Experimental cell research最新文献

Volatile anesthetics that reversibly render humans immobile and unconscious have analogous reversible effects on the motility of non-neural organisms including plants and single cells. The molecular mechanisms by which they do this remain unclear, but are difficult to study in mammals because of complex interactions among diverse tissues and cell types. Here we use freely available software to study cell swimming speeds in the single-celled ciliate species Tetrahymena pyriformis. We first extend previous findings with other volatile anesthetics, to show that isoflurane also reversibly slows swimming speed in Tetrahymena. We then show that prior exposure to the microtubule (MT) stabilizing drug epothilone B (10 nM) confers resistance to isoflurane's anesthetic effect on swimming speed. This result suggests that isoflurane slows swimming in part by interacting with MTs. This conclusion is consistent with our previous experiments supporting that binding to MTs contributes to isoflurane-induced unconsciousness in rats and mice. Our present results are thus consistent with the hypothesis that all ciliates are sensitive to volatile anesthetics, and support conserved molecular mechanisms of anesthetic action in single-celled organisms and mammals. Moreover, our preparation enables inexpensive high-throughput testing of the functional roles played by specific candidate molecular targets of volatile anesthetics in ciliates.

Background: Tendon-bone healing refers to the repair process at the tendon-bone interface following injury, during which the osteogenic differentiation of tendon stem cells (TSCs) plays a critical role. The present research aims to elucidate the role of ADAM8 in regulating the osteogenic differentiation of rat TSCs.

Methods: Rat TSCs were isolated and cultured. ADAM8 overexpression plasmids were constructed to transfect rat TSCs. Cell viability, apoptosis, osteogenic differentiation, and Runx2/OCN mRNA expression were assessed using the CCK-8 assay, flow cytometry, Alizarin Red staining, and quantitative real-time PCR, respectively. Protein expression of Runx2, OCN, as well as the phosphorylation levels of PI3K and Akt were detected using Western blot.

Results: ADAM8 overexpression led to inhibited TSC viability and increased apoptosis. Simultaneously, ADAM8 overexpression also inhibited the osteogenic differentiation capacity of TSCs, evidenced by reduced ALP activity, fewer mineralized nodules, and decreased the expression of the osteogenesis-related genes Runx2 and OCN. Further mechanistic studies demonstrated that ADAM8 overexpression significantly inhibited the phosphorylation levels of PI3K and AKT after osteogenic induction, but these changes were reversed by adding the PI3K agonist 740Y-P. Additionally, 740Y-P was also able to rescue the inhibitory effects of ADAM8 overexpression on TSC proliferation and osteogenic differentiation.

Conclusion: ADAM8 regulates the proliferation and osteogenic differentiation of rat TSCs through the PI3K/AKT signaling.

Background: Atherosclerotic lesions commonly develop in curved or bifurcated arteries, where blood flow exhibits characteristics of low shear stress (LSS). Subjected to LSS continually, endothelial cells (ECs) adopt a pro-atherosclerotic phenotype. Ferroptosis is a recently identified form of controlled cell demise prompted by iron-dependent buildup of cellular reactive oxygen species (ROS), which has been associated with diverse cardiovascular diseases, particularly atherosclerosis (AS). P53 is a broadly acting tumor suppressor that can be activated by diverse stimuli and mediates multiple biological outcomes, including cell cycle arrest, DNA repair, apoptosis, and ferroptosis. However, it remains unknown whether LSS promotes the development of AS by inducing P53-dependent ferroptosis in endothelial cells.

Methods: In our experiments, we induced LSS by partial ligation of the right common carotid artery in high-fat diet-fed (HFD) male ApoE^-/- mice. The application of LSS applied on human umbilical vein endothelial cells (HUVECs) in vitro was through a parallel plate flow chamber configuration.

Results: Our findings demonstrated that LSS induced endothelial ferroptosis, which in turn accelerated AS development both in vivo and in vitro. This effect was partially counteracted by both the ferroptosis inhibitor Fer-1 and endothelium-specific glutathione peroxidase 4 (GPX4) overexpression in ApoE^-/- mice. Mechanistically, LSS was found to promote ferroptosis by driving the upregulation and nuclear translocation of P53 in HUVECs, which transcriptionally repressed xCT. Conversely, silencing or inhibiting P53 mitigated LSS-induced ferroptosis. These findings were corroborated in vivo, where endothelial-specific P53 knockout or inhibition effectively suppressed atherosclerotic plaque formation in mice.

Conclusions: Our experiments suggested that LSS promotes atherosclerosis by inducing endothelial ferroptosis through the P53/xCT signaling pathway.

Neuroblastoma (NB) is a prevalent pediatric tumor, accounting for over 15% of cancer-related fatalities in children. Super-enhancers (SEs), as pivotal cis-regulatory elements known for driving oncogene expression across various tumors, may serve as an innovative strategy for deciphering NB pathogenesis. Here, we meticulously analyzed epigenomic and transcriptomic data to delineate the distinct SE landscape in NB. Our study identified a NB-specific and NB-common SE at the MAB21L2 locus. Functional analyses further underlined MAB21L2's oncogenic role in NB, linking its high expression to poor patient outcomes. MAB21L2 knockdown strikingly inhibited the growth of NB tumor cells in vitro and reduced their proliferation in vivo. Notably, through RNA-seq analysis and experimental verification, we demonstrated that MAB21L2 substantially enhanced the migratory capacity of NB cells. Collectively, these findings underscore the indispensable role of the super-enhancer-MAB21L2 axis in the pathogenesis of NB and provide mechanistic insights into NB progression.

Spinal cord injury (SCI) leads to a cascade of secondary damage responses, including inflammation, apoptosis, and oxidative stress. These processes are crucial in determining the extent of tissue damage and recovery. It is well-established that various molecular mechanisms, such as the regulation of gene expression by non-coding RNAs, contribute significantly to the pathophysiology of SCI. However, the processes behind miRNA-regulated secondary damage are not entirely understood. The SCI mouse model and the cellular model were developed to investigate the effects of miRNAs during SCI. The GEO miRNA expression profile (GSE158195) was retrieved, and the differentially expressed miRNAs were examined using bioinformatics tools. Quantitative real-time polymerase chain reaction (qRT-PCR) was employed to assess the expression levels of miRNA and programmed cell death protein 4 (PDCD4). The Basso, Beattie, and Bresnahan (BBB) scoring system was used to assess neurological function. The concentrations of inflammatory cytokines were quantified via ELISA, whereas the production of reactive oxygen species (ROS) was assessed utilizing commercial kits. Our findings revealed a significant down-regulation of miR-499-5p in the spinal cord tissue of SCI mice. According to the functional study, agomir-miR-499 treatment significantly improved locomotor recovery, reduced tissue damage and edema, and suppressed neuronal death. Agomir-miR-499 also reduced SCI-induced ROS and inflammatory responses in mice. In SCI mice and cell models, miR-499 was discovered to target programmed cell death 4 and regulated its expression at protein and mRNA levels. Furthermore, increasing PDCD4 reversed agomir-miR-499's suppressive effects on the inflammatory response, ROS, and cell death. Agomir-miR-499, meanwhile, has the ability to suppress PDCD4 expression and stimulate the PI3K/AKT signaling pathway in SCI mice. Overall, our research shows that miR-499, a potential therapeutic target for SCI, reduces ROS-induced neuronal death and inflammation through PI3K/Akt signaling in SCI mice.

While the epidermis is a stratified epithelium undergoing continuous turnover, tight junctions (TJs), which are critical barrier structures, form transiently and exclusively within specific cells of the upper stratum. The cytoplasmic-to-membrane translocation of ZO-1, a scaffold protein of TJs, accompanies the assembly of TJs. Previously, we demonstrated that a secreted subset of the nuclear protein High Mobility Group Protein B1 (HMGB1) and the type IV membrane protein epimorphin/syntaxin2 (Stx2) impede, whereas the Stx2 paralogue syntaxin3 (Stx3) promotes, the membrane translocation of ZO-1 in HaCaT keratinocytes. In this study, we observed that HMGB1-knockout (HMGB1-KO) increases membrane-localized ZO-1 in only a restricted subset of cells, accompanied by downregulation of both Stx2 and Stx3. Inducible overexpression of exogenously introduced Stx3 significantly accelerates the membrane localization of ZO-1 in most HMGB1-KO cells, accompanied by upregulation of the PRSS3 gene product mesotrypsin, another supportive element for TJ formation, indicating that nuclear HMGB1 abundance regulates TJ assembly, at least partially, through the downregulation of these syntaxins independent of its extracellular secretion. Given that HMGB1, Stx2, Stx3, and mesotrypsin are all known to be transiently extruded into the extracellular space, these observations elucidate a regulatory mechanism underlying the spatiotemporal formation of TJs by these pleiotropic proteins and provide valuable insights into potential therapeutic strategies for inflammatory skin conditions characterized by compromised barrier function.

Proliferative diabetic retinopathy (PDR) is characterized by pathological angiogenesis and endothelial dysfunction driven by hyperglycemia. Ribosome biogenesis plays a crucial role in endothelial proliferation, yet its involvement in PDR remains unexplored. This study investigates the role of NOP14, a key regulator of ribosome biogenesis, in PDR progression and its interplay with Wnt/β-catenin signaling. NOP14 expression was elevated in PDR models and HG-treated human retinal endothelial cells (HRECs). Knockdown of NOP14 ameliorated retinal damage in PDR mice, decreased angiogenesis-related proteins (CD31, VEGFA, PDGF, ANG2). In vitro, NOP14 knockdown suppressed HG-induced endothelial proliferation, DNA synthesis, mitochondrial activity, and tube formation, accompanied by reduced ribosome biogenesis and promoted cell apoptosis. While overexpression of NOP14 exhibited the opposite effect to NOP14 knockdown on HG-induced HRECs. Mechanistically, NOP14 activated Wnt/β-catenin signaling, as evidenced by increased p-GSK-3β, β-catenin and Cyclin D1 levels and Wnt/β-catenin activity. Activation of Wnt/β-catenin signaling partially rescued the effects of NOP14 knockdown on endothelial dysfunction and ribosome biogenesis. NOP14 promotes PDR progression by driving ribosome biogenesis and endothelial dysfunction through Wnt/β-catenin signaling activation. Targeting the NOP14/Wnt/β-catenin axis offers a promising therapeutic strategy for mitigating pathological angiogenesis in PDR.

Osteoblasts, specialized bone-forming cells, differentiate from mesenchymal stem cells (MSCs). In recent years, stem cell-derived osteoblasts have emerged as potential choices for the treatment of bone-related disorders. A complex network of regulatory elements, including signaling pathways, transcription factors, and non-coding RNAs (ncRNAs), orchestrates MSCs differentiation. Among the key regulators of osteoblast differentiation is Runt-related transcription factor 2 (Runx2), a master transcription factor essential for osteogenic commitment. Elucidating the molecular mechanisms that regulate Runx2 expression and function is critical for the treatment of osteoblast-related disease. Runx2 is regulated through signaling pathways and a complex, post-transcriptional competing endogenous RNA (ceRNA) network. In this network, circular RNAs (circRNAs) and long non-coding RNAs (lncRNAs) sequester microRNAs (miRNAs), thereby fine-tuning Runx2 expression. Signaling pathways can also indirectly regulate Runx2 by inducing the expression of osteo-regulatory miRNAs. This review highlights the regulatory role of Runx2 during osteoblastic differentiation. It also explores how signaling pathways, lncRNAs, circRNAs, and other factors interact with Runx2-regulatory miRNAs involved in this process.