Lipocalin-2 Levels Research Articles

A systematic review of the implications of lipocalin-2 expression in periodontal disease.

Evidence suggests that lipocalin-2 (LCN-2), a bone-derived protein, is upregulated in periodontal diseases. This systematic review aimed to evaluate LCN-2 concentrations in individuals with periodontal diseases, identifying the most suitable body fluids for its detection, the type of periodontal disease with the highest LCN-2 expression, its association with other inflammatory markers and systemic diseases, and whether its expression can be modified by periodontal treatment. A systematic search of Google Scholar, PubMed, and ProQuest up to August 2024 was conducted. The studies were screened and selected by the authors according to specific eligibility criteria. Quality assessment of the included studies was performed according to the study type using STROBE statement for observational studies or the modified Jadad scale for experimental studies. The review was registered in PROSPERO (CRD42023458565). In total, three thousand six hundred and thirty-eight reports were identified, of which twenty-seven were full-text assessed for eligibility, including eleven articles. Seven articles were observational, and four were experimental. Significantly elevated LCN-2 levels were reported in patients with periodontal disease across 9 studies, being higher in periodontitis rather than gingivitis. LCN-2 was mainly detected in gingival crevicular fluid (GCF) and saliva. LCN-2 expression is related to the increment of inflammatory markers, and periodontal therapy decreases LCN-2 concentrations. LCN-2 levels were aggravated when periodontitis was accompanied by obesity and type 2 diabetes. LCN-2 is implicated in periodontal diseases, probably through the inflammation process.

Diagnostic and prognostic value of plasma lipocalin-2 levels in patients with metabolic dysfunction–associated steatotic liver disease

Abstract Background Non-Alcoholic Fatty Liver Disease, recently better recognised as Metabolic Dysfunction–Associated Steatotic Liver Disease, is the most prevalent form of chronic liver disease at present time. It is estimated to impact 32% of the world's population, hence representing a significant health burden. Aim of the work To assess the significance of plasma Lipocalin-2 (LCN2) levels in the diagnosis and prognosis of NAFLD patients. Patients and methods In this retrospective case–control study we recruited 102 subjects aged between 18 and 70 years. The included participants were split into two study groups. Group I: 51 NAFLD patients (61% men, 39% females) and Group II: 51 healthy controls (51% men and 49% females), for whom plasma LCN2 levels were assessed and correlated with NAFLD fibrosis score, FIB4 and fatty liver index. Results In this study, LCN2 levels in NAFLD patients were significantly greater compared to individuals in the control group (p < 0.001), with a mean of 1893.214 ± 1002.852 ng/dL in the cases and a mean of 466.020 ± 397.699 ng/dL in the controls. This suggests the use of LCN2 as a possible diagnostic marker of NAFLD. The mean LCN2 levels in this study also significantly increased as the grade of fatty liver increased from I to III (p < 0.001). This in turn proposes the use of LCN2 as a prognostic marker for NAFLD progression. LCN2 also significantly correlated with the fatty liver index and NAFLD Fibrosis scoring systems, but not with Fib-4. With an area under the ROC of 0.906, it demonstrated excellent diagnostic performance with 84% sensitivity, 90% specificity, 89.6% PPV and 85.2% NPV for the prediction of NAFLD patients. Conclusion Lipocalin-2 performs as a diagnostic and a possible prognostic marker for metabolic dysfunction-associated steatotic liver disease.

Associations between plasma markers and symptoms of anxiety and depression in patients with breast cancer

Background and purposeAmong patients with solid tumors, those with breast cancer (BC) experience the most severe psychological issues, exhibiting a high global prevalence of depression that negatively impacts prognosis. Depression can be easily missed, and clinical markers for its diagnosis are lacking. Therefore, this study in order to investigate the diagnostic markers for BC patients with depression and anxiety and explore the specific changes of metabolism.Method and resultsThirty-eight BC patients and thirty-six matched healthy controls were included in the study. The anxiety and depression symptoms of the participants were evaluated by the 17-item Hamilton Depression Scale (HAMD-17) and Hamilton Anxiety Scale (HAMA). Plasma levels of glial fibrillary acidic protein (GFAP) and lipocalin-2 (LCN2) were evaluated using enzyme linked immunosorbent assay, and plasma lactate levels and metabolic characteristics were analyzed.ConclusionThis study revealed that GFAP and LCN2 may be good diagnostic markers for anxiety or depression in patients with BC and that plasma lactate levels are also a good diagnostic marker for anxiety. In addition, specific changes in metabolism in patients with BC were preliminarily explored.

Lipocalin-2 as a marker of inflammation, bone density, and triglyceride-glucose index for new-onset arthritis patients in Mosul, Iraq.

Lipocalin-2 is an acute phase-associated adipokine that can serve as an inflammatory and biomarker indicator of cartilage deterioration in osteoarthritis. However, its role in the musculoskeletal system remains not fully understood. Hence, this study aimed to evaluate lipocalin-2 and its relationship with markers of inflammation (Interferon-gamma, ESR, and CRP), bone density (vitamin D3 and calcium), and the triglyceride-glucose index in new-onset arthritis patients in Mosul, Iraq. This study included 125 participants aged 20 to 65, divided into two groups. The Arthritis Patient Group comprised 70 participants (37 females and 33 males) attending the Bone Diseases Consultation Unit at the Ibn Sina Teaching Hospital in Mosul, Iraq. The Control Group comprised 31 females and 24 males. Ethical approval was obtained from the Iraqi Ministry of Health - Nineveh Health (No. 2022095). Commercial ELISA kits were used to measure serum lipocalin-2, Interferon-gamma, ESR, and CRP as inflammation markers, vitamin D3, and calcium as bone density markers. Moreover, the Triglyceride Glucose (TYG) Index was evaluated. The findings revealed a significant increase in lipocalin-2 levels in males compared to females, with LCN-2 increasing with age. Arthritis patients showed a significant increase (72%) in lipocalin-2 levels. Inflammatory indicators (erythrocyte sedimentation rate, C-reactive protein, interferon-gamma) displayed significant increases (46%, 1200%, and 581%, respectively). Glucose (23%), triglycerides (71%), and TYG index (21%) also exhibited significant increases. Meanwhile, bone density indicators (vitamin D3 and calcium) found a significant decrease (53% and 20%, respectively) in arthritis patients. Linear correlation coefficient (R) analysis revealed a significant positive relationship between lipocalin-2 and indicators of inflammation, glucose, TG, and TYG index. This study's findings suggest that LCN-2 serum levels were higher in patients with new-onset arthritis than in controls in Mosul, and LCN-2 serum increased in males compared with females and getting older serum LCN-2 increased for the patients and control groups. Furthermore, a significant correlation was found between the Triglyceride Glucose Index, which measures metabolic disorders, and serum LCN-2 levels and inflammatory indicators in new-onset arthritis patients in Mosul, Iraq.

The Role of Nitric Oxide, Lipocalin-2, and Proinflammatory Cytokines on Proteinuria and Insulin Resistance in Type 2 Diabetes Mellitus Subgroups.

Nitric oxide (NO) is a bioactive signaling molecule that mediates various physiological and biological processes. Type 2 diabetes mellitus (T2DM) can be categorized into several subgroups according to fasting plasma glucose (FPG) and hemoglobin A1c (HbA1c) levels. Few studies have closely examined the effect of NO and lipocalin-2 on albuminuria and insulin resistance in T2DM subgroups. This study investigated the role of NO, lipocalin-2, and proinflammatory cytokines on the development of proteinuria and insulin resistance in patients with T2DM subgroups. A total of 256 subjects, including 191 patients with T2DM and 65 non-diabetic healthy individuals, were evaluated. NO metabolites (NOx), lipocalin-2, tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) levels were measured. Patients with T2DM were classified into three subgroups: patients with FPG-defined diabetes (PG-DM), those with HbA1c-defined diabetes (HA-DM), and those who met the criteria for both FPG and HbA1c (PG/HA-DM). The albumin-to-creatinine ratio (ACR) and the homeostasis model assessment of β-cell function (HOMA-B) and insulin resistance (HOMA-IR) were calculated. NOx, lipocalin-2, and TNF-α levels were significantly higher in patients with T2DM than in healthy individuals. Patients with PG/HA-DM had significantly higher NOx levels than those with PG-DM or HA-DM. Of the patients with high NOx levels, patients with lipocalin-2 elevation exhibited higher ACR and HOMA-IR than those without lipocalin-2 elevation. NOx was positively correlated with lipocalin-2, ACR, HOMA-IR, and TNF-α but not with HOMA-B and IL-6. The upper quartile of NOx levels led to a 1.2-fold increase in the risk of albuminuria (odds ratio: 1.215; 95% CI: 1.012-2.418; p < 0.001). NO plays a crucial role in proteinuria and insulin resistance by collaborating with lipocalin-2 and TNF-α, showing significantly higher levels in patients with PG/HA-DM than in those with PG-DM or HA-DM.

GDF15 and LCN2 for early detection and prognosis of pancreatic cancer

BackgroundThe prognosis of pancreatic ductal adenocarcinomas (PDAC) remains very poor, emphasizing the critical importance of early detection, where biomarkers offer unique potential. Although growth differentiation factor 15 (GDF15) and Lipocalin 2 (LCN2) have been linked to PDAC, their precise roles as biomarkers are uncertain. MethodsCirculating levels of GDF15 and LCN2 were examined in human PDAC patients, heathy controls, and individuals with benign pancreatic diseases. Circulating levels of IL-6, CA19-9, and neutrophil-to-lymphocyte ratio (NLR) were measured for comparisons. Correlations between PDAC progression and overall survival were assessed. A mouse PDAC model was employed for comprehensive analyses, complementing the human studies by exploring associations with various metabolic and inflammatory parameters. Sensitivity and specificity of the biomarkers were evaluated. FindingsOur results demonstrated elevated levels of circulating GDF15 and LCN2 in PDAC patients compared to both healthy controls and individuals with benign pancreatic diseases, with higher GDF15 levels associated with disease progression and increased mortality. In PDAC mice, circulating GDF15 and LCN2 progressively increased, correlating with tumor growth, behavioral manifestations, tissue and molecular pathology, and cachexia development. GDF15 exhibited highly sensitive and specific for PDAC patients compared to CA19-9, IL-6, or NLR, while LCN2 showed even greater sensitivity and specificity in PDAC mice. Combining GDF15 and LCN2, or GDF15 and CA19-9, enhanced sensitivity and specificity. InterpretationOur findings indicate that GDF15 holds promise as a biomarker for early detection and prognosis of PDAC, while LCN2 could strengthen diagnostic panels.

Defining age-specific reference intervals for biomarkers distinguishing bacterial from viral infection in paediatrics

Differentiating bacterial from viral infections in children is a common clinical challenge. Novel host immune biomarkers have the potential to aid thediagnosis of infection aetiology and identify children who require antibiotics. Data on novel infection biomarkers gender and age-specific correlations, and reference intervals in healthy paediatrics is lacking. This study reports the plasma levels of three novel biomarkers that can aid in the differentiation of bacterial and viral infection in a healthy group of paediatrics. The levels of (Interferon-Gamma Inducible Protein 10 kDa (IP-10), Lipocalin-2 (LCN2) and TNF-Related Apoptosis-Inducing Ligand (TRAIL) were quantified in 199 plasma samples from healthy paediatrics aged 2 to 16 years old from across the UK. Reference intervals (2.5th and 97.5th) were determined, and biomarker levels were examined for sex and age associations. Reference intervals for IP-10, LCN2 and TRAIL for ages 2–16 years were 36.7–168.1 pg/ml, 14.2–123.3 ng/ml, 57.4–71.4 pg/ml respectively. No biomarker showed an association with sex and IP-10 did not show any association with age. TRAIL levels had a weak continuous negative correlation with age and LCN2 levels had a continuous positive correlation with age. Specific cut-offs for LCN2 in two age categories were identified, while TRAIL did not require age partitions. This study provides age-appropriate reference intervals for three biomarkers of infection in healthy children. These findings have the potential to improve the impact of future research on these biomarkers, the accuracy of clinical decision-making in children with infection, paediatric patient care and outcomes, and antimicrobial stewardship.

Lipocalin-2 as a fundamental protein in type 2 diabetes and periodontitis in mice.

Lipocalin-2 (LCN-2) is an osteokine that suppresses appetite, stimulates insulin secretion, regulates bone remodeling, and is induced by proinflammatory cytokines. The aim of this work was to investigate the participation of LCN-2 in periodontitis associated with type 2 diabetes (T2D) by evaluating alveolar bone loss, glycemic control, inflammation, and femur fragility. A murine model of periodontitis with T2D and elevated LCN-2 concentration was used. Functional LCN-2 inhibition was achieved using an anti-LCN-2 polyclonal antibody, and isotype immunoglobulin G was used as a control. The alveolar bone and femur were evaluated by micro-CT. Glucose metabolism was determined. Tumor necrosis factor (TNF-α) and receptor activator of nuclear factor kappa-B ligand (RANKL) levels in alveolar bone lysates were quantified using ELISA, and serum cytokines were quantified using flow cytometry. A three-point bending test was performed in the femur, and RANKL levels were measured in femur lysates using ELISA. Functional inhibition of LCN-2 in T2D-periodontitis mice decreased alveolar bone loss in buccal and palatal surfaces and preserved the microarchitecture of the remaining bone, decreased TNF-α and RANKL in alveolar bone, reduced hyperglycemia, glucose intolerance, and insulin resistance, and increased insulin production through improving the functionality of pancreatic β cells. Furthermore, this inhibition increased serum free-glycerol levels, decreased serum interleukin (IL)-6, increased serum IL-4, and reduced femur fragility and RANKL expression in the femur. LCN-2 participates in periodontitis associated with T2D. Inhibiting its function in mice with T2D and periodontitis improves pancreatic β-cell function, and glucose metabolism and decreases inflammatory cytokines and bone-RANKL levels, which results in the preservation of femoral and alveolar bone microarchitecture. In this study, we explored the role of a bone protein known as lipocalin-2 (LCN-2) in the connection between periodontitis and type 2 diabetes (T2D). Periodontitis is a destructive gum and alveolar bone disease. LCN-2 levels are increased in both T2D and periodontitis. Using a mouse model of T2D with periodontitis, we examined how blocking LCN-2 function affected various aspects of these two diseases. We found that this inhibition led to significant improvements. First, it reduced alveolar bone loss and preserved bone structure by decreasing local inflammation and bone resorption. Second, it improved glucose and lipid metabolism, leading to better blood-sugar control and decreased insulin resistance. Blocking the functions of LCN-2 also decreased systemic inflammation throughout the body and strengthened bone integrity. Overall, our results suggest that LCN-2 plays a crucial role in the periodontitis associated with T2D. By inhibiting LCN-2 function, we were able to improve pancreatic function, improve glucose metabolism, reduce inflammation, and enhance bone health. Targeting LCN-2 could be a promising strategy for the harmful effects of T2D and periodontitis.

Aloe-emodin alleviates inflammatory bowel disease in mice by modulating intestinal microbiome homeostasis via the IL-4/IL-13 axis

IntroductionInflammatory bowel disease (IBD) is a global health concern. Aloe-emodin (AE) has diverse pharmacological benefits, including anti-inflammatory effects. However, its role in IBD remains unclear, prompting our investigation of its regulatory effects and mechanisms in an IBD mouse model. MethodsWe studied the therapeutic efficacy of AE in alleviating symptoms and modulating cytokine secretion in a murine model of dextran sulfate sodium (DSS)-induced colitis. BALB/c mice were administered DSS to induce colitis and were subsequently treated with varying doses of AE. Changes in body weight, fecal lipocalin-2 (LCN2) levels, colon tissue histology, and serum cytokine concentrations were evaluated to assess the effects of AE treatment. Additionally, 16 S rRNA sequencing was used to analyze alterations in the composition of the gut microbiota following AE intervention. Finally, the database was used to analyze the signaling pathways associated with IBD in AE and to detect the expression levels of interleukin (IL)-4 pathway using real-time quantitative reverse transcription PCR. Exogenous IL-4 was used in rescue experiments to observe its effects on the disease process of IBD under AE regulation. ResultsAE treatment resulted in a dose-dependent mitigation of weight loss, reduction in fecal LCN2 levels, and amelioration of histological damage in DSS-induced colitis in mice. The levels of superoxide dismutase and catalase increased, whereas malondialdehyde decreased following AE treatment, indicating a dose-dependent alleviation of colitis symptoms. Furthermore, AE administration attenuated the secretion of pro-inflammatory cytokines, including IL-17, tumor necrosis factor-alpha (TNF-α), and chemokine ligand 1, while promoting the expression of anti-inflammatory cytokines IL-4 and IL-13. Analysis of the gut microbiota revealed that AE effectively suppressed the overgrowth of colitis-associated bacterial species and restored microbial homeostasis. Finally, we found that overexpression of IL-4 was able to reverse the therapeutic effect of AE for DSS-induced IBD. ConclusionAE shows promise in alleviating colitis severity, influencing inflammatory cytokines, and modulating the gut microbiota in an IBD mouse model via the IL-4/IL-13 pathway, suggesting its potential as a natural IBD remedy.

Lipocalin-2 Level in Patients with Polycystic Ovary Syndrome: Association with Insulin Resistance and Metformin Therapy

Abstract Background PCOS appears to be associated with an increased risk of metabolic aberrations, including insulin resistance and hyperinsulinemia, type 2 diabetes mellitus, dyslipidemia, and cardiovascular disease throughout womens’ lifespan. Aim of the Work The aim of this study is to: Assess the association of Lipocalin-2 with polycystic ovary syndrome in a sample of the Egyptian female. Study the effect of metformin therapy on Lipocalin-2 level in female patients with polycystic ovary syndrome Patients and Methods Our study was conducted on 52 females. Their age ranged between 17-43 years old. They were collected from the outpatient obstetrics and gynaecology clinics of Ain Shams University Hospitals, assess the association of Lipocalin-2 with polycystic ovary syndrome in a sample of Egyptian females and its association with insulin resistance. It was conducted from June 2018 to March 2019. The study was explained to all patients and control subjects, and written consent was obtained before starting the study. Results Serum lipocalin-2 levels did not differ between patients with PCOS and BMI-matched healthy controls (P-value 0.193). In the present study, there was no significant difference between the 2 studied groups as regard HOMA-IR (p = 0.375). Metformin therapy for 3 months in patients with PCOS in the present study, resulted in significant reduction in lipocalin-2 level (p- value&amp;lt;0.01), (mean 54.2±15.3 ng/ml before metformin therapy vs 42.9±14.2 ng/ml after metformin therapy). In our study metformin therapy in PCOS group resulted in significant reduction in weight, BMI, waist circumference and HOMA-IR. Furthermore, linear regression analysis for the parameters affecting lipocalin level in our study, showed that BMI was the only significant confounder affecting lipocalin level. So the reduction in lipocalin level following metformin therapy in PCOS group in our study may be due to weight reduction perse. Conclusion In conclusion, our results show that PCOS per se is not associated with elevated serum lipocalin-2 levels. Weight loss induces a significant reduction in serum lipocalin-2 levels in overweight/obese patients with PCOS. Treatment with metformin of patients with PCOS also induces a reduction in serum lipocalin-2 levels.

Role of Lipocalin-2 Levels in Non-Alcoholic Fatty Pancreatic Disease and its Association with Non-Alcoholic Fatty Liver Disease

Abstract Background Lipocalin-2 (LCN2) is a 25-kDa secretory protein with great importance as a sensitive and specific screening and diagnostic biomarker for hepatic inflammation, acute hepatic injury, hepatic cancer, and radiation-induced early phase hepatic damage and lipid abnormalities. However, little is known about LCN2's functions in the homeostasis and metabolism of hepatic lipids or in the development of steatosis as well as its actual role in the event of inflammation. Aim and Objectives To establish a correlation between the levels of lipocalin as a useful biomarker with increasing severity of steatosis in NAFLD and NAFPD patients. Subjects and Methods This was observational prospective case–control study, was conducted at Hepatology Unit of Ain Shams University Hospital on 50 patients diagnosed with NAFPD and 50 healthy age and sex-matched volunteers without NAFPD during a period of 6 months. All patients were subjected to; Full history taking and thorough clinical examination, Anthropometric measurements, Laboratory investigations, and abdominal ultrasonography. Result There was a significant Positive correlation between Lipocalin 2, BMI, LDL (mg/dL), Cholesterol (mg/dL), Insulin (μU/mL) and HOMA-IR in patient group(P-value ≤ 0.001), Conclusion; lipocalin-2 is a good blood biomarker for detection of hepatic and Pancreatic steatosis. The lipocalin levels increase with increasing severity of steatosis. In addition to this, lipocalin will be useful for early diagnosis of NAFLD, NAFPD and convenient to do with other blood tests. The early intervention may prevent high morbidity and mortality and to manage the disease properly.

Posidonia oceanica (L.) Delile Is a Promising Marine Source Able to Alleviate Imiquimod-Induced Psoriatic Skin Inflammation.

Psoriasis is a chronic immune-mediated inflammatory cutaneous disease characterized by elevated levels of inflammatory cytokines and adipokine Lipocalin-2 (LCN-2). Recently, natural plant-based products have been studied as new antipsoriatic compounds. We investigate the ability of a leaf extract of the marine plant Posidonia oceanica (POE) to inhibit psoriatic dermatitis in C57BL/6 mice treated with Imiquimod (IMQ). One group of mice was topically treated with IMQ (IMQ mice) for 5 days, and a second group received POE orally before each topical IMQ treatment (IMQ-POE mice). Psoriasis Area Severity Index (PASI) score, thickness, and temperature of the skin area treated with IMQ were measured in both groups. Upon sacrifice, the organs were weighed, and skin biopsies and blood samples were collected. Plasma and lesional skin protein expression of IL-17, IL-23, IFN-γ, IL-2, and TNF-α and plasma LCN-2 concentration were evaluated by ELISA. PASI score, thickness, and temperature of lesional skin were reduced in IMQ-POE mice, as were histological features of psoriatic dermatitis and expression of inflammatory cytokines and LCN-2 levels. This preliminary study aims to propose P. oceanica as a promising naturopathic anti-inflammatory treatment that could be introduced in Complementary Medicine for psoriasis.

Quantitative proteomic analysis of circulating exosomes reveals the mechanism by which Triptolide protects against collagen-induced arthritis.

Triptolide (TP), a natural product derived from the herbal medicine Tripterygium wilfordii, exhibits potent immunosuppressive activity. However, the mechanisms underlying its effects in rheumatoid arthritisremain incompletely understood. Collagen-induced arthritis (CIA) model was induced in Sprague-Dawley rats by immunization with bovine type II collagen, and TP was administrated as treatment. The therapeutic effect of TP was evaluated based on paw swelling, histopathology, and serum levels of inflammatory factors. Exosomes isolated from rat serum were characterized by transmission electron microscopy, dynamic light scattering, and western blotanalysis. Proteomic profiling of exosomes was analyzed by direct DIA quantitative proteomics analysis. Gene ontology and the Kyoto Encyclopedia of Genes and Genomes databases were employed for enrichment analysis related to molecular function, biological processes, and signaling pathways. Western blot analysis was used to analyze differentially expressed proteins. TP treatment ameliorated arthritic phenotypes in CIA rats as evidenced by reduced arthritis score, paw swelling, pathological injury severity scores, and serum levels of inflammatory cytokines. The proteomic analysis revealed that TP treatment significantly inhibited complement and coagulation cascades, interleukin-17 signaling pathway, and cholesterol metabolism, which were reactivated in CIA rats. Importantly, lipocalin 2 (LCN2) and myeloperoxidase (MPO) levels were markedly upregulated in the CIA group but suppressed upon TP administration. Furthermore, in synovial tissues, LCN2 and MPO expression levels were also elevated in the CIA group but decreased following TP treatment. Our findings demonstrate that TP alleviates CIA, possibly through modulation of exosomal LCN2 and MPO proteins.

MiR-24-3p and miR-484 are potential biomarkers for neurodegeneration in multiple sclerosis

Multiple sclerosis (MS) is a complex, neurodegenerative chronic disorder. Circulating diagnostic biomarkers for MS have remained elusive, and those proposed so far have limited sensitivity and specificity to MS. Plasma-circulating microRNAs (miRNAs) have advantageous biochemical and physiological attributes that can be utilized in clinical testing and disease monitoring. MS miRNA expression microarray datasets analysis resulted in four candidate miRNAs that were assessed for their expression in a separate MS case-control study. Only miR-24-3p was downregulated in all MS patients compared to healthy controls. MiR-484 was significantly upregulated in relapsing-remitting MS (RRMS) patients compared to healthy controls. Mir-146-5p and miR-484 were significantly downregulated in secondary-progressive MS (SPMS) compared to RRMS. MiR-484 downregulation was associated with worsening disability and increased lipocalin-2 levels. Mir-342-3p and miR-24-3p downregulation were associated with increased semaphorin-3A levels in MS and RRMS patients. In conclusion, mir-24-3p downregulation is diagnostic of MS, and mir-484 upregulation and downregulation are potential biomarkers for RRMS and SPMS conversion, respectively. The differential expression of miR-146a-3p in MS subtypes suggests its potential as an SPMS transition biomarker. The association of downregulated mir-24-3p and mir-484 with increased neurodegeneration biomarkers suggests they play a role in MS pathogenesis and neurodegeneration.

#289 Lipocalin-2 promotes cardiovascular calcification in chronic kidney disease by upregulating STAT3/NCOA4-mediated ferritinophagy and ferroptosis

Abstract Background and Aims Cardiovascular calcification (CVC) is a common complication of chronic kidney disease (CKD) and contributes to cardiovascular morbidity and mortality without any effective therapies. To date, the underlying mechanism of CKD-CVC has not been elucidated. Recent studies suggest that ferroptosis is a novel programmed cell death that may play a role in the process of CKD-CVC, but the mechanism is largely unclear. Ferritinophagy is an important inducer of ferroptosis that is regulated by nuclear receptor coactivator 4 (NCOA4). However, the relationship between ferritinophagy and CKD-CVC has not been explored. Lipocalin-2 (LCN2) is an iron-trafficking protein that has been associated with both osteogenesis and ferroptosis, but the exact role and molecular mechanism of LCN2 in CKD-CVC needs to be explored. Method A cross-sectional clinical study was utilized to examine the association between LCN2 expression and CKD-CVC in serum and calcified radial arteries of CKD patients. LCN2 knockout mice, their wild type littermates and mice infected with VSMCs-targeted adeno-associated virus encoding LCN2 gene or negative control gene were all fed by the high adenine and phosphate diet to induce CKD-CVC.VSMCs transfected with LCN2-overexpressing lentivirus, LCN2-shRNA lentivirus or LCN2 siRNA and recombinant LCN2 stimulated VSMCs were treated under the condition of calcifying medium to investigate the role of LCN2 in CKD-CVC in vitro. Cardiovascular calcification was evaluated by Von-kossa staining and tissue calcium content assay. VSMCs calcification were checked by the Alizarin Red Staining and cellular calcium content assay. Cellular reactive oxygen species (ROS) were measured by the DCFDA assay. Lipid peroxidation was measured by the fluorescence of the fatty acid analog C11-BODIPY581/591 probe. Intracellular labile ferrous iron levels were measured by fluorescence of Phen Green SK probe and ferro-orange probe. Transmission electron microscopy was also used to examine mitochondrial change of ferroptosis. Results The cross-sectional study identified that LCN2 levels in serum and radial arteries were significantly increased as the severity of CKD-CVC increased. High adenine and phosphate diet intake provokes renal fibrosis and cardiovascular calcification in vivo. In vitro, five-days high phosphate sodium stimulation induced calcium deposition and osteoblastic trans differentiation of VSMCs. Meanwhile, high phosphate also increased the levels of ferroptosis in VSMCs as evidenced by decreased cell viability, iron and ROS accumulation, upregulated PTGS2 and MDA levels, reduced glutathione peroxidase 4 (GPX4) levels and significantly mitochondrial damage in VSMCs. Intriguingly, calcium deposition in VSMCs could be reversed by the ferroptosis inhibitor ferrostatin-1. LCN2 expression was significantly upregulated both in the aorta of mice and calcified VSMCs. Compared with WT mice, deletion of LCN2 inhibited cardiovascular calcification whereas VSMC-targeted LCN2 overexpression aggravated CKD-CVC. Consistently, LCN2 knockdown also alleviated calcium deposition of VSMCs. Meanwhile, inhibition of ferroptosis were observed after LCN2 silencing as evidenced by decreased ferrous and lipid oxidation while increased levels of GPX4. Mechanically, we demonstrated high phosphate increased the protein level of NCOA4 and downregulated FTH1, which was reversed by depletion of LCN2. Knockdown of NCOA4 partly alleviated the ferritin reduction, ferroptosis, and calcification in VSMCs, indicating that NCOA4-mediated ferritinophagy was required for VSMCs calcification. Furthermore, we found that the recombinant LCN2 treatment significantly upregulated phosphorylated STAT3. STAT3 signaling inhibitor abolished the recombinant LCN2 induced NCOA4 upregulation and calcification in VSMCs. Conclusion Our findings indicate that the pro-calcific effect of high phosphate is due to VSMCs ferroptosis mediated by LCN2 upregulation. LCN2 silencing alleviates CKD-CVC by suppressing the STAT3/ NCOA4/FTH1 signaling mediated ferritinophagy. LCN2 could serve as a candidate therapeutic strategy for CKD-CVC.

Abstract PO3-26-12: Targeting Lipocalin-2 in HER2+ Inflammatory Breast Cancer Using Herceptin-Conjugated Liposomes

Abstract Inflammatory breast cancer (IBC) is a rare and aggressive subtype of BC that accounts for 1 to 5% of all types of BC. Due to its inflammatory characteristics and the absence of a palpable mass, IBC is usually confounded with a mastitis. Unfortunately, once properly diagnosed, IBC has already metastasized. A high portion of IBC tumors overexpress human epidermal growth factor receptor 2 (HER2). These IBC patients (HER2+) are treated with Trastuzumab, a monoclonal antibody. However, many IBCs become resistant to this therapy. Thus, there is not an optimal treatment against this horrendous disease. One potential target against IBC is Lipocalin-2 (LCN2), a secreted protein involved in iron homeostasis, immune responses, transport of siderophores, and epithelial cell differentiation. In many aggressive tumors, high levels of LCN2 have been associated with increased cancer cell motility, proliferation, angiogenesis, invasion, and metastasis. LCN2 is aberrantly abundant in IBCs compared with non-IBC patients and cell lines. This information indicates that LCN2 could be a target for IBC therapy. Our research team showed that small interference RNAs (siRNAs) targeting LCN2 effectively reduced the cell proliferation and invasion ability of IBC cells. To systemically deliver siRNA specifically to HER2+ IBC cells, we prepared Herceptin-conjugated liposome loaded with siRNA against LCN2. Trastuzumab conjugated liposomes werecharacterized by using dynamic light scattering to measure the size distribution and the zeta potential. Our formulation displayed sizes of 18.3 and 73.7 nm which could correspond to free DSPE-PEG-(2000) trastuzumab and the trastuzumab-liposome, respectively. To determine the optimal timepoint of liposome internalization into IBC cell lines, we incubated HER2+ IBC3 cells with liposomes for different time-points. Optimal internalization was observed at 24 and 48-hr. To further understand the downstream effectors of LCN2 in IBC cells, we performed RNAseq in siRNA-mediated LCN2 knockdown cells. Further bioinformatics with the RNAseq data, revealed 138 dysregulated genes following LCN2 knockdown as compared with a negative control (NC) siRNA. Particularly, increased STAT1 levels has been associated with to distant metastasis and poor clinical prognosis in cancer. In our study, STAT1 levels decreased after LCN2 silencing. Ingenuity Pathway Analysis (IPA) showed 25 canonical pathways altered in the LCN2 knockdown cells as compared with the NC-siRNA. These results indicate that LCN2 activate molecular pathways involved in cell proliferation, invasion and metastasis in IBC cells. Citation Format: Marienid Flores-Colón, Pablo Vivas-Rivera, Pablo E. Vivas-Mejía. Targeting Lipocalin-2 in HER2+ Inflammatory Breast Cancer Using Herceptin-Conjugated Liposomes [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-26-12.

Gut microbiota composition in travellers is associated with faecal lipocalin-2, a mediator of gut inflammation.

We examined the gut microbiota of travellers returning from tropical areas with and without traveller's diarrhoea (TD) and its association with faecal lipocalin-2 (LCN2) levels. Participants were recruited at the Hospital Clinic of Barcelona, Spain, and a single stool sample was collected from each individual to perform the diagnostic of the etiological agent causing gastrointestinal symptoms as well as to measure levels of faecal LCN2 as a biomarker of gut inflammation. We also characterised the composition of the gut microbiota by sequencing the region V3-V4 from the 16S rRNA gene, and assessed its relation with the clinical presentation of TD and LCN2 levels using a combination of conventional statistical tests and unsupervised machine learning approaches. Among 61 participants, 45 had TD, with 40% having identifiable etiological agents. Surprisingly, LCN2 levels were similar across groups, suggesting gut inflammation occurs without clinical TD symptoms. Differential abundance (DA) testing highlighted a microbial profile tied to high LCN2 levels, marked by increased Proteobacteria and Escherichia-Shigella, and decreased Firmicutes, notably Oscillospiraceae. UMAP analysis confirmed this profile's association, revealing distinct clusters based on LCN2 levels. The study underscores the discriminatory power of UMAP in capturing meaningful microbial patterns related to clinical variables. No relevant differences in the gut microbiota composition were found between travellers with or without TD. The findings suggest a correlation between gut microbiome and LCN2 levels during travel, emphasising the need for further research to discern the nature of this relationship.

Tectochrysin ameliorates dextran sulfate sodium-induced chronic colitis by regulating the intestinal flora and inflammatory responses

Tectochrysin (TEC) is a natural flavonoid with anti-inflammatory, antioxidant, antitumor, and wound-healing activity. However, little is known about the therapeutic effects of TEC on inflammatory bowel disease (IBD). This study investigated the anti-inflammatory effect of TEC on IBD. Mice with dextran sulfate sodium (DSS)-induced chronic colitis served as an in vivo model of IBD. Lipopolysaccharide (LPS)-stimulated J774A.1 macrophages and mouse bone marrow-derived macrophages (BMDMs) served as in vitro models of inflammation. In vivo, 0.5% (w/w) TEC ameliorated DSS-induced colitis in mice by decreasing lipocalin-2 levels, disease activity index, colon shorten, weight loss, histological scores, and the apoptosis of colonic mucosal cells; it improved intestinal barrier function by increasing the expression of ZO-1 and occludin, decreasing the Th17/Treg ratio, and reducing the protein expression of NLRP3 and caspase-1 in the colon. Moreover, TEC decreased inflammation by reducing the serum levels of pro-inflammatory cytokines IFN-γ, IL-1β, G-CSF, IL-6, IL-8, iNOS, and TNF-α and increasing the levels of anti-inflammatory cytokines Bcl-2 and IL-10. TEC increased the relative abundance of the beneficial bacteria norank_f_Muribaculaceae in the mouse intestine. In vitro, 500 ng/mL TEC reduced oxidative stress by decreasing LPS-induced mitochondrial ROS production in BMDMs. TEC alleviated the increased levels of IL-1β, IL-6, and TNF-α and increased IL-10 levels in the culture supernatant of LPS-stimulated J774A.1 macrophages and BMDMs. These results indicate that TEC has a protective effect on DSS-induced chronic colitis by inhibiting inflammation, improving intestinal barrier function, and regulating intestinal microbial composition. Therefore, TEC has a high potential to treat IBD.

Cystatin C as an adjunct to HbA1c may prove useful in predicting the development of diabetic complications in children and adolescents with type 1 diabetes

PurposeComplications from diabetes mellitus can occur over time and although glycosylated hemoglobin (HbA1c) is a good biomarker for glycaemic control, other factors also contribute to the development of complications in type 1 diabetes. More markers able to identify the risk of complications are needed. This study aimed to investigate plasma levels of FGF21, Cystatin C, lipocalin-2, and MMP-9 in children and adolescents with different duration of type 1 diabetes and possible correlation to HbA1c to identify potential biomarkers of future complication development.MethodsPatients (n = 244, 0–18 years) with type 1 diabetes, at Helsingborg’s Hospital, Sweden, were included in this study. Circulating levels of FGF21, Cystatin C, lipocalin-2, and MMP-9 were investigated in plasma using automated ELISA with the ELLA™ system and standardised controls.ResultsCystatin C levels were elevated in patients with diabetes duration longer than 5 years (P < 0.001). HbA1c and Cystatin C levels were inversely correlated for all participants (rs = − 0.23, CI95: −0.35-−0.10; P < 0.001). A stepwise multiple regression analysis showed that HbA1c (P < 0.001) and Cystatin C (P = 0.03) were associated to the duration of diabetes at sampling while MMP-9, lipocalin-2, and FGF21 did not reach statistical significance.ConclusionIn conclusion, Cystatin C levels were higher in patients with diabetes duration longer than 5 years, and inverse correlation was found between HbA1c and Cystatin C levels as well as duration of diabetes. Cystatin C may prove useful as an adjunct to HbA1c in predicting eventual development of diabetic complications.

COVID-19 and Pregnancy: A Dangerous Mix for Bone Turnover and Metabolism Biomarkers in Placenta and Colostrum.

Background: In pregnant women, COVID-19 can alter the metabolic environment, cell metabolism, and oxygen supply of trophoblastic cells and, therefore, have a negative influence on essential mechanisms of fetal development. The purpose of this study was to investigate, for the first time, the effects of COVID-19 infection during pregnancy with regard to the bone turnover and endocrine function of several metabolic biomarkers in colostrum and placenta. Methods: One hundred and twenty-four pregnant mothers were recruited from three hospitals between June 2020 and August 2021 and assigned to two groups: Control group and COVID-19 group. Metabolism biomarkers were addressed in placental tissue and colostrum. Results: Lipocalin-2 and resistin levels were higher in the placenta, revealing an underlying pro-inflammatory status in the gestation period for mothers suffering from COVID-19; a decrease in GLP-1 and leptin was also observed in this group. As for adiponectin, resistin, and insulin, their concentrations showed an increase; a decrease in GLP-1, leptin, and PYY was also reported in the colostrum of mothers suffering from COVID-19 compared with the control group. Conclusions: As for bone turnover, placental samples from mothers with COVID-19 showed lower levels of OPG, while DKK-1 increased compared with the control group. Colostrum samples showed higher levels of OPG, SOST, and PTH in the COVID-19 group, a fact that could have noteworthy implications for energy metabolism, fetal skeletal development, and postnatal bone density and mineralization. Further research is needed to explain the pathogenic mechanism of COVID-19 that may affect pregnancy, so as to assess the short-term and long-term outcomes in infants' health.