In Vitro Cellular & Developmental Biology. Animal最新文献

Bergapten (BG), a furanocoumarin compound, has demonstrated diverse pharmacological properties, including the reduction of oxidative stress and inflammation in osteoarthritic chondrocytes. However, the mechanisms underlying BG's effects on chondrocyte differentiation and proliferation remain incompletely understood. This study aimed to elucidate BG's role in chondrocyte differentiation, proliferation, and inflammation prevention using in vitro and in vivo osteoarthritis (OA) models. An OA model was established by treating rabbit primary chondrocytes with sodium nitroprusside (SNP) to induce apoptosis and inflammation. BG treatment significantly upregulated chondrogenic differentiation markers, including type II collagen, SOX9, and aggrecan, and histological staining revealed increased proteoglycan accumulation in OA-induced cells treated with BG. Importantly, BG exhibited pronounced anti-inflammatory effects by modulating the NF-κB pathway: it increased IκBα expression and reduced cyclooxygenase-2 (COX-2) and p50 expression, thereby preventing inflammation in chondrocytes. BG also activated p38 kinase signaling, suggesting its contribution to Chondroprotective effects. Flow cytometry demonstrated that BG reduced SNP-induced apoptosis from 22 to 4%. Western blot analysis confirmed that BG downregulated apoptotic proteins p53 and BAX while upregulating the anti-apoptotic protein Bcl-2. In vivo validation using a zebrafish model showed that BG rescued SNP-induced craniofacial cartilage structural defects. These findings provide novel insights into BG's pharmacological role in promoting chondrocyte differentiation and survival, as well as preventing inflammation, by modulating key inflammatory pathways, apoptotic proteins, and chondrogenic markers. Given its clinical potential, BG may hold promise as a therapeutic agent for OA treatment.

Tissue densification, as a hallmark of development, injury, and fibrosis, alters the mechanical and structural properties of the extracellular matrix (ECM). However, its specific effects on neural cell behavior remain poorly understood. To address this, we developed a 3D in vitro microtissue model composed of collagen and Matrigel, incorporating co-cultures of N2A neuroblastoma and 3T3 fibroblast cells. By modulating fibroblast-driven contraction through scaffold-guided constraint, we generated microtissues with distinct levels of densification with Young's moduli ranging from approximately 0.5 to 1 kPa. Our results demonstrated that increased microtissue densification significantly enhanced N2A migration and aggregate formation, indicating that mechanical compaction facilitates neuronal clustering. Furthermore, higher densification promoted N2A cell proliferation, while apoptosis remained at relatively low baseline levels, suggesting that dense environments support cell expansion without affecting overall viability. Additionally, higher densification suppressed the proportion of neurite-bearing cells without affecting neurite length, implying impaired initiation of neuronal differentiation but not morphological maturation. Together, these findings reveal that ECM densification serves as a critical mechanical cue shaping multiple aspects of neural cell behavior. Compared to conventional hydrogel systems, our collagen-based microtissue model provides a more physiologically relevant in vitro platform for studying neurodevelopmental mechanobiology and guiding biomaterial design for neural tissue engineering.

Polycystic ovary syndrome (PCOS), a common endocrine disorder affecting over 10% of women, is characterized by hyperandrogenism and ovarian dysfunction. While linked to chronic inflammation and granulosa cell apoptosis, its molecular mechanisms remain unclear. In this study, we elucidated the novel role of Krüppel-like factor 5 (KLF5) in the pathogenesis of PCOS and its regulatory role with thioredoxin interacting protein (TXNIP). In a mouse model of PCOS induced by dehydroepiandrosterone (DHEA), KLF5 expression was significantly elevated in ovarian tissues (up-regulated 2.62-fold, P < 0.001), correlating with hyperandrogenism (testosterone: up-regulated 2.83-fold, P < 0.001) and cystic follicle formation. The proliferative capacity of testosterone-treated KGN cells was reduced to 59% after KLF5 was knocked down (P < 0.01), attenuated apoptosis by inhibiting the increase of Bax and Cleaved-caspase 3 proteins and thus attenuated inflammation by down-regulating NLRP3 and Interleukin- (IL-) 1β. Most importantly, dual luciferase assay showed that KLF5 transcriptionally activated TXNIP, resulting in a 3.04-fold enhancement of its promoter activity (P < 0.001). Meanwhile, overexpression of TXNIP reversed the silencing effect of KLF5, resulting in a significant increase in apoptosis and secretion of inflammatory factors. These results reveal a previously unrecognized KLF5/TXNIP axis driving granulosa cell (GC) dysfunction in polycystic ovary syndrome, in which KLF5 transcriptionally upregulates TXNIP to promote apoptosis and NLRP3 inflammasome activation. Our findings provide the first evidence linking KLF5 to the pathogenesis of PCOS and establish this pathway as a potential therapeutic target, bridging a significant gap in understanding the molecular basis of the disease and providing compelling evidence for clinical drug development.

Cisplatin-based chemotherapy is the first-line treatment for lung cancer. However, cisplatin resistance (CR) remains a major challenge, leading to treatment failure. A key driver of CR is enhanced DNA damage repair. Although males absent on the first (MOF) participate in DNA repair, their specific role in mediating CR remains unclear. In this study, CR models were established in PC9 and A549 lung cancer cell lines. Our results showed that high expression of Williams syndrome transcription factor (WSTF) in lung cancer cells was associated with CR. WSTF knockdown inhibited proliferation and promoted apoptosis, DNA damage, and γ-H2AX levels in CR cells. Moreover, MOF was highly expressed in lung cancer cells and regulated by WSTF acetylation. Furthermore, MOF knockdown downregulated H4K16ac levels in CR cells. MOF overexpression significantly upregulated H4K16ac levels, enhanced proliferation, and suppressed apoptosis in CS cells, concomitant with DNA damage repair and reduced γ-H2AX expression. Notably, transfection with the K46R attenuated these MOF-mediated effects in CS cells. Collectively, our study demonstrates that MOF promotes DNA damage repair and enhances CR in lung cancer cells via H4K16ac-mediated WSTF acetylation. These findings provide valuable insights for overcoming chemoresistance and improving patient outcomes.

The skin is the largest organ of the human body, capable of protecting it from external harm. However, due to trauma, paralysis, and other external factors, skin damage can occur, and scars may form. Exosomes have regenerative functions and, as a cell-free therapy, show great potential for wound healing. In this study, we aimed to investigate whether thrombin-preconditioned umbilical cord mesenchymal stem cells (T-pre-UCMSCs) increase the production of exosomes. Different umbilical cord mesenchymal stem cell exosomes can accelerate the healing of skin. In our study, umbilical cord mesenchymal stem cells (UCMCs) were cultured in DMEM/F12 medium without fetal bovine serum (FBS) for 72 h with 200U/ml thrombin. Exosomes were isolated from the supernatant by ultracentrifugation. There are two kinds of exosomes: UCMCs culture supernatant-derived exosomes (UCMSCs-Exos) and T-pre-UCMSCs culture supernatant-derived exosomes (T-UCMSCs-Exos). The skin injury cell model was constructed by treating HaCats with a tip. Additionally, the wound healing capacity of exosomes was evaluated in vivo using a mouse skin injury model. Compared to UCMSCs-Exos, T-UCMSCs-Exos significantly promoted cell proliferation and migration of cells. In vivo experiments demonstrated that T-UCMSCs-Exos can accelerate wound closure and enhance collagen maturation, promoting angiogenesis in the vascularized wound area. These results indicate that T-UCMSCs-Exos have good potential for accelerating wound healing and minimizing scar formation. Our research indicates that thrombin pre-UCMSCs significantly increased the production of exosomes. These findings demonstrate that T-UCMSCs-Exos for skin wounds are a promising cell-free therapy that can be applied in the treatment of skin injuries.

This study aimed to extract and isolate endometrial stromal cells from Arabian mares and investigate their growth and differentiation potential. Endometrial biopsies were obtained from three healthy 6-year-old Arabian mares using a standardized, minimally invasive protocol. The isolated cells were characterized using flow cytometry and differentiation analysis. Flow cytometry revealed mesenchymal markers CD90 (95.2%) and CD105 (97.4%) and hematopoietic markers CD34 (1.17%) and CD45 (0.339%). The cells exhibited differentiation potential into adipocytes, osteoblasts, and chondrocytes. The findings suggest that endometrial cells from Arabian mares represent a promising autologous source of MSCs, particularly suited for regenerative applications in musculoskeletal disorders.

To investigate the protective effect of wogonin against high glucose (HG)-induced ARPE-19 cell injury and to elucidate its mechanism of action. The effects of Wogonin on cell proliferation and apoptosis were systematically evaluated by a high glucose-induced ARPE-19 cell injury model using different doses of Wogonin for intervention. Meanwhile, intracellular reactive oxygen species (ROS) levels, iron ion accumulation and glutathione (GSH) depletion were detected, and the expression changes of apoptosis-related proteins, ferroptosis -related proteins, and cGAS-STING pathway proteins were analyzed. In addition, the cGAS agonist SR-717 was co-administered on the basis of Wogonin intervention to further investigate whether SR-717 could reverse the effects of Wogonin on cells. Wogonin significantly increased cell viability and reduced apoptosis in response to HG. Wogonin also alleviated oxidative stress by decreasing intracellular ROS and iron accumulation while inhibiting glutathione depletion. In addition, wogonin inhibited HG-induced ARPE-19 cell injury by inhibiting the cGAS‒STING signaling pathway and promoting the expression of the cellular GPX4 and SLC7A11 proteins. Wogonin inhibits ferroptosis by suppressing the cGAS-STING signaling pathway and has a protective effect against HG-induced ARPE-19 cell injury, suggesting that it may be used as a therapeutic agent to alleviate DR.

Endothelial cell damage often results in apoptosis and is thus a crucial factor for the development of coronary artery disease (CAD). However, the mechanisms underlying endothelial cell apoptosis remain unclear. Although circular (circ) RNAs have been implicated in apoptosis, the involvement of hsa_circ_0124644 is uncertain. Therefore, the aim of this study was to investigate the effect of hsa_circ_0124644 on endothelial cell apoptosis and to elucidate the underlying molecular processes. We treated endothelial cells with tumor necrosis factor alpha (TNF-α) to simulate the microenvironment of CAD and generate an apoptosis model. We measured cell apoptosis in conjunction with hsa_circ_0124644 expression. After hsa_circ_0124644 overexpression or inhibition, we assessed apoptosis levels using flow cytometry, RT-qPCR, and western blotting. We found that hsa_circ_0124644 overexpression lowered the apoptosis rate and increased cell viability. Similarly, overexpression also upregulated the expression of an anti-apoptotic protein (Bcl-2) and downregulated that of a pro-apoptotic protein (Bax). In conclusion, our findings suggested that hsa_circ_0124644 mediates endothelial cell apoptosis in CAD. These findings have important implications for developing effective treatments of cardiovascular conditions with excessive apoptosis.

The molecular mechanisms underlying growth hormone (GH) therapy in children with idiopathic short stature (ISS) remain incompletely understood. This study investigated how GH promotes bone growth in children with ISS, focusing on insulin-like growth factor-binding protein 2 (IGFBP2) and thrombospondin-1 (THBS1). Analysis of ISS patient plasma showed downregulated IGFBP2, predicted to interact strongly with THBS1. Experiments using human chondrocytes revealed that GH treatment stimulated cell proliferation, accelerated the cell cycle, and induced hypertrophic differentiation, marked by increased expression of proteins like COL10A1, RUNX2, OCN, OPN, and alkaline phosphatase activity. GH also elevated IGFBP2 and insulin-like growth factor-1 (IGF-1) while suppressing THBS1. Crucially, knocking down IGFBP2 blocked these GH effects, reducing proliferation, halting cell cycle progression, decreasing differentiation markers and IGF-1, while increasing THBS1. Conversely, overexpressing IGFBP2 mimicked GH's effects. Importantly, silencing IGFBP2 partially prevented GH-induced proliferation, differentiation, and IGF-1 secretion. This demonstrates that IGFBP2 acts as a key mediator of GH's action by inhibiting THBS1, which subsequently activates the IGF-1 pathway to drive chondrocyte proliferation and hypertrophic differentiation. The IGFBP2-THBS1 axis is thus a core mechanism for GH therapy in ISS, offering a novel therapeutic target for improving treatment.

Research shows that transplanted bone marrow mesenchymal stem cells (BMSCs) have been shown to improve functional outcomes in mice with spinal cord injury (SCI). Many experimental centers have demonstrated that systemic delivery of MSCs in mice can treat neurological diseases, but whether or how it works in acute spinal cord injury is not understood. Various methods such as Basso, Beattie, and Bresnahan (BBB) locomotor rating scale score and biological detection of inflammatory factors were used to test the changes in inflammatory factors of spinal cord injury at different time points in 24 h, 7 d, and 14 d. Twenty-four hours after injury, the functional measurement results of the injured group were significantly weakened compared with the control rats. The functional results of the BMSCs injection injured group were also significantly weakened compared with the control rats. There was no statistical difference between the injured group and the BMSCs injection group. However, the injury group had the highest mortality rate (p < 0.05). Biochemical results showed that compared with the control group, the expression of high-mobility group box 1 (HMGB1) and receptor for advanced glycation end-products (RAGE) and related inflammatory factors in the injury group and BMSCs injection group increased significantly at 7 d after the experiment in Western blot. Similarly, the expression of HMGB1 and RAGE in the injury group was also greater than that in the injection group, and there was a statistical difference in immunohistochemical assessment. Injecting BMSCs into rats with acute spinal cord injury could reduce rat mortality and improve prognostic functional measurements after SCI. BMSCs may promote spinal cord re-repair by inhibiting the HMGB1/RAGE signaling pathway after acute contusive spinal cord injury.