In Vitro Cellular & Developmental Biology. Animal最新文献

Bisphenol P (BPP) is a recognized endocrine disruptor with detrimental effects on human health. This study aimed to evaluate BPP's cytotoxic and genotoxic effects on Madin-Darby bovine kidney (MDBK) cells by examining changes in gene expression, genotoxicity, and cell survival. Various assays were employed, including the MTT assay, comet assay, micronucleus assay, and real-time PCR for gene expression analysis. Among the series of concentrations (0.5 µM, 1 µM, 2 µM, 4 µM, 8 µM, 16 µM, 32 µM, 64 µM, 128 µM, and 256 µM), the treatment with 32 µM BPP (LC₅₀) resulted in 50% cell viability after 24 h via MTT assay. The comet assay revealed a significant increase in comet tail length in BPP-treated groups compared to controls, indicating DNA with the highest damage at the 3xLC_50/2 dose concentration of BPP. The frequency of micronuclei (MNi) was higher than binuclei. A significantly higher level of cytokinesis-block proliferation index (CBPI) was also observed at higher doses than in the negative control group. Gene expression analysis indicated increased levels of OGG1 and HPRT1 in BPP-treated cells compared to untreated controls, with a dose-dependent elevation in OGG1 expression involved in DNA damage response. This study concluded that BPP exhibits both cytotoxic and genotoxic effects on MDBK cells. Expression of DNA repair genes (OGG1, HPRT1) served as biomarkers for genotoxicity. Furthermore, it is recommended that additional studies on BPP's molecular toxicity and its cross-species effects should be explored further to combat its harmful effects.

Probiotics can support the immune function of dairy cows and contribute to the synthesis of milk components in mammary gland tissue. Bovine lactoferrin (bLF) possesses immune-regulating and nutritional properties; however, the impact of probiotics on bLF remains unclear. This study aimed to investigate whether probiotics can enhance the synthesis and secretion of bLF in the mammary gland, with a particular focus on the specific mechanisms by which Lactiplantibacillus plantarum (L. plantarum) regulates bLF. Primary bovine mammary epithelial cells (BMECs) were cultured in six-well plates and treated with various types of probiotics. The expression of bLF was evaluated using quantitative real-time PCR (qRT-PCR), Western blot, and enzyme-linked immunosorbent assay (ELISA). The expression of transcription factors associated with the bLF promoter region, specifically, was analyzed through qRT-PCR and Western blot. Lacticaseibacillus rhamnosus (L. rhamnosus), Streptococcus thermophilus (S. thermophilus), Bifidobacterium (Bifido.), and L. plantarum upregulated bLF gene and protein expression to varying extents, with L. plantarum exhibiting the most pronounced effect. Furthermore, L. plantarum was found to regulate the expression of phosphorylated STAT3 and AP-1. These findings indicate that probiotics can influence the expression of bLF in mammary gland tissue. Additionally, L. plantarum modulates the production of bLF via the STAT3 and AP-1 transcription factor pathways.

Nucleotide-binding oligomerisation domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome activation and pyroptosis exert the pivotal influence on myocardial ischemia/reperfusion (I/R) injury. Ginsenoside Rg1 (Rg1) reportedly has multiple pharmacological actions. However, the cardioprotective potential and underlying mechanism of Rg1 in treating myocardial I/R injury in the context of pyroptosis have not been comprehensively investigated. A rat model of myocardial I/R injury was established by blocking the left anterior descending coronary artery for 30 min followed by reperfusion for 120 min. The prevention of Rg1 against I/R-caused damage and the potential mechanisms were explored. In our study, NLRP3 overexpression abolished the cardioprotective effect of Rg1, and Rg1 treatment improved myocardial function and changes in histological morphology and suppressed I/R-induced cytotoxicity as well as cardiomyocyte pyroptosis by reducing the pyroptosis-related proteins. These results indicate that Rg1 mitigated I/R-induced myocardial damage and pyroptosis by dramatically suppressing NLRP3 inflammasome activation and may provide new insights for the treatment of ischemic heart disease.

This study aimed to investigate the role of mesenchymal homeobox 2 (MEOX2) on breast cancer cell metastasis and its underlying mechanism. Overexpression of MEOX2 in human lymphatic endothelial cell (HLEC) lines was established to assess the adhesion and transendothelial migration of MCF7 and MDA-MB-231 cells to the HLEC cells. After being treated with the oxidative stress inducer H₂O₂ and the antioxidant N-acetylcysteine (NAC), cell viability, reactive oxygen species (ROS) levels, adhesion, and transendothelial migration of MCF7 and MDA-MB-231 cells to HLEC cells were detected. Tumor volume changes were observed in the xenograft model. The expression of C-X-C chemokine receptor type 4 (CXCR4), C-C chemokine receptor type 7 (CCR7), MEOX2, and G protein signal transduction regulator 5 (RGS5) in tumor tissues and ROS levels were detected. MEOX2 was lowly expressed in breast cancer tissues. Upregulated MEOX2 inhibited the proliferation of lymphatic endothelial cells and the adhesion and transendothelial migration of MCF7 and MDA-MB-231 cells to HLEC cells. After MCF7 and MDA-MB-231 cells were treated with oxidative stress inducer H₂O₂, ROS levels increased, and cell viability and MEOX2 expression decreased. After NAC or overexpressed MEOX2 treatment, MEOX2 expression increased, ROS and RGS5 levels, adhesion, and transendothelial migration ability decreased in HLEC cells. Overexpression of MEOX2 resulted in smaller tumor volume, lower ROS levels, and lower CXCR4 and CCR7 expression levels. MEOX2 and RGS5 are pivotal in regulating breast cancer metastasis, offering valuable insights into potential therapeutic strategies for breast cancer metastasis.

Mitochondrial dysfunction, often linked to the deregulation of mitochondrial biogenesis, plays a significant role in the progression of neurological diseases. Dexmedetomidine (Dex), a selective alpha-2 adrenergic agonist utilized for anesthesia and sedation, has a largely unexplored impact on mitochondrial function. In this study, cells were treated with Dex at concentrations of 10 μg/mL and 20 μg/mL. Mitochondrial function was assessed by measuring mitochondrial membrane potential, adenosine triphosphate (ATP) production, and oxygen consumption rates. The expression levels of key mitochondrial genes and proteins were analyzed using quantitative polymerase chain reaction (qPCR) and Western blot. To investigate the role of AMP-activated protein kinase α (AMPK), cells were co-treated with the AMPK inhibitor Compound C. Our results demonstrate that treating cells with Dex significantly enhances mitochondrial membrane potential, ATP production, and oxygen consumption rates. Additionally, Dex increases the expression of vital mitochondrial genes, including Mitochondrially Encoded NADH: Ubiquinone Oxidoreductase Core Subunit 6 (mtND6), Mitochondrially Encoded Cytochrome c Oxidase II (mtCO2), and Mitochondrially Encoded ATP Synthase 6 (mtATP6), while also improving the mtDNA-to-nDNA ratio. The treatment raises Messenger Ribonucleic Acid (mRNA) and protein levels of essential mitochondrial biogenesis regulators such as Nuclear Respiratory Factor 1(Nrf1), Mitochondrial Transcription Factor A (TFAM), Peroxisome Proliferator-Activated Receptor Gamma Coactivator-1α (PGC-1α), and phosphorylated AMP-Activated Protein Kinase α (p-AMPKα). However, when cells are co-treated with the AMPK inhibitor compound C, these positive effects are lost, highlighting the necessity of AMPK activation for the mitochondrial enhancements induced by Dex. These findings suggest a promising therapeutic potential for Dex in supporting neuronal function through mitochondrial pathways.

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.