In Vitro Cellular & Developmental Biology. Animal最新文献

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.

SERPINA3G participates in the antiadipogenesis and insulin resistance induced by TNF-α in 3T3-F442A murine cells. Here, we show that the human orthologue SERPINA3 is expressed in human subcutaneous and visceral adipose depots of normal-weight individuals and that TNF-α and RA induced the overexpression of SERPINA3 mRNA in cultured human subcutaneous and visceral adipocytes, although only TNF-α induced the expression of serpin A3 protein. We also demonstrate that vitamin B6 abrogated the expression of the SERPINA3 gene and diminished the anti-adipogenic effects of TNF-α on mature adipocytes. Our results indicate that SERPINA3 is expressed in human adipose tissues and modulates the antiadipogenic effects of TNF-α, and suggest serpin A3 could be a promissory target in the inflammatory processes linked to obesity and other adipose dysfunctions.

Protein kinase Cβ (PRKCB) is expressed in THP-1 cells and has been found upregulated in periodontitis. Exploring the specific molecular mechanisms that promote the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) is beneficial to optimizing periodontal regeneration. THP-1 cells were induced to differentiate into macrophages, and the levels of PRKCB in macrophages with different phenotypes were examined, including PKC activity. The effect of pg-LPS induction on osteogenic differentiation of hPDLSCs was measured by measuring alkaline phosphatase, osteocalcin, osteogenic-related proteins, and mineralized nodules. Ruboxistaurin, an inhibitor of PRKCB, was used to treat M1 macrophages to examine its effect on macrophage polarization. Additionally, the cascade effect of ruboxistaurin on osteogenic differentiation was investigated by co-incubating hPDLSCs with medium from macrophages. The results indicated that PRKCB upregulation and increased PKC activity were induced in M1 macrophages upon stimulation with LPS/IFN-γ. pg-LPS resulted in decreased levels of osteogenic-related genes in hPDLSCs, accompanied by a decrease in mineralized nodules. PRKCB inhibitor reduced PKC activity, inhibited macrophage M1 polarization, and reduced M1-related inflammatory cytokine secretion. Exposure of hPDLSCs to M1 macrophage-derived conditioned medium impaired their osteogenic differentiation potentials, which was significantly attenuated by pretreatment of M1 macrophages with ruboxistaurin. Together, inhibition of PRKCB suppressed inflammatory M1 macrophage polarization, thus attenuating M1 macrophage-induced impairment in the osteogenic differentiation of hPDLSCs. These results provide a theoretical and scientific basis for optimizing the potential clinical application of hPDLSC therapy in periodontal regeneration.

Yes-associated protein (YAP), an important downstream mediator of the Hippo pathway, exhibits nuclear translocation when it is dephosphorylated. However, its interplay with other intracellular substances has not yet been clarified. Here, we explored mechano-induced YAP nuclear translocations and its interplay with importin-β (Impβ), one of the nucleocytoplasmic transport receptors, through live imaging of the spatiotemporal distribution of intracellular YAP and Impβ following mechanical stimulation, represented by the release of intercellular tension. YAP nuclear translocation was impeded by inhibition of Impβ and F-actin polymerization. Specifically, inhibiting F-actin polymerization not only impeded the nuclear import of YAP but also prevented its translocation from the near regions, adjacent to the wounded cell, toward the nucleus. These results provide a fundamental basis for further research into mechanotransduction, particularly mechano-induced YAP translocation, and may potentially be exploited as a therapeutic target for wound healing.

This study was to investigate the mechanism of miR- 34c- 5p in alleviating kidney injury in diabetic nephropathy (DN) rats by targeting ROCK1 and MAPK/ERK signaling pathway. The rat model of DN was established and fasting blood glucose, 24-h proteinuria, blood urea nitrogen, and serum creatinine were measured to quantify kidney injury. Kidney tissue was dissected for H&E and TUNEL staining. Renal injury factor KIM- 1 was measured by Western blot analysis. Retinal Muller cells (RMCs) were treated with high glucose and transfected. Cell viability was detected by CCK- 8 and apoptosis by flow cytometry. Inflammatory factors in DN rats and RMCs were analyzed by ELISA. The targeting effect of miR- 34c- 54p on ROCK1 was demonstrated by RNA pull-down and dual-luciferase reporter gene. Finally, ROCK1, p-MEK1/2, and p-ERK were assessed by Western blot. Elevating miR- 34c- 5p could inhibit DN kidney injury and high glucose-induced cell injury, and reduce inflammation in kidney tissue of DN rats and RMCs. miR- 34c- 5p targeted to regulate ROCK1 expression, and restoring ROCK1 abolished the therapeutic effect of elevating miR- 34c- 5p. Phosphorylated MEK and ERK were increased in DN rats and RMCs induced by high glucose. miR- 34c- 5p can inhibit kidney injury induced by DN by targeting ROCK1, and the MAPK/ERK pathway may represent the pathological mechanism of DN.