Huwang Mingmu decoction (HWMMT) is a traditional Chinese herbal formula used clinically for diabetic retinopathy (DR), but its neuroprotective mechanisms remain unclear. This study aimed to investigate whether HWMMT protects retinal neurons in early DR by attenuating glutamate excitotoxicity via the IL-1β/c-Jun/glutamine synthetase (GS) signaling axis. Diabetes was induced in Sprague-Dawley rats by streptozotocin injection, and animals were randomized into control, diabetic model (DM), and HWMMT-treated diabetic (DM + HWMMT) groups. HWMMT was orally administered for 8 wk, with body weight and blood glucose monitored weekly. At weeks 4 and 8, retinal tissues and serum were collected for qRT-PCR, Western blotting, and ELISA. In vitro, primary rat retinal Müller glial cells (Müller) were cultured and assigned to five groups: blank control, HWMMT serum control, high-glucose (HG), HG + HWMMT, and HG + HWMMT + IL-1β (rescue group). Cell viability, LDH release, glutamate secretion, apoptosis, and expression of IL-1β, c-Jun, and GS were evaluated. In vivo, DM rats showed slower weight gain, sustained hyperglycemia, upregulated retinal IL-1β and c-Jun expression, downregulated GS, and elevated serum glutamate; HWMMT treatment significantly reversed these abnormalities. In vitro, HG exposure decreased Müller cell viability, increased apoptosis, LDH leakage and glutamate release, and induced IL-1β/c-Jun upregulation with GS downregulation; co-treatment with HWMMT-containing serum reversed all these changes. Notably, exogenous IL-1β supplementation significantly attenuated the cytoprotective and anti‑apoptotic effects of HWMMT and reversed its modulation of IL-1β, c-Jun, GS, and glutamate homeostasis. Collectively, these findings demonstrate that HWMMT exerts neuroprotection in early DR by suppressing the IL-1β/c-Jun signaling cascade and restoring GS expression and function in Müller cells, thereby reducing extracellular glutamate accumulation and mitigating excitotoxic damage. The rescue experiments further support that HWMMT acts specifically through this pathway, providing mechanistic insight and preclinical evidence for HWMMT as a potential therapeutic intervention for early diabetic retinopathy.

Osteoarthritis (OA) is a chronic degenerative joint disease characterized by progressive articular cartilage degradation. Punicic acid (PA), a plant-derived n-3 polyunsaturated fatty acid, has been reported to exert anti-inflammatory and anti-apoptotic effects. However, the molecular mechanisms underlying its effects in OA remain incompletely understood. In this study, bioinformatics analysis was performed to identify apoptosis-related differentially expressed genes (DEGs) and enriched signaling pathways associated with OA. An in vitro OA-like model was established using interleukin-1β (IL-1β)-stimulated SW1353 cells, which were subsequently treated with different concentrations of PA. PA suppressed IL-1β-induced expression of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), matrix metalloproteinase-3 (MMP-3),matrix metalloproteinase-13 (MMP-13), and A Disintegrin And Metalloproteinase with Thrombospondin Motifs 5 (ADAMTS-5) and attenuated apoptosis by modulating Bcl-2-associated X protein (BAX) and B-cell lymphoma 2 (BCL-2) expression. Notably, target prediction and dual-luciferase reporter assays confirmed that mitogen-activated protein kinase kinase 6 (MAP2K6) is a direct target of microRNA-29a-3p (miR-29a-3p), and PA increased miR-29a-3p expression in the in vitro OA-like model. Furthermore, PA inhibited MAP2K6-mediated activation of the p38 mitogen-activated protein kinase (p38 MAPK) pathway, thereby attenuating downstream inflammatory and apoptotic signaling. These findings suggest that PA exerts chondroprotective effects by modulating the miR-29a-3p/MAP2K6/p38 MAPK signaling axis, highlighting its therapeutic potential and supporting the development of miRNA-based therapeutic strategies for OA.

Neurogenic bladder (NB) severely impacts patients' quality of life. This study was designed to explore the molecular mechanisms underlying the pathophysiology of NB-associated bladder dysfunction. A rat model of NB was established via spinal cord injury (SCI). Bladder function was evaluated by urodynamic testing and hematoxylin-eosin staining of tissue sections. The expression levels of SPI1, HCN1, neuronal restrictive silencing factor (NRSF), and endoplasmic reticulum (ER) stress-related proteins in bladder tissues or cells were examined using immunohistochemistry, Western blot, and reverse transcription quantitative polymerase chain reaction. Cell viability was measured with the Cell Counting Kit-8 assay. Apoptosis was assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining and flow cytometry. Potential binding sites between SPI1 and the NRSF promoter were predicted using the JASPAR database and then validated by dual-luciferase reporter and chromatin immunoprecipitation assays. The role of ER stress in NB was investigated using the inhibitor 4-phenylbutyric acid or the inducer tunicamycin. Bladder tissues from NB rats exhibited increased apoptosis and a significant upregulation of ER stress-related proteins. Furthermore, expression of SPI1 and NRSF was elevated, whereas the expression of hyperpolarization-activated cyclic nucleotide-gated channel 1 (HCN1) was reduced in bladder tissues from NB rats. Further analysis revealed that SPI1 binds to the NRSF promoter and upregulates its expression, thereby enhancing the repression of HCN1. Knockdown of SPI1 significantly alleviated bladder dysfunction, attenuated ER stress, and inhibited apoptosis. These effects were reversed by overexpression of NRSF. This study demonstrates that SPI1 contributes to bladder dysfunction by upregulating NRSF, which subsequently potentiates the inhibition of HCN1 and triggers ER stress. The findings identify promising molecular targets and signaling pathways for the intervention and treatment of NB.

Pv11 cells, derived from Polypedilum vanderplanki, uniquely tolerate complete desiccation (anhydrobiosis). Although a CRISPR/Cas9-based precise integration method (CRIS-PITCh) has been developed for Pv11 cells, a CRISPR-free strategy that fixes both the genomic locus and transgene copy number has not yet been established. Here, we implement recombinase-mediated cassette exchange (RMCE) in Pv11 cells and generate master cell lines that retain anhydrobiosis following genetic engineering. We first evaluated the activity of multiple site-specific recombinases in Pv11 cells using a transient two-plasmid reporter assay. Flp, Cre, and Bxb1 recombinases all excised a test cassette and activated a green fluorescent protein (GFP) reporter, whereas phiC31 integrase mediated recombination at the DNA sequence level but did not induce reporter expression under our construct configuration. To enable genomic RMCE, we inserted an FRT/FRT3-landing pad (LP) into a previously identified genomic safe-harbor locus using CRIS-PITCh and isolated clonal master cell lines by single-cell sorting. Using the established master line, Flp-based RMCE achieved site-specific cassette exchange at the LP, producing HaloTag fluorescence and drug resistance upon successful exchange. In addition, the expected post-exchange sequence was confirmed by sequencing. We further established an all-in-one RMCE vector combining the Flp recombinase and donor cassette on a single plasmid. Together, these results demonstrate locus-defined, single-copy transgene integration in anhydrobiotic Pv11 cells via RMCE and provide a standardized, CRISPR-free workflow for routine genetic manipulation in this unique cell line. This workflow facilitates both fundamental research and applied biotechnological applications using desiccation-tolerant cells.

Diabetic vascular complications are a leading cause of disability in diabetic. This study aimed to investigate the functional improvement effects and molecular mechanisms of internalizing RGD (iRGD) peptide-modified ferulic acid-targeted liposomes (Lp-iRGD@FA) on high glucose-induced injured endothelial progenitor cells (EPCs). Lp-iRGD@FA was prepared using the thin-film hydration method and subsequently characterized. In vitro experiments were conducted using human peripheral blood-derived EPCs, in which a high glucose injury model was established. Cells were treated with free ferulic acid, non-targeted liposomes, and targeted liposomes, respectively. In vivo, a diabetic hindlimb ischemia mouse model was established, and Lp-iRGD@FA, non-targeted liposomes, or Lp-iRGD were intramuscularly injected into the ischemic muscles. The high glucose environment significantly inhibited EPC proliferation, promoted apoptosis, and impaired tube formation ability, accompanied by inhibited AMPK phosphorylation and downregulated expression of KLF4 and FAM3A. Intervention with the targeted liposomes effectively reversed these changes. However, the AMPK inhibitor Compound C blocked the Lp-iRGD@FA-induced upregulation of KLF4 and FAM3A and the associated functional improvements, whereas KLF4 supplementation partially rescued the decreased FAM3A expression and impaired tube formation caused by AMPK inhibition. In vivo, Lp-iRGD@FA treatment in diabetic mice enhanced angiogenesis in a hindlimb ischemia model and activated the AMPK/KLF4/FAM3A pathway in ischemic muscles. This study demonstrates that Lp-iRGD@FA efficiently delivers ferulic acid to restore FAM3A expression by activating the AMPK/KLF4/FAM3A signaling axis, subsequently upregulating VEGFA/VEGFR2 levels and improving the function of EPCs under high glucose stress. These findings provide a novel nanomedicine strategy and potential molecular targets for the treatment of diabetic vascular complications.

Given the involvement of long non-coding RNA-differentiation antagonizing non-protein coding RNA (lncRNA DANCR) in osteoporosis (OP), this study aimed to investigate its role in osteogenic differentiation (OD) of bone marrow mesenchymal stem cells (BMSCs) and underlying mechanisms. BMSCs were intervened with sh-DANCR, oe-DANCR, sh-phosphatase and tensin homolog (PTEN) lentivirus, miR-19a-3p agomir, or MK-2206 dihydrochlorode. The number of mature osteoblasts, osteogenic ability, and alkaline phosphatase (ALP) activity were assessed by ALP staining, alizarin red staining, and kit assay. Bioinformatics prediction, dual-luciferase assay, and RNA pull-down assay were conducted to assess the binding between lncRNA DANCR and miR-19a-3p and that between PTEN and miR-19a-3p. The levels of lncRNA DANCR, osteogenesis-related proteins (osteocalcin (OCN), runt-related transcription factor 2 (RUNX2), osteopontin (OPN)), PTEN, phosphorylated-protein kinase B (p-AKT), AKT, phosphorylated-mammalian target of rapamycin (p-mTOR), and mTOR were determined by RNA quantitation and western blot techniques. Silencing of lncRNA DANCR potentiated the OD capability of BMSCs, as evidenced by increased ALP activity, calcium nodule formation, and elevated expression of OPN, RUNX2, and OCN. In contrast, lncRNA DANCR overexpression inhibited the OD function by downregulating miR-19a-3p. Mechanistically, lncRNA DANCR regulated PTEN expression by competitively binding to miR-19a-3p. Additionally, PTEN silencing partially reversed the anti-OD effect of lncRNA DANCR. LncRNA DANCR knockdown activated the AKT/mTOR pathway, whereas lncRNA DANCR overexpression or MK-2206 treatment disrupted this activation. Silencing of lncRNA DANCR facilitates BMSC OD by binding to miR-19a-3p and further regulating the PTEN/AKT/mTOR pathway.

Unlike mammals, teleost fish exhibit lifelong skeletal muscle growth, characterized by continued fiber hypertrophy and the formation of new muscle fibers maintained by a persistent progenitor cell population. However, the limited availability of stable muscle progenitor cell lines from commercially important species such as Atlantic salmon (Salmo salar) constrains mechanistic studies and emerging applications in cellular aquaculture. Here, we report the establishment and characterization of a novel embryonic-derived salmon muscle progenitor cell line, termed SsEC. These cells were derived from late embryonic stages and exhibited a spindle-shaped morphology, robust proliferative capacity, and sustained expansion beyond 30 passages under defined culture conditions. SsECs demonstrated a distinct extracellular matrix preference, with vitronectin supporting long-term maintenance and expansion. Molecular characterization confirmed stable expression of canonical myogenic markers, including myf5 and myod1, while transcriptomic profiling revealed enrichment of genes associated with muscle development and sarcomere organization relative to a non-myogenic salmon cell line. Directed differentiation to muscle, using a two-step protocol, induced efficient formation of multinucleated myotubes expressing myosin heavy chain and sarcomeric α-actinin, with upregulation of key differentiation markers such as myog and tnnt3a. Together, these findings establish SsECs as a robust in vitro model cell line for studying salmon muscle development and provide a novel platform for applications in aquaculture research and cellular seafood production.

Ginsenoside Rc (G-Rc), a major active ingredient of Panax ginseng, has protective effects against various pathological alterations associated with diseases. Nonetheless, the potential benefits of G-Rc in treating cerebral ischemia-reperfusion (CIR) injury remain inadequately explored. In the present study, we evaluated the potential effects of G-Rc on CIR injury via cellular and animal models and explored the underlying mechanisms. Oxygen-glucose deprivation and reoxygenation (OGD/R)-induced neurons treated with G-Rc exhibited a marked reduction in apoptosis, oxidative stress, and inflammation. Rats treated with G-Rc presented significant reductions in neurological deficits, cerebral infarction, and pathological alterations resulting from CIR injury. Moreover, neuronal death, oxidative stress damage, and the inflammatory response in the brains of rats with CIR injury were markedly ameliorated by G-Rc treatment. Molecular docking, molecular dynamics simulations, and relevant experiments validated Sirtuin 1 (Sirt1) as a target of G-Rc. G-Rc increased Sirt1 expression and augmented its enzymatic activity. Notably, G-Rc promoted the activation of nuclear factor-erythroid 2-related factor 2 (Nrf2) by targeting Sirt1. G-Rc failed to induce Nrf2 activation in Sirt1-silenced neurons. Furthermore, the benefits of G-Rc in defending against OGD/R damage were strongly negated by pharmacologically suppressing Nrf2. These observations indicate that G-Rc offers considerable benefits in treating CIR injury in experimental models. These beneficial effects are achieved by enhancing Nrf2 activation via the targeting of Sirt1. This study emphasizes the potential utilization of G-Rc as a pharmaceutical candidate for treating CIR damage.

The southern green stink bug, Nezara viridula L., is a highly polyphagous global pest that causes substantial economic losses on a broad range of crops. Current management strategies rely heavily on chemical insecticides, which can promote resistance development and negatively affect non-target beneficial organisms. The lack of a tractable in vitro cell culture system has limited the development of more targeted and sustainable control approaches for this species. In this study, we identified the optimal egg age for culture initiation and evaluated seven insect cell culture media or medium combinations, as well as several supplements, for their ability to support embryo-derived N. viridula primary cultures. A total of 200 primary cultures were initiated using ~ 6,000 dissected N. viridula eggs over 16 months. Of the media tested, GS medium, a combination of CLG#2 and Shields and Sang medium, supplemented with 9% heat-inactivated fetal bovine serum produced, on average, 40% more attached cell clusters than CLG#2, the next best medium, although this difference was not statistically significant. GS medium also supported the long-term survival and growth of N. viridula primary cells for more than 22 months. Several chemical additives previously reported to enhance insect cell proliferation did not improve N. viridula primary cell growth or survival. The optimized culture conditions described here provide a foundation for the establishment of continuous N. viridula cell lines and support future studies of host-pathogen interactions, insect physiology, and the development of novel pest management strategies.

Equine sarcoids are the most common skin tumors in horses and are characterized by local invasiveness, high recurrence rates, and inconsistent responses to current therapies. Oncolytic virotherapy represents a promising alternative approach that exploits defects in antiviral signaling pathways in tumor cells. Newcastle disease virus (NDV), an avian paramyxovirus that is nonpathogenic in mammals, has demonstrated tumor-selective activity in several species; however, its effects on equine sarcoids have not been previously investigated. In this study, primary cell cultures derived from equine sarcoid tumors were established to evaluate the selective cytotoxicity of NDV. Sarcoid tissues from six horses generated thirteen primary sarcoid cultures, while three primary equine fibroblast cultures served as non-tumor controls. Bovine papillomavirus (BPV) DNA was assessed by quantitative polymerase chain reaction. Cells were infected with a green fluorescent protein-expressing Newcastle disease virus (NDV-GFP), and cell viability was measured after seventy-two hours using a metabolic viability assay. Sarcoid cultures exhibited significantly greater sensitivity to viral infection than normal fibroblasts, with substantially lower half-maximal inhibitory concentrations. Viral infectivity, quantified by fluorescence intensity, correlated with increased susceptibility to virus-induced cytotoxicity. Notably, the oncolytic activity of Newcastle disease virus was observed in both bovine papillomavirus-positive and bovine papillomavirus-negative sarcoid cultures. These findings demonstrate that NDV-GFP induces stronger cytotoxic effects in equine sarcoid cells than in normal fibroblasts, supporting its potential as a novel therapeutic strategy in equine oncology.

Sedentary behaviour, ageing and chronic diseases represent major public health burdens associated with impaired skeletal muscle function and increased oxidative stress. Identifying bioactive compounds capable of mimicking the molecular adaptations induced by physical exercise therefore constitutes a relevant pharmacological strategy. Phlorotannins, polyphenolic compounds exclusively biosynthesised by brown macroalgae, are promising candidates owing to their antioxidant and metabolic properties. This study aimed to characterise a phlorotannin-enriched ethyl acetate extract from Halidrys siliquosa (HS extract) and assess its pharmacological properties in L6 rat myoblasts, a well-established skeletal muscle cell model. Chemical profiling by LC-MS and NMR identified carmalol- and fuhalol-type derivatives, two subclasses of phlorotannins, as predominant constituents, with strong radical scavenging activity in the DPPH assay. HS extract was non-cytotoxic at 1 and 10 µg/mL and did not alter basal ROS levels after 24-h treatment. At the molecular level, HS extract significantly increased the P-AMPK/AMPK ratio and PGC-1α transcript levels, two key mediators of exercise-induced metabolic adaptation, and enhanced glutathione peroxidase (GPx) enzymatic activity, indicating reinforced antioxidant defences. In a preventive setting, HS extract pretreatment markedly reduced H2O2-induced cell death and ROS overproduction. Collectively, these findings demonstrate that HS extract activates exercise-associated metabolic signaling pathways in rat skeletal muscle cells. These results suggest that HS extract may represent a promising source of bioactive compounds capable of modulating exercise-related molecular responses, warranting further investigation in differentiated muscle models and in vivo studies.

Lung cancer remains one of the most lethal and prevalent malignancies worldwide. Although E3 ubiquitin ligase SYVN1 is frequently amplified in lung cancer, its functional role has not been fully elucidated. This study investigates the mechanistic involvement of SYVN1 in regulating the GATA3/NRP1 axis in A549 cells to provide novel insights into potential therapeutic strategies for lung cancer. Expression levels of SYVN1 were assessed in BEAS-2B, A549, NCI-H647, NCI-H2170, and NCI-H520, NCI-H69 cells. Functional assays were conducted after overexpressing or silencing SYVN1, GATA3, and/or NRP1 in A549 cells to examine their interdependent roles. The cells' proliferation, wound healing, invasion and migration abilities were evaluated through CCK-8, scratch assay and Transwell assays, respectively. The results indicated that SYVN1 was upregulated in A549 cells and significantly promoted cell proliferation, invasion, and migration. Mechanistically, SYVN1 interacted with GATA3 and regulated its ubiquitination. Knockdown of SYVN1 suppressed A549 cell proliferation, wound healing, invasion, and migration, and these effects could be reversed by overexpressing GATA3. Similarly, silencing GATA3 reduced these malignant characteristics, but these effects could be restored by overexpressing NRP1, and the knockdown of NRP1 could abrogate the tumor-promoting effects of SYVN1 overexpression. SYVN1 overexpression counteracted the inhibitory effects of NRP1 silencing on A549 tumor progression. Taken together, all these findings prove that SYVN1 could promote the malignant characteristics of A549 cells by modulating the GATA3/NRP1 axis. Thus, targeting SYVN1 could represent a promising therapeutic approach for treating lung cancer.

Systemic sclerosis (SSc) is characterized by progressive fibrosis and complex alterations in extracellular matrix dynamics; however, experimental modeling of fibrotic processes remains limited by poor control of baseline signaling and spontaneous phenotypic drift in vitro. Such variability complicates the interpretation of perturbation-driven responses and reduces the reliability of antifibrotic and toxicological studies. In this study, we developed and characterized a three-dimensional (3D) fibrotic in vitro platform designed to maintain a stable and controlled baseline state across distinct microenvironmental conditions. Primary dermal fibroblasts derived from a bleomycin-induced scleroderma model and human idiopathic pulmonary fibrosis fibroblasts (CCL-191) were embedded in collagen-fibrinogen-based bioinks (Matrigel and Cellink Skin) and fabricated using extrusion-based 3D bioprinting. Across all conditions, the 3D constructs preserved structural integrity, sustained cell viability, and maintained consistent myofibroblast features, including α-SMA expression, fibrotic gene transcription (TGF-β, IL-6, IL-1, collagen I), and canonical signaling activity (pSmad2/3). Importantly, no evidence of spontaneous overactivation or loss of fibrotic signaling was observed, indicating tight regulation of baseline pathway activity independent of bioink composition. These findings demonstrate that fibrotic signaling can be stabilized within a controlled 3D microenvironment, minimizing baseline drift and enabling reproducible molecular and biochemical readouts. By establishing a non-fluctuating fibrotic baseline, this platform provides a robust framework for future perturbation-based studies and enhances the interpretability of antifibrotic and toxicological screening approaches.

Coronary artery disease (CAD) involves intricate immune-related pathways; however, the contribution of epigenetic mechanisms remains inadequately defined. The fat mass and obesity-associated protein (FTO), which functions as an N[6]-methyladenosine (m[6]A) demethylase, has been implicated in the progression of atherosclerotic conditions. This study sought to elucidate how FTO downregulates proteinase 3 (PRTN3) through m[6]A demethylation and inhibits neutrophil activation via the C-X-C motif chemokine ligand 9/C-X-C motif chemokine receptor 3 (CXCL9/CXCR3) signaling pathway in the context of CAD. RNA sequencing was carried out on peripheral blood mononuclear cells (PBMCs) obtained from CAD patients and healthy individuals to detect gene expression differences. Functional enrichment analyses, including Gene Ontology and Kyoto Encyclopedia of Genes and Genomes, were performed along with experimental validation in endothelial progenitor cells (EPCs). The regulatory relationship between FTO and PRTN3 was examined via luciferase reporter assays, RNA immunoprecipitation, and methylated RNA immunoprecipitation quantitative polymerase chain reaction. Neutrophil activation was evaluated by measuring CXCL9/CXCR3 expression, tracking cell migration, and assessing reactive oxygen species (ROS) generation in HL-60 cells. For in vivo validation, apolipoprotein E-deficient (ApoE[-]/[-]) mice were maintained on a high-fat diet and treated with the AAV9 vector carrying FTO via tail vein injection to evaluate effects on atherosclerotic development. Findings indicated that PRTN3 is significantly upregulated in CAD patients, which was corroborated in EPCs. FTO was shown to directly bind to PRTN3 and decrease its expression by reducing m[6]A methylation. Overexpression of FTO enhanced proliferation and migration of EPCs and reduced apoptosis, whereas FTO silencing produced opposing outcomes. Furthermore, PRTN3 was found to stimulate the CXCL9/CXCR3 axis, leading to increased neutrophil migration and ROS production. In vivo, FTO inhibits the activation of neutrophils by down-regulating the expression of PRTN3, reduces the inflammatory response, and protects the occurrence and development of atherosclerosis in mice. These results uncover a novel regulatory pathway involving FTO, m[6]A, PRTN3, and CXCL9/CXCR3 in CAD pathogenesis, highlighting FTO as a promising target for therapeutic intervention.