TLR4 Expression Research Articles

Pomegranate polyphenol punicalin ameliorates lipopolysaccharide-induced memory impairment, behavioral disorders, oxidative stress, and neuroinflammation via inhibition of TLR4-NF-кB pathway.

Neuroinflammation may play an important role in the development of Alzheimer's disease (AD). Previous studies have reported that lipopolysaccharide (LPS)-induced neuroinflammation causes memory impairments and behavioral disorders. We investigated the potential preventive effects of punicalin (PUN), a polyphenolic component of pomegranate, on LPS-induced memory deficiency and anxiety- and depression-like behaviors, along with the underlying mechanisms. LPS-treated cultured microglial BV2 cells and BV2 cell/Neuro-2a (N2a) cell coculture system were investigated for anti-neuroinflammatory effects of PUN in vitro. The in vivo experiments involved mice administered a 4-week course of oral gavage with 1500 mg/kg/d PUN before intraperitoneal LPS (250 mg/kg daily 7 times) injections. The in vitro results demonstrated that PUN inhibited the LPS-induced inflammatory cytokine (IL-18, IL-1β, TNF-ɑ, and IL-6) production in BV2 cells and protected N2a cells from synaptic damage mediated by BV2 microglia-induced neuroinflammation. In in vivo studies, it was observed that PUN improved memory impairment and anxiety- and depression-like behaviors caused by LPS and reduced the expression of inflammatory proteins such as iNOS, COX-2, IL-1β, IL-2, IL-6, and TNF-α. Furthermore, PUN inhibited the LPS-induced production of MDA; increased the activities of CAT, SOD, and GSH-Px, and inhibited LPS-induced Aβ1-42 generation through down-regulation of APP and BACE1 expression. Moreover, PUN also suppressed the expression of TLR4, IRAK4, TRAF6, IKK-β, NF-κB, p65, and HMGB1 in LPS-treated mouse brain and cultured microglial BV-2 cells. These results suggest that PUN inhibits LPS-induced memory impairment via anti-inflammatory and anti-amylogenic mechanisms through inhibition of TLR4-NF-kB activation.

An Exploration of Some Predictors of Quality of Life-Related to the Innate Immune System, Inflammation, and Disease Activity in Patients with Behcet's Syndrome: An Analytical Cross-sectional Study.

Behçet's disease (BD) has a growing prevalence in Silk Road countries. The aim of our cross-sectional study was to explore the clinical and molecular predictors of quality of life in BD patients. One hundred and fifty consecutive Iranian BD patients with an age range between 20-50 years were included. The Leeds Behçet's disease quality of life (BDQoL) in Persian form was fulfilled to evaluate the quality of life. Anthropometric measurements were carried out using the calibrated scales. Iranian Behcet's Disease Dynamic Activity Measure (IBDDAM), Behcet's disease current activity form (BDCAF), and Total Inflammatory Activity Index (TIAI) were used to assess BD activity. mRNA expression of toll-like receptors 2 and 4 (TLR2 and TLR4) and tumor-necrosis-factor-alpha (TNF-α) levels in serum were measured by real-time polymerase chain reaction (PCR) and ELISA, respectively. Multiple linear backward regression at P = 0.1 was used to study the potential predictors of quality of life. TLR2 and BDCAF were shown to be the most important predictors of quality of life in BD patients by 22%. There were positive associations between them (β = 0.326, p = 0.013 for BDCAF; β = 0.366, p = 0.006 for TLR2) and BDQoL value. Higher TLR2 expression as a key protein in recognizing pathogens by innate immunity and BDCAF value as a comprehensive BD assessing scale contribute to poor quality of life among BD patients. Emphasizing therapeutically, approaches associated with lower TLR2 expression and BDCAF value can be considered in future studies.

The involvement of oxidative stress and the TLR4/NF-κB/NLRP3 pathway in acute lung injury induced by high-altitude hypoxia

ObjectiveThis study investigated the effect of oxidative stress and the TLR4/NF-κB/NLRP3 pathway on the pathogenesis of acute lung injury (ALI) induced by high-altitude hypoxia. MethodsRats were placed in an animal hyperbaric oxygen chamber to establish a rat model of ALI induced by high-altitude hypoxia after treatment with N-acetylcysteine (NAC; a reactive oxygen species [ROS] inhibitor) or/and MCC950 (an NLPR3 inflammasome inhibitor). After modeling, the wet-to-dry weight ratio (W/D) of rat lung tissues was calculated. In lung tissues, ROS levels were detected with immunofluorescence, the enzyme activity was tested with the kit, and the expression of TLR4/NF-κB/NLRP3 pathway-related genes and proteins was measured with western blotting and qRT-PCR. The levels of inflammatory factors in the serum were quantified with ELISA. ResultsAfter modeling, rats showed significantly increased W/D, ROS levels, and Malondialdehyde (MDA) concentrations and markedly diminished Superoxide dismutase (SOD) and Glutathione (GSH) concentrations in lung tissues (all P < 0.01), accompanied by substantially enhanced serum levels of TNF-α, IL-6, and IL-1β, significantly reduced serum levels of IL-10, and remarkably augmented TLR4, NLRP3, p-NF-κB p65, NF-κB p65 mRNA, and Caspase-1 expression in lung tissues (all P < 0.01). Furthermore, treatment with NAC or MCC950 alone or in combination prominently lowered the W/D of lung tissues (P < 0.01), serum levels of TNF-α (P < 0.05), IL-6 (P < 0.05), and IL-1β (P < 0.01), and NF-κB p65 expression and phosphorylation (P < 0.05, P < 0.01) while significantly increasing SOD and GSH concentrations (P < 0.05, P < 0.01) and serum levels of IL-10 (P < 0.01) in modeled rats. Meanwhile, treatment of NAC alone or combined with MCC950 significantly reduced MDA concentration and ROS levels (P < 0.05, P < 0.01) in modeled rats, and treatment of MCC950 alone or combined with NAC considerably declined TLR4, NLRP3, and Caspase-1 expression in modeled rats (P < 0.05, P < 0.01). ConclusionInhibition of oxidative stress and the TLR4/NF-κB/NLRP3 pathway can ameliorate ALI in rats exposed to high-altitude hypoxia.

Abstract 148: Loss Of Tlr4 Signaling Inhibits Thrombus Resolution In A Non-monocyte Dependent Manner

Background: Loss of TLR4 signaling reduces Venous Thrombosis (VT) resolution. Leukocyte expression of TLR4 is elevated in humans undergoing acute to chronic DVT transformation. We hypothesized that TLR4 signaling in monocytes directs thrombus clearance. Methods: Wild type, Tlr4 -/- , LysM cre+/- Tlr4 fl/fl mice underwent VT via IVC stasis at 4/7/14d timepoints. Thrombi subtypes were identified by flow cytometry. Thrombus size was assessed, and fibrinolytic mediators were assayed in splenic/bone marrow Mφs. Whole blood from mice was stimulated with TLR2/4 and assayed for thrombolytic potential via HALO assay. Immunohistochemistry was performed for cellular infiltrate. Results: We observed elevated TLR4 cell surface expression on CD11b+/Ly6C- Mo/Mφ from wild type thrombi. Thrombus weight in Tlr4 -/ mice was elevated at 4d stasis, confirming impaired thrombus clearance. In basal conditions, circulating Mo/Mφ saw decreased Urokinase &amp; MMP9 mRNA levels in LysM cre+/- mice. In post thrombotic conditions, we saw decreased protein levels of Urokinase and MMP9 in Cre+ bone marrow-derived Mφs. Whole blood thrombolysis efficiency increased with TLR4 agonism in Cre- mice compared to Cre+. Efficiency was restored with LPS-mediated dual TLR2/4 agonism. Total thrombus weight was unchanged. No difference was identified in monocyte infiltrate into the thrombus (Fig. 1) Conclusions: Genetic deficiency of Mo/Mφ TLR4 signaling alters induction of fibrinolytic/matrix remodeling gene expression. Ex vivo TLR4 agonism increased whole blood thrombolysis in Cre- but not Cre+ mice. Additional TLR2 agonism of Cre+ mice restored thrombolytic activity, suggesting TLR2 as a compensatory pathway that may account for the unchanged in vivo phenotype in LysMcreTlr4fl/fl mice. Further clarifying these cellular pathways may provide an alternate pathway to target VT resolution.

ANGPTL3 accelerates atherosclerotic progression via direct regulation of M1 macrophage activation in plaque

IntroductionThe N-terminal domain of angiopoietin-like protein 3 (ANGPTL3) inhibits lipoprotein lipase activity. Its C-terminal fibrinogen-like (FBN) domain is a ligand of macrophage integrin αvβ3. ObjectivesANGPTL3 might home to plaque where it directly regulates macrophage function via integrin αvβ3 for atherosclerosis progression. MethodsLdlr-/- mice on a high-fat diet and ApoE-/- mice on a chow diet were received adeno-associated virus (AAV)-mediated Angptl3 gene transfer and followed up for 12 weeks. ApoE-/- mice were injected AAV containing FLAG-tagged Angptl3 cDNA for tracing. Atherosclerotic features were compared between Angptl3-/-ApoE-/- mice and ApoE-/- littermates. THP-1 cells were exposed to 0 or 50 μg/ml ANGPTL3 FBN domain for 24 h to evaluate Toll-like receptor (TLR)4 expression using western blot analysis and circulating cytokine and chemokine profiles by the MILLIPLEX MAP assay. Phospho-proteomic profile was established in ANGPTL3-treated macrophages. Integrin β3 deficient THP-1 cells were obtained by sgRNAs targeting RGD sequence using Lentivirus-Cas9 system. ResultsAngptl3 overexpression increased atherosclerotic progression and CD68+ macrophages in plaque (p < 0.05 for all). By immunostaining, FLAG+ cells were identified in plaque of gene transferred ApoE-/- mice. Fluorescent immunostaining detected co-localisation of Angptl3 and CD68 in plaque macrophages. Phospho-proteomic analysis revealed that Angptl3 induced phosphorylation of proteins that were involved in the IL-17 signalling pathway in THP-1 cells. In vitro, ANGPTL3 treatment increased the production of interleukin (IL)-1β and tumour necrosis factor-α in THP-1 cells (p < 0.05 for both). Exposure of ANGPTL3 to THP-1 cells induced Akt phosphorylation which was weakened in integrin β3 deficient ones. ANGPTL3 elevated TLR4 expression via Akt phosphorylation. In response to lipopolysaccharide, nuclear factor-κB activity was 2.2-fold higher in THP-1 cells pre-treated with ANGPTL3 than in untreated cells (p < 0.05). ConclusionsTargeting ANGPTL3 could yield a dual benefit of lowering lipid levels in the blood and suppressing macrophage activation in plaque.

Bifidobacterium bifidum inhibits the TNF-α induced increase in intestinal epithelial tight junction permeability via a TLR-2/TLR-6 and PPAR-γ dependent inhibition of NF-κB p50/p65

Background: Bifidobacterium bifidum (BB) is one of the most widely used probiotic bacterial species and BB has been shown to have a protective effect against intestinal inflammation by an enhancement of the intestinal epithelial tight junction (TJ) barrier. Previous studies from our laboratory showed that a specific BB strain, referred to as BB1, caused a marked enhancement of intestinal epithelial TJ barrier, while strain BB4 had no effect. TNF-α contributes to the development of intestinal inflammation in inflammatory conditions of the gut, in part by increasing intestinal permeability and antigenic penetration. TNF-α induced increase in intestinal epithelial TJ permeability is mediated by an NF-κB p50/p65 activation of myosin light chain kinase (MLCK) gene and MLCK-induced opening of the intestinal TJ barrier. Peroxisome proliferator-activated receptor gamma (PPAR-γ) is an important nuclear receptor known to have anti-inflammatory activity by interfering with NF-κB activation. However, the protective effect of BB1 against TNF-α induced increase in intestinal TJ permeability remain unclear. Aims: The major purpose of this study was to delineate the protective effect of BB1 against the TNF-α induced increase in intestinal TJ permeability and to elucidate the intracellular mechanism involved. Methods: Filter-grown Caco-2 monolayers ( in vitro) and recycling intestinal perfusion of live mice ( in vivo) were the model systems used to assess intestinal TJ permeability. Results: 1) TNF-α caused an increase Caco-2 TJ permeability, as assed by Caco-2 trans-epithelial resistance (TER) and trans-epithelial flux of paracellular marker, FITC-Dextran 10kD. 2) Addition of BB1 (1x108 CFU/ml) to the apical membrane compartment inhibited the TNF-α increase in Caco-2 epithelial TJ permeability, while strain BB4 had no effect. 3) BB1 but not BB4, caused an increase in Caco-2 TLR-2 expression on the apical membrane surface, and siRNA induced knockdown of TLR-2 prevent the BB1 inhibition of TNF-α induced increase in Caco-2 TJ permeability. 4) TNF-α caused a rapid phosphorylation and degradation of Caco-2 IKK-α, as well as and phosphorylation and nuclear translocation of NF-κB p50/p65, indicating NF-κB activation. BB1, but not BB4, inhibited the NF-κB activation. 5) TNF-α activation of NF-κB was associated with an increase in MLCK gene (increase in MLCK promoter activity and mRNA transcription) and kinase activity. BB1, but not BB4, inhibited the TNF-α induced increase in MLCK gene and kinase activity. 6) BB1 but not BB4, caused a rapid PPAR-γ activation (PPAR-γ phosphorylation and nuclear translocation), and siRNA induced knockdown of PPAR-γ inhibited the TNF-α induced activation of NF-κB and increase in Caco-2 TJ permeability. 7) Intraperitoneal injection of TNF-α (5 mg) also caused time-dependent increase in mouse small intestinal permeability (IP) to FITC-Dextran 10kD. TNF-α caused a degradation of IkB-α in intestinal tissue and nuclear translocation of NF-kB in mouse enterocyte. 8) BB1, but not BB4, selectively activated PPAR-γ in mouse enterocytes and inhibited the TNF-α-induced activation of NF-κB, resulting in a decrease in mouse IP. 9) BB1 inhibition of TNF-α activation of mouse enterocyte NF-κB and increase in IP was prevented in mice with intestinal epithelial cell-specific knockout of PPAR-γ Villin-cre and TLR-2 Villin-cre mice. Conclusion: BB1 protects against the TNF-α induced increase in intestinal TJ permeability in a strain-specific manner. BB1 protects against the TNF-α induced increase in intestinal TJ permeability via a novel intracellular mechanism involving BB1/TLR-2 pathway activation of PPAR-γ, and PPAR-γ mediated inhibition of NF-κB p50/p65 and MLCK gene in enterocyte. This research project was supported by the National Institute of Diabetes and Digestive and Kidney Diseases R01-DK-121073-01. 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.

Exploring the efficacy and mechanism of Bailing capsule to improve polycystic ovary syndrome in mice based on intestinal-derived LPS-TLR4 pathway

Ethnopharmacological relevancePolycystic ovary syndrome (PCOS) is a common endocrine disorder associated with reproductive dysfunction and metabolic abnormalities, particularly characterized by insulin resistance and chronic low-grade inflammation. Multiple clinical studies have clearly demonstrated the significant efficacy and safety of the combination of Bailing capsules (BL) in the treatment of PCOS, but its pharmacological effects and mechanisms still require further study. Aim of the studyTo evaluate the effect of BL on improving PCOS in mice and explore the mechanism. MethodsIn this study, Dehydroepiandrosterone (DHEA) injection was administered alone and in combination with a high-fat and high-sugar diet to induce PCOS-like mouse. They were randomly divided into five groups: normal group (N), PCOS group (P), Bailing capsule low-dose group (BL-L), Bailing capsule high-dose group (BL-H) and Metformin + Daine-35 group (M + D). Firstly, the effects of BL on ovarian lesions, serum hormone levels, HOMA-IR, intestinal barrier function, inflammation levels, along with the expression of IRS1, PI3K, AKT, TLR4, Myd88, NF-κB p65, TNF-α, IL-6, and Occludin of the ovary, liver and colon were investigated. Finally, the composition of the gut microbiome of fecal was tested. ResultsThe administration of BL significantly reduced body weight, improved hormone levels, improved IR, and attenuated pathological damage to ovarian tissues, up-regulated the expression of IRS1, PI3K, and AKT in liver. It also decreased serum LPS, TNF-α, and IL-6 levels, while downregulating the expression of Myd88, TLR4, and NF-κB p65. Additionally, BL improved intestinal barrier damage and upregulated the expression of Occludin. Interestingly, the abundance of norank_f__Muribaculacea and Lactobacillus was down-regulated, while the abundance of Akkermansia was significantly up-regulated. ConclusionThe results of the study showed that BL exerts a treatment PCOS effect, which may be related to the modulation of the gut microbiota, the improvement of insulin resistance and the intestinal-derived LPS-TLR4 inflammatory pathway. Our research will provide a theoretical basis for the clinical treatment of PCOS.

Artemisia annua L. polysaccharide improves the growth performance and intestinal barrier function of broilers challenged with Escherichia coli.

With the high intensification of poultry breeding, a series of diseases caused by pathogenic bacteria threaten the health of poultry and human. Among them, poultry diseases induced by Escherichia coli cause significant economic loss every year. The aim of this study was to investigate the effects of dietary supplementation with Artemisia annua L. polysaccharide (AAP) on the growth performance and intestinal barrier function of broilers with Escherichia coli (E. coli) challenge. A total of 256 one-day-old chicks were randomly assigned to four treatment groups: control group (fed basal diet), AAP group (fed basal diet supplemented with AAP), E. coli group (fed basal diet and orally administered E. coli), AAP + E. coli group (fed basal diet supplemented with AAP and orally administered E. coli). Dietary AAP supplementation elevated the BW, ADG and ADFI in non-challenged broilers. AAP also increased the apparent metabolic rate of EE and Ca in E. coli-challenged broilers. Moreover, AAP not only enhanced the serum IgA content but also decreased the serum and jejunum content of IL-6, as well as the jejunum level of IL-1β in non-challenged broilers. AAP also down-regulates the mRNA level of inflammatory factors (IL-1β, IL-6, and TNF-α) by inhibiting the mRNA expression of TLR4 and MyD88 in intestinal NF-κB signaling pathway of E. coli-challenged broilers. Meanwhile, AAP up-regulates the activity and mRNA level CAT by down-regulating the mRNA level of Keap1 in intestinal Nrf2 signaling pathway of E. coli-challenged broilers, and decreased serum MDA concentration. AAP significantly elevated the mRNA level of CAT, SOD and Nrf2 in jejunal of non-challenged broilers. Interestingly, AAP can improve intestinal physical barrier by down-regulating serum ET content, increasing the jejunal villus height/crypt depth (VH/CD) and ZO-1 mRNA level in broilers challenged by E. coli. AAP also elevated the VH/CD and the mRNA level of Occludin, ZO-1, Mucin-2 in non-challenged broilers. Importantly, AAP reshaped the balance of jejunum microbiota in E. coli-challenged broilers by altering α diversity and community composition. In summary, AAP ameliorated the loss of growth performance in broilers challenged with E. coli, probably by regulating the intestinal permeability and mucosa morphology, immune function, antioxidant ability, and microbiota.

Fusobacterium periodonticum BCT protein targeting glucose metabolism to promote the epithelial-mesenchymal transition of esophageal cancer cells by lactic acid

BackgroundThe cancer microbiota was considered the main risk factor for cancer progression. We had proved that Fusobacterium periodonticum (F.p) was higher abundance in Esophageal cancer(EC)tissues. Bioinformation analysis found that BCT was a key virulence protein of F.p. However, little is known about the role and mechanism of BCT in EC. This study aimed to recognize the key virulence protein of F.p and explore the mechanism of BCT in promoting EC.MethodsWe constructed a eukaryotic expression vector and purified the recombinant protein BCT. CCK8 used to analyzed the activity of EC after treated by different concentration of BCT. UPLC-MS/MS and ELISA used to detect the metabonomics and metabolites. The ability of migration and invasion was completed by transwell assay. RT-QPCR, WB used to analyze the expression of relevant genes.ResultsOur data showed that BCT was higher expression in EC tumor tissues (p < 0.05) and BCT in 20 µg/mL promoted the survival, invasion and migration of EC cells (EC109) (p < 0.05). Meanwhile, UPLC-MS/MS results suggested that BCT resulted in an augmentation of hypotaurine metabolism, arachidonic acid metabolism, glycolysis/gluconeogenesis, tryptophan metabolism, citrate cycle activity in EC109. The metabolic changes resulted in decreasing in glucose and pyruvate levels but increase in lactate dehydrogenase (LDH) activity and lactic acid (LA) as well as the expression of glucose transporter 1, Hexokinase 2, LDH which regulated the glycolysis were all changed (p < 0.05). The BCT treatment upregulated the expression of TLR4, Akt, HIF-1α (p < 0.05) which regulated the production of LA. Furthermore, LA stimulation promoted the expression of GPR81, Wnt, and β-catenin (p < 0.05), thereby inducing EMT and metastasis in EC109 cells.ConclusionAltogether, these findings identified that impact of BCT in regulation of glycolysis in EC109 and its involves the TLR4/Akt/HIF-1α pathway. Meanwhile, glycolysis increasing the release of LA and promote the EMT of EC109 by GPR81/Wnt/β-catenin signaling pathway. In summary, our findings underscore the potential of targeting BCT as an innovative strategy to mitigate the development of EC.

Antiperiodontitis impact of extract from edible herb Aster glehni and its bioactive compound, 3,5-dicaffeolyquinic acid

Periodontitis is a chronic oral disease caused by periodontal pathogenic bacteria, such as Porphyromonas gingivalis. The lipopolysaccharide of P. gingivalis (PgLPS) is a crucial virulence factor that contributes to the pathogenesis of periodontitis. The present study investigated the anti-inflammatory effects of extract from Aster glehni (AGE), a region-specific edible herb in Korea, and its main bioactive compound, 3,5-dicaffeolyquinic acid (3,5-DCQA), on PgLPS-induced inflammatory responses and the associated intracellular mechanisms. AGE significantly suppressed the expression of interleukin (IL)-1β and IL-6, while moderately inhibiting monocyte chemoattractant protein-1 (MCP-1) expression at both mRNA and protein levels in RAW 264.7 cells stimulated by PgLPS. Furthermore, AGE effectively attenuated mitogen-activated protein kinase (MAPK) phosphorylation and nuclear factor-κB (NF-κB) activation induced by PgLPS. In contrast, 3,5-DCQA partially inhibited the expression of IL-1β, IL-6, and MCP-1 at both mRNA and protein levels. Additionally, 3,5-DCQA appeared to attenuate MAPK phosphorylation and NF-κB activation without exhibiting clear dose-dependency. Moreover, AGE dose-dependently attenuated TLR2 and TLR4 expression, while 3,5-DCQA decreased both TLR2 and TLR4 expression without clear dose-dependency. AGE demonstrated effective inhibition of PgLPS-induced inflammatory responses, with 3,5-DCQA playing a partial role in the inhibitory potential of AGE.

The NeuroProtect Formula: A Preventive Approach to AD Targeting the HIF-1/PI3K-AKT Signaling Pathway Evaluated through In Vivo, In Vitro, and Network Pharmacology Approaches.

Alzheimer's Disease (AD) is a progressive neurodegenerative disorder with limited options for reversing its middle-to-late stages. Early intervention is crucial to slow down disease progression. This study aimed to investigate the potential of the NeuroProtect (NP) formula, a combination of geniposide and Panax notoginseng saponins, in preventing AD. We evaluated the effects of the NP formula on amyloid plaque accumulation, neuronal degeneration, and molecular signaling pathways using in vivo and in vitro models. To predict functional pathways and potential downstream targets of NP intervention, we employed network pharmacology. The preventative impact of the NP formula was assessed using APP/PS1 mice. We conducted HE staining, ELISA assay, Golgi staining, and immunohistochemistry to detect the protective effect of NP. Additionally, cell experiments were performed to assess cell activity and target protein expression. Network pharmacology analysis revealed 145 drug-disease interactions and identified 5 core active targets associated with AD. Molecular docking results demonstrated strong binding affinity between the components of the NP formula (GP, GN-Rb1, GN-Rg1, NS-R1) and target proteins (STAT3, HIF1A, TLR4, mTOR, VEGFA). Notably, the binding energy between NS-R1 and mTOR was -11.4kcal/mol. Among the top 10 enriched KEGG pathways, the HIF-1 and PI3K-AKT signaling pathways were highlighted. In vivo experiments demonstrated that the NP formula significantly ameliorated pathological changes, decreased the Aβ42/Aβ40 ratio in the hippocampus and cortex, and increased dendritic spine density in the CA1 region during the early stage of AD. In vitro experiments further illustrated the NP formula's ability to reverse the inhibitory effects of Aβ25-35 on cell viability and regulate the expression of Tlr4, Mtor, Hif1a, Stat3, and Vegfa. Our findings suggest that NP exhibits neuroprotective effects during the early stages of AD, positioning it as a potential candidate for AD prevention. The NP formula may exert its preventive effects through the HIF-1/PI3K-AKT signaling pathway, with mTOR identified as a key target.

Investigation of Potential Gut Health Biomarkers in Broiler Chicks Challenged by Campylobacter jejuni and Submitted to a Continuous Water Disinfection Program.

The exploration of novel biomarkers to assess poultry health is of paramount importance, not only to enhance our understanding of the pathogenicity of zoonotic agents but also to evaluate the efficacy of novel treatments as alternatives to antibiotics. The present study aimed to investigate potential gut health biomarkers in broiler chicks challenged by Campylobacter jejuni and subjected to a continuous water disinfection program. A total of 144 one-day-old hatched broiler chicks were randomly allocated to four treatment groups with four replicates each, according to the following experimental design: Group A received untreated drinking water; Group B received drinking water treated with 0.01-0.05% v/v Cid 2000™ (hydrogen peroxide, acetic acid and paracetic acid); Group C was challenged by C. jejuni and received untreated drinking water; and Group D was challenged by C. jejuni and received drinking water treated with 0.01-0.05% v/v Cid 2000™. The use of Cid 2000™ started on day 1 and was applied in intervals until the end of the experiment at 36 days, while the C. jejuni challenge was applied on day 18. Potential biomarkers were investigated in serum, feces, intestinal tissue, intestinal content, and liver samples of broilers. Statistical analysis revealed significant increases (p < 0.001) in serum cortisol levels in C. jejuni-challenged broilers. Serum fluorescein isothiocyanate dextran (FITC-d) increased significantly (p = 0.004) in broilers challenged by C. jejuni and treated with drinking water disinfectant, while fecal ovotransferrin concentration also increased significantly (p < 0.001) in broilers that received the drinking water disinfectant alone. The gene expression levels of occludin (p = 0.003) and mucin-2 (p < 0.001) were significantly upregulated in broilers challenged by C. jejuni, while mucin-2 significantly increased in birds that were challenged and received the drinking water disinfectant (p < 0.001). TLR-4 expression levels were significantly (p = 0.013) decreased in both groups that received the drinking water disinfectant, compared to the negative control group. Finally, the C. jejuni challenge significantly increased (p = 0.032) the crypt depth and decreased (p = 0.021) the villus height-to-crypt-depth ratio in the ileum of birds, while the tested disinfectant product increased (p = 0.033) the villus height in the jejunum of birds. Furthermore, the counts of C. jejuni in the ceca of birds (p = 0.01), as well as its translocation rate to the liver of broilers (p = 0.001), were significantly reduced by the addition of the water disinfectant. This research contributes to novel insights into the intricate interplay of water disinfection and/or C. jejuni challenge with potential intestinal biomarkers. In addition, it emphasizes the need for continued research to unveil the underlying mechanisms, expands our understanding of broiler responses to these challenges and identifies breakpoints for further investigations.

Competitive Inhibition of Okanin against Plasmodium falciparum Tyrosyl-tRNA Synthetase.

Malaria is a severe disease that presents a significant threat to human health. As resistance to current drugs continues to increase, there is an urgent need for new antimalarial medications. Aminoacyl-tRNA synthetases (aaRSs) represent promising targets for drug development. In this study, we identified Plasmodium falciparum tyrosyl-tRNA synthetase (PfTyrRS) as a potential target for antimalarial drug development through a comparative analysis of the amino acid sequences and three-dimensional structures of human and plasmodium TyrRS, with particular emphasis on differences in key amino acids at the aminoacylation site. A total of 2141 bioactive compounds were screened using a high-throughput thermal shift assay (TSA). Okanin, known as an inhibitor of LPS-induced TLR4 expression, exhibited potent inhibitory activity against PfTyrRS, while showing limited inhibition of human TyrRS. Furthermore, bio-layer interferometry (BLI) confirmed the high affinity of okanin for PfTyrRS. Molecular dynamics (MD) simulations highlighted the stable conformation of okanin within PfTyrRS and its sustained binding to the enzyme. A molecular docking analysis revealed that okanin binds to both the tyrosine and partial ATP binding sites of the enzyme, preventing substrate binding. In addition, the compound inhibited the production of Plasmodium falciparum in the blood stage and had little cytotoxicity. Thus, okanin is a promising lead compound for the treatment of malaria caused by P. falciparum.

S1PR3 suppresses the inflammatory response and extracellular matrix degradation in human nucleus pulposus cells.

Sphingosine 1-phosphate receptor 3 (S1PR3) participates in the inflammatory response in multiple types of diseases. However, the biological role of S1PR3 in intervertebral disc degeneration and the underlying mechanism are unclear. The aim of the present study was to investigate the functional role and the mechanism of S1PR3 in lipopolysaccharide (LPS)-induced human nucleus pulposus cells. The expression of S1PR3 and Toll-like receptor (TLR) 2 in LPS-induced nucleus pulposus (NP) cells was investigated using western blotting. The Cell Counting Kit-8 assay was used to detect cell proliferation, and the levels of inflammatory factors were detected using ELISA. Flow cytometry and western blotting were used for the assessment of apoptosis. The deposition of extracellular matrix (ECM) proteins was investigated using reverse transcription-quantitative PCR and western blotting. In addition, western blotting was used to investigate the protein expression levels of phosphorylated (p)-STAT3, STAT3, p-JNK, JNK, p-ERK, ERK, p-p38 and p38associated with STAT3 and MAPK signaling. S1PR3 expression was reduced, while TLR2 expression was elevated in LPS-induced human nucleus pulposus cells (HNPC). S1PR3 overexpression increased HNPC viability, inhibited the inflammatory response and suppressed apoptosis. Meanwhile, S1PR3 overexpression regulated the expression of ECM-related proteins. Additionally, overexpression of S1PR3 inhibited the expression of the TLR2-regulated STAT3 and MAPK pathways in LPS-induced HNPCs. Furthermore, TLR2 overexpression partially offset the impacts of S1PR3 overexpression on HNPC viability, apoptosis level, inflammation and as ECM degradation. In conclusion, STAT3 overexpression suppressed viability injury, the inflammatory response and the level of apoptosis and alleviated ECM protein deposition in HNPCs through the TLR2/STAT3 and TLR2/MAPK pathways, which may offer a promising candidate for the amelioration of intervertebral disc degeneration.

Exploring Pyroptosis-related Signature Genes and Potential Drugs in Ulcerative Colitis by Transcriptome Data and Animal ExperimentalValidation.

Ulcerative colitis (UC) is an idiopathic, relapsing inflammatory disorder of the colonic mucosa. Pyroptosis contributes significantly to UC. However, the molecular mechanisms of UC remain unexplained. Herein, using transcriptome data and animal experimentalvalidation, we sought to explore pyroptosis-related molecular mechanisms, signature genes, and potential drugs in UC.Gene profiles (GSE48959, GSE59071, GSE53306, and GSE94648) were selected from the Gene Expression Omnibus (GEO) database, which contained samples derived from patients with active and inactive UC, as well as health controls. Gene Set Enrichment Analysis (GSEA), Weighted Gene Co-expression Network Analysis (WGCNA) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed on microarrays to unravel the association between UC and pyroptosis. Then, differential expressed genes (DEGs) and pyroptosis-related DEGs were obtained by differential expression analyses and the public database. Subsequently, pyroptosis-related DEGs and their association with the immune infiltration landscape were analyzed using the CIBERSORT method. Besides, potential signature genes were selected by machine learning (ML) algorithms, and then validated by testing datasets which included samples of colonic mucosal tissue and peripheral blood. More importantly, the potential drug was screened based on this. And these signature genes and the drug effect were finally observed in the animal experiment.GSEA and KEGG enrichment analyses on key module genes derived from WGCNA revealed a close association between UC and pyroptosis. Then, a total of 20 pyroptosis-related DEGs of UC and 27 pyroptosis-related DEGs of active UC were screened. Next, 6 candidate genes (ZBP1, AIM2, IL1β, CASP1, TLR4, CASP11) in UC and 2 candidate genes (TLR4, CASP11) in active UC were respectively identified using the binary logistic regression(BLR), least absolute shrinkage and selection operator (LASSO), random forest (RF) analysis and artificial neural network (ANN), and these genes also showed high diagnostic specificity for UC in testing sets. Specially, TLR4 was elevated in UC and further elevated in active UC. The results of the drug screen revealed that six compounds (quercetin, cyclosporine, resveratrol, cisplatin, paclitaxel, rosiglitazone) could target TLR4, among which the effect of quercetin on intestinal pathology, pyroptosis and the expression of TLR4 in UC and active UC was further determined by the murine model.These findings demonstrated that pyroptosis may promote UC, and especially contributes to the activation of UC. Pyroptosis-related DEGs offer new ideas for the diagnosis of UC. Besides, quercetin was verified as an effective treatment for pyroptosis and intestinal inflammation. This study might enhance our comprehension on the pathogenic mechanism and diagnosis of UC and offer a treatment option for UC.

OA24 Age-associated T-bet+ B cells in healthy and autoimmune pregnancy

Abstract Background/Aims Inflammatory rheumatic diseases (IRD), including systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA), affect females of reproductive age and are characterised by loss of immune tolerance and an increased burden of adverse pregnancy outcomes (APO). Mechanisms by which IRD may affect immune adaptations of healthy pregnancy to maintain host and fetal defence whilst tolerating male fetal antigens are poorly characterised. This study aimed to investigate B cell subpopulations in healthy and IRD in pregnancy, with a focus on IgD-CD27- (double negative, DN) T-bet+ B cells, considered pathogenic in IRD. Given disparate relationships between disease activity and risk of APO in SLE and RA pregnancy, the objective of this pilot study was to determine if pregnancy alters the frequency and/or the phenotype of DN T-bet+ B cells in IRD compared with healthy control pregnancies. Methods Peripheral blood mononuclear cells were obtained from IRD pregnancy (SLE n = 5; RA n = 8), IRD non-pregnancy (SLE n = 12; RA n = 10), and healthy pregnant (n = 8) people at University College London Hospital by informed consent. Cells were analysed by flow cytometry to identify B cell subpopulations and their expression of multiple markers including T-bet, IFN-γR, TLR7, TLR9, Ki67, CD38 and CD19 was assessed. Results There were no significant differences in B cell subpopulations, categorised based on the expression of IgD and CD27, between the cohorts. In healthy pregnancy, the median frequency (%) of CD19+T-bet+, IgD-CD27+T-bet+, DNT-bet+ cells was 5, 1.3 and 2, respectively. In contrast, the median frequency of CD19+T-bet+ cells was significantly elevated in SLE pregnancy at 13% compared with 5% (p &amp;lt; 0.05) in healthy pregnancy[GI3] [RV4] . Similarly, the median frequency of DNT-bet+ B cells was elevated in SLE pregnancy at 6% compared with 2% (p = 0.009) in healthy pregnancy. RA pregnancy, but not SLE pregnancy, had significantly lower frequency of Ki67+ DN T-bet+ cells compared to non-pregnant RA (p=0.04) and SLE (p=0.07). ROC analysis demonstrated that for both RA (p = 0.016, AUC = 0.84, 95% CI [0.65, 1.00]) and SLE (p = 0.015, AUC=0.88) the frequency of DN T-bet+ Ki67+ B cells can discriminate between pregnancy and non-pregnancy samples. In pooled analysis (n = 43), but not in single cohorts, DN T-bet+ cells had significantly high TLR7 and TLR9 expression. High frequencies of CD19hi DN T-bet+ cells were found but were unaffected by pregnancy. Clustering revealed canonical DN T-bet+ clusters and CD38hi T-bet+ clusters enriched in SLE and pregnancy-altered. Conclusion We have immunophenotyped B cell populations in healthy pregnancy and identified different patterns of alteration in frequency and proliferation of DNTbet+ cells in IRD compared with healthy pregnancy that are disease specific. This pilot study reveals previously unrecognized immunological changes in disease pregnancy warranting further larger studies of their mechanistic basis and clinical relevance. Disclosure S. Benton: None. C. Raine: None. K. Shah: None. S. Luong: None. A. Venkatkrishnan: None. M. Leandro: None. M. Castelino: None. D. Sen: None. D.A. Isenberg: None. A. Akbar: None. I. Giles: Grants/research support; IG has received an unrestricted research grant, travel and speaker fees from UCB Pharma. V.R. Reddy: None.

P160 High levels of CD19 expression is a characteristic feature of age-associated B cells in ageing and autoimmunity

Abstract Background/Aims Recent evidence implicates an important role for age-associated B cells (ABCs), defined as CD19+IgD-CD27-T-bet+ B cells, in healthy ageing, following viral infection and in active SLE. We know that co-receptors of the B cell receptor (BCR) such as CD19 and other activating receptors can influence B cell activation through distinct pathways. Therefore, we compared the expression of CD19 in B cell subpopulations including ABCs in ageing and SLE. Methods Peripheral blood was obtained from six young (&amp;lt;45 years) healthy participants, seven elderly (&amp;gt;65 years) healthy participants and eight patients with SLE (25-65 years). We performed flow cytometry on peripheral blood mononuclear cells to analyse the expression of extracellular and intracellular markers. We performed statistical analysis using GraphPad Prism v7. Results CD19hi B cell frequency was at least 10-fold higher in T-bet+ cells as a whole and particularly enriched in IgD-CD27-T-bet+, ABCs, in all groups, p &amp;lt; 0.05. The geometric mean fluorescent intensity (gMFI, expression) of CD40 was significantly lower in IgD-CD27-T-bet+ subpopulations compared to IgD-CD27-T-bet- B cell subpopulations in all groups (Table). In all groups, in CD19+T-bet+ cells, compared to CD19+T-bet- cells, there was a significantly increased frequency of IFNγR+ cells, TLR4+ cells, TLR7+ cells and TLR9+ cells (p &amp;lt; 0.05 for all) with remarkable difference noted in SLE patients for TLR7 and TLR9, p &amp;lt; 0.01 (Table). In the switched memory IgD-CD27+ and ABCs, TLR7 expression was significantly higher (p &amp;lt; 0.01) in SLE patients (mean 43.8% and 18.5% respectively) compared to elderly participants (mean 13.5% compared to 4.3% respectively). Conclusion High levels of expression of CD19 is a characteristic feature of ABCs and CD19hiT-bet+ cells predominantly belong to the IgD-CD27-T-bet+ ABC subpopulation. CD19hi IgD-CD27-T-bet+ cells also had significantly lower expression of CD40 and higher expression of IFNγR and TLR7 and TLR9, suggesting that CD19 plays a significant role in moderating activation of ABCs triggered through T-independent, IFNγR-TLR-T-bet pathway. Taken together with the significant role of CD19 in regulating signalling threshold of B cell activation, and its stable expression across different B cell developmental stages, these data provide compelling mechanistic rationale for targeting CD19 to deplete B cells, including potentially pathogenic age-associated, in autoimmune disease such as SLE. Disclosure K. Shah: None. V. Poon: None. S.J. Taylor: None. C. Klein: Corporate appointments; CK is employed by Roche. Shareholder/stock ownership; CK has patents and stock ownership at Roche. F. Kollert: Corporate appointments; FK is employed by Roche. Shareholder/stock ownership; FK has stock ownership at Roche. F. Schuler: Corporate appointments; FS is employed by Roche. Shareholder/stock ownership; FS has patents and stock ownership at Roche. S. Mathur: None. M. Castelino: None. D. Sen: None. J. Cambridge: None. M. Cragg: None. M. Leandro: None. A. Akbar: None. V. Reddy: None.

Exosomes from MicroRNA-125b-Modified Adipose-Derived Stem Cells Promote Wound Healing of Diabetic Foot Ulcers.

Exosomes derived from Adipose-Derived Stem Cells (ADSCs-Exo) have been implicated in the enhancement of wound repair in Diabetic Foot Ulcers (DFU). The current research was designed to explore the therapeutic potential and underlying mechanisms of ADSCs-Exo modified with microRNA-125b (miR-125b) in the context of DFU. Rat models with DFU and human umbilical vein endothelial cells (HUVECs) subjected to high glucose (HG) conditions served as experimental systems and were administered miR-125b-engineered ADSCs-Exo. Then, the expressions of CD34, Ki-67, angiogenesis-related factors (VEGF and TGFβ-1), angiogenesis inhibitor DLL-4, and inflammation-related proteins (TLR-4 and IL-6) were detected. MiR-125b was upregulated in ADSCs-Exo. MiR-125b-mimics transfection in ADSCs- Exo reduced inflammatory infiltration and promoted granulation formation and wound healing in wound tissues. MiR-125b-mimics-modified ADSCs-Exo injection increased the expression of CD34, Ki-67, VEGF, and TGFβ-1, whereas decreased the expression of DLL-4, TLR-4, and IL-6 in wound tissues of DFU rats. In addition, miR-125b-mimics-ADSCs-Exo injection reversed the negative effects of HG on the proliferation, migration, and angiogenesis of HUVECs, as well as the positive effects of cell apoptosis. Moreover, miR-125b-inhibitor-ADSCs-Exo injection had the opposite effects to miR-125b-mimics-ADSCs-Exo. ADSCs-Exo promoted wound healing of DFU rats, especially when overexpressing miR-125b.

Infection of Human Macrophage-Like Cells by African Swine Fever Virus.

The African swine fever (ASF) virus (ASFV) and ASF-like viral sequences were identified in human samples and sewage as well as in different water environments. Pigs regularly experience infections by the ASFV. The considerable stability of the virus in the environment suggests that there is ongoing and long-term contact between humans and the ASFV. However, humans exhibit resistance to the ASFV, and the decisive factor in developing infection in the body is most likely the reaction of target macrophages to the virus. Therefore, this study aimed to characterize the responses of human macrophages to the virus and explore the distinct features of the viral replication cycle within human macrophages. The ASFV Armenia/07 strain was used in all experiments. In this study, quantitative real-time polymerase chain reaction (qRT-PCR) was used to determine the ASFV gene expression; flow cytometry analysis was performed to evaluate the effects of the inactive and active ASFV (inASFV and aASFV) treatments on the phenotype of THP-1-derived macrophages (Mφ0) and inflammatory markers. Moreover, other methods such as cell viability and apoptosis assays, staining techniques, phagocytosis assay, lysosome-associated membrane protein (LAMP-1) cytometry, and cytokine detection were used during experiments. Our findings showed that the virus initiated replication by entering human macrophages. Subsequently, the virus shed its capsid and initiated the transcription of numerous viral genes, and at least some of these genes executed their functions. In THP-1-derived macrophages (Mφ0), the ASFV implemented several functions to suppress cell activity, although the timing of their implementation was slower compared with virus-sensitive porcine alveolar macrophages (PAMs). Additionally, the virus could not complete the entire replication cycle in human Mφ0, as indicated by the absence of viral factories and a decrease in infectious titers of the virus with each subsequent passage. Overall, the infection of Mφ0 with the ASFV caused significant alterations in their phenotype and functions, such as increased TLR2, TLR3, CD80, CD36, CD163, CXCR2, and surface LAMP-1 expression. Increased production of the tumor necrosis factor (TNF) and interleukin (IL)-10 and decreased production of interferon (IFN)-α were also observed. Taken together, the virus enters human THP-1-derived macrophages, starts transcription, and causes immunological responses by target cells but cannot complete the replicative cycle. These findings suggest that there may be molecular limitations within human macrophages that at least partially restrict the complete replication of the ASFV. Understanding the factors that hinder viral replication in Mφ0 can provide valuable insights into the host-virus interactions and the mechanisms underlying the resistance of human macrophages to the ASFV.

Experimental study on the inhibition of the NLRP3 signaling pathway with Shengsan Jiedu Huayu decoction to alleviate inflammatory injury in rats with acute-on-chronic liver failure

Objective: To observe the therapeutic effect of Shengsan Jiedu Huayu decoction in alleviating inflammatory liver injury in rats with acute-on-chronic liver failure (ACLF) and its effect on the activation intensity for the NLRP3 signaling pathway. Methods: 63 SD rats were randomly divided into a blank group, a model group, and low-, medium-, and high-dose groups of Shengsan Jiedu Huayu decoction (7.29 g/kg/d, 14.58 g/kg/d, and 29.16 g/kg/d). The ACLF rat model was replicated using carbon tetrachloride combined with d-galactosamine and lipopolysaccharide. Different dose gradients of the Shengsan Jiedu Huayu decoction were used for a five-day intervention treatment, and then rat serum and tissue samples were collected. A biochemical analyzer was used to detect the serum levels of ALT, AST, and TBIL in rats. ELISA was used to detect serum IL-18 and IL-1β content. HE staining was used to observe histomorphological changes in liver tissue. Immunohistochemistry was used to detect GSDMD expression in liver tissue. Western blot and PCR were used to detect NLRP3, Caspase1, ASC, TLR4, IL-1β, IL-18 protein, and mRNA expression levels.The groups were compared using analysis of variance and the rank-sum test. Results: Compared with the blank group, the model group's rat liver tissue was severely injured. Serum levels of ALT, AST, and TBIL, inflammatory factors IL-1β and IL-18, and the GSDMD protein expression level, NLRP3 expression level, TLR4, caspase 1, ASC, IL-1β, IL-18 protein, and mRNA (P<0.01) were all significantly increased in the model than the blank group (P<0.01). Additionally, compared with the model group, the low-, medium-, and high-dose groups of Shengsan Jiedu Huayu decoction had improved liver tissue injury in ACLF rats, while the serum levels of ALT, AST, TBIL, IL-1β, IL-18, liver tissue GSDMD protein, NLRP3, TLR4, caspase 1, and ASC expressions were all lower in the different dose gradients of the Shengsan Jiedu Huayu decoction than the model group, with the most evident reduction in the high-dose group (P<0.01). Conclusion: Shengsan Jiedu Huayu decoction can weaken the activation intensity of the NLRP3 signaling pathway, alleviate liver tissue pathological injury, reduce inflammatory factor release, and alleviate inflammatory liver injury in ACLF rats.