Search Results (922)

Liver fibrosis is a critical determinant of the progression of chronic liver disease (CLD). Total flavonoids from Carthamus tinctorius L. (TFCTLs) exhibit diverse pharmacological activities while their effect on liver fibrosis remains incompletely understood. This study aimed to elucidate the effects and mechanisms of TFCTLs on liver fibrosis. To this end, we first established a carbon tetrachloride (CCl₄)-induced liver fibrosis model in mice. Histological analysis demonstrated that TFCTL treatment significantly alleviated CCl₄-induced liver collagen deposition (p < 0.001). Meanwhile, TFCTLs could also downregulate the expression levels of fibrosis markers α-SMA and collagen I in a dose-dependent manner (p < 0.05). In vitro, a cellular model of fibrosis was generated by treating HSC-T6 cells with TGF-β1. EdU incorporation assays revealed that TFCTLs significantly suppressed HSC proliferation (p < 0.05). Furthermore, immunofluorescence staining for α-SMA demonstrated a marked reduction in HSC activation upon TFCTL treatment. The inhibitory effect of TFCTLs on cell migration was confirmed by wound healing and transwell assays, which revealed a substantial decrease in the number of migrated cells (p < 0.001). Additionally, flow cytometric analysis indicated that TFCTL treatment promoted HSC apoptosis (p < 0.05). Further mechanistic investigations revealed that TFCTLs exert their antifibrotic effects by inhibiting Hedgehog pathway and activating autophagy process. The antifibrotic effect of TFCTLs was partially reversed by the autophagy inhibitor 3-MA. Furthermore, the Hedgehog agonist PUR not only counteracted the anti-fibrotic actions of TFCTLs but also suppressed TFCTL-induced autophagy activation. In conclusion, our study demonstrated that TFCTLs attenuate liver fibrosis by inhibiting Hedgehog signaling and subsequently promoting autophagy, highlighting their potential as a therapeutic agent for liver fibrosis. Full article

Background: Membrane potential represents one of the fundamental physiological characteristics of cells, playing a critical role in cellular function. Studies have shown that membrane hyperpolarization positively regulates the osteogenic differentiation of mesenchymal stem cells. Optogenetic technology based on the Natronomonas pharaonis halorhodopsin (NpHR) light-activated channel can induce membrane hyperpolarization through optical methods. Given the working principle of optogenetic technology, this study aimed to investigate whether optogenetic activation of NpHR channels could induce membrane hyperpolarization in human dental follicle stem cells (hDFCs)—mesenchymal stem cells derived from dental follicle tissue—to regulate their osteogenic differentiation. Methods: hDFCs were isolated and cultured. Engineered hDFCs expressing the NpHR channels were constructed through lentiviral transduction. Patch clamps were performed to observe the effects of optogenetic activation of NpHR channels on membrane potentials of hDFCs. Single-cell Ca²⁺ imaging were performed to observe the effects of membrane hyperpolarization via modulation of extracellular K⁺ concentration ([K⁺]_e) on the intracellular Ca²⁺ levels of hDFCs. Cell viability assay, transwell chamber assay, wound healing assay, osteogenic differentiation induction, alizarin red staining, alkaline phosphatase (ALP) staining, real-time reverse transcriptase polymerase chain reaction (RT-qPCR) and Western blot (WB) were performed to observe the effects of optogenetic activation of NpHR channels on proliferation, migration, and osteogenic differentiation of NpHR-hDFCs. Results: Reversing membrane hyperpolarization via modulation of extracellular K⁺ concentration ([K⁺]_e) was shown to suppress osteogenic differentiation of hDFCs, whereas promoting membrane hyperpolarization via opening ATP-sensitive K⁺ channels was shown to enhance osteogenic differentiation of hDFCs. Hyperpolarizing cells by decreasing [K⁺]_e increased intracellular Ca²⁺ levels of hDFCs. Optogenetic activation of NpHR channels by an optogenetic system induced membrane hyperpolarization and significantly enhanced the proliferation, migration, and osteogenic differentiation abilities of NpHR-hDFCs. Conclusions: Hyperpolarization by optogenetic activation of NpHR channels can promote hDFCs’ proliferation, migration, and osteogenic differentiation abilities. Full article

Elucidating the interactions among microbial communities in the Sichuan paocai fermentation system is of great significance for ensuring the safety and quality of paocai. In this study, the interaction between Bacillus subtilis Y61 and Weissella paramesenteroides (CWP) was preliminarily verified through the culture of CWP using the cell-free supernatant derived from Y61. Building on this, a transwell chamber was employed to spatially isolate the two bacteria. Combined with transcriptomic and metabolomic profiling, the underlying interaction mechanism was revealed. Weissella paramesenteroides (CWP) exhibited enhanced growth in the cell-free supernatant of Bacillus subtilis Y61, confirming a cross-feeding relationship between the two strains. In the transwell chamber, the promoting effect was most significant when Weissella paramesenteroides (CWP) was in the upper compartment and Bacillus subtilis Y61 in the lower compartment. Transcriptomic analysis showed that Weissella paramesenteroides (CWP) significantly upregulated genes involved in fatty acid synthesis and metabolism while downregulating those related to amino acid anabolism (p < 0.05). Metabolomic analysis further revealed that metabolites secreted by Bacillus subtilis Y61, including the key metabolites arginine and isovaleric acid, were markedly depleted during co-culture. Exogenous supplementation assays revealed that the combination of 0.1 g arginine and 2 mg isovaleric acid exhibited the strongest growth-promoting effect on Weissella paramesenteroides (CWP). Collectively, these results demonstrated that Bacillus subtilis Y61 promoted the growth of Weissella paramesenteroides (CWP) through cross-feeding via the extracellular secretion of the key metabolites arginine and isovaleric acid. Full article

Background/Objective: Bisphenol AF (BPAF), a fluorinated analogue of bisphenol A, is an environmental contaminant associated with hepatotoxicity and metabolic disruption. However, the systematic molecular mechanisms linking early transcriptional events to metabolic dysfunction in the liver remain poorly defined. The aim of this study is to elucidate the association between BPAF exposure and hepatic lipid accumulation by integrating transcriptomics, cellular metabolomics, and targeted phenotypic assays. Methods: We performed RNA-sequencing on livers from mice exposed to BPAF (0.1–10 mg/kg/day, 28 days), and performed non-targeted metabolomics on AML12 murine hepatocytes co-cultured with RAW264.7 macrophages in a Transwell system (0–2500 nM BPAF, 48 h). Key metabolic pathways were identified through integrated bioinformatics and validated using enzymatic assays, qRT-PCR, Western blotting, and phenotypic staining (lipid droplets, ROS). Results: Multi-omics integration revealed significant disruption of PPAR signaling and the tricarboxylic acid (TCA) cycle. A striking dose-dependent accumulation of succinate was observed in exposed cells, concomitant with a significant inhibition of succinate dehydrogenase (SDH) activity (52% reduction at 2500 nM, p < 0.001). Transcriptomic data confirmed the downregulation of mitochondrial fatty acid β-oxidation genes. Phenotypic validation indicated that BPAF exposure is associated with oxidative stress, pro-inflammatory cytokine release (TNF-α, IL-6), and pronounced intracellular lipid droplet accumulation in hepatocytes. Conclusions: This study suggests that BPAF exposure is associated with SDH dysfunction, TCA cycle arrest, and lipid dysregulation. Whether BPAF directly inhibits SDH or acts through upstream mitochondrial targets warrants further structural and kinetic investigation. Full article

Background: The mucosal barrier presents a significant challenge for non-invasive delivery of macromolecular therapeutics, often requiring administration with poor bioavailability and increased toxicity risks. The polymeric immunoglobulin receptor (pIgR) contains an extracellular secretory component (SC) for immunoglobulin binding and a membrane-anchored stem domain capable of apical-to-basolateral transcytosis. We hypothesized that targeting the stem domain could enable active drug transport across mucosal barriers. Methods: Using phage display, we identified four high-affinity nanobodies against human and murine pIgR. Two lead candidates (3LTHMP-4 and 3LTHMP-5) demonstrated efficient apical-to-basolateral transport in vitro (Transwell assays) and in vivo (fluorescence imaging). Engineered bispecific antibodies fusing these nanobodies with anti-IL-5 mAb reslizumab were administered via inhalation in a murine asthma model at one-tenth the intraperitoneal reslizumab dose. Resluts: The bispecific antibodies showed significant therapeutic efficacy, while reslizumab alone at equivalent concentrations failed to demonstrate efficacy. Hydrogen–Deuterium Exchange Mass Spectrometry (HDX-MS) revealed that both 3LTHMP-4 and 3LTHMP-5 specifically bind to the pIgR stem domain (residues 578–612), a region distinct from the dimeric IgA binding site. Conclusions: These findings suggest that stem domain-specific binding may facilitate transport across the mucosal barrier while preserving native receptor physiology, offering a potential strategy for effective transmucosal delivery of biologics. Full article

Heparanase 1 (HPSE1) is the only mammalian endoglycosidase that cleaves heparan sulfate (HS), a glycosaminoglycan (GAG), and is frequently upregulated in cancers, thereby promoting tumor progression. Despite extensive efforts to develop inhibitors of its HS-degrading activity, its non-enzymatic functions limit therapeutic efficacy and pose a major challenge for therapeutic development. Thus, inhibiting HPSE1 expression is critical for controlling its enzymatic and non-enzymatic functions; however, no FDA-approved inhibitors are currently available. Here, we identify auranofin (AUF), an oral gold-containing drug used to treat rheumatoid arthritis, as a potent inhibitor of HPSE1 promoter activity. High-throughput screening revealed that an atypical protein kinase C (aPKC)–NF-κB signaling axis is a key regulator of HPSE1 expression. Notably, AUF treatment reduced HPSE1 expression and significantly suppressed the invasive capacity of MDA-MB-231 cells in a Transwell migration assay. We then investigated the role of HPSE1 in the invasive activity of MDA-MB-231 cells, which produce higher levels of hyaluronan (HA) and HS than non-invasive cells. Neither HS degradation, HA supplementation in Matrigel during Transwell migration, nor HPSE1 overexpression alone was sufficient to drive invasion, suggesting that invasive capacity depends on mesenchymal features and coordinated induction of HPSE1 and GAGs rather than HS degradation. Collectively, our findings demonstrate that AUF-mediated inhibition of aPKC suppresses HPSE1 expression, thereby inhibiting both its enzymatic and non-enzymatic functions and limiting cancer progression, metastasis, and angiogenesis. These results highlight the therapeutic potential of AUF for targeting HPSE1-driven tumor progression and support its repurposing for cancer treatment. Full article

Radix pseudostellariae saponins (RPS) enhance immune responses in animals; however, the regulatory mechanisms of these effects remain unclear. This study observed that 14 days post-intranasal immunization with RPS and a Mycoplasma gallisepticum-attenuated vaccine (MGAV), MGAV-specific antibody titers were significantly increased in the blood, and chemokine (C-C motif) ligand 5 (CCL5) messenger RNA expression was significantly increased in the trachea and blood of chickens. Transcriptomic analysis demonstrated that RPS treatment significantly upregulated specific Kyoto Encyclopedia of Genes and Genomes pathways, notably the cytokine–cytokine receptor interaction pathway, which is linked to immune cell migration and involves chemokine receptor chemokine (C-C motif) receptor 4 (CCR4). This finding was corroborated at the protein level by immunohistochemical evidence showing increased CCL5 expression in tracheal tissue. In vitro studies showed that RPS enhanced the phagocytic capacity of RAW264.7 macrophages against ovalbumin, with immunofluorescence revealing time-dependent and dose-dependent CCL5 in these cells. Transwell and scratch-healing assays confirmed that RPS promoted this migration of both RAW264.7 cells and CCR4-positive lymphocytes. Collectively, the findings revealed that RPS modulated the activation, chemotaxis, and migration of macrophages and lymphocytes and is associated with the promotion of the CCL5/CCR4 signaling axis, providing novel evidence for the immune-enhancing effects of RPS by enhancing immunogenicity. Full article

Recent studies have highlighted the potential of urinary bladder matrix (UBM) derived from decellularized porcine urinary bladder as a bioactive hydrogel. Despite its complex composition of over 100 proteins, Type I collagen is the primary constituent of UBM. Caco-2 cells are widely used as an in vitro model of the intestinal epithelium; however, to date, no published study has evaluated the effects of UBM on Caco-2 cells. In this study, Electric Cell–Substrate Impedance Sensing (ECIS) was used to measure Caco-2 cell attachment and wound-healing migration on UBM-coated microelectrodes. Our results demonstrate that UBM hydrogel coating at 0.2 mg/mL significantly accelerates cell attachment and enhances migration rates compared to uncoated controls. These stimulatory effects were comparable to those observed with 0.2 mg/mL Type I collagen, suggesting that UBM can function as effectively as Type I collagen. We further monitored barrier formation in Caco-2 cells cultured on UBM-coated transwell membrane inserts using TEER measurements and scanning electron microscopy. The TEER values reached 300 Ω·cm² within three days, indicating the rapid establishment of mature tight junctions. Overall, these results show that UBM hydrogel coatings are effective substrates for Caco-2 cells, performing as well as Type I collagen in all our tests. Full article

Atherosclerosis begins with endothelial dysfunction, inflammatory activation, and immune-cell recruitment within a spatially organized vascular wall. Conventional static cultures and Transwell systems are advantageous for isolated readouts, but they fail to effectively recapitulate multicellular compartmentalization, extracellular matrix support, and dynamic perfusion within a singular platform. Here, we present a four-channel microfluidic vascular-wall chip designed to reconstitute an endothelial cell-extracellular matrix-smooth muscle cell arrangement and to model early atherosclerosis-related inflammatory endothelial dysfunction. The device comprises a perfusable endothelial channel, a collagen I hydrogel region embedded with human aortic smooth muscle cells, a cell-free matrix region, and a culture-medium supply channel. Under physiological conditions, HUVECs formed a ZO-1-positive endothelial barrier and maintained high cellular viability. Stimulation with TNF-α and IL-1β (10 ng/mL each) elevated IL-6 secretion, promoted the recruitment of THP-1-derived M0-like macrophages, disrupted ZO-1 continuity, and increased FITC-dextran permeability without causing extensive cell death. The chip was subsequently utilized to evaluate metformin and atorvastatin therapies. The combinational treatment produced a more pronounced attenuation of MCP-1 secretion than either monotherapy under the inflammatory background. While this platform does not recapitulate advanced plaque formation, lipid deposition, foam-cell formation, or disturbed arterial shear, it successfully provides a microfluidic model of early inflammatory endothelial dysfunction to facilitate mechanistic studies and preliminary anti-inflammatory drug evaluation. Full article

Background: Glioblastoma (GBM) is among the malignant tumors with the lowest five-year survival rate. Current treatments offer limited efficacy and first-line options are scarce, highlighting the urgent need for novel drugs. Cariprazine can cross the blood–brain barrier and has been reported to inhibit certain tumors; however, its effect on GBM remains unknown. This study aims to elucidate its anti-GBM effects and mechanisms. Methods: Cell proliferation and apoptosis were assessed by wound healing, Transwell, colony formation assays, flow cytometry and JC−10 staining. Co-immunoprecipitation (Co-IP) examined the effect of cariprazine on D2/D3–ARRB2 interaction. Direct binding of cariprazine to ARRB2 was determined by molecular docking and CETSA. Western blotting and immunofluorescence detected changes in proliferation and apoptosis-related proteins. In vivo anti-GBM activity was evaluated in subcutaneous mouse models. Results: Cariprazine inhibited GBM cell proliferation and migration, promoted apoptosis, and showed low astrocyte toxicity. In mice, it suppressed GBM allograft growth without overt systemic toxicity. These effects were mediated through D2/D3 receptors, as cariprazine disrupted the D2/D3–ARRB2 interaction and thereby inhibited ERK signaling. It also upregulated ARRB2, further inhibiting the growth of GBM. Molecular docking and CETSA confirmed the direct binding of cariprazine to ARRB2 at LEU-245 and PHE-246. Conclusions: This study is the first to repurpose cariprazine for GBM, elucidating a unique ARRB2-centered dual mechanism, thus offering a new therapeutic strategy. Full article

Clear cell renal cell carcinoma (ccRCC) is notorious for its clinical unpredictability. While Aquaporin-1 (AQP1) is a major water channel in healthy kidneys, its specific role and regulatory mechanisms in ccRCC remain unclear. Using bioinformatics analysis of 610 TCGA-KIRC patients (RNA sequencing and DNA methylation), single-cell transcriptomics of 27,402 cells, and experimental validation (CCK-8, scratch, Transwell, and xenograft assays, with Western blotting, HE staining, and immunohistochemistry), we systematically characterized AQP1 expression, regulation, and function. AQP1 was significantly downregulated in ccRCC via promoter hypermethylation, with single-cell analysis confirming tumor cell-specific loss. Low AQP1 correlated with worse prognosis; multivariate Cox regression identified AQP1 as an independent protective factor (HR = 0.510, p < 0.001), and a prognostic nomogram showed good predictive accuracy for 1-, 3-, and 5-year survival. AQP1 overexpression suppressed proliferation, migration, invasion, and xenograft growth, accompanied by upregulation of TNF-α, TNFRSF1A, Bax, and Cleaved Caspase-3 and reduced Vimentin, suggesting activation of TNF-related pro-apoptotic signaling. AQP1 is epigenetically silenced in ccRCC and suppresses tumor growth via TNF-mediated apoptosis, establishing it as an independent prognostic biomarker and candidate therapeutic target. Full article

The link between neutrophil extracellular traps (NETs) and hepatocyte ferroptosis in liver ischemia–reperfusion injury (LIRI) is unclear. Adipose-derived mesenchymal stem cell exosomes (ADSCs-Exo) hold therapeutic potential for LIRI. This study employed miniature pigs to investigate the NETs’ role and ADSCs-Exo’s protection in LIRI. In vitro, established hepatocyte oxygen-glucose deprivation/reoxygenation (OGD/R) model and Transwell co-culture system with polymorphonuclear neutrophils (PMNs). In vivo, a laparoscopic minimally invasive LIRI model was constructed in miniature pigs, followed by ADSCs-Exo intervention. Results demonstrated that NETs exacerbate OGD/R-induced hepatocyte ferroptosis via myeloperoxidase. ADSCs-Exo inhibited NET formation via the NADPH/MAPK pathway, thereby mitigating ferroptosis, and ultimately improved liver histopathology and function. This study is the first to demonstrate in a large animal model that ADSCs-Exo alleviate LIRI by inhibiting NET formation via the NADPH/MAPK pathway, consequently attenuating hepatocyte ferroptosis. These findings provide novel insights into LIRI pathogenesis, support the translational potential of ADSCs-Exo as a cell-free therapeutic strategy, and highlight the value of the miniature pig model in liver research. Full article

Objectives: This study aims to investigate the pharmacological effects and potential mechanisms of dehydrocorydaline, the primary active component of Corydalis yanhusuo W.T. Wang, as a potential therapeutic agent for pancreatic cancer, thereby providing new insights into its treatment. Methods: The pharmacological effects were assessed through MTT assay, colony formation assay, flow cytometry, scratch wound assay, and transwell assay. Potential mechanisms were explored through bioinformatics analysis and Western blot. Results: Dehydrocorydaline was verified to stimulate apoptosis and inhibit the growth, migration, and invasion of pancreatic cancer BxPC-3 cells. These effects may be associated with suppressed HSP90α expression, induced ERBB2 degradation, and subsequent inhibition of STAT3 and PI3K/Akt/mTOR pathway activation, as well as altered expression of multiple downstream proteins. Conclusions: This study demonstrates that dehydrocorydaline is the main active component of Corydalis yanhusuo W.T. Wang with anti-pancreatic cancer activity. Based on protein expression-level evidence, it may exert its effects by inhibiting HSP90α expression and inducing ERBB2 degradation, thereby affecting the PI3K/Akt/mTOR and STAT3 pathways, ultimately suppressing proliferation, migration, and invasion while promoting apoptosis in BxPC-3 cells. These findings justify further investigation of dehydrocorydaline as a potential treatment for pancreatic cancer. Full article

Although schistosome eggs are widely recognized as the principal drivers of hepatic granulomatous inflammation and fibrosis, the independent effects of adult worms may be masked by strong egg antigen-mediated responses. This study aimed to investigate whether adult Schistosoma japonicum worms alter the miRNA expression profile of hepatic stellate cells and to explore the potential relevance of these changes to liver fibrosis and hepatocellular carcinoma-related processes. A non-contact Transwell co-culture system was established using paired Schistosoma japonicum worms or male worms and hepatic stellate cells. Male worms were additionally included to further assess worm-derived effects independent of egg production–related influences. Untreated hepatic stellate cells served as controls. Total RNA was extracted for miRNA sequencing, and differentially expressed miRNAs were identified. Target gene prediction, Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis, and validation using The Cancer Genome Atlas database were subsequently performed. Both paired worms and male worms significantly altered the miRNA expression profile of hepatic stellate cells. Several differentially expressed miRNAs were identified, among which hsa-miR-103a-3p showed relatively stable changes. Pathway enrichment analysis suggested that the potential target genes of hsa-miR-103a-3p were mainly enriched in AMP-activated protein kinase, mechanistic target of rapamycin, tumor necrosis factor, insulin signaling, and cellular senescence pathways. Further analysis using The Cancer Genome Atlas database showed that hsa-miR-103a-3p had diagnostic value in hepatocellular carcinoma and was associated with alpha-fetoprotein level, albumin level, Ishak fibrosis score, pathological stage, histological type, and tumor status. These findings suggest that adult S. japonicum worms may alter the miRNA expression profile of hepatic stellate cells, and that hsa-miR-103a-3p may be associated with fibrogenic responses and may have potential relevance to hepatocellular carcinoma-related processes. However, this inference is based on correlative TCGA data and does not imply a causal role in schistosomiasis-associated hepatocarcinogenesis. Full article

Background/Objectives: Melanoma is a highly aggressive cancer with a strong metastatic potential, and therapeutic resistance remains a major clinical challenge despite advances in targeted therapies and immunotherapies. Secreted frizzled-related protein 1 (sFRP1) exhibits context-dependent roles in cancer; however, its function in metastatic melanoma remains poorly defined. This study investigated the role of sFRP1 in melanoma progression and evaluated the anti-tumor effects of the pharmacological compound WAY-316606. Methods: sFRP1 expression was quantified in metastatic melanoma cell lines, xenograft models, and TCGA datasets. The anti-tumor effects of WAY-316606 on cell viability, cell cycle progression, cell migration and invasion, and expression of extracellular matrix (ECM)-related genes were assessed using WST assays, flow cytometry, wound healing and transwell invasion assays, and quantitative real-time PCR, respectively. Results: sFRP1 expression was consistently elevated in metastatic melanoma cell lines, xenograft models, and TCGA datasets, and high sFRP1 expression was associated with poor overall survival. WAY-316606 selectively suppressed melanoma cell viability with minimal cytotoxic effects on non-tumorigenic cells, and induced G1 phase cell cycle arrest. Furthermore, WAY-316606 markedly impaired the migratory and invasive capacities of metastatic melanoma cells, accompanied by downregulation of key ECM remodeling and fibrosis-related genes, including VIM, CCN2, FN1, and TGFBI. sFRP1 knockdown partially phenocopied the anti-migratory and gene expression effects of WAY-316606. Conclusions: Collectively, our findings identify sFRP1-asscoaited signaling contribute to aggressive melanoma phenotypes and highlight the therapeutic potential of its pharmacological inhibition using WAY-316606. Full article