Search Results (795)

Disruption of the intestinal epithelial barrier is a key driver of gut-derived inflammation in various disorders, yet strategies to preserve or restore barrier integrity remain limited. To address this, we evaluated a four-strain Bifidobacterium mixture—selected for complementary anti-inflammatory potency and industrial scalability—in lipopolysaccharide (LPS)-challenged RAW 264.7 macrophages and a Caco-2/THP-1 transwell co-culture model. Pretreatment with the probiotic blend reduced nitric oxide (NO) release in a dose-dependent manner by 25.9–48.3% and significantly down-regulated the pro-inflammatory markers in macrophages. In the co-culture system, the formulation decreased these markers, increased transepithelial electrical resistance (TEER) by up to 31% at 10⁵ colony-forming unit (CFU)/mL after 48 h, and preserved the membrane localization of tight junction (TJ) proteins. Adhesion to Caco-2 cells (≈ 6%) matched that of the benchmark probiotic Lacticaseibacillus rhamnosus GG, suggesting direct epithelial engagement. These in vitro findings demonstrate that this probiotic mixture can attenuate LPS-driven inflammation and reinforce epithelial architecture, providing a mechanistic basis for its further evaluation in animal models and clinical studies of intestinal inflammatory disorders. Full article

Murine microglia exhibit rapid self-renewal upon removal from the postnatal brain. However, the signaling pathways that regulate microglial repopulation remain largely unclear. To address this knowledge gap, we depleted microglia from mixed glial cultures using anti-CD11b magnetic particles and cultured them for 4 weeks to monitor their repopulation ability in vitro. Flow cytometry and immunocytochemistry revealed that anti-CD11b bead treatment effectively eliminated >95% of microglia in mixed glial cultures. Following removal, the number of CX3CR1-positive microglia gradually increased; when a specific threshold was reached, repopulation ceased without any discernable rise in cell death. Cell cycle and 5-ethynyl-2′-deoxyuridine incorporation assays suggested the active proliferation of repopulating microglia at d7. Time-lapse imaging demonstrated post-removal division of microglia. Colony-stimulating factor 1 receptor-phosphoinositide 3-kinase-protein kinase B signaling was identified as crucial for microglial repopulation, as pharmacological inhibition or neutralization of the pathway significantly abrogated repopulation. Transwell cocultures revealed that resident microglia competitively inhibited microglial proliferation probably through contact inhibition. This in vitro microglial removal system provides valuable insights into the mechanisms underlying microglial proliferation. Full article

RNA-seq analysis of the highly differentiated human retinal pigment epithelial (RPE) cell-line ARPE-19, cultured on transwells for ≥4 months, yielded 44,909 genes showing 83.35% alignment with the human reference genome. These included mRNA transcripts of RPE-specific genes and those involved in retinopathies. Monolayers were fed photoreceptor outer segments (POS), designed to be synchronously internalised, mimicking homeostatic RPE activity. Cells were subsequently fixed at 4, 6, 24 and 48 h when POS were previously shown to maximally co-localise with Rab5, Rab7, LAMP/lysosomes and LC3b/autophagic compartments. A comprehensive analysis of differentially expressed genes involved in proteolysis revealed a pattern of gene orchestration consistent with POS breakdown in the autophagy-lysosomal pathway. At 4 h, these included elevated upstream signalling events promoting early stages of cargo transport and endosome maturation compared to RPE without POS exposure. This transcriptional landscape altered from 6 h, transitioning to promoting cargo degradation in autolysosomes by 24–48 h. Longitudinal scrutiny of mRNA transcripts revealed nuanced differences even within linked gene networks. POS exposure also initiated transcriptional upregulation in ubiquitin proteasome and chaperone-mediated systems within 4–6 h, providing evidence of cross-talk with other proteolytic processes. These findings show detailed evidence of transcriptome-level responses to cargo trafficking and processing in RPE cells. Full article

Accurate in vitro models of intestinal permeability are essential for predicting oral drug absorption. Standard models like Caco-2 cells have well-known limitations, including lack of segment-specific physiology, but are widely used. Emerging models such as organoid-derived monolayers and microphysiological systems (MPS) offer enhanced physiological relevance but require comparative validation. We performed a head-to-head evaluation of Caco-2 cells, human jejunal (J2) and duodenal (D109) enteroid-derived cells, and EpiIntestinal^TM tissues cultured on either static Transwell and flow-based MPS platforms. We assessed tissue morphology, barrier function (TEER, dextran leakage), and permeability of three model small molecules (caffeine, propranolol, and indomethacin), integrating the data into a physiologically based gut absorption model (PECAT) to predict human oral bioavailability. J2 and D109 cells demonstrated more physiologically relevant morphology and higher TEER than Caco-2 cells, while the EpiIntestinal^TM model exhibited thicker and more uneven tissue structures with lower TEER and higher passive permeability. MPS cultures offered modest improvements in epithelial architecture but introduced greater variability, especially with enteroid-derived cells. Predictions of human fraction absorbed (F_abs) were most accurate when using static Caco-2 data with segment-specific corrections based on enteroid-derived values, highlighting the utility of combining traditional and advanced in vitro gut models to optimize predictive performance for F_abs. While MPS and enteroid-based systems provide physiological advantages, standard static models remain robust and predictive when used with in silico modeling. Our findings support the need for further refinement of enteroid-MPS integration and advocate for standardized benchmarking across gut model systems to improve translational relevance in drug development and regulatory reviews. Full article

Cell migration assays provide valuable insights into pathological conditions, such as tumor metastasis and immune cell infiltration, and the regenerative capacity of tissues. In vitro tools commonly used for cell migration studies exploit commercial transwell systems, whose functionalities can be improved through engineering of the pore pattern. In this context, we propose the fabrication of a transwell-like device pursued by combining the proton beam writing (PBW) technique with wet etching onto thin layers of polydimethylsiloxane (PDMS). The resulting transwell-like device incorporates a PDMS membrane with finely controllable pore patterning that was used to study the arrangement and migration behavior of HCMEC/D3 cells, a well-established human brain microvascular endothelial cell model widely used to study vascular maturation in the brain. A comparison between commercial polycarbonate membranes and the PBW-holed membranes highlights the impact of the ordering of the pattern and porosity on cellular growth, self-organization, and transmigration by combining fluorescent microscopy and advanced digital processing. Endothelial cells were found to exhibit distinctive clustering, alignment, and migratory behavior close to the pores of the designed PBW-holed membrane. This is indicative of activation patterns associated with cytoskeletal remodeling, a critical element in the angiogenic process. This study stands up as a novel approach toward the development of more biomimetic barrier models (such as organ-on-chips). Full article

Background: Preeclampsia (PE) is a pregnancy-specific disease and hypertensive disorder with a multifactorial pathogenesis involving complex molecular regulatory networks. Recent studies highlight the critical role of non-coding RNAs, particularly miRNAs and lncRNAs, in PE development. This study investigates the molecular interaction between miR-7151-5p and the lncRNA KCNQ1OT1 and their functional contributions to PE pathogenesis. Methods: An integrative approach combining RNAhybrid-based bioinformatics, dual-luciferase reporter assays, qRT-PCR, Transwell migration and invasion assays, and RNA sequencing was employed to characterize the binding between miR-7151-5p and KCNQ1OT1 and assess their influence on trophoblast cell function and gene expression. Results: A bioinformatic analysis predicted a stable binding site between miR-7151-5p and KCNQ1OT1 (minimum free energy: –37.3 kcal/mol). The dual-luciferase reporter assay demonstrated that miR-7151-5p directly targets KCNQ1OT1, leading to suppressed transcriptional activity. In HTR8/SVneo cells, miR-7151-5p overexpression significantly downregulated both KCNQ1OT1 and Notch1 mRNA, whereas its inhibition showed no significant changes, suggesting additional regulatory mechanisms of Notch1 expression. Transwell assays indicated that miR-7151-5p overexpression suppressed trophoblast cell migration and invasion, whereas its inhibition enhanced these cellular behaviors. RNA-seq analysis further revealed that miR-7151-5p overexpression altered key signaling pathways, notably the TGF-β pathway, and significantly modulates PE-associated genes, including PLAC1, ANGPTL6, HIRA, GLA, HSF1, and BAG6. Conclusions: The regulatory effect of miR-7151-5p on KCNQ1OT1, along with its influence on trophoblast cell dynamics via Notch1 and TGF-β signaling pathways, highlights its role in PE pathogenesis and supports its potential as a biomarker in early PE screening. Full article

Non-small cell lung cancer (NSCLC) is a predominant form of lung cancer that is often diagnosed at an advanced metastatic stage. The processes of cancer cell migration and invasion involve epithelial-to-mesenchymal transition (EMT), which is crucial for metastasis. Targeting cancer aggressiveness with effective plant compounds has gained attention as a potential adjuvant therapy. Eurycomanone (ECN), a bioactive quassinoid found in the root of Eurycoma longifolia Jack, has demonstrated anti-cancer activity against various carcinoma cell lines, including human NSCLC cells. This study aimed to investigate the in vitro effects of ECN on the migration and invasion of human NSCLC cells and to elucidate the mechanisms by which ECN modulates the EMT in these cells. Non-toxic doses (≤IC20) of ECN were determined using the MTT assay on two human NSCLC cell lines: A549 and Calu-1. The results from wound healing and transwell migration assays indicated that ECN significantly suppressed the migration of both TGF-β1-induced A549 and Calu-1 cells. ECN exhibited a strong anti-invasive effect, as its non-toxic doses significantly suppressed the TGF-β1-induced invasion of NSCLC cells through Matrigel and decreased the secretion of MMP-2 from these cancer cells. Furthermore, ECN could affect the TGF-β1-induced EMT process in various ways in NSCLC cells. In TGF-β1-induced A549 cells, ECN significantly restored the expression of E-cadherin by inhibiting the Akt signaling pathway. Conversely, in Calu-1, ECN reduced the aggressive phenotype by decreasing the expression of the mesenchymal protein N-cadherin and inhibiting the TGF-β1/Smad pathway. In conclusion, this study demonstrated the anti-invasive activity of eurycomanone from E. longifolia Jack in human NSCLC cells and provided insights into its mechanism of action by suppressing the effects of TGF-β1 signaling on the EMT program. These findings offer scientific evidence to support the potential of ECN as an alternative therapy for metastatic NSCLC. Full article

Lysolecithin is a performance-enhancing product for livestock. Lysolecithins contain functional phospholipids (PLs) and lysophospholipids (LPLs) and have been used in monogastric feed formulations because they can enhance lipid emulsification, digestion, and absorption (surface chemistry). Another underexplored aspect is that lysolecithin mixtures can serve as signaling via so-called nutritional gene expression-regulating action. The scope of this study was to fully understand the potential of a lysolecithin source derived from soybeans to influence intestinal nutrient transport in the intestinal tract. In this context, in vitro cell culture data with intestinal Caco-2 cells revealed that a lysolecithin-based product can significantly improve intestinal cell viability. Furthermore, a Transwell culture experiment showed that lysolecithins can significantly trigger gene expression. The most significantly affected genes could be correlated with G-coupled protein cascades. Enrichment analyses showed that amino acid transport and lipid metabolism pathways are significantly affected. Furthermore, the polarized cell culture revealed that the studied lysolecithin could affect the abundance of metabolites/nutrients in the basolateral compartment when applied apically, indicating that its action exceeds surface chemistry. In conclusion, the data on intestinal cell viability, gene expression, and metabolite abundance seem to reveal the bioactivities of lysolecithin. The latter data suggest that the specific lysolecithin source used here is more than a biosurfactant; more specifically, it seems to be a potent bioactive mixture of amphiphilic compounds triggering cell signaling pathways. Full article

Background: The mechanisms underlying the metastasis of non-small-cell lung cancer (NSCLC) have long been a focal point of medical research. The anti-tumor effects of chelerythrine (CHE) have been confirmed; however, its ability to inhibit tumor metastasis and the underlying mechanisms remain unknown. The aim of this study was to investigate the inhibitory effects and molecular mechanisms of CHE on transforming growth factor-beta (TGF-β)-induced epithelial–mesenchymal transition (EMT). Methods: Wound healing and Transwell assays were employed to evaluate TGF-β-induced migration in A549 cells and the inhibitory effects of CHE. Ribonucleotide reductase subunit M2 (RRM2) expression levels were detected via Western blot and immunofluorescence staining. Western blot and RT-qPCR were used to examine the expression levels of EMT-related markers. Animal experiments were conducted to analyze the role of RRM2 in the CHE inhibition of TGF-β-induced lung cancer metastasis. Results: This study found that TGF-β treatment enhanced the metastasis of A549 cells, while CHE inhibited the expression of TGF-β-induced EMT-related transcription factors by RRM2, thereby suppressing tumor cell migration (p < 0.05). Furthermore, the oral administration of CHE inhibited the metastasis of A549 cells to the lungs from the tail vein in mice, consistent with in vitro findings. Despite the high doses of CHE used, there was no evidence of toxicity. Conclusions: Our data reveal the mechanism of the anti-metastatic effects of CHE on TGF-β-induced EMT and indicate that CHE can be used as an effective anti-tumor treatment. Full article

Lung adenocarcinoma is a prevalent, aggressive cancer with a poor prognosis due to early metastasis and resistance to treatment. LncRNA AFAP1-AS1 has been shown to be associated with the development of multiple carcinomas. This study investigates the functional role of AFAP1-AS1 in lung adenocarcinoma cell proliferation via miR-508-3p and ZWINT. Human lung adenocarcinoma A549 cells were transfected with siRNA constructs against AFAP1-AS1 (si-AFAP1-AS1) to silence its expression. Cell proliferation was evaluated via CCK-8 and colony-forming assays. Apoptosis was assessed using AO/EB staining, and invasion was determined via Transwell assay. The interaction between AFAP1-AS1, miR-508-3p, and ZWINT was confirmed via dual luciferase reporter assay and qRT-PCR analysis. Data were analysed using appropriate statistical tests. AFAP1-AS1 was significantly upregulated in lung adenocarcinoma cells compared to normal BEAS-2B cells. Silencing of AFAP1-AS1 resulted in a marked reduction in A549 cell proliferation and colony development, as observed in CCK-8 and colony formation assays. The AO/EB assay showed a significant increase in apoptosis (30 ± 4.4%) in si-AFAP1-AS1 transfected cells compared to control si-NC (3 ± 1.2%). In addition, knockdown of AFAP1-AS1 led to an upsurge of pro-apoptotic Bax and decline of anti-apoptotic Bcl-2 expression. The dual luciferase assay established the interaction between AFAP1-AS1 and miR-508-3p. Furthermore, ZWINT, identified as a target of miR-508-3p, was significantly upregulated in lung adenocarcinoma tissues. Overexpression of ZWINT rescued the inhibitory effects of AFAP1-AS1 silencing on cell proliferation, colony formation, and apoptosis, while also reversing the reduction in cell invasion. AFAP1-AS1 accelerates the development of lung adenocarcinoma by cell proliferation, apoptosis, and invasion via the miR-508-3p/ZWINT axis. Thus, targeting AFAP1-AS1 or its downstream regulatory axis could offer novel therapeutic approaches in lung adenocarcinoma treatment. Full article

Hepatocellular carcinoma (HCC) remains a major clinical challenge due to its high recurrence rate and limited response to monotherapies, such as sorafenib—the standard first-line therapy for advanced HCC. This is partly attributed to its cellular heterogeneity. Increasing evidence implies SRC family kinase (SFK) activation in HCC progression, highlighting the potential of SRC-targeted therapies. In this study, we observed that SRC and YES1 were significantly upregulated in clinical HCC specimens compared to its adjacent non-tumoral tissues (p < 0.001), suggesting relevance as therapeutic targets. High SRC expression was noticed in patients with poor prognosis, as confirmed in TCGA cohort. To evaluate the efficacy of dual targeting, we assessed the combination between SRC inhibitors, saracatinib and dasatinib, with sorafenib in six hepatic cell models, representing both S1 and S2 subtypes. Cytotoxicity assays demonstrated reduced cell viability with the combination therapies compared to either monotherapy, irrespective of the HCC subtype. Wound healing and Transwell migration assays revealed inhibition of cell migration and invasion following combination treatment, underscoring its potential to suppress metastatic behavior. RT-qPCR analysis further confirmed downregulation of the expression of MMP2 and MMP9, genes associated with HCC cell invasion. Additionally, combined therapies decreased VEGFA and HIF1A expression compared to sorafenib alone, suggesting a potential to counteract the adaptive resistance mechanisms of cells to sorafenib. In summary, the combination of SFK inhibitors with sorafenib significantly enhances anti-tumor activity, offering a promising strategy to address HCC cellular heterogeneity and improve treatment efficacy. Full article

Age-related macular degeneration (AMD) is the main cause of blindness in Western nations. AMD models addressing specific pathological pathways are desired. Through this study, a best-practice protocol for polarized porcine single-eye retinal pigment epithelium (RPE) preparation for AMD-relevant models of RPE barrier and polarity is established. Single-eye porcine primary RPE cells (from one eye for one well) were prepared in 12-well plates including Transwell inserts. Different coatings (laminin (Lam), Poly-ᴅ-Lysine (PDL), fibronectin (Fn) and collagens) and varying serum contents (1%, 5% and 10%) were investigated to determine optimal culture parameters for this model. Success rates of cultures, cell number (trypan-blue exclusion assay), morphology/morphometry (light and fluorescence microscopy), protein secretion/expression (ELISA, Western blot), gene expression (qPCR), transepithelial electric resistance (TEER) and polar location of bestrophin 1 (BEST1) by cryosectioning (IHC-Fr) were assessed. Cells seeded on Lam exhibited the highest level of epithelial cells and confluence properties. Fn resulted in the highest cell number growth. Lam and Fn exhibited the highest culture success rates. TEER values and vascular endothelial growth factor secretion were highest when Lam was used. For the first time, polar (Transwell) porcine single-eye RPE morphometry parameters were determined. RPE on Lam showed bigger cells with a higher variety of cell shapes. CIV displayed the lowest claudin 19 expression. The highest basolateral expression of BEST1 was achieved with Lam coating. The higher the serum, the better the cell number increase and confluence success. A reduction in serum on Lam showed positive results for RPE morphology, while morphometry remained stable. A five percent serum on Lam showed the highest culture success rate and best barrier properties. RPE65 expression was reduced by using 10% serum. Altogether, the most suitable coating of Transwell inserts was Lam, and a reduction in serum to 5% is recommended, as well as a cultivation time of 28 days. A protocol for the use of polar porcine single-eye cultures with validated parameters was established and is provided herein. Full article

Objectives: This study aims to investigate the impact of Gossypol on human cervical cancer cells and elucidate its mechanism of action to establish a foundation for further clinical investigations. Methods: Cell proliferation, migration, and invasion were evaluated through CCK−8, wound healing, and Transwell assays. Fe₃O₄-BP-Gossypol (Fe₃O₄@Gossypol) conjugates were synthesized by linking Fe₃O₄ with Gossypol using benzophenone crosslinking. Successful conjugation was confirmed through scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FT-IR), and ultraviolet–visible spectrophotometry (UV-Vis). Subsequent to co-incubation with HeLa cell lysates, Fe₃O₄@Gossypol complexes facilitated the magnetic enrichment and purification of target proteins, which were identified using high-resolution mass spectrometry (HR-MS). The identified targets underwent KEGG pathway and GO analyses, followed by molecular docking with Gossypol. HeLa cells were exposed to Gossypol at concentrations of 7.48, 14.96, and 29.92 μmol·L⁻¹ for 48 h, and protein expression levels were quantified via Western blotting. Results: Gossypol notably suppressed cervical cancer cell proliferation, migration, and invasion. The integration of target fishing, network pharmacology, and molecular docking highlighted PIK3R2, MAPK1, and GRB2 as potential therapeutic targets. Western blot analysis revealed a dose-dependent reduction in PIK3R2, GRB2, and MAPK1 expression in Gossypol-treated groups compared to controls (p < 0.05). Conclusions: Gossypol may exhibit anti-cervical cancer effects by modulating the PI3K/AKT signaling pathway. Full article

Colon cancer is one of the leading malignant tumors worldwide, and the membrane protein PAQR3 has been identified as a tumor suppressor in multiple cancers. Notably, the peptide synthesized from 6 to 55 amino acids at the N-terminal of PAQR3 (P6-55) has been shown to effectively inhibit the growth of gastric cancer cells. This study aims to elucidate the mechanism of PAQR3 and explore its therapeutic potential in colon cancer. CCK8 cell viability assays, colony formation assays, and transwell migration assays were employed to systematically assess the inhibitory effects of PAQR3 on the proliferation and migration of colon cancer cells. Furthermore, we confirmed that P6-55 exhibits functional similarities to PAQR3, effectively inhibiting the growth of colon cancer in vitro and in vivo. RNA sequencing revealed that PAQR3 suppresses tumor growth via the PI3K-AKT signaling pathway, providing a strong theoretical foundation for therapeutic strategies targeting PAQR3/P6-55. In conclusion, our findings highlight the therapeutic potential of PAQR3/P6-55 as novel colon cancer inhibitors. Full article

Type II testicular germ cell tumors (TGCTs) are the most common solid malignancies in young men and are classified into seminomas and non-seminomatous subtypes. Seminomas are known for their highly pro-inflammatory tumor microenvironment (TME) with abundant immune cell infiltration. While previous work has demonstrated that the seminoma-derived cell line TCam-2 induces immune cell activation in co-culture and undergoes phenotypic changes itself, the underlying mechanisms remained unclear. To explore the role of direct cell–cell interaction and the effects mediated by soluble mediators such as cytokines, we conducted co-culture experiments of TCam-2 cells with purified human T cells or monocytes, including Transwell assays and treatments with IL-6, TNFα, or their respective blocking antibodies Tocilizumab and Adalimumab. In this way, we found that immune cell activation, indicated by enhanced secretion of pro-inflammatory cytokines and an upregulation of activation markers, strongly depended on direct physical contact between both cell types. Nonetheless, we also unveiled the role of soluble mediators in both immune cell activation and promoting a shift in TCam-2 cells from a seminoma-like phenotype to a more dedifferentiated phenotype, suggesting that cytokines critically shape the TME. These observations highlight the complexity of tumor–immune interactions in the seminoma microenvironment, offering new insight into immune-driven dynamics in TGCTs. Full article