In Vitro Cellular & Developmental Biology. Animal最新文献

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.

Resistance to radiation therapy (RT) poses a significant challenge in managing non-small cell lung cancer (NSCLC). Despite research into how tumor-sourced exosome (Exo) miRNAs influence tumor RT resistance and macrophage M2 polarization, the process through which Exos with miR-616-3p modulate macrophage polarization to impact NSCLC RT resistance is still not well understood. The objective of this research was to investigate the molecular processes by which RT regulates M2 polarization of macrophages via the Exos miR-616-3p derived from NSCLC cells. Identification of Exos was conducted using transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). Flow cytometry, immunofluorescence, and ELISA were employed to verify the macrophage phenotype. The expression of miR-616-3p was identified using RT-qPCR, and the targeting relationship between miR-616-3p and PTEN was confirmed through dual-luciferase reporter gene tests and RIP identification. In NSCLC, miR-616-3p showed high expression levels and was linked to RT and M2 polarization in macrophages. Subsequent research indicated that RT prompted the influx of Exos-miR-616-3p from NSCLC cells into macrophages. Both the H1299 lung cancer cell line and the M0 macrophages underwent co-culture. Findings indicated that NSCLC cells induced by RT and Exos elevated the proportion of CD163 + CD206 + positive cells in macrophages via miR-616-3p and augmented Arg1, IL-10, TGF-β1, and VEGF levels, and enhanced M2 polarization in macrophages. Regarding the molecular process, miR-616-3p suppressed PTEN protein expression while concurrently boosting the levels of p-PI3K/PI3K and p-AKT/AKT; either amplifying PTEN or suppressing PI3K could markedly weaken the impact of ionizing radiation (IR), inhibiting the impact of NSCLC cell Exos on macrophages' M2 polarization. This research reveals that in NSCLC cells induced by IR, Exos with miR-616-3p expression reduce PTEN levels and enhance the PI3K/AKT signaling pathway, leading to increased M2 polarization in macrophages and worsening NSCLC progression.

This study aimed to evaluate the effects of conditioned medium derived from murine skeletal muscle (SMCM) on oxidative stress and testicular morphology in vitro. Initially, Wistar rats underwent treadmill familiarization and a maximal incremental test (MIT). Animals were then submitted to a single exercise session at 60% of the maximum speed established by MIT. In Experiment 1, femoral muscles from trained animals were cultured in αMEM supplemented with 1.25 mg/mL BSA to produce SMCM. In Experiment 2, testes from sedentary rats were fragmented and cultured for 24 h in αMEM alone or αMEM added to irisin at 100 ng/mL or αMEM added to SMCM at 25, 50, 75, or 100%. HPLC confirmed the presence of irisin in SMCM. Oxidative stress analyses demonstrated catalase activity was higher in irisin and 75% of SMCM treatments, while glutathione peroxidase (GPX) activity was significantly higher in the irisin when compared to fresh control. It is important to highlight that 25% of SMCM was similar to fresh control in GPX activity and thiol content. Histological assessment revealed structural alterations in cultured testes, although overall tubular organization was preserved. These findings suggest that skeletal muscle SMCM modulates oxidative balance and testicular structure, with the 25% concentration yielding the most favorable antioxidant profile.

Obesity resulting from chronic overnutrition and physical inactivity promotes the development of metabolic disorders by disrupting physiological processes in metabolically active organs, including skeletal muscles. To investigate whether skeletal muscle stem cells (satellite cells, SCs) are affected by systemic metabolic stress, we established primary SC cultures from male mice fed a high-fat diet (HFD) for 8 wk, and from control mice fed a standard chow (CTL). This model allowed us to assess diet-induced obesity (DIO)-related changes in SC-specific molecular and cellular signatures. Although body weight, body fat composition, and adipose tissue-associated macrophages differed significantly between DIO and CTL ex vivo, we observed no differences in the in vitro behaviour of primary SC-derived myoblasts from either group. Parameters such as proliferation and differentiation following serum deprivation were comparable. Expression levels and distribution patterns of myogenic regulatory factors (MRF), SC-specific markers (Pax7, CD56, Itga7), and hallmarks for senescence (GLB1), autophagy (p62, LC3B), and oxidative stress (ALDH1A1, ALDH1A3) remained unchanged. Thus, potential differences in the signatures of SC-derived myoblasts after 8 wk of a high-fat diet cannot be depicted in vitro. However, future experiments should address whether prolonged and metabolically more susceptible diets will exert long-term effects on myogenesis in vitro or not. Overall, we propose that primary SC cultures are better suited for acute in vitro testing regarding the molecular and cellular plasticity in metabolic shifts as induced by pharmacological treatments or genetical modifications, rather than for modeling long-term dietary effects.