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.
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
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 N⁶-methyladenosine (m⁶A) demethylase, has been implicated in the progression of atherosclerotic conditions. This study sought to elucidate how FTO downregulates proteinase 3 (PRTN3) through m⁶A 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 m⁶A 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, m⁶A, PRTN3, and CXCL9/CXCR3 in CAD pathogenesis, highlighting FTO as a promising target for therapeutic intervention.
Hepatocellular carcinoma (HCC) has a poor prognosis and limited treatment options. This study investigated the role and mechanism of Jiedu Xiaozheng Yin (JXY) in HCC using network pharmacology and in vivo/in vitro experiments. Active components of JXY and their potential therapeutic targets for HCC were identified via network pharmacology. A protein–protein interaction (PPI) network was constructed to identify core targets, and bioinformatics analysis confirmed their association with HCC. LC–MS/MS was used to isolate major components, which were then subjected to molecular docking with core targets. The effects of JXY on HCC were further validated through in vivo and in vitro experiments. Analysis of 171 core target genes was performed using PPI network screening and enrichment analysis. Quercetin was identified as the primary active component of JXY. Molecular docking confirmed its interaction with CYP1A2, which is overexpressed in HCC tissues. In vivo, JXY and quercetin significantly increased CYP1A2 and PTEN expression, while decreasing p-PI3K and p-Akt levels in HCC-bearing mice, thereby suppressing tumor growth and lung metastasis and promoting apoptosis. In vitro, both JXY and quercetin inhibited HCC cell proliferation and migration. CYP1A2 knockdown partially reversed, while its overexpression enhanced, these inhibitory effects. Quercetin, the main active component of JXY, upregulates CYP1A2 to promote PTEN expression, thereby inhibiting the PI3K/Akt signaling pathway and suppressing HCC progression.
Intramuscular fat deposition is a key determinant of meat quality in livestock species; however, yak (Bos grunniens) stromal vascular cells (SVCs) exhibit relatively low adipogenic differentiation efficiency under conventional in vitro conditions. In this study, we established a three-dimensional (3D) spheroid culture system to improve adipogenic differentiation of yak SVCs and compared it with traditional two-dimensional (2D) monolayer culture. Yak SVCs were isolated from longissimus dorsi muscle and characterized prior to differentiation. Cells were subjected to adipogenic induction under both 2D and 3D conditions. Adipogenic differentiation was evaluated by morphological observation, Oil Red O staining and quantification, and quantitative real-time PCR (RT-qPCR) analysis of adipogenic marker genes. Additionally, Western blot analysis of key adipogenic proteins was performed specifically on yak SVCs. To validate the induction system, murine C3H10T1/2 cells were included as a parallel control group and evaluated using the aforementioned non-protein assays. The results showed that 3D culture significantly enhanced lipid accumulation and increased the expression of adipogenic marker genes compared with 2D culture in yak SVCs. Consistently, protein levels of PPAR γ were significantly elevated in the 3D group of yak SVCs, while FABP4 showed an upward trend. Furthermore, the C3H10T1/2 control group exhibited successful adipogenic differentiation with similarly enhanced lipid accumulation and gene expression under 3D conditions, confirming the reliability of our system. In conclusion, 3D spheroid culture improves adipogenic differentiation of yak SVCs and provides a useful in vitro model for studying adipose development in yak. This system may facilitate future studies on the regulation of intramuscular fat deposition in livestock species.
Utilizing single-cell RNA sequencing and network pharmacology, this study explored the mechanism by which Bletilla striata polysaccharide (BSP) mitigates peri-implantitis-associated osteolysis. Integrated analyses revealed a significant expansion of pro-inflammatory macrophages and osteoclasts in diseased tissues, with differentially expressed genes enriched in glycolytic pathways. Mechanistic exploration indicated that BSP suppresses osteoclastogenesis primarily by targeting matrix metalloproteinase-9 (MMP9), thereby inhibiting proteolytic activity and cytoskeletal remodeling. Preliminary rescue experiments supported this notion, showing that while BSP effectively downregulated LPS-induced MMP9 upregulation and suppressed macrophage glycolytic metabolism, forced overexpression of MMP9 partially restored MMP9 levels and reversed the inhibitory effect of BSP on osteoclast formation. These findings suggest a potential “metabolism-protease” interplay, where BSP concurrently modulates immune metabolism and protease activity to alleviate inflammatory bone loss, providing a foundational basis for developing plant-derived polysaccharide therapeutics.
The aim of this study was to isolate human periapical cyst-derived mesenchymal stem cells (PC-MSCs) and compare their immunophenotypic profiles, clonogenic capacity, metabolic activity, migration capacity, and differentiation characteristics with those of dental pulp mesenchymal stem cells (DP-MSCs). PC-MSCs and DP-MSCs were isolated from six periapical cysts and six impacted third molars, respectively. The cells were characterized by flow cytometry and trilineage differentiation assays (osteogenic, adipogenic, chondrogenic). Proliferation was assessed using the MTT assay and colony-forming unit-fibroblast (CFU-F) efficiency. Migration potential was evaluated using an in vitro scratch assay. The secretion of odontogenic markers, dentin sialophosphoprotein (DSPP) and dentin matrix acidic phosphoprotein (DMP1), was quantified using enzyme-linked immunosorbent assay (ELISA). Both cell types expressed characteristic mesenchymal stem cell markers. PC-MSCs exhibited significantly higher colony-forming efficiency (CFU-F, p < 0.05) and superior migration capacity compared to DP-MSCs (p < 0.05). Both MSC populations demonstrated differentiation capacity. DP-MSCs showed more extensive Alizarin Red S staining, while PC-MSCs showed relatively stronger Oil Red O staining. ELISA analysis revealed no statistically significant differences in DSPP and DMP1 secretion levels between the two groups (p > 0.05). Although DP-MSCs exhibited superior mineralizing ability, PC-MSCs displayed higher clonogenic and migratory potential, while no statistically significant differences were observed in DSPP and DMP1 secretion levels between the groups. These findings suggest that periapical cysts, often regarded as pathological waste, may represent a promising and readily accessible source of MSC-like cells with potential relevance for regenerative endodontic research.
Mild thermal stimulation enhances skeletal muscle differentiation; however, its underlying metabolic basis remains unclear. Here, we demonstrate that thermal stimulation promotes myogenic differentiation through enhanced glucose uptake and transient lipid droplet (LD) accumulation in C2C12 myoblasts. Thermal stimulation at 39 °C induced a transient increase in LD formation during early differentiation. Early transient LD accumulation, particularly on days 1 and 2, was positively correlated with the day 5 fusion index, suggesting that early LD formation is associated with subsequent myogenic differentiation. Suppression of LD formation by Plin2 knockdown impaired myotube formation, indicating that LD formation is functionally required for myogenic differentiation. Thermal stimulation enhanced glucose uptake, accompanied by increased Slc2a4 and Ppargc1a expression and increased GLUT4 fluorescence intensity. Moreover, higher glucose availability further supported LD formation and myogenic differentiation even under serum-free conditions. Notably, mitochondrial mass, ATP content, and oxidative capacity remained largely unchanged, suggesting that enhanced glucose uptake is associated with LD accumulation without a corresponding increase in mitochondrial oxidative capacity. These findings identify transient LD formation as a key metabolic event driving myogenic differentiation under thermal stimulation.
Colorectal cancer (CRC) is a malignant tumor with a high global incidence. Tianma granule (TMG) is a traditional Chinese medicine compound. However, its pharmacodynamic material basis and mechanism of action against CRC remained unclear. This study systematically analyzed the anti-CRC mechanism of TMG by integrating metabolomics, network pharmacology, and experimental validation. HPLC-MS identified the main active components of TMG. Core targets and pathways were predicted by network pharmacology. The binding affinity between components and targets was evaluated by molecular docking. In vitro experiments utilized the HCT116 and HCT-8 cells model, where the effects of TMG and its key component Apigenin on cell activity. Apigenin was identified as a key flavonoid component in TMG by metabolomics. Network pharmacology analysis indicated that TMG intervened in CRC through multi-target actions, with the Wnt/β-catenin pathway being the core regulatory pathway. In vitro experiments demonstrated that both TMG and Apigenin inhibited HCT116 and HCT-8 cell proliferation, promoted apoptosis, and weakened invasion and migration capabilities. Apigenin promotes β-catenin degradation and suppresses its nuclear translocation by upregulating GSK3β and APC expression. The ablation or mutation of GSK3β or APC abolishes this effect, whereas β-catenin knockdown alone recapitulates the anti-cancer effects of TMG and Apigenin, demonstrating that their actions are strictly dependent on β-catenin. Apigenin, as the core active ingredient of TMG, was demonstrated to target GSK3β/APC to promote β-catenin degradation and inhibit the activation of the Wnt/β-catenin pathway, thereby exerting anti-CRC effects.
The present study examined the impact of conjugated linoleic acid (CLA) on the differentiation and proliferation of bovine intramuscular preadipocyte (BIP) cells, specifically exploring the role of G protein-coupled receptor 41 (GPCR41). Cells were exposed to CLA at various concentrations ranging from 50 to 150 μmol/L. Flow cytometry revealed that treatment with 100 μmol/L CLA led to a significant increase in the proliferation rate. During differentiation, 100 and 150 μmol/L CLA markedly enhanced lipid droplet accumulation. Maximal cytoplasmic lipid content was observed when CLA was combined with insulin and dexamethasone. CLA treatment significantly enhanced the transcript abundance of the adipogenic transcription factors peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT-enhancer binding protein alpha (C/EBPα). Knockdown of GPCR41 via RNA interference in CLA-treated cells led to a significant suppression of GPCR41 expression, accompanied by decreased PPARγ mRNA levels and diminished cytoplasmic lipid droplet formation, indicating impaired differentiation. Taken together, these results show that CLA enhances both differentiation and proliferation of bovine intramuscular preadipocytes, possibly via activation of C/EBPα and PPARγ, with GPCR41 playing a critical role in mediating this adipogenic effect.
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.
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.
