In Vitro Cellular & Developmental Biology. Animal最新文献

Ferroptosis is a novel form of programmed cell death characterized by the accumulation of lipid peroxides and associated with neuropathic diseases. However, the molecular mechanisms remain unclear. This study aimed to investigate the potential mechanism of DDX3 in pentylenetetrazole (PTZ)-induced ferroptosis in human neuronal SH-SY5Y cells. PTZ induced SH-SY5Y cells to simulate the neuropathic disease model in vitro. Western blot analysis was used to assess DDX3, β-catenin, β-catenin phosphorylated at Ser37/Thr41, GPX4, and ACSL4 expression. Nuclear accumulation of β-catenin was tested by IF. MMP-7, c-Myc, cyclin D1, LEF1, and Axin2 were detected by qRT-PCR. Cell viability was measured by CCK-8. Apoptosis was detected by flow cytometry. Total antioxidant status (TAS) and total oxidant status (TOS) levels were detected by biochemical kit. ROS production was detected by flow cytometry. Biochemical kits were used to detect MDA, 4-HNE, Fe²⁺, and GSH levels. Our results showed that DDX3 expression was decreased in PTZ-induced SH-SY5Y cells. DDX3 overexpression promoted PTZ-induced SH-SY5Y cell viability, inhibited apoptosis, promoted TAS and GSH expression, and inhibited TOS, MDA, 4-HNE, Fe²⁺, and ROS levels, indicating that DDX3 reduced PTZ-induced SH-SY5Y cell ferroptosis. DDX3 knockdown reduced total β-catenin protein, nuclear accumulation of β-catenin, Wnt target genes (MMP-7, c-Myc, cyclin D1, LEF1, and Axin2), and GPX4 expression in PTZ-induced SH-SY5Y cells, while increasing β-catenin phosphorylated at Ser37/Thr41 and ACSL4 expression. The effect of DDX3 overexpression on the above indexes was opposite to that of DDX3 knockdown. β-catenin overexpression and Wnt/β-catenin signaling activator CHIR99021 increased total β-catenin protein, nuclear accumulation of β-catenin, MMP-7, c-Myc, cyclin D1, LEF1, and Axin2 expression in PTZ-induced SH-SY5Y cells, while decreasing β-catenin phosphorylated at Ser37/Thr41expression and ACSL4 expression. In addition, β-catenin overexpression and CHIR99021 increased cell viability, reduced apoptosis, and upregulated TAS, GSH, and GPX4 expression, while decreasing TOS, MDA, 4-HNE, Fe²⁺, ROS, and ACSL4 levels. GPX4 knockdown and ACSL4 overexpression reversed β-catenin overexpression effects. Further results showed that DDX3 inhibited PTZ-induced SH-SY5Y cell ferroptosis by activating Wnt/β-catenin signaling. Our results suggested that DDX3 inhibited PTZ-induced ferroptosis in SH-SY5Y cells through activation of Wnt/β-catenin signaling. Our findings may provide new molecular targets for the treatment of neuropathic diseases.

Diet has an important impact on intestinal homeostasis, and the establishment of appropriate experimental models to study the effect of food compounds is of interest. The organoid model can be used to check the positive protective role of active food compounds on intestinal tissue. In the current study, mouse intestinal organoids were used to model air-liquid interface (ALI), lipopolysaccharide (LPS)-induced inflammation, and macrophage co-culture-based inflammation modelling. The activity of hesperidin, capsaicin, allicin, and 18β-glycyrrhetinic acid (18β-GA) was determined in organoid culture. Morphology, crypt number, area, and intensity were analyzed. mRNA expression analysis and immunostaining analysis were performed for inflammation and proliferation markers. The ALI model exerted a suitable organoid culture system to mimic intestinal growth based on our results. Hesperidin, capsaicin, and allicin demonstrated positive effects on LPS-induced inflammation. All of the food compounds showed positive effects in macrophage co-culture for organoid structure and growth but not for macrophage proliferation and viability. All compounds reduced the inflammatory gene expression and increased stem cell marker and proliferation-related gene expression in the ALI model. In addition, capsaicin showed positive effects on organoid growth and maturation. This study generated an experimental model system to test food components and might be used in further research.

ARL4C, a member of the small GTP-binding protein superfamily, is overexpressed in the tumor lesions of various cancers. Antisense oligonucleotide (ASO) therapy targeting ARL4C (ASO-1316-A), a 15-mer ASO, has been shown to inhibit xenograft tumor formation. To enhance therapeutic efficacy and reduce off-target effects, fourteen 18-mer ARL4C-targeting ASOs were developed. Among them, ASO-2025-A/L demonstrated superior suppression of ARL4C mRNA and protein expression compared to ASO-1316-A. In vitro, ASO-2025-A/L inhibited the proliferation, migration, and adhesion abilities of PANC-1 and S2-CP8 pancreatic cancer cells as well as PC-9 lung cancer cells more effectively than ASO-1316-A. ASO-2025-A/L was predicted to reduce the expression of 26 potential off-target genes, compared to 2824 potential off-target genes affected by ASO-1316-A in PANC-1 cells. While ASO-2025-A/L downregulated 317 genes, which were confirmed by RNA-sequence analysis, the majority was involved in cell adhesion pathways downstream of ARL4C signaling, and only two genes corresponded to off-target genes. In vivo, intravenous administration of ASO-2025-A/L inhibited xenograft tumor growth induced by PC-9 and PANC-1 cells, accompanied by a reduction in ARL4C mRNA levels in tumors without elevating serum liver toxicity markers. These findings highlight the development of an improved 18-mer ARL4C-targeting ASO with enhanced therapeutic efficacy and reduced off-target effects.

Direct reprogramming, involving overexpression of transcription factors in combination with specific small-molecule treatments to induce cell fate conversion, represents a promising strategy for regenerative medicine and disease modeling. Monocistronic expression of four transcription factors (CRX, NEUROD, RAX, and OTX2) has been demonstrated to induce the expression of phototransduction-associated genes and confer light responsiveness in fibroblasts. In contrast, polycistronic expression of the same four factors resulted in the upregulation of only two cone-specific genes, indicating that optimization of reprogramming conditions is required to achieve complete photoreceptor differentiation. In this study, we focused on Müller glial cells, which exhibit regenerative potential in the retina of lower vertebrates, and explored their reprogramming into photoreceptor-like cells. We thus combined polycistronic expression of the aforementioned transcription factors and a pharmacological reprogramming approach utilizing a defined cocktail of small molecules. Transcriptomic analysis revealed that transcription factor introduction alone led to the upregulation of the genes related to neuronal identity and extracellular matrix components. Notably, the combination of transcription factor expression and chemical treatment induced photoreceptor-specific gene expression. However, no genes associated with phototransduction were detected, suggesting that further refinement is required to promote full differentiation into functionally mature light-responsive photoreceptors. Overall, our findings demonstrate that immortalized Müller glia can partially activate neuronal and photoreceptor genes through reprogramming, suggesting their potential for scalable drug screening and disease modeling. Clinical trial number: not applicable.

The prognosis for patients diagnosed with oral mucosal melanoma is poor, and the etiology of this disease remains to be elucidated. The underlying reason for this is that no malignant melanoma (MM) cell line derived from oral mucosal tissue has been successfully established to date. The establishment of a human MM cell line designated OS-MM from a tissue sample of a patient diagnosed with palatal mucosal melanoma was undertaken. For a period exceeding three decades, tumor cells have undergone uninterrupted proliferation, exhibiting a spindle-like morphology. Chromosomes exhibit a low-triploid pattern with an observed mode of 47. Heterotransplantation of the cells into SCID mice has resulted in the formation of tumor masses. The cells expressed vascular endothelial growth factor (VEGF) and its receptors. The addition of exogenous recombinant VEGF₁₆₅ did not alter proliferation but enhanced motility. A genetic analysis of OS-MM cells revealed the absence of BRAF and NRAS mutations; however, a GNAS mutation was identified. The OS-MM cell line should contribute to advances in personalized therapy for malignant oral mucosal melanoma.

The regeneration of the sponge Hymeniacidon heliophila was investigated using the in vitro microexplant model. This model differs from cell dissociation, surface ablation, and fragment culture models because it preserves the choanosome attached to the substrate, maintaining the apical-basal axis while entirely lacking the ectosome and the water flow polarity (ostia-osculum). The complete ectosome regenerative development was assessed using stereoscopy and scanning electron microscopy until complete adult formation. The observed stages of ectosome regeneration from the preserved choanosome align with existing data on sponge regeneration. Epithelization begins ubiquitously across the microexplant surface and expands to fully encase the microexplant. Two key processes were noticed: (1) the formation of an extracellular layer at the surface and (2) the appearance of discrete patches of pinacoderm over the extracellular matrix, probably by mesenchymal-to-epithelial transition. In more advanced regeneration stages, ectosomal spicules align transversely to the pinacoderm, with their tips protruding outward. These spicules provide structural support for the developing subdermal space while the choanosome concurrently becomes functional. Additionally, outer cells were identified in both adult microexplants and naturally growing individuals. Further efforts are needed to distinguish whether these outer cells are epibionts or autologous. As final considerations, the data were discussed from an evolutionary perspective that regeneration in sponges is an inherent feature linked to the emergence of multicellularity in animals. This process represents a primordial mechanism for stabilizing metazoan multicellular organization.

This study aims to elucidate the ameliorative effects and potential mechanisms of formononetin (FN) on airway inflammation in asthmatic rats. An ovalbumin-induced asthmatic rat model was established and treated with FN. Lung function, histopathology, apoptosis, inflammatory cytokine levels, and lipid profiles were assessed. In parallel, LPS-stimulated BEAS-2B cells were treated with FN and the cAMP/PKA pathway activator coleonol (CLE). Cell proliferation, inflammatory cytokine expression, and cAMP/PKA pathway activity were evaluated. FN significantly improved lung function, reduced airway inflammation and pathological damage, and inhibited apoptosis in asthmatic rats. It also decreased serum levels of IgE, IFN-γ, IL-4, and TNF-α, and improved lipid metabolism by lowering TC, TG, and LDL-C while increasing HDL-C. In BEAS-2B cells, FN promoted proliferation and suppressed inflammation under LPS stimulation. These effects were mediated through the inhibition of the cAMP/PKA signaling pathway, as evidenced by reduced cAMP levels and PKA phosphorylation. The protective effects of FN were partially reversed by CLE. FN ameliorates airway inflammation in asthma by modulating lipid metabolism and suppressing the cAMP/PKA signaling pathway, as demonstrated in both rat models and human bronchial epithelial cells. These findings highlight FN as a promising therapeutic candidate for asthma treatment.

Over the past five decades, numerous treatment approaches have been developed to target either the inhibition of bone resorption or the promotion of bone formation. However, therapeutic strategies for osteoporosis still face limitations and safety concerns. Bentamapimod, initially designed for neurological disorders, is a novel JNK inhibitor that has demonstrated anti-tumor and immunomodulatory properties. In this study, we aim to investigate the protective role of bentamapimod against bone loss in mice suffering from estrogen deficiency. In vivo, we investigated that bentamapimod alleviated bone loss in mice suffering from estrogen deficiency. According to the micro-CT and histomorphometry assays, bentamapimod inhibits bone resorption as well as bone formation, but the effect of bentamapimod on bone resorption is better than that of bone formation, which ultimately results in ameliorating bone loss caused by ovariectomy. Moreover, we confirmed that bentamapimod can attenuate receptor activator of nuclear factor-kB ligand (RANKL)-induced osteoclast differentiation via inactivating the JNK, while anisomycin (a JNK agonist) partially confronts this effect. Our study demonstrated that bentamapimod would be able to be used as a promising new drug strategy for osteoporosis via inhibiting osteoclast differentiation both in vitro and in vivo.

This review emphasizes the significance of insect cell lines in transcriptomic research, highlighting their role as vital tools for uncovering cellular and molecular mechanisms of insect physiology, immune responses, and adaptation to environmental stressors. Cell lines derived from tissues such as the midgut, fat body, nervous system, and reproductive organs enable researchers to examine gene expression changes in a controlled setting, making discoveries that are difficult to achieve through whole-organism studies. High-throughput sequencing and single-cell RNA sequencing (scRNA-seq) have identified genes linked to detoxification, stress response, development, and immune defense, offering valuable insights for future applications in agriculture, pest control, and biotechnology. To organize this information clearly, we have summarized key findings in a table, providing an accessible overview of each cell line's important roles in transcriptomic research. This method not only highlights the adaptability of insect cell lines in functional genomics but also underscores their usefulness as model systems in pest management, virology, and bioengineering. Through utilizing transcriptomics, insect cell lines continue to advance our understanding of insect biology and foster the development of innovative strategies for sustainable crop protection and biotechnological use.

KIF11 is a mitotic kinesin responsible for the formation and maintenance of bipolar spindles, and it has high expression in the hepatocellular carcinoma (HCC). However, the role of the KIF11 gene in the hepatitis B virus (HBV)-related HCC remains unknown. Thus, this study aims to explore the function of transcription factor HOXA4 binding to KIF11 in HBV-related HCC, with the goal of providing a novel gene therapy approach for its treatment. HBV-positive (HepG2.2.15 cells) and HBV-negative (HepG2 cells) liver cancer cells were used to investigate the expression of KIF11 and HOXA4. HepG2 cells or HepG2.2.15 cells were transfected with the pc3.1-HBx, si-KIF11, and si-HOXA4, or co-transfected with the si-HOXA4 and oe-KIF11 for subsequent analysis. The binding of HOXA4 protein to the KIF11 gene promoter was examined based on a ChIP assay. The characteristics of HepG2.2.15 cells and HepG2 cells were assessed using CCK-8 and flow cytometry. HBV transcription and replication levels were detected via Northern and Southern blotting. The secretion level of HBV antigens in the HepG2.2.15 cell supernatant was measured by ELISA. KIF11 and HOXA4 were highly expressed in the HepG2.2.15 cells. Silencing KIF11 inhibited cell viability and HBV replication and transcription, reduced HBsAg and HBeAg levels in cell supernatants, promoted apoptosis, and downregulated p-PI3K/PI3K and p-AKT/AKT protein expression in HepG2.2.15 cells. pc3.1-HBx promoted cell viability, inhibited apoptosis, and upregulated p-PI3K/PI3K and p-AKT/AKT protein expression in HepG2 cells, which was reversed by si-KIF11. ChIP assays confirmed that HOXA4 bound to the KIF11 gene promoter. Silencing HOXA4 suppressed cell viability and HBV replication and transcription, decreased HBsAg and HBeAg levels in cell supernatants, enhanced apoptosis, and downregulated p-PI3K/PI3K and p-AKT/AKT protein expression in HepG2.2.15 cells. Silencing HOXA4 inhibited cell viability, promoted apoptosis, and downregulated p-PI3K/PI3K and p-AKT/AKT protein expression in HepG2 cells with pc3.1-HBx transfection. Overexpression of KIF11 counteracted the effects of si-HOXA4 in the HepG2.2.15 cells or HepG2 cells with pc3.1-HBx transfection. In conclusion, silencing of HOXA4 inhibited HBV replication and HCC proliferation by downregulating KIF11, providing a novel gene-assisted therapeutic approach for HBV-related HCC.