In Vitro Cellular & Developmental Biology. Animal最新文献

5-Fluorouracil (5-FU) is a cornerstone chemotherapeutic agent commonly employed in colorectal cancer (CRC) treatment. Prolonged use of 5-FU can trigger drug resistance, primarily through the upregulation of thymidylate synthase (TS). Consequently, strategies targeting TS suppression could enhance 5-FU's therapeutic potential in resistant CRC cases. Short-chain fatty acids (SCFAs), derived from the fermentation of dietary fibers by gut microbiota, are implicated in various disease mechanisms, including cancer. Among SCFAs, sodium butyrate (NaB) is known to inhibit TS expression, reduce CRC cell viability, and promote apoptosis. However, the potential of sodium propionate (NaP), another SCFA, to exhibit similar effects remains under investigation. This study reveals that NaP, when combined with 5-FU, synergistically decreases CRC cell survival and enhances apoptosis. Furthermore, NaP counteracts the 5-FU-induced upregulation of TS, amplifying its inhibitory effects on drug-resistant CRC cells. These results suggest that NaP may serve as an effective adjunct in improving the therapeutic outcomes of 5-FU-based CRC treatments.

Ginsenoside Rb1 ameliorates renal fibrosis, yet its effects on myocardial fibrosis (MF) remain unclear. In this study, we aimed to explore the role of ginsenoside Rb1 in chronic heart failure (CHF) and MF. To explore the correlation between endothelial-mesenchymal transition (EndMT) in endothelial cells and IGFBP2 expression in M1 macrophages, M1 macrophages were polarized and co-cultured with myocardial microvascular endothelial cells (MMVECs). IGFBP2 levels in the macrophages and levels of endothelial-specific markers and EndMT-related indexes in MMVECs were measured. Additionally, we treated the macrophages with ginsenoside Rb1. The CHF mice model was established using transverse aortic constriction (TAC) and then treated with ginsenoside Rb1. The effects of Rb1 on cardiac function, MF, and cardiomyocyte hypertrophy in CHF mice were assessed. We observed the successful differentiation of M1 macrophages using in vitro experiments. M1 macrophages co-cultured with MMVECs demonstrated the ability to enhance the EndMT effect in MMVECs, as evidenced by elevated levels of IGFBP2 in the macrophages and a reduction in the viability of MMVECs. This decrease in cell viability was mitigated following the knockdown of IGFBP2. Rb1 treatment significantly suppressed the expression of IGFBP2 and inhibited the occurrence of the EndMT in MMVECs. The in vivo experiment findings showed that ginsenoside Rb1 notably enhanced cardiac function, attenuated cardiomyocyte hypertrophy, and alleviated MF in CHF mice. Furthermore, ginsenoside Rb1 inhibited M1 macrophage polarization, reduced IGFBP2 expression in the myocardium, and suppressed the EndMT effect of MMVECs in mice. Ginsenoside Rb1 alleviated MF in mice with CHF by inhibiting M1 macrophage IGFBP2-mediated EndMT.

Skeletal muscle regeneration depends on satellite cells that maintain tissue homeostasis through self-renewal and the production of myoblasts that differentiate into mature myofibers. Dysregulation of these processes can lead to muscle degeneration, highlighting the need to elucidate their molecular mechanisms. In this study, we investigated the role of the Grb2/Sos1 signaling pathway in regulating satellite cell self-renewal and differentiation using C2C12 cells. Knockdown of either Grb2 or Sos1 significantly reduced the formation of Bcl-2-positive reserve cells and increased the proportion of differentiated myotubes. Conversely, forced expression of Grb2 increased the number of reserve cells, whereas the Grb2 P49L mutant, which disrupts its interaction with Sos1, decreased reserve cell formation and resulted in thinner myotubes. Although forced expression of Sos1 alone did not significantly increase reserve cell numbers, the chimeric protein cSos-SH2, which combines elements of Grb2 and Sos1, produced a pronounced increase of reserve cells. These results demonstrate that a precise balance between Grb2 and Sos1, along with their coordinated subcellular localization, is critical for controlling reserve cell populations. Activated by growth factor receptor tyrosine kinases and extracellular matrix/integrin interactions, the Grb2/Sos1 signaling pathway is critical for maintaining the muscle satellite cell pool, thereby playing an essential role in muscle regeneration.

The declining population of the Antillean manatees has prompted interest in developing conservation strategies, including somatic cell cryopreservation. However, the type and concentration of intracellular cryoprotectant agents (CPAs) are limiting factors for its success. Therefore, we evaluated three concentrations (5, 8, 10%) of dimethyl sulfoxide (Me₂SO) and ethylene glycol (EG) to assess if reducing CPA concentration is efficient for the cells of these animals. Cells not subjected to cryopreservation were used as a control. All cells were analyzed for morphology, viability, metabolism, proliferative activity (PDT), apoptosis, levels of reactive oxygen species (ROS), and mitochondrial membrane potential (ΔΨm). Regardless of the solution used, the cryopreservation did not change frozen-thawed cells' morphology, metabolism, and apoptosis levels compared to control group cells (p > 0.05). Immediately after thawing, cells derived from the 8% Me₂SO group-maintained viability similar to the control; after in vitro culture of thawed cells, this positive response of viability was observed only in cells cryopreserved in solutions containing 5% and 8% CPA, regardless the type of CPA. Interestingly, cells frozen in 8% Me₂SO showed a higher PDT value than the other groups (p < 0.05). Cells frozen with 10% EG showed higher ROS than the control group (p < 0.05). Additionally, regardless of the solution used, cryopreservation resulted in a change in ΔΨm. In summary, reducing the concentration of CPAs (5 and 8%) helps with somatic cell quality, regardless of the CPA type used in Antillean manatees.

The skin is a vital organ that regulates the temperature, nutrient absorption, and perception of sensations. Wound healing is a complex biological process in multicellular systems that consists of four key phases: hemostasis, inflammation, proliferation, and remodeling. This study develops a new approach for synthesizing dihydropyrimidinones (DHPM) named Biginelli scaffolds via a simple, rapid, eco-friendly, and cost-effective solvent-free Biginelli reaction for wound healing activities. The synthesis involved a one-pot three-component coupling reaction of β-ketoester derivatives, anisaldehyde, and simple urea in a domestic microwave oven. The synthesized (B1-B4) scaffolds were characterized using melting point, UV-Vis, FT-IR, HRMS, 2D-NMR (NOESY), and proton/carbon NMR spectroscopies. The molecular docking results showed that the synthetic scaffolds (B1-B4) had strong binding abilities, with B3 and B4 having the best interactions in the group, similar to the control compound (curcumin). It exhibited less cytotoxic effects up to 80 µg/mL in Tilapia gill (TG) cells in the MTT assay. The synthesized scaffolds (60 µg/mL) enhanced TG cell growth and had potential applications in wound healing. Biginelli (B1-B4) scaffolds showed good antioxidant properties in the DPPH assay. RT-qPCR analysis indicated that TG cells exposed to different (B1-B4) scaffold concentrations had significantly increased VEGF gene expression. The scaffolds showed no toxic effects on adsorption, distribution, metabolism, excretion, and toxicity (ADMET) analysis, and the structure was optimized using the DFT-B3LYP-6311G-(d,p) hybrid basis set. This method has wide applications in future research and provides insights into tissue engineering and biomedical applications.

To investigate the role of miR-944 in the progression of bladder cancer (BC) and explore its potential as a therapeutic target. In this study, we collected 12 pairs of BC tissues and paracancerous tissues and subcutaneously injected T24 cells into BALB/c nude mice at 1 × 10⁶/mouse to establish the BC animal model for experimental investigation. RT-qPCR and western blot were used to detect the expression of related genes and proteins, and the malignant progression of T24 cells and BC was detected by CCK-8, Transwell, scratch wound, and immunohistochemistry. This study found that miR-944 expression was low in BC clinical samples and cell lines. Overexpression of miR-944 inhibited the proliferation, migration, and invasion of BC cells and inhibited BC tumor growth in vivo. Mechanistically, overexpression of miR-944 downregulated ATIC by inhibiting SHMT1, thereby activating the AKT/FOXO3A signaling pathway and promoting the expression of autophagy-related proteins LC3II/I and Beclin1. At the same time, it can inhibit the expression of epithelial-mesenchymal transition (EMT)-related proteins vimentin, fibronectin, and N-cadherin, ultimately inhibiting the proliferation, migration, and invasion of BC cells, and increasing the apoptosis level of BC cells to improve the development of BC. Our study confirmed that the upregulation of miR-944 may become a new target for the treatment of BC.

This study explores the mechanism of lncRNA TDRG1 in high glucose (HG)-induced human retinal microvascular endothelial cell (hRMEC) injury. hRMECs were cultured in HG medium, followed by the detection of cell viability, proliferation, migration, and angiogenesis using CCK-8, EdU, Transwell, and tube formation assays. LncRNA TDRG1, miR-7-5p, G3BP2, VEGFA, and CD31 expression in hRMECs was detected by RT-qPCR or western blot. After transfection with lncRNA TDRG1 siRNA or miR-7-5p inhibitor or G3BP2 pcDNA3.1, hRMEC injury induced by HG was evaluated. Dual luciferase, RIP, or RNA pull-down assays were performed to verify the binding of lncRNA TDRG1, miR-7-5p, and G3BP2. HG treatment notably elevated the expressions of lncRNA TDRG1 and G3BP2 in hRMECs but diminished the expression of miR-7-5p. Low expression of lncRNA TDRG1 restrained the proliferation, migration, and angiogenesis of hRMECs while diminishing VEGFA and CD31 expression. Mechanistically, lncRNA TDRG1 upregulated the transcription level of G3BP2 by competitively binding to miR-7-5p. Low expression of miR-7-5p or overexpression of G3BP2 weakened the inhibitory effect of lncRNA TDRG1 silencing on HG-induced hRMEC injury. In conclusion, lncRNA TDRG1 upregulates the transcription level of G3BP2 by competitively binding to miR-7-5p, thus exacerbating HG-induced hRMEC injury.

The functionality and structural integrity of the cardiovascular system are critically dependent on vascular smooth muscle cells (VSMCs). Human mesenchymal stem cells (hMSCs) have significant potential for differentiating into VSMCs, making them a valuable resource in regenerative medicine and the development of vascular grafts. This study explored the synergistic effects of micropatterned substrates and TGF-β1 on the differentiation of hMSCs into VSMCs. HMSCs were cultured on both micropatterned and flat substrates for a duration of 6 days, with some groups receiving TGF-β1 treatment, after which cell morphology and the expression of specific smooth muscle markers were evaluated through Western blotting, immunofluorescence staining, and RT-qPCR. Results indicated that hMSCs on micropatterned substrates treated with TGF-β1 exhibited significantly elevated protein levels of smooth muscle myosin heavy chain (MYH11) compared with hMSCs on flat substrates without TGF-β1 (p < 0.001). Additionally, MYH11 expression was markedly enhanced in samples cultured on micropatterned substrates with TGF-β1. Furthermore, hMSCs treated with TGF-β1 on flat substrates exhibited increased cadherin-11 mRNA expression compared with both micropatterned and flat substrates lacking TGF-β1 (p < 0.05). Interestingly, KLF4 protein levels were significantly higher in hMSCs on flat substrates without TGF-β1 compared to those cultured on micropatterned substrates with TGF-β1 treatment (p < 0.001). In conclusion, this study demonstrated that the combination of micropatterned substrates and TGF-β1 treatment preferentially enhances MYH11 expression, indicative of advanced smooth muscle cell organization, along with modulating KLF4 levels and upregulating cadherin-11 expression in hMSCs. These findings provide critical insights into the differentiation pathways of MSCs into VSMCs and may inform the design of improved vascular grafts that better replicate the properties of native blood vessels.

Type 2 diabetes mellitus (T2DM) affects over 90% of diabetic patients and is characterized by insulin resistance (IR), primarily due to impaired GLUT4 function and abnormalities in insulin signaling within adipose and skeletal muscle cells. Dysfunctional adipose tissue elevates triglyceride and fatty acid levels, worsening IR. Photobiomodulation therapy (PBMT), which employs low-power light, has emerged as a potential treatment by enhancing glucose metabolism and reducing inflammation through the activation of the PI3K/AKT signaling pathway. Key factors influencing IR include FOXO1, GFAT-2, and PTP1B, which play significant roles in insulin signaling and glucose homeostasis. In this study, 3T3-L1 preadipocytes were cultured in high glucose DMEM with FBS and antibiotics, with differentiation induced using dexamethasone and insulin, followed by laser treatment. The viability of preadipocytes and adipocytes was assessed using the MTT assay, while oil red O staining quantified lipid droplet formation. An insulin resistance model was established, and glucose levels and gene expression were analyzed through qRT-PCR. The findings indicated that PBMT did not adversely affect cell viability and significantly reduced triglyceride levels and glucose uptake in IR models. Additionally, PBMT altered gene expression related to adipogenesis, suggesting its potential in managing IR and adipocyte function. Overall, while the mechanisms of PBMT require further investigation, the therapy shows promise in alleviating insulin resistance and its associated metabolic consequences.

The self-renewal capacity of chondrocytes in osteoarthritis (OA) joints is limited, and mesenchymal stem cells (MSCs) are crucial in disease treatment. This study established an OA model from equine bone marrow-derived mesenchymal stem cells (eBMSCs). The eBMSCs were cultured and differentiated into chondrocytes to generate cartilage pellets, which were induced for 7 d with inflammatory cytokines, interleukin-1 beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) to mimic OA conditions. Treated culture medium was collected to estimate enzyme activity (MMP-2, MMP-3, and MMP-9) using zymography, and the cartilage pellets were collected to estimate both anabolic gene (COL2A1) and catabolic gene expression (MMP2, MMP3, and MMP9) using qRT-PCR. Cartilage degradation was observed when induced with IL-1β + TNF-α on cartilage pellets. IL-1β + TNF-α decreased the expression levels of COL2A1 and MMP2 genes, and enhanced their enzymatic activities, while Alcian blue-positive glycosaminoglycan in cartilage pellets induced by IL-1β + TNF-α groups decreased. These results suggested that IL-1β + TNF-α induced on cartilage pellets from eBMSCs could be used as an in vitro OA model in horses.