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Diabetic retinopathy is one of the most important complications of diabetes. It is a leading cause of visual loss in the world. While adequate control of hyperglycemia, hypertension and hyperlipidemia can decrease the prevalence of diabetic retinopathy, 60% of patients with type 2 diabetes develop this complication whereas it is estimated that most patients with type 1 diabetes will develop diabetic retinopathy. Upregulation of various pro-inflammatory molecules and vascular endothelial growth factor (VEGF) play a central role in the pathogenesis of the disease. Here we review the role of CD40 as an upstream inducer of these abnormalities and the development of diabetic retinopathy.
Psoriasis is a chronic immune-mediated disease driven by genetic susceptibility and environmental factors, including microbial exposure. While HLA-C-linked variants represent the strongest genetic risk factors, their relationship with the cutaneous microbiome remains incompletely understood. This study aimed to investigate host-microbiome interactions in psoriasis through integrative multi-omics analysis. Skin microbiome profiling using 16S rRNA sequencing and targeted genotyping of psoriasis-associated single-nucleotide polymorphisms (SNPs) was performed in lesional and non-lesional skin from patients with plaque psoriasis and in healthy controls. Integrated analysis was conducted using supervised multivariate modeling (DIABLO) to identify coordinated genetic and microbial features associated with disease status. Combined genetic and microbial signatures differentiated lesional, non-lesional, and healthy skin. Variants within the HLA-C susceptibility region, including rs12191877, rs10484554, and rs4406273, showed contributions to group separation and demonstrated positive associations with Peptostreptococcus anaerobius. Associations involving ERAP1 variants linked antigen-processing pathways with inflammation-associated microbial taxa in lesional skin. Importantly, genotype-microbiome correlations were also detected in clinically non-lesional skin, where an increased psoriasis risk allele dosage co-varied with a higher relative abundance of P. anaerobius and Aerococcus urinae. In contrast, commensal-associated taxa were enriched in healthy controls and formed genotype-linked clusters only in non-lesional skin. These findings suggest that psoriasis is characterized by coordinated host genetic and microbial interaction patterns centered on antigen presentation pathways. The presence of a genotype-microbiome coupling in non-lesional skin may indicate that genetically determined immune configurations could shape microbial community structure prior to visible lesion development. Rather than reflecting uniform dysbiosis, psoriasis may represent a dynamic host-microbe ecosystem in which genetic susceptibility influences microbial persistence and inflammatory readiness.
Anti-IgLON5 disease is an autoimmune disease, in which autoantibodies (AABs) against the neuronal cell surface protein IgLON5 lead to profound brain dysfunction and Tau pathology. How α-IgLON5 AABs cause neuronal Tau protein pathology and neurodegeneration remains unclear. We find that patient-derived α-IgLON5 AABs cluster IgLON5 proteins with other cell surface proteins, leading to neuronal hyperactivity that triggers pathological Tau missorting and phosphorylation, typically observed early in Tau-related neurodegenerative diseases. In wild-type mice, α-IgLON5 AABs induce hippocampal Tau phosphorylation and neuroinflammatory responses. Our findings establish a causal link between the α-IgLON5 AABs and Tau pathology in anti-IgLON5 disease patients and highlight the role of neuronal hyperactivity as a disease-overarching driver of Tau pathology and provide a potential target for therapeutic intervention.
Lupus nephritis (LN) is a severe immune-mediated renal disorder causing significant morbidity and mortality in patients with systemic lupus erythematosus (SLE). Traditional biomarkers (serum complement components C3 and C4 and anti-dsDNA antibodies) have limited sensitivity and specificity for detecting renal flares; thus, new markers are needed to improve relapse detection and therapeutic response monitoring. We conducted a cross-sectional study including 71 women with SLE and 20 age- and sex-matched controls. Clinical data were collected, global activity was evaluated using the SLEDAI, and renal activity was evaluated using the renal SLEDAI (rSLEDAI). Serum syndecan-1 (SDC-1) and anti-dsDNA were measured using ELISA, and 24 h proteinuria was quantified. According to the rSLEDAI, 38 patients (53.5%) had LN flare, with a mean SDC-1 level of 108.5 ± 69.3 ng/mL and anti-dsDNA level of 113.1 ± 148.8 IU/mL. SDC-1 was correlated with the rSLEDAI (r = 0.32; p = 0.006), prednisone dose (r = 0.37; p = 0.002), proteinuria (r = 0.33; p = 0.005), and anti-dsDNA (r = 0.33; p = 0.006), while anti-dsDNA was positively correlated with proteinuria (r = 0.39; p = 0.001 and SDC-1 (r = 0.33; p = 0.006) and negatively correlated with age (r = -0.33; p = 0.006). High SDC-1 (cutoff ≥ 89 ng/mL) had higher sensitivity for detecting renal flares than anti-dsDNA (66% vs. 45%). In the multivariable analysis, high SDC-1 levels had around a 3-fold higher risk of being associated with LN flares, independently of anti-dsDNA and complement component levels. These results support serum SDC-1 as a promising biomarker for identifying renal flares in SLE patients, and it should be combined with traditional biomarkers to increase its value as a clinical tool. Follow-up studies are required to determine its value for predicting long-term renal outcomes.
Toll-like receptor 7/8 agonists (TLR7/8a), such as resiquimod (R848), are highly potent in activating dendritic cells and thus hold promise for T cell-mediated tumor immunotherapies. However, the short half-life of these small molecules in the lesion and the associated systemic immunotoxicity post-leakage of the drug into the circulation make their clinical application challenging. To overcome these shortcomings, we tested prolonged TLR7/8a therapy by intratumoral infusion of R848 for 25 h using a micropump to achieve durable therapeutic effects while minimizing the proinflammatory cytokine levels in the plasma post leakage of the drug into the circulation. The results showed that prolonged immunotherapy with R848 (as low as 1 μg) significantly suppressed tumor growth (inhibition rates up to 98%, p < 0.01) in treated mice compared to control mice receiving regular intratumoral injection of R848. Higher levels of CD86+or CD11c+ D.C.s, CD4+/CD8+/OX40+ T cells, and cytokines (TNF-α/IFN-γ) were observed in the tumors and spleens of the mice in the treated group compared to the sham group (p < 0.05), indicating efficient activation of local and abscopal immunity by prolonged therapy with R848. Furthermore, the R848 functional concentration assay demonstrated that the micropump prolonged the treatment time of R848 drugs in tumors and reduced the requirement for higher doses, enhancing safety. Taken together, this study provides new insights into TLR7/8a immunotherapy for improved clinical performance, with potential benefits for patients with superficial tumors amenable to prolonged intratumoral infusion via micropump.
Immune checkpoint inhibitors have transformed the treatment of advanced melanoma, yet many patients develop primary or acquired resistance. Although most work has focused on adaptive checkpoints (PD-1 and CTLA-4), accumulating evidence implicates innate immune suppression and stem-like, drug-resistant melanoma cell states. CD24, a small, heavily glycosylated glycosylphosphatidylinositol (GPI)-anchored surface protein, sits at the intersection of these processes and is emerging as a context-dependent biomarker and potential mediator of aggressive, therapy-resistant melanoma states. In this review, we synthesize evidence indicating that CD24 is both a tumour-intrinsic and tumour-extrinsic regulator in melanoma. We summarize the structure, glycosylation and regulation of CD24, then discuss its role in melanoma, supporting phenotypic plasticity, sustaining stem-like populations and promoting resistance to BRAF-targeted and cytotoxic therapies through SOX2/STAT3-linked programmes. We then examine the CD24-Siglec-10 axis as an innate immune checkpoint that suppresses macrophage and dendritic cell function, promotes immune-excluded 'cold' tumour microenvironments and may shape responses to immunotherapy among CD24+ melanoma cells. We highlight CD24 in tumour tissue, blood and extracellular vesicles as potential biomarkers of prognosis and pathway activity, and review CD24-axis interventions, including anti-CD24 antibodies, Siglec-10 antagonists and CD24-targeted CAR-T/CAR-NK cells, with rational combinations alongside PD-1/CTLA-4 blockade and MAPK-targeted therapy. We propose that biomarker-driven trials targeting this axis could open a new front in melanoma immunotherapy.
Palmitoylation is a reversible lipid modification, regulating protein localization and signaling in neurons. Growth-associated protein 43 (GAP43) requires palmitoylation for axonal development and synaptic plasticity; however, its depalmitoylase and regulation in neural circuits remain unknown. We investigated whether palmitoyl-protein thioesterase 1 (PPT1) is the principal depalmitoylase for GAP43 and examined how disrupted PPT1-GAP43 signaling affects neuronal morphology and circuit function. Using biochemical assays, structural modeling, CRISPR-Cas9-generated GAP43 point mutation mice (GAP43-PM), PPT1-knockout mice (PPT1-KO), electrophysiology, and behavior, we demonstrated that PPT1 interacts with GAP43 at Cys3 and Cys4 to mediate its depalmitoylation, disruption of this interaction causes GAP43 hyperpalmitoylation that drove excessive dendritic arborization and aberrant growth cone expansion, enhanced glutamatergic transmission, and hippocampal network hyperexcitability, resulting in cognitive deficits without lysosomal storage pathology. Exogenous PPT1 reduced these morphological/synaptic abnormalities. Our findings establish the PPT1-GAP43 depalmitoylation pathway as essential for neuronal circuit homeostasis; its dysfunction contributes to neurodevelopmental disorders, identifying a potential therapeutic target for palmitoylation-related neurodevelopmental disorders.
Rengyolone, a cyclohexylethanol derivative isolated from Incarvillea mairei (Bignoniaceae), exhibited a potent anti-inflammatory effect in LPS-stimulated BV-2 cells by inhibiting the release of the inflammatory mediators nitric oxide (NO) and prostaglandin E2 (PGE2), as well as downregulating the levels of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) and elevating the level of the anti-inflammatory cytokine IL-10. Further investigation revealed that rengyolone exerts its anti-inflammatory effect in microglia by suppressing the protein expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2), as well as by blocking the TLR4/NF-κB signaling pathway. These results suggest that rengyolone may be a promising lead compound for the treatment of neuroinflammation.
Widespread uses of nuclear materials increase the risk of accidental or intentional radiation exposure, which can result in acute radiation syndrome (ARS). Hematopoietic ARS (H-ARS) occurs at relatively low doses and is potentially lethal without intervention. While several FDA-approved cytokine-based radiomitigators exist, many require repeated dosing, complicating deployment in mass-casualty scenarios. This study evaluated a novel long-acting, murine-reactive granulocyte-macrophage colony-stimulating factor (LA-GM-CSF; mPDM608) as a prophylactic and mitigative countermeasure for H-ARS. Male and female C57BL/6 mice were exposed to lethal or sublethal total body irradiation (TBI) and treated with LA-GM-CSF using single- or multi-dose regimens administered before or after TBI. Safety, 30-day survival, hematologic recovery, bone marrow cellularity, serum GM-CSF pharmacokinetics, endothelial injury markers, and cytokine profiles were assessed using standard hematology, histopathology, ELISA, and multiplex assays. LA-GM-CSF was well tolerated at doses up to 30 mg/kg. Single or limited dosing conferred significant survival benefits compared with vehicle controls, with optimal efficacy observed at lower doses (3 mg/kg). Post-TBI administration as a single dose 24 h after exposure markedly improved survival in both sexes, with stronger hematopoietic recovery in males. LA-GM-CSF accelerated recovery of neutrophils, red blood cells, platelets, hematocrit, and sternal megakaryocytes, prolonged circulating GM-CSF levels, and favorably modulated endothelial injury markers and select cytokines. LA-GM-CSF demonstrates strong potential as a next-generation radiation countermeasure, providing robust survival benefit and hematopoietic recovery with minimal dosing. The results shown here support further development for H-ARS management under the FDA Animal Rule.
The redox mechanisms of RAW 264.7 macrophages exposed to 2.45 GHz RF-EMF at subthermal specific absorption rates and to lipopolysaccharide (LPS) and/or the SARS-CoV-2 spike protein (CSP) were investigated. To this end, cellular responses (lysosomal and mitochondrial activity, nitric oxide (NO) production, and cell survival/death) were measured after 6, 24, and 48 h. Selective loss of viability in cells exposed to RF and LPS was observed at 6 h, consistent with early defects in membrane permeability. Lysosomal activity was significantly enhanced in cells treated with RF + LPS. Mitochondrial activity decreased in cells exposed to RF + LPS at 6 h and increased in cells treated with RF + CPS/LPS. Cell viability decreased greatly in cells treated with LPS and CSP + LPS after 24, particularly after 48 h. Nitrite levels peaked in non-irradiated cells treated with RF + LPS and in CSP + LPS at 24 h and decreased in irradiated cells after 48 h. Irradiation affected selection of the death mode: apoptosis decreased or remained unchanged in cells subjected to any of the treatments, while necrosis increased in cells treated with CPS, LPS, or both for 48 h. The combination of RF-EMF and infectious agents reprogrammed the interaction between mitochondria/lysosomes/nitric oxide (NO)/cell death in macrophages in a time- and stimulus-dependent manner.
A single nucleotide substitution in codon 118 of HLA-DQB1*03:01:01:01 results HLA-DQB1*03:615N.
Killer cell immunoglobulin-like receptors (KIR) and their cognate HLA ligands regulate the functions of natural killer (NK) cells. However, extensive sequence homology within the KIR family limits the ability of monoclonal antibodies to selectively recognise individual receptors, and no KIR2DS1-specific reagent is available to date. Here, we comprehensively delineate the binding profile of the monoclonal antibody S22019F. Using Ba/F3 cells, NK cell clones, and primary NK cells, we demonstrate that S22019F selectively binds KIR2DS1. Moreover, functional assays reveal that binding of S22019F to KIR2DS1 is preserved upon activation by its HLA-C ligand. We further confirm that S22019F does not cross-react to KIR2DL3*005 allotypes and we extend its application to the analysis of KIR2DS1+ T cells. Collectively, these findings underscore the value of S22019F as a reagent that selectively recognises KIR2DS1, enabling improved analyses of KIR2DS1-bearing lymphocytes in fundamental and clinical research.
Introduction: Alterations of the microbiota-gut-brain axis, including increased intestinal permeability (IP), changes in microbial activity, and immune activation, are central to the pathophysiology of irritable bowel syndrome with diarrhea (IBS-D). The low-fermentable oligo-di-monosaccharides and polyols (FODMAP) diet (LFD) is an established therapy for IBS, yet its systemic effects, particularly in patients with elevated depressive symptoms, remain incompletely characterized. Methods: This single-arm pre-post study investigated associations between depressive symptom severity and markers of small IP (s-IP), endotoxin exposure, inflammation, and erythrocyte membrane polyunsaturated fatty acid (PUFA) composition in 43 IBS-D patients undergoing a 12-week personalized LFD. Patients were classified using the Symptom Checklist-90-Revised depression subscale into those with (d+, n = 23) and without (d-, n = 20) clinically elevated depressive symptoms. Results: At baseline, d+ patients exhibited higher s-IP, circulating lipopolysaccharide levels, inflammatory markers, and a more pro-inflammatory PUFA profile. Following LFD, significant improvements in symptoms and several biological parameters were observed in the overall cohort. Greater absolute changes in d+ patients were consistent with their higher baseline values rather than indicating differential responsiveness. Baseline depressive symptoms were not significantly associated with the magnitude of post-intervention changes in IP or inflammatory markers. Conclusions: These findings suggest that elevated depressive symptoms identify an IBS-D subgroup characterized by greater baseline biological burden. Results should be interpreted as associative given the single-arm design, absence of a control group, and the concurrent reduction in body weight, which may have influenced the observed changes. Randomized controlled studies are needed to clarify the role of dietary interventions in modulating gut-brain axis-related pathways in IBS-D.
Transforming growth factor-β1 (TGF-β1) and tumor necrosis factor-α (TNF-α) are central regulators of vascular inflammation and remodeling in coronary artery disease. However, their cell-type-specific and context-dependent effects in primary human coronary artery endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) remain incompletely defined. Primary human coronary artery endothelial cells (pHCAECs) and smooth muscle cells (pHCASMCs) were stimulated with TGF-β1 (10 ng/mL), TNF-α (100 ng/mL), or their combination. Canonical SMAD2/3 activation, Krüppel-like factor 11 (KLF11) expression, cytoskeletal and junctional remodeling, vascular cell adhesion molecule-1 (VCAM-1) expression, migration dynamics (wound healing and confluent assays), and endothelial tube formation were assessed using immunofluorescence microscopy, live-cell imaging, and quantitative trajectory analysis. Both cytokines were associated with increased nuclear pSMAD2/3 signal in ECs and VSMCs, consistent with functional interplay between inflammatory and TGF-β-related signaling pathways. In pHCAECs, TNF-α robustly induced VCAM-1 functional expression and disrupted VE-cadherin continuity, whereas TGF-β1 primarily promoted cytoskeletal remodeling without strong inflammatory activation. TGF-β1 increased endothelial migration velocity and accumulated distance. In contrast, TNF-α preferentially enhanced Euclidean displacement and directional persistence, shifting the migratory pattern toward more directed movement most evident under combined TGF-β1 + TNF-α stimulation. Notably, TGF-β1 significantly reduced endothelial tube formation, indicating impaired network organization rather than proangiogenic activity. In pHCASMCs, TGF-β1 enhanced migratory activity, particularly in confluent monolayers, whereas TNF-α enhanced directional displacement. KLF11 was induced by TGF-β1 in both pHCAECs and pHCASMCs. In pHCAECs, TNF-α also increased KLF11 and co-stimulation promoted nuclear enrichment, whereas in pHCASMCs TNF-α alone was not effective and combined treatment amplified the TGF-β1 response, supporting cell-type-specific integration of inflammatory and TGF-β-dependent signals. TGF-β1 and TNF-α differentially regulate the inflammatory activation and migration of primary human coronary vascular cells in a cell-type- and structural-context-dependent manner. TGF-β1 enhances migratory force generation, whereas TNF-α reinforces directional polarization, and their integration determines effective vascular repair dynamics. Canonical SMAD2/3 activation does not uniformly predict functional outcome, and KLF11 was identified as a context-sensitive transcription-associated factor showing differential nuclear localization in response to cytokine stimulation, representing a hypothesis-generating observation for future mechanistic studies.
Owing to the multifactorial nature of inflammatory bowel disease (IBD) pathogenesis, conventional two-dimensional (2D) models inadequately recapitulate the complex in vivo microenvironment. This study sought to develop an immune-microenvironment-integrated intestinal-on-a-chip model to overcome these limitations. A microfluidic chip was engineered to co-culture intestinal epithelial (Caco-2) cells and macrophages, facilitating the simulation of IBD pathological conditions for mechanistic investigations. Following inflammatory stimulation, M0 macrophages polarized into the M1 phenotype, concomitant with the upregulation of pro-inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and interleukin-1 beta (IL-1β). This induction disrupted the expression of tight junction proteins (e.g., zonula occludens-1 [ZO-1]) in Caco-2 cells, thereby compromising epithelial barrier integrity. Infliximab was used as a model drug to inhibit TNF-α and modulate macrophage polarization within the chip, effectively rescuing impaired epithelial barrier integrity. This study establishes a reliable intestinal-on-a-chip model that recapitulates macrophage-epithelial interactions in IBD, providing a robust platform for elucidating the mechanisms underlying intestinal barrier dysfunction and developing targeted therapeutic strategies.
Urinary epidermal growth factor (uEGF) and clusterin (uCLU) are emerging biomarkers in chronic kidney disease (CKD), but rigorous analytical validation is required before clinical implementation. We evaluated intra-individual variability and long-term storage stability of uEGF and uCLU in CKD. In the prospective, multicenter UVALID study, 60 adults with CKD stages 2-4 underwent urine sampling at three visits over 8 weeks. First-morning and 24-h urine samples were collected to assess intra-individual variability over 24 h, 3 days and 8 weeks. Biomarkers were measured in duplicate by ELISA and normalized to urinary creatinine (/Cr). Inter-laboratory performance was assessed using quality control samples. Stability after 12 and 15 months of storage at -20 °C and -80 °C and the influence of pH were evaluated. Over 24 h, 3 days, and 8 weeks, uEGF/Cr demonstrated low variability and remained stable after long-term storage at both temperatures. In contrast, uCLU/Cr showed greater variability and pronounced instability at -20 °C, whereas stability was preserved at -80 °C. Samples with pH > 6 partially preserved uCLU stability at -20 °C. Inter-laboratory reproducibility was acceptable for uEGF but suboptimal for uCLU at low concentrations. Thus, uEGF showed robust analytical performance, supporting its potential clinical applicability in CKD, whereas uCLU exhibited important analytical and pre-analytical limitations, warranting further assay optimization. These findings underscore the need for rigorous validation to facilitate biomarker implementation in clinical practice.
Plant polysaccharides can exert immunomodulatory activities. In this study we provided chemical characterization of wheat cell culture-derived polysaccharides (WCCPS) and assessed their capacity to modulate inflammatory responses in mouse macrophages. The total sample (T-010) contained arabinogalactans, arabinans, glucans and xyloglucans. Fractionation by anion-exchange chromatography rendered a bound acidic fraction (B-010) and an unbound neutral fraction (UB-010). The B-010 fraction was enriched in arabinogalactans and arabinans, with some galactans, homogalacturonans, and arabinoxylans. The neutral UB-010 fraction was composed of glucans and xyloglucans. None of the WCCPS preparations triggered cytokine production on their own, but each potentiated different macrophage responses to bacterial lipopolysaccharide (LPS). The total WCCPS in T-010 increased LPS-induced tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-6 secretion, whereas the acidic arabinogalactan-rich fraction B-010 boosted IL-6 release and selectively upregulated nitric oxide synthase 2 (Nos2) and cholesterol 25-hydroxylase (Ch25h) expression in response to LPS. In contrast, the neutral UB-010 fraction enhanced IL-6 levels and induced Nos2 expression without altering Ch25h expression. These results suggest that WCCPS can modulate distinct aspects of the inflammatory response, with their effects shaped by their composition and structural features. Future research will focus on elucidating the molecular mechanisms underlying the immunomodulatory activity of WCCPS.
The pathogenesis of IgA nephropathy is mediated by B-cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL). Telitacicept is a fusion protein that targets and neutralizes both BAFF and APRIL and, as such, might be effective in IgA nephropathy. We now report a prespecified interim analysis of a phase 3, multicenter, double-blind, randomized, placebo-controlled trial, which enrolled adults with biopsy-proven IgA nephropathy and persistent proteinuria (protein level, ≥1.0 g per day), despite appropriate supportive care. Patients were randomly assigned in a 1:1 ratio to receive subcutaneous once-weekly telitacicept (240 mg) or matching placebo. The primary end point was the geometric mean ratio of the 24-hour urinary protein-to-creatinine ratio at 39 weeks relative to baseline. Safety was also evaluated. A total of 318 patients were assigned to receive telitacicept or placebo (159 in each group). At week 39, the percentage change in the 24-hour urinary protein-to-creatinine ratio was -58.9% with telitacicept and -8.8% with placebo, which corresponded to a relative difference (based on the ratio of geometric mean reductions between the two groups) of -55.0% (95% confidence interval [CI], -61.3 to -47.6; P<0.001) in favor of active medication. The percentage change in the estimated glomerular filtration rate relative to baseline was -1.0% (95% CI, -3.2 to 1.2) with telitacicept and -7.7% (95% CI, -9.9 to -5.4) with placebo. Adverse events were more common with telitacicept than with placebo (in 89.3% vs. 78.6% of patients), although serious adverse events were less common (in 2.5% vs. 8.2%). No unexpected safety findings were reported with telitacicept. In patients with IgA nephropathy at high risk for progression, 39 weeks of treatment with telitacicept led to a greater reduction in the 24-hour urinary protein-to-creatinine ratio than placebo. (Funded by RemeGen; TELIGAN ClinicalTrials.gov number, NCT05799287.).
This study aimed to optimize the ultrasonic-assisted extraction of polyphenols from Archidendron clypearia and to evaluate their anti-hyperuricemic effects. Polyphenols from medicinal plants have attracted increasing attention due to their potential roles in regulating uric acid metabolism. In this study, single-factor experiments combined with Box-Behnken response surface methodology were employed to optimize extraction conditions, and an entropy weighting method was applied to integrate total polyphenols and Archidendrin I into a comprehensive evaluation index. The bioactivity of the obtained extract was further assessed through in vitro assays and a hyperuricemic mouse model. The optimal extraction conditions were determined to be 50% ethanol, a liquid-to-material ratio of 30, and 31 min of sonication, yielding 175 mg GAE/g DW of total polyphenols and 80.34 mg/g DW of Archidendrin I. The extract exhibited significant xanthine oxidase inhibitory activity, reduced serum uric acid levels, regulated urate transporters (URAT1, GLUT9, and ABCG2), and alleviated renal and hepatic injury in hyperuricemic mice. These findings indicate that the optimized process enables efficient extraction of polyphenols from Archidendron clypearia, and the resulting extract exerts beneficial regulatory effects on uric acid metabolism, highlighting its potential as a natural agent for hyperuricemia management.
This research presents the design, synthesis, and thorough evaluation of a new series of benzofuran-1,4-dihydropyridine hybrids (4a-i) as potential multi-target anti-inflammatory drugs. These compounds were created through an optimized multistep process and examined using a combination of in silico, in vitro, and in vivo methods. Molecular docking revealed a potential dual-target profile, with the leading compounds showing strong predicted binding to both COX-2 and TNF-α. Notably, compound 4i demonstrated the highest affinity for COX-2 (MolDock score = -181.002 kcal/mol), with good selectivity over COX-1 (Δ = -21.35), and also showed one of the strongest affinities for TNF-α (-174.847 kcal/mol). Molecular dynamics simulations confirmed the stability of the 4i-COX-2 complex. This in silico effectiveness was supported by strong in vitro anti-inflammatory activity (eg. 10.09 µg/mL for IC50 of 4b in hypotonicity-induced hemolysis). Notably, the compounds showed excellent in vivo activity in a carrageenan-induced paw edema model, with 4i achieving 91.5% inhibition at 90 min. Histological analysis confirmed that the lead compounds were highly gastrointestinal (GI)-sparing, consistent with their predicted COX-2 selectivity. No liver damage was observed, and platelet count remained normal (4i). Overall, this study identifies compound 4i as a potent, orally active, and safe antinflammatory candidate with potential dual mechanisms.