Gastric cancer is a common digestive system malignancy worldwide, and its high metastatic potential is one of the main reasons for poor patient prognosis. Dihydrocapsaicin, a natural derivative of capsaicin found in chili peppers, has recently been shown to possess anti-tumor potential. However, the effects and underlying mechanisms of dihydrocapsaicin on gastric cancer cell migration and invasion remain unclear. This study aims to investigate the impact of dihydrocapsaicin on the migratory and invasive abilities of gastric cancer cells and to elucidate the regulatory role of TRPV1 in this process. Human gastric cancer cell lines NCI-N87 and HGC-27 were treated with different concentrations of dihydrocapsaicin. The changes in the migration and invasion abilities of gastric cancer cells were detected. Meanwhile, WB was used to detect the differential expression of TRPV1, E-cadherin, N-cadherin, Vimentin, and Snail. Subsequently, the expression of TRPV1 was interfered in the dihydrocapsaicin-treated gastric cancer cell lines, and the differential expressions of the above cell migration, invasion, and E-cadherin, N-cadherin, Vimentin, and Snail proteins were detected. Dihydrocapsaicin could significantly and dose-dependently inhibit the migration and invasion of gastric cancer cells NCI-N87 and HGC-27. In addition, dihydrocapsaicin treatment significantly increased the expression of TRPV1 and E-cadherin proteins in gastric cancer cells, and significantly inhibited the expression of N-cadherin, Vimentin, and Snail proteins in gastric cancer cells. Functional rescue experiments confirmed that when the expression of TRPV1 was inhibited, the inhibitory effect of dihydrocapsaicin on the migration and invasion of gastric cancer cells was significantly weakened. Dihydrocapsaicin can effectively inhibit the migration and invasion abilities of gastric cancer cells by upregulating the expression of TRPV1.

The characteristics of diabetic vascular complications are impaired angiogenesis, which leads to hindlimb ischemia. Although astragaloside IV (AS-IV) can promote angiogenesis, its poor targeting to endothelial progenitor cells (EPCs) limits its therapeutic effect. Here, we developed liposomes modified with iRGD to load AS-IV (Lp-iRGD@AS-IV) to enhance its delivery and explored its mechanism. Lp-iRGD@AS-IV and fluorescently labeled liposomes were prepared, and their phenotypic characteristics were detected. The study results showed that the uptake efficiency of Lp-iRGD@AS-IV by EPCs was higher than that of Lp@AS-IV. In a diabetic mouse hindlimb ischemia model induced by streptozotocin, Lp@AS-IV and Lp-iRGD@AS-IV improved cell damage, increased capillary density, and reduced reactive oxygen species accumulation. However, the therapeutic effect of Lp-iRGD@AS-IV was more significant than that of Lp@AS-IV. Lp-iRGD@AS-IV attenuated high glucose-induced inhibitory effect on cell viability, migration, and invasion capability of EPCs. Additionally, we analyzed the regulatory effects of the Hippo-YAP/TAZ signaling pathway in diabetic vascular complications. AS-IV increased the expression of vascular growth factors (VEGFa, VEGFb, VEGFc, FGF, and Ang-1), eNOS, and osteopontin, and enhanced glucose-lipid metabolism, via activating the upstream Hippo signaling pathway while inactivating the downstream YAP/TAZ activity. In conclusion, Lp-iRGD@AS-IV significantly enhanced the delivery of AS-IV to EPCs and improved hindlimb ischemia in diabetic mice. AS-IV can restore diabetes-associated impaired angiogenesis through the Hippo-YAP/TAZ pathway. Lp-iRGD@AS-IV may be a targeted therapy for diabetic vascular complications.

Cisplatin-induced acute kidney injury (AKI) limits its clinical use, and effective renoprotective agents are lacking. Daphnetin (DAP), a natural coumarin, exhibits anti-inflammatory and antioxidant properties, but its role and mechanism in cisplatin nephrotoxicity remain unclear. This study investigates whether DAP protects against cisplatin-induced renal tubular injury and elucidates the involvement of the HIF-1α signaling pathway. A rat model of cisplatin-induced AKI and an in vitro HK-2 cell injury model were used. Renal function, histopathology, injury markers (KIM-1, NGAL), inflammatory cytokines (TNF-α, IL-1β, IL-6, TGF-β), cell viability, proliferation, apoptosis, and HIF-1α expression were assessed. Bioinformatics analysis and rescue experiments using the HIF-1α activator ML228 were performed. DAP significantly ameliorated cisplatin-induced renal dysfunction, pathological damage, and inflammatory responses in rats. In HK-2 cells, DAP enhanced viability and proliferation, reduced apoptosis, and downregulated KIM-1, NGAL, and pro-inflammatory cytokines. HIF-1α protein and mRNA level was upregulated by cisplatin, and DAP reversed this effect. Bioinformatics and KEGG analysis identified the HIF-1 pathway as the most enriched. Activation of HIF-1α by ML228 abolished DAP's protective effects, confirming the pathway's critical role. DAP alleviates cisplatin-induced AKI by inhibiting the HIF-1α signaling pathway. This natural compound represents a promising candidate for preventing chemotherapy-related renal injury.

Dental pulp stem cells (DPSCs) play a critical role in maintaining dental pulp homeostasis and supporting dentin-pulp complex regeneration, while tumor necrosis factor-α (TNF-α)-mediated inflammation severely impairs their biological functions. This study explored the role of the miR-335-5p/DKK1/autophagy axis in TNF-α-induced DPSCs dysfunction. Human DPSCs were stimulated with 20 ng/mL TNF-α; miR-335-5p overexpression and DKK1 silencing were achieved via transfection. qRT-PCR, Western blot, SA-β-gal staining, immunofluorescence, transmission electron microscopy, ALP/ARS staining, and dual-luciferase assay were used to detect related indicators. Results showed TNF-α downregulated miR-335-5p, upregulated DKK1, inhibited autophagy, induced senescence, disrupted cytoskeleton, and suppressed osteogenesis; miR-335-5p directly targeted DKK1's 3'-UTR. Overexpressing miR-335-5p or silencing DKK1 restored DPSCs' autophagic flux, alleviated senescence, and rescued osteogenic potential. In conclusion, the miR-335-5p/DKK1/autophagy axis mediates TNF-α-induced DPSCs dysfunction, and targeting this axis may improve stem cell-based dental pulp and bone reconstruction under inflammation.

Poor tendon-bone healing is a major challenge after anterior cruciate ligament reconstruction (ACLR). TFRD, the total flavonoids of Rhizoma Drynariae, has pharmacological effects in promoting bone formation, but it remains less understood whether it accelerates tendon-bone healing by regulating the osteogenic differentiation of tendon-derived stem cells (TDSCs). TDSCs were isolated from the Achilles tendons of rats and characterized for their stem cell properties through flow cytometry and multilineage differentiation assays. The effects of TFRD on TDSC proliferation, migration, and osteogenic differentiation were evaluated using the cell counting kit-8 assay, scratch wound healing assays, Alizarin Red S staining, and Western blot analysis. Gene and protein expression levels associated with the transforming growth factor-β/bone morphogenetic protein (TGF-β/BMP) signaling pathway were assessed by real-time quantitative polymerase chain reaction and Western blot. A rat ACLR model was created and divided into four experimental groups: Sham, ACLR, TFRD, and TFRD + Noggin. Tendon-bone healing was analyzed using hematoxylin and eosin staining, Masson's trichrome staining, bone mineral density measurements, and immunohistochemistry. Noggin was used to inhibit BMP signaling for pathway validation. TFRD (50-100 μg/mL) significantly enhanced the proliferation, migration, and osteogenic differentiation of TDSCs, and increased the expression of key osteogenic markers, including runt-related transcription factor 2 (RUNX2) and alkaline phosphatase (ALP). Mechanistically, TFRD activated the TGF-β/BMP signaling pathway, leading to elevated levels of TGF-β1, BMP-2, and phosphorylated Smad1/5/8. Moreover, TGF-β1 extended the duration of BMP-2 signaling and suppressed Noggin expression. The osteogenic effects of TFRD were diminished when Noggin was applied, indicating its role in modulating the BMP pathway. In vivo, TFRD significantly improved collagen fiber alignment, calcification, and bone mineral density at the tendon-bone junction, while also upregulating RUNX2 and ALP expression. TFRD promotes osteogenic differentiation of TDSCs by activating the TGF-β/BMP signaling pathway, improves tendon-bone healing after ACLR, and provides experimental support for its clinical application.

This work establishes a robust and reproducible 96-well plate-based 3D HepG2 tumour spheroid model for drug screening applications. A 7-d culture protocol was optimised to generate HepG2 spheroid model using 96-well ultra-low attachment plates with 5 seeding densities (500, 1000, 2500, 5000 and 10,000 cells/well). Spheroids were evaluated for morphology, viability and proliferation. The optimized model was treated with doxorubicin and sorafenib at their respective IC50 values (2.4 µM and 5.3 µM, determined in HepG2 monolayer). Drug-specific responses were assessed via spheroid growth, viability and expression of apoptotic and drug resistance markers. Spheroids seeded at 500 cells/well exhibited optimal characteristics, including sustained proliferative capacity (up to 30-fold increase relative to day 1) and progressive spheroidal growth (1.85-fold-change in diameter); hence, it was selected for further experiments. Day 4 was identified as the optimal treatment point based on morphology, viability and diameter progression. The 72-h IC50 values were determined to be 2.4 µM for doxorubicin and 5.3 µM for sorafenib; these concentrations were chosen for subsequent treatments on HepG2-derived spheroids. Doxorubicin induced a delayed but potent cytotoxic response, while sorafenib triggered a slower, sustained effect, distinct from its rapid action in monolayer cultures. Furthermore, spheroid diameter did not correlate with viability or proliferation decline, highlighting its limitation as a sole readout. Molecular profiling revealed drug-specific responses in which ABCB1 was upregulated by doxorubicin, while ABCC2 was selectively induced by sorafenib. Sorafenib progressively reduced BAX/BCL2 ratio, suggesting potential resistance development. This study presents an optimized HepG2 spheroid model with validated culture and treatment parameters, capturing drug-specific cellular and molecular responses for therapeutic screening and mechanistic studies in hepatocellular carcinoma.

The setup of experimental designs in basic bone research begins with selecting appropriate osteogenic supplements to induce differentiation. Many studies describe the use of the supplements ascorbic acid, dexamethasone, and β-glycerophosphate in varying combinations and concentrations. To clarify their individual and combined effects on MC3T3-E1 cells, we conducted a detailed study focusing on cell morphology, mineralization, and gene expression of key osteogenic markers at four time points. Cell morphology was analyzed using light and scanning electron microscopy. Mineralization was assessed via Alizarin Red staining for calcium and von Kossa staining for phosphate. Additionally, energy-dispersive X-ray analysis was used to detect calcium and phosphorus in the cell layer. Sirius Red staining was applied to characterize the fiber formation observed under electron microscopy. To evaluate the influence of the supplements on gene expression, real-time quantitative PCR was performed for the osteogenic markers alkaline phosphatase, bone sialoprotein, bone morphogenetic protein 2, collagen 1a2, osteocalcin, osterix, and runt-related transcription factor 2. Osteogenic differentiation of MC3T3-E1 cells could be induced by application of ascorbic acid and β-glycerophosphate leading to upregulation of osteogenic markers, changes in cellular morphology, and mineralizing activity. Nevertheless, this effect could be further increased by additional use of dexamethasone. While most osteogenic marker genes were already upregulated on day 7, we did not see the first signs of mineralization until day 14. We recommend using ascorbic acid, β-glycerophosphate, and dexamethasone in order to get an osteogenic phenotype of MC3T3-E1 on day 14 at the earliest.

Marine invertebrate cell cultures are a potential source for diverse biotechnological applications, given the wide range of bioactive compounds they synthesize and accumulate. Yet, the number of established marine invertebrate cell culture systems remains limited compared with those of insects and vertebrates, particularly with respect to adherent cell cultures. Here we studied the in vitro adherence of circulating blood cells from the colonial ascidian Botryllus schlosseri. Two experimental approaches were employed, seeding blood cells either alone (setup 1) or in combination with tissue fragments (setup 2), using three basal media (DMEM, DMEM/F-12, RPMI) on culture plates coated with either Poly-L/D-lysine, gelatin, collagen, or laminin. Each experiment lasted for up to 3 d. Setup 1 results reveal that Botryllus cells remain viable and can adhere to coated surfaces in all tested media. Collagen- and laminin-coated plates supported longer-term cultures, whereas Poly-D (or L)-lysine coatings were more suitable for short-term studies. Among the basal media, RPMI and DMEM/F12 most effectively supported cell attachment. Setup 2 plates consistently showed higher cell adherence compared to setup 1, suggesting that tissue-derived factors may enhance attachment. Overall, circulating Botryllus cells demonstrate the capacity for substrate adhesion in vitro, offering a foundation for the development of adherent cell cultures.

RecQ-mediated genome instability protein 2 (RMI2), a component of the RecQ helicase complex, has been shown to be highly expressed in various cancers and is often associated with poorer patient survival. However, its specific role in ovarian cancer (OC) remains unclear. This study investigates whether RMI2 contributes to OC progression via the ATR/CHK1 pathway. Bioinformatics analysis and experimental validation revealed that RMI2 was highly expressed in OC tissues and cell lines compared with normal controls. Functional assays demonstrated that knockdown of RMI2 inhibited OC cell proliferation and colony formation, induced G2/M phase arrest, and promoted apoptosis. Furthermore, the knockdown of RMI2 led to increased DNA damage (manifested as the accumulation of γH2AX) and downregulated homologous recombination repair proteins (BRCA1, NBS1, RAD51). Under DNA damage stress induced by Olaparib, a Poly (ADP-ribose) Polymerase (PARP) inhibitor, RMI2 knockdown synergistically enhanced DNA damage and apoptosis. Mechanistically, RMI2 directly interacted with ATR, and its knockdown reduced ATR phosphorylation and the expression of its downstream effector CHK1. Additionally, overexpression of ATR partially rescued the inhibitory effects of RMI2 knockdown on OC cell growth and DNA damage. In conclusion, RMI2 may promote OC development by activating the ATR/CHK1 pathway to sustain genomic stability, highlighting its potential as a prognostic biomarker and therapeutic target in OC.

Fish cell lines serve as ideal in vitro models for investigating the toxicity of environmental pollutants in fish. In this study, a novel cell line (PmOVC) derived from the ovary of red seabream (Pagrus major) was successfully established. Using polystyrene microplastics (PS-MPs)-a typical environmental pollutant-as the test substance, the toxicity sensitivity of the PmOVC cell line was systematically assessed from the dimensions of particle size intake and cell activity. The results showed that the PmOVC cells exhibited robust growth in M199 medium supplemented with 15% FBS at 23°C and high exogenous gene transfection efficiency and showed high sensitivity to the toxic effects of microplastics. Specifically, the smaller the particle size of microplastics, the higher the internalization efficiency of cells, among which 100 nm PS-MPs could be completely endocytosed into the cytoplasm; the concentration of PS-MPs was significantly positively correlated with the apoptosis rate of cells (R[2] ≥ 0.8). In summary, the established PmOVC cell line provides a critical in vitro tool for in-depth investigation of the toxicological responses and underlying mechanisms of fish to environmental pollutants.

Osteoarthritis (OA) is a common degenerative joint disease characterized by the progressive degeneration of articular cartilage (AC), resulting in pain, stiffness, and reduced joint function. Despite its widespread prevalence, effective treatments for OA remain elusive, partly due to challenges in accurately modeling the disease. In the present study, adhesion energy, collagen (COL), and total glycosaminoglycans (GAG), the main extracellular matrix (ECM) components of AC, and their potential correlations were evaluated in human chondrocytes derived from both healthy and OA male donors. A model surface of silicon nitride was used to investigate adhesion energy using atomic force microscopy (AFM), while colorimetric assays were applied to quantify COL and GAG at five different time points during monoculture (days 0, 1, 7, 14, and 21). Our results indicate significant heterogeneity in adhesion energies at both donor and phenotype levels. Chondrocytes isolated from healthy donors consistently display higher adhesion energies, along with greater heterogeneity, compared to those derived from OA donors. These differences in adhesion energy and heterogeneity can be attributed to greater diversity and density of cellular surface molecules in cells from healthy donors. In contrast, total GAG and COL levels were significantly elevated in OA chondrocytes compared to those in healthy chondrocytes. Moreover, the study found a positive correlation between adhesion energy and GAG production in OA-derived chondrocytes, but no significant correlations with DNA or COL levels. No correlation was observed between adhesion energy and GAG, DNA, or COL levels in healthy cells. In conclusion, these findings highlight the importance of considering individual donor characteristics when developing OA disease models, therapeutics, or engineered tissues.

The extensive use of commercial pesticide formulations, often containing multiple active ingredients, has raised growing concerns about their potential environmental and human health impacts. Among the most widely applied pesticides are imidacloprid (IMI), λ-cyhalothrin (LCT), deltamethrin (DMN), and thiamethoxam (TMX). This study investigated the cytotoxic effects of acute exposure to commercial formulations of these compounds on a seabass spinal cord (SBSC) cell line. Cytotoxicity was evaluated using MTT and Neutral Red (NR) uptake assays, in conjunction with assessments of reactive oxygen species (ROS) generation, plasma membrane staining, lysosomal acidity, fluorescence-based cellular labelling, and effects on neurite-like outgrowth. Effective concentrations (EC10 and EC50) were determined for each pesticide, demonstrating that even low-dose exposure (EC10) significantly disrupted neurite-like outgrowth in SBSC cells. These findings suggest that low-level pesticide exposure may pose a substantial risk to neurodevelopment.

Umbilical cord blood HSCs transplantation is limited by low cell numbers and engraftment efficiency. The bone marrow endothelial cell (EC) niche is vital for HSCs. Adenoviral E4ORF1 protein enables ECs (e.g., HUVECs) to support HSCs expansion, but functional differences between E4ORF1 from distinct serotypes (respiratory Ad5 vs. enteric Ad52) are unknown. This study compared Ad5- and Ad52-E4ORF1 effects on engineered HUVECs and their support of hematopoietic stem/progenitor cells (HSPCs). Transcriptome analysis confirmed relevant receptor/metabolism gene expression in HUVECs. Sequence alignment revealed no significant nucleotide/amino acid homology between Ad5- and Ad52-E4ORF1. Engineered HUVECs expressing Ad5- or Ad52-E4ORF1 were co-cultured with mouse HSPCs. Outcomes were assessed via in vitro expansion, flow cytometry phenotyping, and competitive transplantation into irradiated mice evaluating engraftment and multilineage reconstitution. In vitro, Ad5-E4ORF1 promoted greater total hematopoietic cell expansion, while Ad52-E4ORF1 better maintained stemness, yielding a significantly higher proportion of ST-HSCs. Competitive transplantation revealed Ad5-E4ORF1 co-cultured HSPCs achieved higher early engraftment and superior initial myeloid reconstitution. Conversely, Ad52-E4ORF1 supported HSPCs exhibited significantly enhanced and sustained late-phase T-cell production, though B-cell levels declined after 12 weeks. Ad5 and Ad52 E4ORF1 exert serotype-specific regulation on HUVECs, differentially impacting HSPC fate: Ad5-E4ORF1 favors expansion and early myeloid engraftment, while Ad52-E4ORF1 preserves ST-HSCs and enhances short-term T-lymphopoiesis. These differences correlate with high E4ORF1 sequence divergence and are crucial for optimizing HSCs transplantation strategies.