We first confirmed podophyllotoxin as the principal bioactive constituent in Qinghai-derived Sinopodophyllum hexandrum. Then, using the purified compound, we evaluated its inhibitory effects and mechanism in MFC gastric cancer cells and a syngeneic subcutaneous tumor model. Podophyllotoxin from Qinghai Sinopodophyllum hexandrum was identified by FT-UHR LC-MS. Its cytotoxicity against MFC gastric cancer cells was measured by CCK-8 assay. Cell proliferation, migration, invasion, and apoptosis were determined by colony formation, wound healing, Transwell, and flow cytometry assays, respectively. A syngeneic subcutaneous tumor model was established in 32 BALB/c mice for in vivo evaluation. Transcriptome sequencing was performed to profile gene expression in treated MFC cells, and Reverse transcription quantitative real-time PCR (RT-qPCR) verified key genes in MFC cells and tumor tissues. Podophyllotoxin significantly suppressed MFC cell proliferation, colony formation, migration, and invasion (P < 0.05), and induced early apoptosis (P < 0.05). In vivo, it inhibited tumor growth in a dose-dependent manner (P < 0.05). Transcriptome analysis showed that podophyllotoxin affected the DNA damage response pathway. RT-qPCR verified altered expression of DNA damage-related genes including Id3, Id4, Polq, Ddias, Gadd45a, Cip2a, Recql4, Rad54b, Mastl, and Fanci. Western blot confirmed downregulation of ID4 protein. Podophyllotoxin inhibits the growth of MFC gastric cancer cells and syngeneic subcutaneous tumors, probably by modulating the expression of DNA damage response-related genes. ID4 downregulation may serve as a potential target in this experimental model.

Three-dimensional (3D) cell culture models, including tumor spheroids and organoids, more accurately recapitulate the structural and physiological complexity of tissues than conventional monolayer cultures, making them valuable platforms for cancer research and drug screening. Volume is a biologically relevant parameter for monitoring growth dynamics and treatment response; however, its quantification often relies on manual image analysis, which is labor-intensive, subjective, and prone to inter-operator variability. Here, we introduce SpheroQuaNNt, a web-based application for automated spheroid segmentation and volume estimation using a U-Net Convolutional Neural Network (CNN). SpheroQuaNNt enables analysis of heterogeneous morphologies through image-based segmentation and provides a web-based interface for volume measurement. The model was trained and validated using 615 microscopy images representing diverse 3D culture systems, including monoculture and hybrid spheroids generated from human and canine tumor cell lines and human embryonic kidney cells, as well as tumor organoids derived from human melanoma lesions. After 30 training epochs, the model achieved 99.35% accuracy during training and 98.56% on the validation dataset, with an average Dice Score of 0.9478 for supervised validation. The method accurately captured volume variations across heterogeneous spheroids, although those presenting marked density heterogeneity between the core and periphery with a dark peripheral margin remained more challenging. Estimated volumes showed strong agreement with measurements performed independently by two experts, supporting its reliability. Overall, SpheroQuaNNt provides a standardized solution for volumetric characterization of spheroids and organoids, reducing operator-dependent variability and supporting high-throughput drug screening and quantitative analysis of 3D cell culture models.

The role of cyclin-dependent kinase inhibitor 1B (CDKN1B) in non-alcoholic fatty liver disease (NAFLD) and its underlying mechanism remain unclear. In the present study, NAFLD models were constructed in high-fat diet (HFD)-fed mice and free fatty acid (FFA)-treated HepG2 cells. The expression of CDKN1B, markers of liver injury, inflammation, and mitophagy (PINK1, Parkin, Beclin1, and the LC3II/I ratio) were assessed. CDKN1B overexpression and knockdown were performed in HepG2 cells to investigate the functional roles of CDKN1B. The phase separation potential of CDKN1B was predicted bioinformatically and validated via in vitro droplet assays, immunofluorescence, and fluorescence recovery after photobleaching (FRAP). To determine whether the protective effects of CDKN1B depend on its phase separation property, the phase separation inhibitor 1,6-hexanediol (1,6-HD) was applied at graded concentrations. The results indicated that CDKN1B was downregulated in both in vivo and in vitro NAFLD models, accompanied by lipid accumulation, inflammation, mitochondrial dysfunction, and impaired mitophagy. CDKN1B overexpression effectively reversed these pathologies, whereas CDKN1B knockdown exacerbated them. Moreover, CDKN1B contains an intrinsically disordered region and undergoes liquid-liquid phase separation (LLPS) both in vitro and in HepG2 cells. Critically, 1,6-HD abolished the protective effects of CDKN1B overexpression on mitophagy, mitochondrial integrity, lipid metabolism, and inflammation in a concentration-dependent manner. Collectively, CDKN1B alleviates NAFLD by promoting mitophagy, and this protective function is dependent on its ability to undergo LLPS. This study unveils a novel phase-separation-dependent mechanism of CDKN1B in regulating mitochondrial function and mitophagy, presenting CDKN1B condensates as a potential new target for NAFLD therapy.

Epilepsy is a chronic neurological disorder, more common in children and the elderly. Although treatments have advanced, significant challenges remain. This study focuses on its molecular mechanisms to provide new insights and strategies for its treatment. Gene expression omnibus (GEO) database analysis, enrichment analysis, and the establishment of protein-protein interaction (PPI) networks were conducted for gene screening. HT-22 cells were treated with glutamate (Glu) to establish an in vitro epilepsy model. Gene knockdown and overexpression were achieved through cell transfection. Cell viability was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, while gene expression at the protein and messenger RNA (mRNA) levels was detected by western blot (WB) and real-time quantitative polymerase chain reaction (RT-qPCR). Flow cytometry was used to analyze apoptosis and mitochondrial membrane potential. Lactate dehydrogenase (LDH), reactive oxygen species (ROS), and Fe[2+] levels were measured using commercial kits. Kainic acid (KA) was administered intraperitoneally to mice to establish an epilepsy animal model, which was used to verify the role of the gene in epilepsy development. Transcription factor binding sites in target gene promoters were predicted using the JASPAR database and validated by chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays. Tissue inhibitor of metalloproteinases 1 (Timp1) was highly expressed in tissue samples from the epilepsy mouse models of GSE88992 and GSE73878, as well as in the Glu-induced HT-22 cell model. In these models, Timp1 knockdown increased cell viability, reduced apoptosis, decreased LDH, promoted mitochondrial membrane potential, and lowered ROS, Fe[2+], and acyl-CoA synthetase long-chain family member 4 (Acsl4) levels. In KA-induced animal models, Timp1 knockdown reduced seizure score, cleaved caspase-3 (C-caspase3)/total caspase-3, and Acsl4 expression. cAMP response element-binding protein 1 (Creb1) transcriptionally activated Timp1. In the Glu-induced HT-22 cell model, Creb1 knockdown decreased Timp1 protein expression, increased cell viability and mitochondrial membrane potential, and reduced apoptosis, LDH, ROS, Fe[2+], and Acsl4 levels, while Timp1 overexpression partially reversed these effects. Taken together, Creb1 promotes the progression of epilepsy by activating Timp1, providing a new perspective and potential targets for epilepsy treatment.

[This retracts the article DOI: 10.1007/s10616-015-9885-6.].

Neuroinflammation represents a key pathological process in early brain injury (EBI) following subarachnoid hemorrhage (SAH) and contributes significantly to poor outcomes. Silymarin, known for its anti-inflammatory and antioxidant activities, has been shown to exert neuroprotective effects. Nevertheless, the molecular mechanisms underlying these effects remain to be clarified. An in vivo endovascular perforation model and an in vitro hemoglobin-stimulated model were used to investigate the pharmacological effects of silymarin in SAH. Neurological function was assessed using SAH scores and neurobehavioral scores. Neuronal apoptosis was evaluated by TUNEL staining. Neuroinflammation was measured by ELISA or qPCR, and microglial polarization status was determined by assessing CD86 and CD206 expression via Western blotting (WB) or immunofluorescence (IF) staining. Ruxolitinib and AS1517499 were applied to block the JAK1/STAT6 pathway. Silymarin attenuated early brain injury (EBI) after SAH, as indicated by improved neurological scores, reduced neuronal apoptosis, and lower brain water content. In vivo, silymarin treatment was associated with JAK1/STAT6 activation, elevated M2 markers, and decreased pro‑inflammatory cytokines. In vitro, silymarin activated the JAK1/STAT6 pathway, promoted M2 microglial polarization, and suppressed inflammatory cytokine production. In addition, pharmacological blockade of JAK1/STAT6 reversed silymarin's protective effects, confirming a causal role of this pathway in the cellular context. Our in vitro data demonstrate that silymarin directly stimulates JAK1/STAT6 to drive M2 polarization and anti‑inflammatory responses. In vivo, we observed a correlation between pathway activation and neuroprotective benefits. Together, these findings provide a rationale for further exploring silymarin‑based therapies in SAH.

Impaired fracture healing remains a significant clinical challenge. Bone marrow mesenchymal stem cells (BMSCs) and the exosomes (BMSC-Exos) play a pivotal role in bone regeneration, partly by transferring functional microRNAs (miRNAs). A-to-I RNA editing can profoundly alter miRNA function, but its role in fracture healing, particularly for miRNA-99a-5p, is underexplored. We first profiled differentially edited miRNAs in serum from 50 fracture patients and healthy controls. A murine fracture model was established and treated with BMSC-Exos, where either the edited or wild-type form of miRNA-99a-5p was knocked down. In vitro studies involved transfecting BMSCs with ADAR expression vectors to investigate editing mechanisms, and overexpressing/inhibiting edited/wild-type miRNA-99a-5p in MC3T3-E1C osteoblasts to elucidate its direct functional impacts on osteogenic differentiation, apoptosis, proliferation, and migration. Validation of target genes were performed by dual-luciferase reporter assays, qPCR, and Western blot. Key readouts included alkaline phosphatase (ALP) activity, Alizarin Red S (ARS) staining, flow cytometry, Transwell migration, CCK-8 proliferation assays and HE staining. Clinical analysis revealed significantly lower A-to-I edited miRNA-99a-5p levels in fracture patients, correlating with delayed union. In mice, BMSC-Exos promoted fracture healing, an effect attenuated by edited miRNA-99a-5p knockdown. In vitro, edited miRNA-99a-5p enhanced osteogenesis and inhibited apoptosis, while the wild-type form had opposing effects. ADAR2 mediated this editing. Edited miRNA-99a-5p directly targeted and suppressed Growth Differentiation Factor 11 (GDF11) expression. Importantly, GDF11 overexpression reversed the pro-osteogenic effects of edited miRNA-99a-5p, in both direct and exosome-mediated contexts. Reduced A-to-I editing of miRNA-99a-5p is associated with impaired fracture healing. BMSC-Exos facilitate bone repair by delivering ADAR2-edited miRNA-99a-5p, which promotes osteogenic differentiation and bone formation through the specific targeting and downregulation of GDF11. This study uncovers a novel exosome-mediated RNA editing pathway critical for fracture healing and highlights edited miRNA-99a-5p and GDF11 as potential therapeutic targets for bone repair.

Chronic obstructive pulmonary disease (COPD) is characterized by persistent oxidative stress and airway epithelial injury. However, the molecular mechanisms linking iron metabolism and cellular stress responses in COPD remain unclear. This study aimed to investigate the role of iron regulatory protein 2 (IRP2) in COPD-associated epithelial injury. 16HBE and A549 cells were exposed to cigarette smoke extract (CSE) under a COPD-associated hypoxic microenvironment. IRP2 was silenced using shRNA, and Sestrin2 restoration experiments were performed. Cellular injury was evaluated by cell viability, ROS production, inflammatory cytokines, intracellular Fe²⁺ accumulation, ferroptosis-related proteins, and NRF2 activation. CSE exposure under a COPD-associated hypoxic microenvironment induced epithelial injury, accompanied by increased oxidative stress, inflammation, iron accumulation, and ferroptosis-related changes. IRP2 silencing alleviated these alterations and enhanced NRF2 nuclear activation with increased expression of antioxidant proteins. Restoration of Sestrin2 partially reversed the protective effects induced by IRP2 depletion. Furthermore, pharmacological inhibition of ferroptosis attenuated epithelial injury. IRP2 contributes to COPD-associated epithelial injury by regulating oxidative stress, iron homeostasis, and ferroptosis-related responses, providing new insights into the molecular mechanisms underlying COPD progression.

Electrical stimulation has emerged as a promising strategy for promoting cardiogenic differentiation of mesenchymal stem cells; however, the available evidence remains highly heterogeneous across stimulation parameters, experimental platforms, and outcome measures. This systematic review synthesized the available in vitro evidence regarding the effects of electrical stimulation on MSC cardiogenic differentiation, with particular emphasis on stimulation parameters, experimental platforms and biological outcomes related to early cardiogenic commitment and structural maturation. A systematic search was conducted in seven electronic databases in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) 2020 guidelines. Fifteen preclinical in vitro studies met the eligibility criteria. Human-derived MSCs were the most frequently investigated cell source and carbon-based electrodes were the most commonly reported conductive material. Across the included studies, electrical stimulation was frequently associated with morphological changes and increased expression of markers related to early cardiogenic commitment and structural maturation, although findings were inconsistent for some outcomes. A stimulation frequency of 1 Hz, pulse widths of 5 ms, and stimulation periods of approximately two weeks were the most frequently employed conditions. Nevertheless, substantial heterogeneity in stimulation parameters, experimental platforms and outcome measures precluded direct comparisons among studies and prevented identification of an optimal stimulation protocol. Overall, the available evidence suggests that electrical stimulation may represent a promising strategy for promoting the cardiogenic differentiation of mesenchymal stem cells in vitro; however, greater methodological standardization, standardized outcome assessment and functional characterization are required before definitive conclusions regarding its effectiveness and optimal stimulation protocols can be established.

Colorectal cancer (CRC) remains a major contributor to cancer mortality. Cell adhesion molecules (CAMs) hold an essential role in CRC malignant progression. However, how to leverage CAMs-related genes to predict CRC prognosis and guide its treatment is still unclear. Gene expression data and clinical information of CRC patients were obtained from TCGA and GEO databases. Subsequently, a series of analyses were utilized to obtain 7 CAM-related feature genes and set up a prognostic risk model. The riskscore was obtained, and patients were stratified into high-risk (HR) and low-risk (LR) groups. Significant survival differences were observed (P < 0.001). According to the clinicopathological features and risk score, the ROC curve demonstrated that the riskscore showed good prognostic performance. Significant differences were in the immune infiltration of different groups. Patients in the LR group had a higher rate of gene mutation. Through enrichment analysis, the differential genes of the two groups were mainly involved in the Ras signaling pathway, signaling receptor activator activity, and growth factor activity. In addition, CGP-60,474, CMK, Dasatinib, and Imatinib may be targeted drugs in the HR group, while the targeted drugs in the LR group may be Erlotinib, Gemcitabine, JW-7-52-1, and Lapatinib. Expression validation of the 7 model genes was performed using both TCGA transcriptomic data and in vitro qRT-PCR, with consistent trends observed between the two approaches. In summary, the prognostic model can be applied to predict CRC prognosis.

Myocardial ischemia-reperfusion injury (MIRI) is characterized by exacerbated myocardial damage following the restoration of blood flow after myocardial ischemia, owing to factors such as increased production of reactive oxygen species, calcium overload, and inflammatory responses. Induction of cellular autophagy has been reported to alleviate MIRI. Kisspeptin-10 (Kp-10) is a short peptide produced by hydrolysis of the precursor protein encoded by the KISS1 gene and has shown promising autophagy-inducing properties. Here, the effects of Kp-10 on OGD/R-induced injury were investigated in H9c2 cardiomyocytes using an in vitro model relevant to MIRI. H9c2 cardiomyocytes were treated with OGD/R, followed by incubation with 50 or 100 nM Kp-10. Markedly reduced cell viability, an increased apoptotic rate and MDA levels, and diminished SOD and CAT activities were observed in OGD/R-stimulated H9c2 cardiomyocytes. These changes were accompanied by downregulated LC3-II/I, ATG5, and Beclin1, decreased nuclear levels of YY1 and TFEB, and reduced transcriptional activities of YY1 and TFEB, all of which were remarkably reversed by Kp-10. Moreover, the effects of Kp-10 on cell viability, oxidative stress, and the expression of autophagy-related proteins were substantially abolished by silencing YY1 or TFEB. Collectively, Kp-10 attenuated OGD/R-induced H9c2 cardiomyocyte injury in vitro, possibly by enhancing autophagy through the YY1/TFEB axis. These findings provide a cellular basis for further investigation of Kp-10 in MIRI, although validation in animal models is required before its clinical relevance can be assessed.

Golgi protein 73 (GP73) has been identified as a potential biomarker for liver diseases. Numerous studies have consistently shown that GP73 expression is significantly elevated in the serum of patients suffering from various types of liver injury. Nevertheless, the underlying regulatory mechanisms by which GP73 contributes to liver fibrosis remain largely undefined. A liver fibrosis model was established by treating LX-2 cells with TGF-β1 for 24 h. LX-2 cells were subsequently cultured and subjected to various treatments, including transfection with plasmids (sh-NC, sh-GP73, oe-GP73) and administration of inhibitors (3-MA and LY294002). Western blot analysis was conducted to quantify protein levels of GP73, fibrosis markers, autophagy-related proteins, and components of the PI3K/AKT/mTOR signaling pathway. Quantitative PCR (qPCR) was utilized to measure GP73 mRNA expression across different samples. Additionally, immunofluorescence assays were performed to evaluate autophagic activity in LX-2 cells. Both mRNA and protein levels of GP73 in LX-2 cells were upregulated following TGF-β1 induction. Activation of the PI3K/AKT/mTOR pathway, as evidenced by increased phosphorylation of PI3K, AKT, and mTOR, along with enhanced autophagy, was observed upon treatment with TGF-β1 or GP73 overexpression. Conversely, silencing GP73 mitigated TGF-β1-induced fibrotic responses. Furthermore, inhibition of the PI3K/AKT/mTOR signaling axis using LY294002 alleviated GP73-mediated suppression of autophagy. The findings suggest that GP73 is associated with autophagy-related changes and fibrotic responses in LX-2 cells, potentially involving modulation of the PI3K/AKT/mTOR signaling pathway.

[This corrects the article DOI: 10.1007/s10616-026-00964-2.].

Asthma is a chronic airway disease where androgens play a regulatory role. Dickkopf-1 (DKK1), a secreted glycoprotein, is positively correlated with androgens. This study aims to explore whether androgens upregulate DKK1 to inhibit Th2 response, potentially alleviating asthma. First, an asthma model was established using immunodeficient male mice. These mice were then divided into five groups and administered dihydrotestosterone (DHT) and/or the DKK-1 inhibitor WAY-262,611. Airway inflammation and lung tissue damage were assessed in each group. Furthermore, bronchoalveolar lavage fluid (BALF) inflammatory markers, the helper T cell 1/helper T cell 2 (Th1/Th2) ratio, and relevant parameters in serum and lung tissue were evaluated. Next, we established an asthma cell model using airway epithelial cells (BECs) and dust mites (HDM) stimulation. After treating with negative control (si-NC) and DKK1-targeted small interfering RNA (si-DKK1), five cell groups were established, with DHT treatment included. We assessed DHT's impact on DKK1 expression in BECs, then co-cultured these five cell groups with CD4[+] T cells to verify links to Th1/Th2-related factors. Firstly, the in vivo experiments showed that DHT attenuated asthma-induced lung damage and inflammation, reduced Th2's proportion, and normalized Th1/Th2 balance. Co-treatment with WAY-262,611 reversed DHT's protection impact. Subsequently, in vitro cell experiments confirmed that DHT suppressed Th2 response; siRNA-mediated DKK1 knockdown abrogated this suppression. Androgens promote the expression of DKK1 in BECs, thereby inhibiting Th2 response and ultimately improving asthma.

This study aims to investigate the neuroprotective effects and underlying molecular mechanisms of baicalein (Bai) in a mouse model of Parkinson's disease (PD) induced by 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Using an MPTP-induced PD mouse model, the effects of Bai intraperitoneal injection on motor function and dopaminergic neuronal injury were assessed. Adeno-associated virus serotype 9 (AAV9)-mediated mitogen-activated protein kinase 1 overexpression (oe-MAPK1) was employed for gain-of-function experiments. Western blot technique was adopted to measure the levels of MAPK1, ribosomal protein S6 kinase 1 (RSK1), phosphorylated RSK1, PTEN-induced putative kinase 1 (PINK1), Parkin, microtubule-associated protein 1 light chain 3 (LC3)-II/I, and p62. Additionally, mitochondrial membrane potential and mitochondrial ultrastructure were observed to assess mitochondrial function. The binding between Bai and MAPK1 was examined using molecular docking and cellular thermal shift assay. Bai intraperitoneal injection markedly alleviated motor deficits, mitigated dopaminergic neuronal loss, raised mitochondrial membrane potential, and reduced ultrastructural mitochondrial damage post MPTP-induced PD in mice. Meanwhile, Bai upregulated PINK1, Parkin, and the LC3-II/I ratio while downregulating p62, indicating enhanced mitophagy. With a binding energy of -7.8 kcal/mol, Bai could directly bind to MAPK1, which destabilized MAPK1 thermostability and suppressed MAPK1 expression as well as subsequent RSK1 phosphorylation. Notably, MAPK1 overexpression repressed mitophagy, aggravated motor dysfunction and neuronal damage, and partially abrogated the neuroprotective efficacy of Bai. Bai binds directly to MAPK1, suppresses the MAPK1/RSK1 signaling cascade, triggers mitophagy, and ultimately ameliorates motor impairment and neuronal degeneration, conferring neuroprotection against PD in mice.