Expression Of IL-1β Research Articles

The Potential Roles of IL-1β, IL-6, and RIPK3 in the Pathogenesis of Stevens–Johnson Syndrome/Toxic Epidermal Necrolysis

Background/Objectives: Stevens–Johnson Syndrome and Toxic Epidermal Necrolysis (SJS/TEN) are rare but severe skin conditions, often triggered by medications, that can be life-threatening. These conditions frequently affect the eyes, causing ocular surface disease, which can result in visual impairment or blindness. Although the exact mechanisms behind SJS/TEN remain unclear, key inflammatory mediators such as IL-1β, IL-6, and RIPK3 are believed to play critical roles in inflammation, necroptosis, and regulatory processes. Investigating these factors offers new insights into the disease’s underlying mechanisms and potential targets for treatment. This study aims to determine the roles of IL-1β, IL-6, and RIPK3 in the pathogenesis of SJS/TEN. Methods: The study examined the expression levels of IL-1β, IL-6, and RIPK3 in skin biopsies from patients with biopsy-confirmed SJS/TEN, using lichen planus as a positive control and normal skin as a baseline control. Immunohistochemistry was employed for this analysis. Additionally, the impact of SJS/TEN patient plasma on mitochondrial function was assessed in platelets and human corneal epithelial (H-CET) cells. Using a fluorescent plate reader, mitochondrial activity and superoxide ion levels were measured, comparing plasma from SJS/TEN patients to normal human plasma. Results: Skin biopsies from SJS/TEN patients showed a significantly higher expression of IL-1β, IL-6, and RIPK3 compared to both lichen planus and normal controls. Furthermore, plasma from SJS/TEN patients significantly reduced platelet viability and increased mitochondrial and total cellular superoxide ions, as demonstrated by elevated levels of MitoSOX Red and CellROX Red. Conclusions: These findings suggest that IL-1β, IL-6, and RIPK3 may contribute to the pathogenesis of SJS/TEN and highlight their potential as targets for therapeutic intervention.

Glabridin Suppresses Macrophage Activation by Lipoteichoic Acid In Vitro: The Crucial Role of MAPKs-IL-1β-iNOS Axis Signals in Peritoneal and Alveolar Macrophages

Inflammation, a fundamental response to infection and injury, involves interactions among immune cells and signaling molecules. Dysregulated inflammation contributes to diseases such as autoimmune disorders and cancer. Interleukin-1 beta (IL-1β), produced by macrophages in response to lipoteichoic acid (LTA) from Gram-positive bacteria, is a key inflammatory mediator. Glabridin (GBD), a bioactive compound from licorice root, exhibits anti-inflammatory properties. This study investigates GBD’s effects on LTA-induced proinflammatory signaling in RAW 264.7 murine macrophages and alveolar macrophages, MH-S, focusing on IL-1β expression and signaling pathways. Cell viability assays confirmed that 20 μM GBD was non-cytotoxic. Confocal microscopy and quantitative PCR showed that GBD significantly reduced IL-1β fluorescence intensity, mRNA, and protein levels. GBD also inhibited inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production. Further analysis revealed that GBD suppressed NF-κB p65 nuclear translocation and selectively modulated MAPK pathway activation by reducing JNK and p38 MAPK phosphorylation without affecting ERK. Studies using specific inhibitors demonstrated that IL-1β production reduction was mechanistically linked to MAPK pathway inhibition. These findings highlight GBD’s potential as a therapeutic agent for inflammatory diseases through its ability to modulate critical inflammatory mediators and signaling pathways.

P0033 GPR84, which is increased in Ulcerative Colitis patients, regulates the cytokine secretion of macrophages

Abstract Background Inflammatory Bowel Disease (IBD) is a gastrointestinal disorder comprising Ulcerative Colitis (UC) and Crohn´s Disease (CD) characterized by an exacerbated and chronic immune response. Although GPR84 has emerged as a proinflammatory receptor in some pathologies such as atherosclerosis or reflux esophagitis, its role in IBD remains unclear. We aim to analyze the protein expression of GPR84 in IBD patients and its role in the cytokine secretion in macrophages. Methods Surgical resections from UC (n=5) and CD patients (n=5) were obtained. Healthy colon and ileum from colon-carcinoma patients (n=10) were used as controls. Protein expression of GPR84 and CD68 was analyzed by WesternBlot and immunohistochemistry. U937 monocytes were differentiated into macrophages with PMA (0,01μM) during 48 hours and treated with the GPR84 agonist decanoic acid (50-250μM) and a proinflammatory cocktail (0,1μM LPS + 20ng/mL IFNγ) during 24 hours. Some of them were treated with the GPR84 antagonist (10μM) or transiently silenced with a specific siRNA-GPR84. RNA was isolated and gene expression of pro- and anti-inflammatory cytokines was quantified by qPCR. Cytokine levels in supernatant were also quantified by Luminex. Data were expressed as fold induction vs control (mean±SEM) and compared by t-test. A p-value<0.05 was considered statistically significant. Results Protein levels of GPR84 were significantly increased in UC patients (150.3±11.75 vs 100.0±11.85), while no differences were observed in CD patients vs healthy ileums. Immunohistochemistry revealed a slight staining of this receptor in intestinal epithelial cells, whereas several cells of the lamina propria were strongly stained. Double immunohistochemistry confirmed that intestinal CD68 macrophages expressed GPR84 in both UC and CD patients. U937-derived macrophages treated with decanoic acid showed a significant increased expression of IL1b (2.68±0.86) and IL8 (2.44±0.48) vs vehicle-treated macrophages (0.998±0.23 and 1.05±0.17, respectively). In addition, IL1b levels in supernatant from macrophages treated with decanoic acid were significantly increased compared with the supernatant from vehicle-treated macrophages (4.50±1.05 vs 1.00±0.19). Moreover, the cotreatment with decanoic acid and GPR84 antagonist significantly reduced the expression of IL1b (0.99±0.29) vs vehicle-treated macrophages (1.94±0.26) and the transiently silencing of this receptor significantly reduced the expression of IL8 (0.89±0.25) vs siCtrl macrophages (4.02±1.59). Conclusion GPR84 is increased in UC patients and expressed in intestinal CD68+ macrophages. It regulates the expression of several cytokines pointing GPR84 as a promising pharmacological target for UC patients.

Tissue Perfusion and Biomarkers Assessment Following Root Coverage Procedures.

To assess tissue perfusion changes and wound healing biomarker levels after root coverage procedures with coronally advanced flap in combination with the cross-linked xenogeneic collagen matrix (CCMX), loaded either with a placebo or recombinant human platelet-derived growth factor-BB (rhPDGF). This study was designed as a secondary analysis from a previously published clinical trial, and it assessed the tissue perfusion changes over 6 months around multiple gingival recession defects, treated with either with CCMX alone (control) or with CCMX + rhPDGF (test). High frequency Doppler ultrasonography (HFUS) scans were obtained at sites of interest at baseline, 2 weeks, 3 months, and 6 months after surgery. Dynamic tissue perfusion measurements (DTPMs) were performed at the midfacial, interproximal, and transverse aspects of the teeth by an operator, blinded to treatment allocation, using a software package. The expression of different wound healing biomarkers from the gingival crevicular fluid was also assessed. The regression analyses showed similar tissue perfusion changes between the two groups throughout the majority of the 6 months. DTPMs at 2 weeks showed the test group to have significantly higher perfusion relief intensity (pRI, p < 0.001), mean perfused area (pA, p < 0.001), mean blood flow intensity (FImean, p = 0.021), and total blood flow intensity (FItot, p = 0.021) at the graft region of interest (ROI) compared to control sites. The test sites also exhibited significantly greater pA (p = 0.033) and blood flow intensity "blue" (FIblue, meaning flow away from the transducer, p = 0.035) at the level of the flap compared to the control sites. At 2 weeks, FIblue of the graft was directly correlated with the final mean root coverage (p = 0.008) and complete root coverage (p = 0.003). FImean and FItot of the graft exhibited a direct correlation with volume gain at 6 months (p = 0.031 for both parameters). The final GT gain was correlated to the early DTPMs (pA and FIblue) of the graft and the flap. The two groups exhibited different expressions of IL-1β, PDFG-BB, and VEGF over 3 months, with the 1-week levels of PDGF-BB that were associated with time to recovery. HFUS allowed exquisite assessment of tissue perfusion occurring at the entire surgical reconstructive regions and also within the flap and the graft. Sites treated with CCMX + rhPDGF exhibited higher DTPMs, primarily within the graft and flap ROIs at the 2-week timepoint compared to sites augmented with CCMX + saline. Early DTPMs at the graft and flap ROIs showed associations with PROMs and the final clinical outcomes. ClinicalTrials.gov: NCT04462237.

P0098 Effect of the dietary emulsifier κ-carrageenan on the inflammatory state and permeability of intestinal epithelial cells from patients with Crohn’s disease

Abstract Background The consumption of ultra-processed foods has increased worldwide and is associated with the rise in inflammatory bowel disease (IBD)1. However, any causative factors and their underlying mechanisms are yet to be identified. Interestingly, our group found a higher consumption of the dietary emulsifier carrageenan (CGN) in Belgian IBD patients, compared to healthy controls2. This study aimed to elucidate whether different types of CGN can alter the permeability and inflammatory state of the intestinal epithelium in patients with Crohn’s disease (CD). Methods Confluent Caco-2/HT29-MTX cocultures (n=4) were exposed to either κ-, ι-, or λ-CGN (100 µg/ml) for 24 hours. Confluent epithelial monolayers derived from colonic organoids3 of 5 CD patients were exposed to κ-CGN (100 µg/ml), the most abundant subtype in foods, for 48 hours. Two independent experiments were performed per patient (n = 10). In both models, inflammation was established by adding an inflammatory mix (100 ng/mL TNF-α, 20 ng/mL IL-1β, and 1 µg/mL flagellin)4 to the basolateral side, 24 hours prior to CGN exposure. Changes in permeability were measured by transepithelial electrical resistance (TEER) at predetermined timepoints (0, 12, 24, 48 hours). In organoid-derived monolayers, cytokines were quantified in the apical and basolateral supernatant using the V-PLEX Proinflammatory Panel 1 kit (Meso Scale Diagnostics) and gene expression was analysed with RT-qPCR (Taqman probes, run on Viia7) (n = 8; 2 samples excluded due to low RNA concentrations). Results In organoid-derived monolayers, κ-CGN increased the expression of TNF, IL8 and IL1B, both in inflamed and non-inflamed monolayers (n=8; Repeated measures (RM) one-way ANOVA), compared to the control (Fig. 1A). Release of the cytokines IL-6, IL-13, IL-4, IL-2, and IL-10 in the apical supernatant was increased after 48 hours of κ-CGN stimulation (n=8; RM one-way ANOVA) (Fig. 1B). κ-CGN exposure did not affect TEER (n=10; RM one-way ANOVA) (Fig. 2B+C), but induced an upregulation of tight junction markers ZO1 and OCLN, and resulted in a downregulation of MUC5AC and upregulation of MUC5B (n=8; RM one-way ANOVA or Friedman test) (Fig. 2D). Additionally, none of the CGN subtypes altered permeability of non-inflamed or inflamed Caco-2/HT29-MTX cocultures (n=4; Kruskal-Wallis test), compared to the unexposed control (Fig. 2A). Conclusion Dietary κ-CGN caused upregulation of inflammatory markers and affected cytokine release of intestinal epithelial cells from patients with CD, while permeability remained unaltered. When inflammation was already present, this pro-inflammatory effect was more pronounced, suggesting a role for dietary CGN in perpetuating inflammation during active CD.

DOP114 LDN-071, the novel amino acid-based PAI-1 inhibitor, significantly reduces the severity of acute and chronic inflammation in colitis models

Abstract Background Over recent decades, significant progress has been made in the pharmacological management of Inflammatory Bowel Diseases (IBD). However, the sustained efficacy of existing anti-inflammatory therapies remains suboptimal, highlighting the urgent need for novel treatments. Recent studies have implicated the coagulation pathway, particularly Plasminogen Activator Inhibitor 1 (PAI-1), as a crucial link between epithelium and inflammation in IBD. Genetic deletion of PAI-1 has been shown to reduce experimental colitis severity in mice. Based on these findings, we aimed to develop and evaluate novel PAI-1 inhibitors for IBD treatment. Methods Using the A3SMO® platform, we synthesized LDN-071, an amino acid-based PAI-1 inhibitor, and assessed its inhibitory potential and cytotoxicity in vitro. Acute and chronic colitis models were induced in mice using DSS, with 10 mg/bwkg LDN-071 administered orally. Body weight, spleen weight, and colon length were monitored, while colon histology was analysed. Cytokine expression was measured via ELISA and qRT-PCR, and collagen deposition was quantified using a hydroxyproline assay. Human colon organoid (HCO) and fibroblast (FB) cultures from IBD patients were used to evaluate LDN-071’s effects. Toxicity was assessed in vivo in mice. Results LDN-071 effectively inhibited PAI-1 in vitro without cytotoxicity (1µM–1mM). In DSS-treated mice, 10 mg/bwkg LDN-071 prevented body weight loss, bloody diarrhea, colon shortening, and spleen enlargement. Histological analysis showed that PAI-1 inhibition significantly reduced severe inflammation, ulceration, crypt structure loss, and goblet cell depletion in acute and chronic colitis models. Additionally, PAI-1 inhibition reduced mucosal TNF-α, IL-1β, and IL-6 expression during acute inflammation. In chronic colitis, LDN-071 decreased inflammation, epithelial erosion, and fibrosis while significantly lowering collagen deposition. Importantly, no toxicity was observed at 100 mg/bwkg LDN-071 for seven days. In vitro, LDN-071 reduced pro-inflammatory cytokine expression in HCOs from IBD patients and significantly decreased fibrosis-related gene expression in patient-derived FB cultures. Conclusion Our results showed that LDN-071, a novel amino acid-based inhibitor of PAI-1, significantly ameliorated the severity of acute and chronic colitis in mice. However, the LDN-071 reduced the expression of inflammation- and fibrosis-related genes and proteins in vitro human models. We propose that the inhibition of PAI-1 could be a potential novel therapeutic strategy in IBD.

P0124 Therapeutic Potential of Adipose-Derived Extracellular Vesicles for Inflammation Modulation and Mucosal Healing in Crohn’s Disease

Abstract Background Crohn’s disease (CD) is a chronic, idiopathic inflammatory bowel disease characterised by discontinuous lesions and transmural inflammation that can affect the entire gastrointestinal tract. Despite recent therapeutic advancements, around 50% of patients with CD require at least one surgical intervention within ten years of diagnosis, with 80% of these patients developing new lesions following surgery.1 Emerging evidence highlights that the therapeutic efficacy of mesenchymal stem cells (MSCs) is predominantly attributed to the extracellular vesicles (EVs) they secrete.2 These nanovesicles have gained recognition as critical mediators of anti-inflammatory responses and tissue repair. This research seeks to develop MSC-derived EV-based acellular therapy to regulate the immune response, heal the intestinal mucosa, restore gut microbiota balance, and ultimately reduce disease severity and recurrence. Methods Inclusion criteria for selecting healthy donors for obtaining EVs derived from subcutaneous adipose tissue MSCs include sex, age &amp;lt;40 years, and BMI &amp;lt;25 kg/m². Exclusion criteria include smokers, alcohol consumers, and patients with cancer or other chronic conditions. EV isolation was performed using size-exclusion chromatography, and characterisation was conducted via flow cytometry and electron microscopy (Cryo-TEM). To assess the therapeutic properties of the EVs, biopsies of inflamed perilesional adipose tissue and intestinal mucosa from CD patients were treated with a dose of 150 µg/mL for 20 hours. Finally, the gene expression profile of pro-inflammatory markers (TNFα, IL1B, IL6, and CCL2) and anti-inflammatory markers (IL10) was analysed by qPCR. Results The isolation and characterization of EVs were successfully demonstrated, along with their functional efficacy in both adipose and intestinal explant models. Explants treatment with EVs resulted in a significant reduction in pro-inflammatory cytokines, with IL-1β and TNF-α expression decreased by up to 200-fold compared to untreated explants, while also promoting a notable increase in the anti-inflammatory marker IL-10. Conclusion Adipose tissue-derived EVs could regulate inflammation and promote the regeneration of the intestinal barrier, thus reducing the severity of CD and lowering the risk of post-surgical recurrence. References 1Golovics PA, Mandel MD, Lovasz BD, Lakatos PL. Inflammatory bowel disease course in Crohn’s disease: Is the natural history changing? World J Gastroenterol. 2014;20(12):3198-3207. 2.Zhou Y, Yamamoto Y, Xiao Z, Ochiya T. The immunomodulatory functions of mesenchymal stromal/stem cells mediated via paracrine activity. J Clin Med. 2019;8(1)

P0170 The presence of creeping fat in a TNBS-induced colitis model is associated with greater acute inflammation and bacterial translocation

Abstract Background The hyperplasia of the mesenteric adipose tissue in Crohn’s disease, known as creeping fat, is associated with transmural lesions and the translocation of microbial antigens. In a rat model of TNBS-induced colitis, 50% of the animals presented adipose hyperplasia. Macroscopic evaluations showed that these animals had a more advanced progression of the disease, and colonoscopy could predict the presence of fat two days before euthanasia (L Clua et al, ECCO, 2024). Methods Colitis was induced using TNBS (n=10; half female), with the animals being sacrificed after five days. Tissue from the lesion along with mesenteric adipose tissue was obtained for histopathological staining and evaluation (haematoxylin-eosin, Masson’s trichrome, and Gram). Samples of intestinal mucosa and mesenteric fat were preserved in RNAlater to assess the expression of inflammatory markers by qRT-PCR. Results were statistically compared between animals with and without creeping fat, including sex. Results In histopathological analysis, using a validated scoring system, animals with creeping fat showed significantly greater severity of acute inflammation at the transmural level, with an increase in cryptitis and a more pronounced and extensive fibrosis pattern. Additionally, colonies of Gram-positive cocci were detected in all animals with creeping fat. In contrast, animals without fat hyperplasia exhibited appreciable adipogenesis in only two individuals, with an average of 105 adipocytes per field, compared to 185 in animals with creeping fat. qRT-PCR revealed higher expression of Il1b, Il6, Il10, Nod2, and Tlr2 in the intestinal mucosa and hyperplastic mesenteric fat of animals with creeping fat. A significant increase in Tnfa was also observed in hyperplastic adipose tissue, while the expression of Tgfbr1 was higher in the intestinal mucosa of animals without creeping fat. No differences were found between sexes. Conclusion The results indicated a more intense acute inflammatory and fibrotic component in animals with creeping fat, consistent with the macroscopic observations. The hyperplasia and inflammation observed in the mesenteric adipose tissue may be provoked by bacterial translocation, whereas animals without hyperplasia present a component influenced by TGF-β1 activity that could help counteract the inflammation. References L Clua, R Suau, M Guasch, D Monfort-Ferré, I Vañó, C Serena, E Domènech, R Bartolí, J Manyé, P022 Development of a novel experimental model for studying creeping fat in Crohn’s Disease, Journal of Crohn’s and Colitis, Volume 18, Issue Supplement_1, January 2024, Pages i275–i276, https://doi.org/10.1093/ecco-jcc/jjad212.0152

Investigation of the Mechanism of Pachyman against Gout Arthritis with Network Pharmacology Analysis and Verification In Vivo.

Pachyman, derived from Poria cocos, has been used to treat gouty arthritis (GA) for thousands of years, although its precise role and mechanisms remain unclear. Herein, we investigate the therapeutic effects of pachyman on GA and explore their underlying mechanisms. Network pharmacology and experimental methods were employed to investigate the therapeutic mechanisms of pachyman against GA. The protein-protein interaction network of shared targets between pachyman and gout was constructed. Furthermore, we elucidated the functions and mechanisms of pachyman against GA. Subsequently, we validated the predicted mechanisms from an experiment on rats. The treatment of GA with pachyman primarily related to tumor necrosis factor (TNF), matrix metalloproteinases (MMP), and relaxation factor signaling pathways. In the experimental validation, pachyman were found to regulate the expression of IL-1β, TNF-α, TGF-β, superoxide dismutase, and glutathione peroxidase of hyperuricemic rats. These collective findings suggest that pachyman holds promise as an alternative treatment for GA.

NLRP3 is a BMI-independent mediator of stable COPD

PurposeThe inflammatory response in animal models of chronic obstructive pulmonary disease (COPD) is activated by the NLR-family-pyrin-domain-containing-3 (NLRP3) inflammasome pathway, which is also known to play a role in obesity-related inflammation. The NLRP3/caspase-1/interleukin (IL)-1β pathway might be involved in the progression of COPD with increasing body mass index. To our knowledge, no previous studies have explored the role of NLRP3 inflammasome markers in linking COPD and obesity. Here, we aim to investigate this potential connection by examining levels of NLRP3, caspase-1, IL-1β, and IL-17A and to provide additional data on the expression of these molecules in relation to smoking status and COPD severity.MethodsA case‒control study was conducted between July 2020 and March 2023. Peripheral blood mononuclear cells were isolated, and total RNA was extracted for real-time quantitative polymerase chain reaction (qPCR) analysis to measure the expression levels of inflammasome molecules.Results29 subjects who were diagnosed with stable COPD and 32 controls were included in the data analysis. NLRP3 and IL-17A but not caspase-1 or IL-1β expression was significantly greater in the COPD group than in the control group. We detected a significant increase in NLRP3 levels in the smoker COPD group (p = 0.009) and nonsmoker COPD group (p = 0.045) compared with those in the nonsmoker control group. There was no significant correlation between BMI and the inflammasome markers.ConclusionAs proinflammatory biomarkers, NLRP3 and IL-17A are prominent in stable COPD patients. Smoking may trigger NLRP3-mediated inflammation in stable COPD patients. The expression levels of NLRP3 inflammasome molecules did not differ in terms of disease severity or BMI.Graphical

Shear-Stress Initiates Signal Two of NLRP3 Inflammasome Activation in LPS-Primed Macrophages through Piezo1.

The innate immune system is tightly regulated by a complex network of chemical signals triggered by pathogens, cellular damage, and environmental stimuli. While it is well-established that changes in the extracellular environment can significantly influence the immune response to pathogens and damage-associated molecules, there remains a limited understanding of how changes in environmental stimuli specifically impact the activation of the NLRP3 inflammasome, a key component of innate immunity. Here, we demonstrated how shear stress can act as Signal 2 in the NLRP3 inflammasome activation pathway by treating LPS-primed immortalized bone marrow-derived macrophages (iBMDMs) with several physiologically relevant magnitudes of shear stress to induce inflammasome activation. We demonstrated that magnitudes of shear stress within 1.0 to 50 dyn/cm2 were able to induce ASC speck formation, while 50 dyn/cm2 was sufficient to induce significant calcium signaling, gasdermin-D cleavage, caspase-1 activity, and IL-1β secretion, all hallmarks of inflammasome activation. Utilizing NLRP3 and caspase-1 knockout iBMDMs, we demonstrated that the NLRP3 inflammasome was primarily activated as a result of shear stress exposure. Quantitative polymerase chain reaction (qPCR), ELISA, and a small molecule inhibitor study aided us in demonstrating that expression of Piezo1, NLRP3, gasdermin-D, IL-1β, and CCL2 secretion were all upregulated in iBMDMs treated with shear stress. This study provides a foundation for further understanding the interconnected pathogenesis of chronic inflammatory diseases and the ability of shear stress to play a role in their progression.

Priming and release of cytokine IL-1β in microglial cells from the retina.

The P2X7 receptor (P2X7R) for extracellular ATP is implicated in several forms of retinal degeneration, including diabetic retinopathy, age-related macular degeneration, and glaucoma. P2X7R stimulation can trigger release of master cytokine IL-1β from microglia in the brain and from macrophages, but evidence of release from retinal microglia is indirect. Isolated mouse and rat retinal microglia, and wholemounts from CX3CR1+/GFP mice, were examined to determine if ATP induced IL-1β release directly from retinal microglial cells and if it also primed expression of IL-1β on an mRNA and protein level. Isolated retinal microglia were ramified and expressed low levels of polarization markers unless provoked. Over 90% of isolated microglial cells expressed P2X7R, with cytoplasmic Ca2+ elevation following receptor stimulation. ATP induced a dose-dependent release of IL-1β from primed microglial cells that was blocked by P2X7R antagonist A839977 and emulated by agonist BzATP. P2X7R stimulation also primed Il1b mRNA in isolated microglia cells. BzATP increased IL-1β immunostaining and GFP fluorescence throughout lamina of retinal wholemounts from CX3CR1+/GFP mice. Some of the IL-1β and GFP signals colocalized, particularly in the outer retina, and in projections extending distally through photoreceptor layers. The inner retina had more microglia without IL-1β, and more IL-1β staining without microglia. Substantial IL-1β release was also detected from rat retinal microglial cells, but not optic nerve head astrocytes. In summary, this study implicates microglial cells as a key source of released IL-1β when levels of extracellular ATP are increased following retinal damage, and suggest a greater participation in the outer retina.

C-type natriuretic peptide suppresses VEGFa gene expression by attenuating IL6-STAT3 signal pathway in primary synovial fibroblasts from rat knee.

C-type natriuretic peptide (CNP) can be a new disease-modifying anti-osteoarthritis drug (DMOAD) candidate because intraarticular injection of CNP attenuates both articular cartilage degradation and persistent pain in a rat knee arthritis model. This study aimed to elucidate the underlying molecular mechanisms by which CNP protects the knee joint from osteoarthritic changes. Gene expression analyses indicated that CNP did not interfere with the expression of IL1β -responsive genes in rat primary synovial fibroblasts or the monocytic cell line, RAW264.7cells. In contrast, total RNA sequence analyses indicated that CNP negatively regulated the IL6-STAT3 signaling pathway and VEGFa gene expression in rat synovial fibroblasts. As previously indicated, IL6 induced phosphorylation of 705Tyr residue of STAT3 and its nuclear translocation to activate VEGFa gene expression; however, in this study, we showed that CNP induced phosphorylation of 727Ser residue and inhibited IL6-induced nuclear translocation of STAT3. Since the IL6 pathway has been shown to accelerate articular cartilage degradation and induce knee pain, our data suggest that CNP can act as a DMOAD by negatively regulating IL6-mediated proinflammatory signals in the knee joint.

High glucose induces pro-inflammatory polarization of macrophages by inhibiting immune-responsive gene 1 expression.

To investigate the effect of high glucose on macrophage polarization and the role of immune-responsive gene 1 (IRG1) in mediating its effect. RAW264.7 cells were transfected with IRG1-overexpressing plasmid or IRG1 siRNA via electroporation and cultured in either normal or high glucose for 72 h to observe the changes in cell viability and morphology using CCK-8 assay and phase contrast microscopy. The protein levels of IRG1, iNOS, Arg-1, IL-1β and IL-10 in the treated cells were detected with Western blotting, and the fluorescence intensities of iNOS and Arg-1 were detected using immunofluorescence assay. The protein levels of IL-1β and IL-10 in the culture medium were determined with ELISA. High glucose exposure significantly reduced IRG1 and Arg-1 expressions, increased iNOS and IL-1β expressions and IL-1β secretion, and decreased IL-10 level in RAW264.7 cells. Transfection with the IRG1-overexpressing plasmid provided the cells with obvious resistance to high glucose-induced changes in iNOS, Arg-1, IL-1β and IL-10, whereas IRG1 knockdown further enhanced the effects of high glucose exposure on Arg-1 expression and the expression and secretion of IL-10. High glucose promotes M1 polarization of the macrophages possibly through a mechanism to inhibit the expression of IRG1 protein, thus leading to chronic inflammatory response.

Anti-inflammatory Effects of the Fucoidan from Sea Cucumber Apostichopus japonicus.

Fucoidan from Apostichopus japonicus (Aj-FUC) has shown anti-inflammatory activity, whereas its mechanism was not explicated. This study investigated the anti-inflammatory potential and mechanism of the fucoidan from green and purple A. japonicus (G-FUC and P-FUC) in lipopolysaccharide (LPS)-treated RAW264.7 cells. Results showed that Aj-FUCs at 25-400µg/mL had no toxicity to cells after 24h stimulation and promoted cell phagocytic activity. ELISA results indicated that Aj-FUC reduced the nitric oxide (NO), tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), and IL-6 levels and increased IL-10 level. The Aj-FUC suppressed transcription of inflammatory-related genes (tnf-α, il-1β, il-6, nlrp3, inos, cox-2, tlr4, trif, and nf-κb) in LPS-treated RAW264.7 cells, among which G-FUC had stronger anti-inflammatory effects. Moreover, Aj-FUC upregulated the mRNA expression of autophagic genes (beclin1, lc3II, and lamp2). The immunoblotting and immunofluorescence analyses of Beclin-1 and LC3II supported that Aj-FUC enhanced autophagy activity. After autophagy inhibited by 3-methyladenine, the mRNA expressions of tnf-α, il-6, il-1β, and nlrp3 were significantly upregulated in LPS-induced cells treated with Aj-FUC, suggesting the suppressed inflammation by Aj-FUC mediated via autophagy. Summarily, the present study demonstrated that Aj-FUC showed anti-inflammatory effects by elevating autophagy activity in LPS-induced macrophages.

Exploring the Active Constituents of Andrographis paniculata in Protecting the Skin Barrier and the Synergistic Effects with Collagen XVII.

Andrographis paniculata is mainly used to treat skin inflammations, wounds, and infections. In this study, Andrographis Herba, the aerial part of the plant, was proven to increase the viability of UVB-damaged HaCat cells and reduce reactive oxygen species levels. The chemical composition of Andrographis Herba extract (AHE) was analyzed using UPLC-Q-TOF-MS, and diterpene lactones were identified as its primary constituents. Then, the fraction of diterpene lactones was prepared and exhibited similar effects to AHE. AHE, its diterpene lactones component, and its representative constituent andrographolide all decreased the expression of IL-1β, IL-6, and CDKN1A. Furthermore, the protective effects of AHE and its active ingredients on UVB-damaged epidermal stem cells were investigated. Notably, the combined treatment with andrographolide and collagen XVII enhanced the viability of UVB-damaged epidermal stem cells, increased the expression of stemness markers integrin β1 and p63, and decreased the expression of the differentiation marker keratin 10. This combination demonstrated significant synergy in maintaining skin homeostasis, which provides evidences for the development of skin-protective products.

Phlorofucofuroeckol-A: A Natural Compound with Potential to Attenuate Inflammatory Diseases Caused by Airborne Fine Dust.

Background and Objectives: Persistent exposure to airborne fine dust (FD) particles contributing to air pollution has been linked to various human health issues, including respiratory inflammation, allergies, and skin diseases. We aimed to identify potential seaweed anti-inflammatory bioactive reagents and determine their effects on systemic inflammatory responses induced by FD particles. Materials and Methods: While exploring anti-inflammatory bioactive reagents, we purified compounds with potential anti-inflammatory effects from the seaweed extracts of Ecklonia cava, Ecklonia stolonifera, and Codium fragile. Structural analyses of the purified compounds siphonaxanthin (Sx), fucoxanthin (Fx), dieckol (Dk), and phlorofucofuroeckol-A (PFF-A) were performed using NMR and LC-MS/MS. Results: Notably, these compounds, especially PFF-A, showed significant protective effects against FD-induced inflammatory responses in RAW 264.7 cells without cytotoxicity. Further investigation of inflammatory-associated signaling demonstrated that PFF-A inhibited IL-1β expression by modulating the NF-κB/MAPK signal pathway in FD-induced RAW 264.7 cells. Additionally, gene profiling revealed the early activation of various signature genes involved in the production of inflammatory cytokines and chemokines using gene profiling. Treatment with PFF-A markedly reduced the expression levels of pro-inflammatory and apoptosis-related genes and even elevated the Bmp gene families. Conclusions: These results suggested that PFF-A is a potential natural therapeutic candidate for managing FD-induced inflammatory response.

Methyl 3-Bromo-4,5-dihydroxybenzoate Attenuates Inflammatory Bowel Disease by Regulating TLR/NF-κB Pathways.

Inflammatory bowel disease (IBD) is characterized by uncontrolled, chronic relapsing inflammation in the gastrointestinal tract and has become a global healthcare problem. Here, we aimed to illustrate the anti-inflammatory activity and the underlying mechanism of methyl 3-bromo-4,5-dihydroxybenzoate (MBD), a compound derived from marine organisms, especially in IBD, using a zebrafish model. The results indicated that MBD could inhibit the inflammatory responses induced by CuSO4, tail amputation and LPS in zebrafish. Furthermore, MBD notably inhibited the intestinal migration of immune cells, enhanced the integrity of the gut mucosal barrier and improved intestinal peristalsis function in a zebrafish IBD model induced by trinitro-benzene-sulfonic acid (TNBS). In addition, MBD could inhibit ROS elevation induced by TNBS. Network pharmacology analysis, molecular docking, transcriptomics sequencing and RT-PCR were conducted to investigate the potential mechanism. The results showed that MBD could regulate the TLR/NF-κB pathways by inhibiting the mRNA expression of TNF-α, NF-κB, IL-1, IL-1β, IL6, AP1, IFNγ, IKKβ, MyD88, STAT3, TRAF1, TRAF6, NLRP3, NOD2, TLR3 and TLR4, and promoting the mRNA expression of IL4, IκBα and Bcl-2. In conclusion, these findings indicate that MBD could be a potential candidate for the treatment of IBD.

Astragaloside IV inhibits retinal pigment epithelial cell senescence and reduces IL-1β mRNA stability by targeting FTO-mediated m6A methylation.

Resistance to senescence in retinal pigment epithelial (RPE) cells can delay the progression of age-related macular degeneration (AMD). However, the mechanisms underlying RPE cell senescence remain inadequately understood, and effective therapeutic strategies are lacking. While astragaloside IV (Ast) has demonstrated anti-aging properties, its specific effects on RPE cell senescence and potential mechanisms are not yet fully clarified. This study aimed to explore the impacts of Ast on RPE cell senescence and to uncover the molecular mechanisms involved. The therapeutic efficacy of Ast was assessed using sodium iodate (NaIO3)-induced adult retinal pigment epithelial cell line 19 (ARPE-19) cell models and an AMD mouse model. To investigate the mechanisms by which Ast mitigated RPE cell senescence, RNA sequencing (RNA-seq), drug affinity responsive target stability-mass spectrometry (DARTS-MS), cellular thermal shift assay (CETSA), reverse transcription quantitative PCR (RT-qPCR), as well as western blotting were conducted. Ast significantly inhibited NaIO3-treated ARPE-19 cell senescence and protected against NaIO3-induced AMD in mice. RNA-seq analysis revealed that Ast significantly attenuated inflammation-related signaling pathways and reduced the mRNA levels of interleukin-1 beta (IL-1β). Specifically, Ast decreased the stability of IL-1β mRNA while enhancing its N6-methyladenosine (m6A) methylation. Furthermore, Ast directly interacted with fat mass and obesity-associated protein (FTO). Knockdown or pharmacological inhibition of FTO mitigated the senescence and IL-1β expression in NaIO3-treated ARPE-19 cells. FTO was essential for Ast to inhibit cellular senescence and IL-1β expression. Additionally, inhibition or knockdown of FTO conferred also provided resistance to AMD in the murine model. Our results indicated that Ast significantly attenuated RPE cell senescence and showed anti-AMD properties. FTO was demonstrated to be a promising therapeutic target for AMD treatment. These findings may provide a deeper understanding of the molecular mechanisms underlying RPE cell senescence in AMD and offer potential strategies for its prevention and management.

Modulation of Intestinal Inflammation and Protection of Dopaminergic Neurons in Parkinson’s Disease Mice through a Probiotic Formulation Targeting NLRP3 Inflammasome

Emerging evidence highlights the significance of peripheral inflammation in the pathogenesis of Parkinson’s disease (PD) and suggests the gut as a viable therapeutic target. This study aimed to explore the neuroprotective effects of the probiotic formulation VSL#3® and its underlying mechanism in a PD mouse model induced by MPTP. Following MPTP administration, the striatal levels of dopamine and its metabolites, as along with the survival rate of dopaminergic neurons in the substantia nigra, were significantly reduced in PD mice. MPTP also significantly increased the mRNA expression of pro-inflammatory cytokines TNF-α and IL-1β, while reducing anti-inflammation mediators, like glia cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) in the striatum. These pathological changes were notably mitigated by VSL#3® treatment, suggesting its neuroprotective and anti-inflammatory effects in the brain. Additionally, VSL#3® significantly lowered the circulating levels of pro-inflammatory cytokines, and reduced TNF-α and IL-1β mRNA expression in the liver, indicating an inhibition of cytokine transfer. In the intestine, the probiotic treatment markedly decreased the mRNA expression of pro-inflammatory cytokines, (TNF-α, IL-1β, IL-6 and IL-17), and the other two key components of the NLRP3 inflammasome, caspase-1 and NLRP3, demonstrating an inhibition of VSL#3® on gut NLRP3 inflammasome. VSL#3® exerts neuroprotective effects in PD mice through the suppression of intestinal inflammation, particularly inhibiting the intestinal NLRP3 inflammasome. This study supports the therapeutic potential of targeting intestinal inflammation and utilizing probiotics in PD treatment.