Leukotriene B4 Research Articles

Effects of oral hyaluronic acid on monosodium iodoacetate-induced osteoarthritis in rats: mechanistic insights and therapeutic implications

AbstractThis study aimed to meticulously assess the effectiveness of hyaluronic acid (HA) in mitigating symptoms associated with monosodium iodoacetate (MIA)-induced osteoarthritis (OA) symptoms in rodent models and to investigate the underlying mechanistic pathways. Eight-week-old rats were randomly allocated to a normal control group and three experimental groups (n = 10 per group). The normal group did not undergo any treatment. The experimental groups were administered MIA for 1 week to induce osteoarthritis, and orally administered distilled water (control group), 2 mg/kg indomethacin (INDO group), or 20 mg/kg HA (HA20 group) daily for 4 weeks. The HA20 group showed a significant improvement in hind-paw weight-bearing distribution after 4 weeks compared to the control group. HA suppressed inflammatory responses by reducing the overproduction of prostaglandin E2 and leukotriene B4 and protected the vital components of the articular ECM, including glycosaminoglycans and aggrecan. HA treatment effectively reduced inflammation, protected cartilage by inhibiting MMP expression, and suppressed inflammatory mediator production. This study demonstrates that HA has potential to alleviate OA symptoms in a rodent model stimulated with MIA, rendering it a promising therapeutic agent for OA.

Neutrophil swarming is crucial for limiting oral mucosal infection by Candida albicans.

Oral mucosal colonization by C. albicans (Ca) is benign in healthy people but progresses to deeper infection known as oropharyngeal candidiasis (OPC) that may become disseminated when combined with immunosuppression. Cortisone use and neutropenia are risk factors for invasive mucosal fungal infections, however the mechanisms are poorly understood. Here we identify in vivo neutrophil functional complexes known as swarms that are crucial for preventing Ca epithelial invasion. Anti-Ly6G antibody treatment impaired swarm formation and increased fungal infection depth confirming the role of neutrophil swarms in limiting Ca invasion. Neutrophil swarm function could be disrupted by administration of resolvins, and required leukotriene B4 receptor 1 (BLT1) expression so that administration of a leukotriene synthesis inhibitor reduced neutrophil swarm size permitting Ca invasion beyond the basement membrane. Cortisone treatment similarly reduced neutrophil swarming behavior and BLT1 expression and delayed expression of epithelial cytokines and chemokines. Thus, swarm structures have an important function in preventing deep invasion by C. albicans within the oral mucosa and represent a mechanism for increased disease severity under immune deficient clinical settings.

Salidroside attenuates sepsis-induced acute lung injury by inhibiting ferroptosis-dependent pathway.

Sepsis triggers a systemic inflammatory response that can lead to acute lung injury (ALI). Salidroside (SAL) has many pharmacological activities such as antiinflammatory and anti-oxidation. The objective of the study was to explore the mechanism of SAL on ALI caused by sepsis. A model of ALI in septic mice was established by cecal ligation and puncture. Following SAL treatment, the effect of SAL on the ferroptosis pathway in mice was analyzed. The pathological damage of lung tissue, the levels of inflammatory factors and apoptosis in bronchoalveolar lavage fluid (BALF) of mice were evaluated, and the changes of gene expression level and metabolite content abundance were explored by combining transcriptomics and metabolomics analysis. The effect of SAL on ferroptosis in mice with lung injury was observed by intraperitoneal injection of ferroptosis activator Erastin or ferroptosis inhibitor Ferrostatin-1 to promote or inhibit ferroptosis in mice. SAL significantly alleviated the pathological damage of lung tissue, decreased the number of TUNEL positive cells and the levels of TNF-α, IL-1β, IL-6 in BALF, and increased the level of IL- 10 in lung injury mice. Moreover, the Fe2+ content and malondialdehyde decreased significantly, the reactive oxygen species and glutathione content increased significantly, and the arachidonic acid metabolites 20-hydroxyeicosatetraenoic acid (20- HETE), (5Z, 8Z, 10E, 14Z)-12-Oxoeicosa-5,8,10,14-tetraenoic acid (12-OxOETE), (5Z, 8Z, 10E, 14Z)-(12S)-12-Hydroxyeicosa-5,8,10,14-tetraenoic acid (12(S)-HETE), (5Z, 8Z, 14Z)-11,12-Dihydroxyeicosa-5,8,14-trienoic acid (11,12-DHET), (5Z, 11Z, 14Z)-8,9- Dihydroxyeicosa-5,11,14-trienoic acid, Leukotriene B4, Leukotriene D4 were significantly up-regulated after SAL treatment. Salidroside alleviates ALI caused by sepsis by inhibiting ferroptosis.

Acute inflammation upregulates FAHFAs in adipose tissue and in co-cultured adipocytes

Since the discovery of fatty acid hydroxy fatty acids (FAHFAs), significant progress has been made in understanding their regulation, biochemistry, and physiological activities. Here, we contribute to this understanding by revealing that inflammation induces the production of fatty acid hydroxy stearic acids (FAHSAs) and fatty acid hydroxyoctadecadienoic acids (FAHODEs) in white adipose tissue depots and in adipocytes co-cultured with macrophages. In LPS-induced co-culture systems, we confirm that adipose triglyceride lipase (ATGL) is required for inflammation-induced FAHFA generation and demonstrate that inflammation is necessary for producing hydroxy fatty acids. Chemically synthesized FAHODEs show anti-inflammatory activities in vivo, but only at supraphysiological concentrations. While endogenous FAHFAs are unlikely to be anti-inflammatory due to their low concentrations, conversion of pro-inflammatory hydroxy fatty acids into FAHFAs may modulate inflammation. We test this concept by showing the pro-inflammatory lipids—hydroxyeicosatetraenoic acids (HETEs) and leukotriene B4 (LTB4)—are converted into FAHFAs in cell culture, and that two LTB4-derived FAHFAs have are modestly anti- not pro-inflammatory. Further research is needed to establish whether these increased FAFHA levels have a role in inflammation or are simply markers of inflammation, but the discovery of significant increases in FAHFA upon acute inflammation advances our knowledge of FAHFAs.

Immunomodulatory metabolites in IgE-mediated food allergy and oral immunotherapy outcomes based on metabolomic profiling.

The immunometabolic mechanisms underlying variable responses to oral immunotherapy (OIT) in patients with IgE-mediated food allergy are unknown. To identify novel pathways associated with tolerance in food allergy, we used metabolomic profiling to find pathways important for food allergy in multiethnic cohorts and responses to OIT. Untargeted plasma metabolomics data were generated from the VDAART healthy infant cohort (N = 384), a Costa Rican cohort of children with asthma (N = 1040), and a peanut OIT trial (N = 20) evaluating sustained unresponsiveness (SU, protection that lasts after therapy) versus transient desensitization (TD, protection that ends immediately afterward). Generalized linear regression modeling and pathway enrichment analysis identified metabolites associated with food allergy and OIT outcomes. Compared with unaffected children, those with food allergy were more likely to have metabolomic profiles with altered histidines and increased bile acids. Eicosanoids (e.g., arachidonic acid derivatives) (q = 2.4 × 10-20) and linoleic acid derivatives (q = 3.8 × 10-5) pathways decreased over time on OIT. Comparing SU versus TD revealed differing concentrations of bile acids (q = 4.1 × 10-8), eicosanoids (q = 7.9 × 10-7), and histidine pathways (q = .015). In particular, the bile acid lithocholate (4.97 [1.93, 16.14], p = .0027), the eicosanoid leukotriene B4 (3.21 [1.38, 8.38], p = .01), and the histidine metabolite urocanic acid (22.13 [3.98, 194.67], p = .0015) were higher in SU. We observed distinct profiles of bile acids, histidines, and eicosanoids that vary among patients with food allergy, over time on OIT and between SU and TD. Participants with SU had higher levels of metabolites such as lithocholate and urocanic acid, which have immunomodulatory roles in key T-cell subsets, suggesting potential mechanisms of tolerance in immunotherapy.

Distinct mechanisms of type 3 secretion system recognition control LTB4 synthesis in neutrophils and macrophages.

Leukotriene B4 (LTB4) is an inflammatory lipid produced in response to pathogens that is critical for initiating the inflammatory cascade needed to control infection. However, during plague, Yersinia pestis inhibits the timely synthesis of LTB4 and subsequent inflammation. Using bacterial mutants, we previously determined that Y. pestis inhibits LTB4 synthesis via the action of the Yop effector proteins that are directly secreted into host cells through a type 3 secretion system (T3SS). Here, we show that the T3SS is the primary pathogen associated molecular pattern (PAMP) required for production of LTB4 in response to both Yersinia and Salmonella. However, we also unexpectantly discovered that T3SS-mediated LTB4 synthesis by neutrophils and macrophages require the activation of two distinctly different host signaling pathways. We identified that phagocytosis and the NLRP3/CASP1 inflammasome significantly impact LTB4 synthesis by macrophages but not neutrophils. Instead, the SKAP2/PLC signaling pathway is required for T3SS-mediated LTB4 production by neutrophils. Finally, while recognition of the T3SS is required for LTB4 production, we also discovered that a second unrelated PAMP-mediated signal activates the MAP kinase pathway needed for synthesis. Together, these data demonstrate significant differences in the host factors and signaling pathways required by macrophages and neutrophils to quickly produce LTB4 in response to bacteria. Moreover, while macrophages and neutrophils might rely on different signaling pathways for T3SS-dependent LTB4 synthesis, Y. pestis has evolved virulence mechanisms to counteract this response by either leukocyte to inhibit LTB4 synthesis and colonize the host.

Leukotrienes E4 and B4 and vascular endothelium – New insight into the link between vascular inflammation and peripheral arterial

Leukotrienes are proinflammatory mediators that participate in the process of atherogenesis and contribute to the development of symptomatic peripheral arterial disease. The aim was to evaluate the relationship between leukotriene E4 (LTE4) and B4 (LTB4) with parameters reflecting endothelial vascular function in patients with chronic lower limb ischemia. This prospective observational study enrolled 50 consecutive patients undergoing endovascular treatment due to chronic lower limb ischemia (Rutherford 3). All participants were followed-up for one year (after 1, 3, 6 and 12 months), with a sequential assessment of urinary LTE4 and LTB4, as well as measures of endothelial and vascular function: Flow-Mediated Dilatation (FMD), Intima-Media Thickness (IMT), corrected Augmentation Index (AI75), Shear Rate (SR), Ankle-Brachial Index (ABI), Toe-Brachial Index (TBI). There was a significant relationship between LTE4 and measures of vascular function: FMD (R2 = 0.69, P < 0.001), IMT (R2 = 0.12, P < 0.01), AI75 (R2 = 0.43, P < 0.001), SR (R2 = 0.48, P < 0.001). Similar findings were noted for LTB4: FMD (R2 = 0.47, p < 0.001), IMT (R2 = 0.23, P < 0.001), AI75 (R2 = 0.61, P < 0.001) and SR (R2 = 0.33, P < 0.001). Alterations in parameters were significantly related: ΔLTE4 vs ΔFMD(R2 = 0.63, P < 0.001), ΔSR (R2 = 0.42, P < 0.001) and ΔLTB4 vs AI75(R2 = 0.40, P < 0.001), SR(R2 = 0. 29, P < 0.001). We conclude, that increasing concentrations of LTE4 and LTB4 are associated with impairment of vascular and endothelial function, which may lead to worse endovascular treatment clinical outcomes.

Leukotriene B4 is elevated in diabetes and promotes ventricular arrhythmogenesis in guinea pig.

Diabetes (DM) patients have an increased risk (~50%) for sudden cardiac death (SCD), mostly as a result of ventricular arrhythmias. The molecular mechanisms involved remain partially defined. The potent proinflammatory lipid mediator leukotriene (LT) B4, is pathologically elevated in DM compared to nondiabetic patients, resulting in increased LTB4 accumulation in heart, leading to an increased risk for life-threatening proarrhythmic signatures. We used electrophysiology, immunofluorescence, and confocal microscopy approaches to evaluate LTB4 cellular effects in guinea pig heart and ventricular myocytes. We have observed that LTB4 is increased in multiple mouse models (C57BL/6 J/Lepob/ob and PANIC-ATTAC) of DM, promotes profound cellular arrhythmogenesis (spontaneous beats and early after depolarizations, EADs), and severely depresses the rapidly activating delayed rectifier K current (hERG1/IKr) density in HEK293 cells and guinea pig ventricular myocytes. We have further found that guinea pigs challenged with LTB4 displayed a significantly prolonged QT interval, and that this can be prevented with LTB4R inhibition, suggesting that preventing such LTB4R effects may be therapeutically beneficial in DM. Our data suggests that a further elucidation of LTB4 vulnerable substrates, and how this leads to ventricular arrhythmias, is likely to lead to continued improvements in management options, and the development of new therapies for prevention of SCD in DM patients.

Neutrophils on the mast cell menu.

A recent article in Cell reported a new peculiar interaction between mast cells and neutrophils. Upon IgE/Ag-mediated degranulation, mast cells (MCs) produce leukotriene B4 (LTB4), which attracts migrating neutrophils. Some neutrophils are able to establish close contact with MCs and end up trapped inside the MC, forming a cell-in-cell structure. While the neutrophil eventually dies inside the vacuole, the MC benefits from the remains of its prisoner, using it as a nutrient reserve and reusing its antimicrobial weapons.

Leukotriene B4 receptor 1 (BLT1) activation by leukotriene B4 (LTB4) and E resolvins (RvE1 and RvE2)

Leukotriene B4 (LTB4) is a lipid inflammatory mediator derived from arachidonic acid (AA). Leukotriene B4 receptor 1 (BLT1), a G protein-coupled receptor (GPCR), is a receptor of LTB4. Nonetheless, the resolution of inflammation is driven by specialized pro-resolving lipid mediators (SPMs) such as resolvins E1 (RvE1) and E2 (RvE2). Both resolvins are derived from omega-3 fatty acid eicosapentaenoic acid (EPA). Here, long-term molecular dynamics simulations (MD) were performed to investigate the activation of the BLT1 receptor using two pro-resolution agonists (RvE1 and RvE2) and an inflammatory agonist (LTB4). We have analyzed the receptor's activation state, electrostatic interactions, and the binding affinity the Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA) approach. The results showed that LTB4 and RvE1 have kept the receptor in an active state by higher simulation time. MD showed that the ligand-receptor interactions occurred mainly through residues H94, R156, and R267. The MMPBSA calculations showed residues R156 and R267 were the two mainly hotspots. Our MMPBSA results were compatible with experimental results from other studies. Overall, the results from this study provide new insights into the activation mechanisms of the BLT1 receptor, reinforcing the role of critical residues and interactions in the binding of pro-resolution and pro-inflammatory agonists.

Volatile Organic Compounds (VOC) and Serum Leukotriene B4 between COPD Patients and COPD with Lung Cancer Patients

Introduction: Chronic obstructive pulmonary disease (COPD) is estimated to become the third leading cause of death worldwide in 2030. COPD can affect the lungs and cause chronic systemic inflammation. Leukotriene B4 (LTB4) is involved in COPD and lung cancer pathogenesis. There has been the development of non-invasive methods for detecting lung disease in the last few decades, such as the examination of volatile organic compounds (VOC). This study aimed to analyze the serum LTB4 and the difference of VOCs in exhaled breath of stable COPD patients and COPD with lung cancer patients. Methods: This case-control study recruited 20 stable COPD patients and 20 patients with COPD and lung cancer. An exhaled breath sample was collected in Tedlar bags and analyzed using an arrayed sensor breath analyzer to check the concentration of VOCs. Meanwhile, a venous blood sample was collected to examine the level of LTB4 using an ELISA kit. Independent t-test and Mann-Whitney test were used to analyze the data. Results: The carbon dioxide (CO2), nitrogen dioxide (NO2), carbon monoxide (CO), benzene (C6H6), and propane (C3H8) levels were significantly different (p &lt;0.05) in COPD-only patients compared to COPD with lung cancer patients. Serum LTB4 increased in both groups. Conclusion: CO2, CO, and C3H8 levels increased, but the NO2 level decreased in COPD patients with lung cancer compared to COPD-only patients. Serum LTB4 increased in COPD with lung cancer patients.

Application of Ensemble Machine Learning Methods for QSAR Classification of Leukotriene A4 Hydrolase Inhibitors in Drug Discovery

Inflammatory diseases such as asthma, rheumatoid arthritis, and cardiovascular conditions are driven by overproduction of leukotriene B4 (LTB4), a potent inflammatory mediator. Leukotriene A4 hydrolase (LTA4H) plays a critical role in converting leukotriene A4 into LTB4, making it a prime target for drug discovery. Despite ongoing efforts, developing effective LTA4H inhibitors has been challenging due to the complex binding properties of the enzyme and the structural diversity of potential inhibitors. Traditional drug discovery methods, like high-throughput screening (HTS), are often time-consuming and inefficient, prompting the need for more advanced approaches. Quantitative Structure-Activity Relationship (QSAR) modeling, enhanced by ensemble machine learning techniques, provides a promising solution by enabling accurate prediction of compound bioactivity based on molecular descriptors. In this study, six ensemble machine learning methods—AdaBoost, Extra Trees, Gradient Boosting, LightGBM, Random Forest, and XGBoost—were employed to classify LTA4H inhibitors. The dataset, comprising 636 compounds labeled as active or inactive based on pIC50 values, was processed to extract 450 molecular descriptors after feature engineering. The results show that the LightGBM model achieved the highest classification accuracy (83.59%) and Area Under the Curve (AUC) value (0.901), outperforming other models. XGBoost and Random Forest also demonstrated strong performance, with AUC values of 0.890 and 0.895, respectively. The high sensitivity (95.24%) of the XGBoost model highlights its ability to accurately identify active compounds, though it exhibited slightly lower specificity (61.36%), indicating a higher false-positive rate. These findings suggest that ensemble machine learning models, particularly LightGBM, are highly effective in predicting bioactivity, offering valuable tools for early-stage drug discovery. The results indicate that ensemble methods significantly enhance QSAR model accuracy, making them viable for identifying promising LTA4H inhibitors, potentially accelerating the development of anti-inflammatory therapies.

Aspergillus fumigatus-derived gliotoxin impacts innate immune cell activation through modulating lipid mediator production in macrophages.

Gliotoxin (GT), a secondary metabolite and virulence factor of the fungal pathogen Aspergillus fumigatus, suppresses innate immunity and supports the suppression of host immune responses. Recently, we revealed that GT blocks the formation of the chemotactic lipid mediator leukotriene (LT)B4 in activated human neutrophils and monocytes, and in rodents in vivo, by directly inhibiting LTA4 hydrolase. Here, we elucidated the impact of GT on LTB4 biosynthesis and the entire lipid mediator networks in human M1- and M2-like monocyte-derived macrophages (MDMs) and in human tissue-resident alveolar macrophages. In activated M1-MDMs with high capacities to generate LTs, the formation of LTB4 was effectively suppressed by GT, connected to attenuated macrophage phagocytic activity as well as human neutrophil movement and migration. In resting macrophages, especially in M1-MDMs, GT elicited strong formation of prostaglandins, while bacterial exotoxins from Staphylococcus aureus evoked a broad spectrum of lipid mediator biosynthesis in both MDM phenotypes. We conclude that GT impairs functions of activated innate immune cells through selective suppression of LTB4 biosynthesis, while GT may also prime the immune system by provoking prostaglandin formation in macrophages.

Immune, Oxidative, and Morphological Changes in the Livers of Tibetan Sheep after Feeding Resveratrol and β-Hydroxy-β-methyl Butyric Acid: A Transcriptome-Metabolome Integrative Analysis.

This study investigated the effects of dietary resveratrol (RES) and β-Hydroxy-β-methyl butyric acid (HMB) on immune, oxidative, and morphological changes in the livers of Tibetan sheep using transcriptomics and metabolomics. One hundred and twenty male Tibetan lambs of a similar initial weight (15.5 ± 0.14 kg) were randomly divided into four groups with thirty lambs per treatment: (1) H group (basal diet without RES or HMB); (2) H-RES group (1.5 g/day of RES); (3) H-HMB group (1250 mg/day of HMB); (4) H-RES-HMB group (1.5 g/day of RES and 1250 mg/day of HMB). The experiment was conducted for 100 days, including a pre-test period of 10 days and a formal period of 90 days. The results showed significantly increased concentrations of glutathione peroxidase, superoxide dismutase, and IgM in the H-RES-HMB group (p < 0.05), while the malondialdehyde levels were significantly decreased (p < 0.05). The glycolytic indices including creatinine kinase (CK), malate dehydrogenase (MDH), and succinate dehydrogenase (SDH) were significantly increased in the H-RES-HMB group compared with the others (p < 0.05). A histological analysis showed that the hepatic plate tissue in the H-RES-HMB group appeared normal with multiple cells. The transcriptomic analysis showed that the expression of genes associated with the calcium signaling pathway (MYLK2, CYSLTR2, ADCY1, HRH1, ATP2B2, NOS2, HRC, ITPR1, and CAMK2B) and the NF-κB signaling pathway (BCL2 and CARD14) in the H-RES-HMB group were upregulated. The key differential metabolites (d-pyroglutamic acid, DL-serine, DL-threonine, fumarate, and glyceric acid) were enriched in the pathways associated with D-amino acid metabolism, the citrate cycle (TCA cycle), and carbon metabolism. The combined transcriptomic and non-targeted metabolomic analyses showed the co-enrichment of differential genes (NOS2 and GLUD1) and metabolites (fumarate) in arginine biosynthesis-regulated glycolytic activity, whereas the differential genes (ME1, SCD5, FABP2, RXRG, and CPT1B) and metabolites (Leukotriene b4) co-enriched in the PPAR signaling pathway affected the immune response by regulating the PI3K/AKT and cGMP/PKG signaling. In conclusion, the dietary RES and HMB affected the hepatic antioxidant capacity, immune response, and glycolytic activity through modulating the transcriptome (BCL2, CAMK2B, ITPR1, and IL1R1) and metabolome (DL-serine, DL-threonine, fumaric acid, and glycolic acid).

A mouse protozoan boosts antigen-specific mucosal IgA responses in a specific lipid metabolism- and signaling-dependent manner

IgA antibodies play an important role in mucosal immunity. However, there is still no effective way to consistently boost mucosal IgA responses, and the factors influencing these responses are not fully understood. We observed that colonization with the murine intestinal symbiotic protozoan Tritrichomonas musculis (T.mu) boosted antigen-specific mucosal IgA responses in wild-type C57BL/6 mice. This enhancement was attributed to the accumulation of free arachidonic acid (ARA) in the intestinal lumen, which served as a signal to stimulate the production of antigen-specific mucosal IgA. When ARA was prevented from undergoing its downstream metabolic transformation using the 5-lipoxygenase inhibitor zileuton or by blocking its downstream biological signaling through genetic deletion of the Leukotriene B4 receptor 1 (Blt1), the T.mu-mediated enhancement of antigen-specific mucosal IgA production was suppressed. Moreover, both T.mu transfer and dietary supplementation of ARA augmented the efficacy of an oral vaccine against Salmonella infection, with this effect being dependent on Blt1. Our findings elucidate a tripartite circuit linking nutrients from the diet or intestinal microbiota, host lipid metabolism, and the mucosal humoral immune response.

Oxylipins in Aqueous Humor of Primary Open-Angle Glaucoma Patients.

Investigate the oxylipin profiles in the aqueous humor of primary open-angle glaucoma (POAG) patients. Aqueous humor samples were collected from 17 POAG patients and 15 cataract subjects and subjected to a liquid chromatography/mass spectrometry (LC-MS) analysis to detect the oxylipins. The prediction potential of the differential abundant oxylipins was assessed by the receiver operating characteristic (ROC) curves. Pathway and correlation analyses on the oxylipins and clinical and biochemical parameters were also conducted. The LC-MS analysis detected a total of 76 oxylipins, of which 29 oxylipins reached the detection limit. The multivariate analysis identified five differential abundant oxylipins, 15-keto-prostaglandin F2 alpha (15-kPGF2α), Leukotriene B4 (LTB4), 12,13-Epoxyoctadecenoic acid (12,13-Epome), 15-Hydroxyeicosatetraenoic acid (15-HETE) and 11-Hydroxyeicosatetraenoic acid (11-HETE). The five oxylipins are enriched in the arachidonic acid metabolism and linoleic acid metabolism pathways. Pearson correlation analysis showed that 11-HETE was positively correlated with intraocular pressure and central corneal thickness and negatively with cup/disk area ratio in the POAG patients. In addition, 15-kPGF2α was moderately and positively correlated with the mean deviation (MD) of visual field defect, and LTB4 was moderately and negatively correlated with macular thickness. This study revealed the oxylipin profile in the aqueous humor of POAG patients. Oxylipins involved in the arachidonic acid metabolism pathway could play a role in POAG, and anti-inflammatory therapies could be potential treatment strategies for POAG.

Impact of Epithelial Claudin-4 and Leukotriene B4 Receptor 2 in Normoganglionic Hirschsprung Disease Colon on Post Pull-through Enterocolitis

PurposeTo investigate whether Leukotriene B4 receptor 2 (BLT-2), an upstream regulator of tight junction protein (TJP) Claudin-4, and TJPs could be etiologic factors in Hirschsprung-associated enterocolitis (HAEC) after pull-through (PT) for Hirschsprung disease (HD). MethodsNormoganglionic colon (HD-N) and aganglionic rectum (HD-A) specimens from rectal/rectosigmoid (R/RS) or descending/transverse (D/T) HD were assessed using quantitative polymerase chain reaction (qPCR) for Occludin, TJP-1, TJP-2, Junctional adhesion molecule (JAM)-1, JAM-2, Claudin-1, Claudin-3, Claudin-4, and BLT-2 and immunoblotting for Claudin-4 using fresh specimens obtained intraoperatively (2021–2024; n = 17; R/RS = 15 and D/T = 2). Claudin-4 immunohistochemistry was also evaluated quantitatively using preserved (n = 29; R/RS = 20 and D/T = 9; 2009–2021) and fresh HD specimens for comparison with anorectal malformation patients having colostomy closure as controls (n = 42) and between HD-A versus HD-N, R/RS versus D/T, and HAEC (+) versus HAEC (−). Technically inadequate or transitional zone PT were excluded. ResultsSubjects were 123 PT cases. Mean ages at PT/colostomy closure (years) were R/RS: 2.7 ± 2.9, D/T: 1.6 ± 2.2, and controls: 1.4 ± 0.7. Postoperative HAEC occurred 18 times in 14 PT cases (grade I = 5, grade II = 13). Post-PT HAEC was significantly more frequent in D/T (50.0% versus 6.4%; p < 0.001); Claudin-4 was significantly lower in HD-N from post-PT HAEC cases, especially D/T (p < 0.05) on immunohistochemistry. Claudin-4 was significantly lower in HD-N/HD-A compared with controls on immunoblotting (p < 0.05) and immunohistochemistry (p < 0.001). qPCR showed TJP-1, TJP-2, JAM-1, JAM-2, Claudin-4, and BLT-2 were significantly lower in HD-N/HD-A compared with controls. ConclusionsLower Claudin-4 and BLT2 in post-PT HAEC HD-N (especially D/T) suggests generalized epithelial barrier derangement with possible etiologic implications for HAEC. Level of EvidenceⅡ

Biological Evaluation of Lysionotin: a Novel Inhibitor of 5-Lipoxygenase for Anti-glioma.

To explore the potential mechanism of lysionotin in treating glioma. First, target prediction based on Bernoulli Naïve Bayes profiling and pathway enrichment was used to predict the biological activity of lysionotin. The binding between 5-lipoxygenase (5-LO) and lysionotin was detected by surface plasmon resonance (SPR) and molecular docking, and the inhibitory effects of lysionotin on 5-LO and proliferation of glioma were determined using enzyme inhibition assay in vitro and cell viability analysis, respectively. Furthermore, the pharmaceutical effect of lysionotin was explored by cell survival rate analysis and liquid chromatography with tandem mass spectrometry (LC-MS/MS). The protein expression, intracellular calcium ion concentration and cytoskeleton detection were revealed by Western blot, flow cytometry and fluorescence labeling, respectively. Target prediction and pathway enrichment revealed that lysionotin inhibited 5-LO, a key enzyme involved in the arachidonic acid metabolism pathway, to inhibit the proliferation of glioma. Molecular docking results demonstrated that 5-LO can be binding to lysionotin through hydrogen bonds, forming bonds with His600, Gln557, Asn554, and His372. SPR analysis further confirmed the interaction between 5-LO and lysionotin. Furthermore, enzyme inhibition assay in vitro and cell survival rate analysis revealed that 50% inhibition concentration of lysionotin and the median effective concentration of lysionotin were 90 and 16.58 µmol/L, respectively, and the results of LC-MS/MS showed that lysionotin inhibited the production of 5S-hydroperoxy-eicosatetraenoic acid (P<0.05), and moreover, the LC-MS/MS results indicated that lysionotin can enter glioma cells well (P<0.01) and inhibit their proliferation. Western blot analysis demonstrated that lysionotin can inhibit the expression of 5-LO (P<0.05) and downstream leukotriene B4 receptor (P<0.01). In addition, the results showed that lysionotin affected intracellular calcium ion concentration by inhibiting 5-LO to affect the cytoskeleton, as determined by flow cytometry and fluorescence labeling. Lysionotin binds to 5-LO could suppress glioma by inhibiting arachiodonic acid metabolism pathway.

Integration of proteomics and metabolomics analysis investigate mechanism of As-induced immune injury in rat spleen

Arsenic (As) is a widespread metalloid and human carcinogen found in the natural environment, and multiple toxic effects have been shown to be associated with As exposure. As can be accumulated in the spleen, the largest peripheral lymphatic organ, and long-term exposure to As can lead to splenic injury. In this study, a Sprague-Dawley (SD) rat model of As-poisoned was established, aiming to explore the molecular mechanism of As-induced immune injury through the combined analysis of proteomics and metabolomics of rats’ spleen. After feeding the rats with As diet (50 mg/kg) for 90 days, the spleen tissue of the rats in the As-poisoned group was damaged, the level of As was significantly higher than that of the control group (P < 0.001), and the level of inflammatory cytokine interleukin-6 (IL-6) was decreased (P < 0.01). Proteomics and metabolomics results showed that a total of 134 differentially expressed proteins (DEPs) (P < 0.05 and fold change > 1.2) and 182 differentially expressed metabolites (DEMs) (VIP >1 and P < 0.05) were identified in the spleens of the As poisoned group compared to the control group (As/Ctrl). The proteomic results highlight the role of hypoxia-inducible factors (HIF), natural killer cell mediated cytotoxicity, and ribosomes. The major pathways of metabolic disruption included arachidonic acid (AA) metabolism, glycerophospholipid metabolism and folate single-carbon pool. The integrated analysis of these two omics suggested that Hmox1, Stat3, arachidonic acid, phosphatidylcholine and leukotriene B4 may play key roles in the mechanism of immune injury to the spleen by As exposure. The results indicate that As exposure can cause spleen damage in rats. Through proteomic and metabolomic analysis, the key proteins and metabolites and their associated mechanisms were obtained, which provided a basis for further understanding of the molecular mechanism of spleen immune damage caused by As exposure.

Combining the Vaginal Microbiome and Serum Metabolome to Screen for Potential Biomarkers of Early Pregnancy in Cows.

Early pregnancy diagnostic techniques are of significant importance in livestock farming, particularly in dairy farming. This study aimed to screen artificially inseminated cows for potential biomarkers at day 21 of pregnancy using microbiota-metabolomics analysis. The microbiome analysis revealed significant changes (p < 0.05) in the composition and abundance of the vaginal microbiota in cows after pregnancy. Notably, there was an increase in the abundance of [Eubacterium]_hallii_group (p < 0.05) associated with the production of short-chain fatty acids in the pregnant group compared with the non-pregnant group. Furthermore, significant alterations were observed in the serum metabolome, with notable changes in the concentrations of prolyl-hydroxyproline (Pro-Hyp) (p < 0.01) and bonactin (p < 0.01). The majority of differential metabolites clustered within the pathways of amino acid metabolism and lipid metabolism, with lipid metabolism exhibiting a higher proportion and playing a pivotal role in early pregnancy. An enzyme-linked immunosorbent assay was employed to quantify three key metabolites of the arachidonic acid pathway. The results demonstrated significant decreases in serum concentrations of leukotriene B4 (LTB4) (p < 0.05) and prostaglandin F2α (PGF2α) (p < 0.01) and no significant changes in arachidonic acid (AA) (NS) concentrations after 21 days of gestation in cows. Spearman's correlation analysis was utilized to investigate the interrelationship between the vaginal microbiota and serum metabolites. In conclusion, the present study demonstrated that biomaterials such as bonactin, Pro-hyp, LTB4, PGF2α in serum metabolites and [Eubacterium]_hallii_group in the vaginal flora of cows could be utilized as potential biomarkers for 21 days of gestation in cows.