reduce DKD injury and exerts anti-DKD effects by inhibiting the NLRP3-mediated NLRP3 inflammasome activation and pyroptosis in vitro and in vivo.

Injection of Collagen Binding Domain-Brain Derived Neurotrophic Factor Promotes SIRT1 Expression: Improving Neuroinflammation in Experimental Subarachnoid Hemorrhage.

BACKGROUND: Subarachnoid hemorrhage (SAH) is a severe cerebrovascular disease, often leading to neuroinflammation and neuronal damage. Activation of the Nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 (NLRP3) inflammasome is closely associated with post-SAH neuroinflammation, while activation of Nicotinamide Adenine Dinucleotide (NAD)-dependent deacetylase sirtuin-1 (SIRT1) has neuroprotective effects. This study aimed to investigate the impact of injectable Collagen Binding Domain-Brain Derived Neurotrophic Factor (CBD-BDNF) on neuroinflammation and neuronal damage following SAH. METHODS: After establishing the SAH model, experimental animals were divided into three groups: sham surgery group (Sham), SAH group, and SAH+neuroregenerative scaffold (CBD-BDNF treatment) group. Behavioral performance was evaluated using neurofunctional deficit, beam balance, and Y-maze tests. Expression of inflammatory factors and essential proteins was quantitatively analyzed using Enzyme-Linked Immunosorbent Assay (ELISA) kits and immunoblotting. Terminal deoxynucleotidyl transferase dUTP Nick End Labeling (TUNEL) staining was used to assess cell apoptosis. To further investigate the mechanism of action of CBD-BDNF on SIRT1, the model animals were treated with EX527 (SIRT1 inhibitor) for comparative studies. RESULTS: Neurological deficit tests, CBD-BDNF improves functional outcomes after SAH. Compared to the SAH group, the SAH+neuroregenerative scaffold group showed significantly increased expression of SIRT1 protein and significantly decreased expression of NLRP3, Apoptosis-associated speck-like protein containing a CARD (ASC), and c-caspase-1. The inflammatory cytokines Interleukin-1 beta (IL-1ß), IL-6, and IL-18 levels also significantly decreased in the SAH+neuroregenerative scaffold group. Additionally, animals in the SAH+neuroregenerative scaffold group showed better neurofunctional recovery in neurofunctional deficit and beam balance tests. The number of apoptotic cells significantly decreased in the SAH+neuroregenerative scaffold group compared to the SAH group. However, when SIRT1 was inhibited with EX527, the aforementioned neuroprotective effects were reversed, indicating the involvement of CBD-BDNF through SIRT1 activation. CONCLUSION: This study demonstrates that injectable CBD-BDNF can significantly alleviate neuroinflammation and neuronal damage resulting from SAH by blocking NLRP3 inflammasome activation and promoting SIRT1 expression. These findings provide a new therapeutic strategy for neuroprotection after SAH and reveal the mechanism of action of CBD-BDNF as a potential therapeutic agent. Future research will further explore the long-term efficacy and safety of CBD-BDNF.

The molecular effects underlying the pharmacological activities of daphnetin.

As an increasingly well-known derivative of coumarin, daphnetin (7,8-dithydroxycoumarin) has demonstrated various pharmacological activities, including anti-inflammation, anti-cancer, anti-autoimmune diseases, antibacterial, organ protection, and neuroprotection properties. Various studies have been conducted to explore the action mechanisms and synthetic methods of daphnetin, given its therapeutic potential in clinical. Despite these initial insights, the precise mechanisms underlying the pharmacological activities of daphnetin remain largely unknown. In order to address this knowledge gap, we explore the molecular effects from the perspectives of signaling pathways, NOD-like receptor protein 3 (NLRP3) inflammasome and inflammatory factors; and try to find out how these mechanisms can be utilized to inform new combined therapeutic strategies.

Osteoblast-specific down-regulation of NLRP3 inflammasome by aptamer-functionalized liposome nanoparticles improves bone quality in postmenopausal osteoporosis rats.

Rationale: NLRP3 inflammasome is critical in the development and progression of many metabolic diseases driven by chronic inflammation, but its effect on the pathology of postmenopausal osteoporosis (PMOP) remains poorly understood. Methods: We here firstly examined the levels of NLRP3 inflammasome in PMOP patients by ELISA. Then we investigated the possible mechanisms underlying the effect of NLRP3 inflammasome on PMOP by RNA sequencing of osteoblasts treated with NLRP3 siRNA and qPCR. Lastly, we accessed the effect of decreased NLRP3 levels on ovariectomized (OVX) rats. To specifically deliver NLRP3 siRNA to osteoblasts, we constructed NLRP3 siRNA wrapping osteoblast-specific aptamer (CH6)-functionalized lipid nanoparticles (termed as CH6-LNPs-siNLRP3). Results: We found that the levels of NLRP3 inflammasome were significantly increased in patients with PMOP, and were negatively correlated with estradiol levels. NLRP3 knock-down influenced signal pathways including immune system process, interferon signal pathway. Notably, of the top ten up-regulated genes in NLRP3-reduced osteoblasts, nine genes (except Mx2) were enriched in immune system process, and five genes were related to interferon signal pathway. The in vitro results showed that CH6-LNPs-siNLRP3 was relatively uniform with a dimeter of 96.64 ± 16.83 nm and zeta potential of 38.37 ± 1.86 mV. CH6-LNPs-siNLRP3 did not show obvious cytotoxicity and selectively delivered siRNA to bone tissue. Moreover, CH6-LNPs-siNLRP3 stimulated osteoblast differentiation by activating ALP and enhancing osteoblast matrix mineralization. When administrated to OVX rats, CH6-LNPs-siNLRP3 promoted bone formation and bone mass, improved bone microarchitecture and mechanical properties by decreasing the levels of NLRP3, IL-1ß and IL-18 and increasing the levels of OCN and Runx2. Conclusion: NLRP3 inflammasome may be a new biomarker for PMOP diagnosis and plays a key role in the pathology of PMOP. CH6-LNPs-siNLRP3 has potential application for the treatment of PMOP.

Sodium Tanshinone IIA Sulfonate alleviates vascular senescence in diabetic mice by modulating the A20-NFκB-NLRP3 inflammasome-catalase pathway.

Diabetes accelerates vascular senescence, which is the basis for atherosclerosis and stiffness. The activation of NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome and oxidative stress are closely associated with the deteriorative senescence in endothelial cells (ECs) and vascular smooth muscle cells (VSMCs). For decades, Sodium Tanshinone IIA Sulfonate (STS) has been utilized as a cardiovascular medicine with acknowledged anti-inflammatory and anti-oxidative properties. Nevertheless, the impact of STS on vascular senescence remains unexplored in diabetes. Diabetic mice, primary ECs and VSMCs were transfected with the NLRP3 overexpression/knockout plasmid, the tumor necrosis factor alpha-induced protein 3 (TNFAIP3/A20) overexpression/knockout plasmid, and treated with STS to detect senescence-associated markers. In diabetic mice, STS treatment maintained catalase (CAT) level and vascular relaxation, reduced hydrogen peroxide probe (ROSgreen) fluorescence, p21 immunofluorescence, Senescence ß-Galactosidase Staining (SA-ß-gal) staining area, and collagen deposition in aortas. Mechanistically, STS inhibited NLRP3 phosphorylation (serine 194), NLRP3 dimer formation, NLRP3 expression, and NLRP3-PYCARD (ASC) colocalization. It also suppressed the phosphorylation of IkappaB alpha (IκBα) and NFκB, preserved A20 and CAT levels, reduced ROSgreen density, and decreased the expression of p21 and SA-ß-gal staining in ECs and VSMCs under HG culture. Our findings indicate that STS mitigates vascular senescence by modulating the A20-NFκB-NLRP3 inflammasome-CAT pathway in hyperglycemia conditions, offering novel insights into NLRP3 inflammasome activation and ECs and VSMCs senescence under HG culture. This study highlights the potential mechanism of STS in alleviating senescence in diabetic blood vessels, and provides essential evidence for its future clinical application.

Enhanced Cardiomyocyte NLRP3 Inflammasome-Mediated Pyroptosis Promotes d-Galactose-Induced Cardiac Aging.

BACKGROUND: Cardiac aging represents an independent risk factor for aging-associated cardiovascular diseases. Although evidence suggests an association between NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome formation and numerous cardiovascular diseases, its role in cardiac aging remains largely unclear. METHODS AND RESULTS: The longevity of mice with wild-type and NLRP3 knockout (NLRP3-/-) genotypes was assessed, with or without d-galactose treatment. Cardiac function was evaluated using echocardiography, and cardiac histopathology was examined through hematoxylin and eosin and Masson's trichrome staining. Senescence-associated ß-galactosidase (SA-ß-gal) staining was employed to detect cardiac aging. Western blotting was used to assess aging-related proteins (p53, p21) and pyroptosis-related proteins. Additionally, dihydroethidium staining, lactate dehydrogenase release, and interleukin-1ß ELISA assays were performed, along with measurements of total superoxide dismutase and malondialdehyde levels. In vitro, H9c2 cells were exposed to d-galactose for 24 hours in the absence or presence of N-acetyl-l-cysteine (reactive oxygen species inhibitor), BAY-117082 (nuclear factor κ-light-chain enhancer of activated B cells inhibitor), MCC950 (NLRP3 inhibitor), and VX-765 (Caspase-1 inhibitor). Immunofluorescence staining was employed to detect p53, gasdermin D, and apoptosis-associated speck-like protein proteins. Intracellular reactive oxygen species levels were assessed using fluorescence microscopy and flow cytometry. Senescence-associated ß-galactosidase staining and Western blotting were also employed in vitro for the same purpose. The results showed that NLRP3 upregulation was implicated in aging and cardiovascular diseases. Inhibition of NLRP3 extended life span, mitigated the aging phenotype, improved cardiac function and blood pressure, ameliorated lipid metabolism abnormalities, inhibited pyroptosis in cardiomyocytes, and ultimately alleviated cardiac aging. In vitro, the inhibition of reactive oxygen species, nuclear factor κ-light-chain enhancer of activated B cells, NLRP3, or caspase-1 attenuated NLRP3 inflammasome-mediated pyroptosis. CONCLUSIONS: The reactive oxygen species/nuclear factor κ-light-chain enhancer of activated B cells/NLRP3 signaling pathway loop contributes to d-galactose-treated cardiomyocyte senescence and cardiac aging.

[2, 6-dimethoxy-1, 4-benzoquinone alleviates septic shock in mice by inhibiting NLRP3 inflammasome activation].

OBJECTIVE: To investigate the mechanism of 2, 6-dimethoxy-1, 4-benzoquinone (DMQ), an active ingredients in fermented wheat germ extract, for inhibiting NLRP3 inflammasome activation and alleviating septic shock in mice. METHODS: Cultured murine bone marrow-derived macrophages (BMDM) stimulated with lipopolysaccharide (LPS) were treated with DMQ, followed by treatment with Nigericin, ATP, and MSU for activating the canonical NLRP3 inflammasome; the noncanonical NLRP3 inflammasome was activated by intracellular transfection of LPS, and AIM2 inflammasome was activated using Poly A: T.In human monocytic THP-1 cells, the effect of Nigericin on inflammasome activation products was examined using Western blotting and ELISA.Co-immunoprecipitation was performed to explore the mechanism of DMQ-induced blocking of NLRP3 inflammasome activation.In a male C57BL/6J mouse model of LPS-induced septic shock treated with 20 and 40 mg/kg DMQ, the levels of IL-1ß and TNF-α in the serum and peritoneal lavage fluid were determined using ELISA, and the survival time of the mice within 36 h was observed. RESULTS: Treatment with DMQ effectively inhibited LPS-induced activation of canonical NLRP3 inflammasome in mouse BMDM and human THP-1 cells and also inhibited non-canonical NLRP3 inflammasome activation in mouse BMDM, but produced no significant effect on AIM2 inflammasome activation.DMQ significantly blocked the binding between ASC and NLRP3.In the mouse models of septic shock, DMQ treatment significantly reduced the levels of IL-1ß in the serum and peritoneal fluid and obviously prolonged survival time of the mice. CONCLUSION: DMQ can effectively block ASC-NLRP3 interaction to inhibit NLRP3 inflammasome activation and alleviate LPSinduced septic shock in mice.

Ameliorative effect of nodakenin in combating TNBS-induced ulcerative colitis by suppressing NFƙB-mediated NLRP3 inflammasome pathway.

Ulcerative colitis (UC) is an enduring and complex inflammatory bowel disease that is clinically prevalent, progressive, and debilitating. As of now, the few effective medical treatments for UC have unacceptably high side effects. It is crucial to find safer and more effective UC treatments. Nodakenin possesses anti-inflammatory and antioxidant activity by suppressing several pro-inflammatory mediators. In the present study, we aimed to evaluate the colonoprotective effect of nodakenin in combating colitis through the NFƙB-mediated NLRP3 inflammasome pathway. In mice, UC was induced by 2,4,6-trinitrobenzene sulfonic acid (TNBS). Nodakenin (10, 20, and 40 mg/kg) was introduced intragastrically, and disease activity index (DAI) score was calculated. Malondialdehyde (MDA), myeloperoxidase (MPO), superoxide dismutase (SOD), nitric oxide (NO) levels, tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) concentration were evaluated in colon homogenate. Colon samples were used for histopathological investigation and mRNA expression studies involving nuclear factor kappa B (NFƙB), cyclooxygenase-2 (COX-2), inducible nitric oxide (iNOS), nucleotide-binding receptor domain 3 (NLRP3), interleukin-1ß (IL-1ß), and interleukin-18 (IL-18). Nodakenin treatment was found effective in lowering the DAI score, histological score, MPO, MDA, and NO levels while elevating SOD levels as compared to the model control group, showcasing its anti-inflammatory and antioxidant properties. Nodakenin (40 mg/kg) significantly downregulated the expression of TNF-α, IL-6, NFƙB (1.24-fold), iNOS (1.2-fold), COX-2 (1.98-fold), NLRP3 (1.78-fold), IL-1ß (1.29-fold), and IL-18 (1.17-fold) conferring its great anti-inflammatory potential in combating colitis. Taking together, nodakenin presumably alleviated TNBS-induced colitis by NFƙB-mediated NLRP3 inflammasome pathway and reduced colon damage by downregulating various transcriptional genes and pro-inflammatory mediators.

Renoprotective effect of a novel combination of 6-gingerol and metformin in high-fat diet/streptozotocin-induced diabetic nephropathy in rats via targeting miRNA-146a, miRNA-223, TLR4/TRAF6/NLRP3 inflammasome pathway and HIF-1α.

BACKGROUND: MiRNA-146a and miRNA-223 are key epigenetic regulators of toll-like receptor 4 (TLR4)/tumor necrosis factor-receptor-associated factor 6 (TRAF6)/NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome pathway, which is involved in diabetic nephropathy (DN) pathogenesis. The currently available oral anti-diabetic treatments have been insufficient to halt DN development and progression. Therefore, this work aimed to assess the renoprotective effect of the natural compound 6-gingerol (GR) either alone or in combination with metformin (MET) in high-fat diet/streptozotocin-induced DN in rats. The proposed molecular mechanisms were also investigated. METHODS: Oral gavage of 6-gingerol (100 mg/kg) and metformin (300 mg/kg) were administered to rats daily for eight weeks. MiRNA-146a, miRNA-223, TLR4, TRAF6, nuclear factor-kappa B (NF-κB) (p65), NLRP3, caspase-1, and hypoxia-inducible factor-1 alpha (HIF-1α) mRNA expressions were measured using real-time PCR. ELISA was used to measure TLR4, TRAF6, NLRP3, caspase-1, tumor necrosis factor-alpha (TNF-α), and interleukin-1-beta (IL-1ß) renal tissue levels. Renal tissue histopathology and immunohistochemical examination of fibronectin and NF-κB (p65) were performed. RESULTS: 6-Gingerol treatment significantly reduced kidney tissue damage and fibrosis. 6-Gingerol up-regulated miRNA-146a and miRNA-223 and reduced TLR4, TRAF6, NF-κB (p65), NLRP3, caspase-1, TNF-α, IL-1ß, HIF-1α and fibronectin renal expressions. 6-Gingerol improved lipid profile and renal functions, attenuated renal hypertrophy, increased reduced glutathione, and decreased blood glucose and malondialdehyde levels. 6-Gingerol and metformin combination showed superior renoprotective effects than either alone. CONCLUSION: 6-Gingerol demonstrated a key protective role in DN by induction of miRNA-146a and miRNA-223 expression and inhibition of TLR4/TRAF6/NLRP3 inflammasome signaling. 6-Gingerol, a safe, affordable, and abundant natural compound, holds promise for use as an adjuvant therapy with metformin in diabetic patients to attenuate renal damage and stop the progression of DN.

Successful Treatment With High-Dose Colchicine of a 101-Year-Old Patient Diagnosed With COVID-19 After an Emergency Cholecystectomy.

There are multiple factors associated with increased morbidity and mortality in COVID-19 patients, and advanced age is one such independent prognostic factor. It is well established that the multiorgan failure and death in COVID-19 patients are due to the hyperactivation of the NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome and the ensuing cytokine storm. Colchicine, a well-known anti-inflammatory drug, has been shown to inhibit the NLRP3 inflammasome in micromolar concentrations potently. It has the unique property of accumulating in leukocytes, which is the primary cause of the abnormal activation of the NLRP3 inflammasome in COVID-19. It has been shown that achieving inhibitory concentrations of colchicine in leucocytes requires treatment with higher doses. Our recent studies showed that treatment with higher doses of colchicine in both outpatient and inpatient settings is safe and results in remarkable cure rates and significantly decreased mortality rates, even in the most severely affected patients with multiple comorbidities and risk factors. The main risk factor for severe COVID-19 is age, especially over 85 years. Here, we present a unique case of a 101-year-old male who underwent two major emergency abdominal surgeries and contracted COVID-19 while in the hospital. Laboratory tests showed increased values of markers for severe COVID-19, including CRP, D-dimer, and ferritin. Increased opacities bilaterally paracardially and moderate right-side pleural effusions were detected on the chest X-ray. We initiated our high-dose colchicine treatment regimen, resulting in the patient's complete recovery and discharge. We are convinced that the administration of high-dose colchicine to high-risk COVID-19 patients should be mandatory.

S-glutathionylation in hepatocytes is involved in arsenic-induced liver fibrosis through activation of the NLRP3 inflammasome, an effect alleviated by NAC.

Arsenic, a toxicant widely distributed in the environment, is considered as a risk factor for liver fibrosis. At present, the underlying mechanism still needs to be explored. In the present study, we found that, for mice, chronic exposure to arsenic induced liver fibrosis, activated the NLRP3 inflammasome, and increased the levels of reactive oxygen species (ROS). After hepatocytes were co-cultured with hepatic stellate cells (HSCs), we observed the arsenic-activated NLRP3 inflammasome in hepatocytes, and the co-cultured HSCs were activated. Further, we found that, in livers of mice, arsenic disturbed GSH metabolism and promoted protein S-glutathionylation. A 3D molecular docking simulation suggested that NLRP3 binds with GSH, which was confirmed by immunoprecipitation experiments. N-acetylcysteine (NAC) increased the levels of GSH in hepatocytes, which suppressed the S-glutathionylation of NLRP3 and blocked arsenic-induced activation of the NLRP3 inflammasome. Mechanistically, an imbalance of the redox state induced by arsenic promotes the S-glutathionylation of NLRP3, which regulates activation of the NLRP3 inflammasome, leading into the activation of HSCs. Moreover, NAC increases the levels of GSH to block arsenic-induced S-glutathionylation of NLRP3, thereby blocking arsenic-induced liver fibrosis. Thus, via activating HSCs, the S-glutathionylation of NLRP3 in hepatocytes is involved in arsenic-induced liver fibrosis, and, for hepatocytes, NAC alleviates these effects by increasing the levels of GSH. These results reveal a new mechanism and provide a possible therapeutic target for the liver fibrosis induced by environmental factors.