In Vitro Cellular & Developmental Biology. Animal最新文献

Podocyte injury is a pivotal factor in the advancement of diabetic nephropathy (DN). The present study aimed to delineate the influence of disrupted thyroxine signaling on podocyte apoptosis in DN mouse models. We employed bioinformatics analyses, coupled with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment assessments, to identify differentially expressed genes (DEGs) associated with thyroxine signaling in both human and murine DN datasets. Subsequently, we elucidated the function of thyroid hormone receptor α1 (THRA1) and nuclear receptor co-repressor 1 (NCOR1) on glomerular injury and podocytes apoptosis under hypothyroid and hyperglycemic conditions, respectively. Our findings highlight that hypothyroidism significantly alters glomerular gene expression profiles in DN mice leading to increased podocyte apoptosis. This effect occurs through a dual mechanism: on one hand, the upregulation of THRA1 expression induced by DN results in direct glomerular injury, which was further aggravated by hypothyroidism; on the other hand, the downregulation of NCOR1 expression thereby increases THRA1 activity levels. Our data suggests that disturbed thyroxin signals could trigger podocyte apoptosis and glomerular injury in DN mice, offering new insights into DN pathogenesis while laying groundwork for innovative therapeutic strategies.

In many visual disorders, the neural retinal cells are irreversibly damaged. Because of the low proliferation and repair capacity of these cells in humans, the visual acuity can be highly destroyed in patients, and this is while the current therapies can only partially reduce the extent of this disorder. Although retinal progenitor cells (RPCs) derived from human embryonic stem cells (hESCs) are among one of the most considered sources for the treatment of retinal degenerative disorders, their limited proliferation rate is a significant challenge to provide a sufficient number of cells for transplantation therapy. Thus, this study aims to provide an efficient approach for monitoring RPCs to optimize in vitro maintenance conditions in the future. This was designed by introducing a stable line of hESCs expressing EGFP reporter driven by the human RAX promoter (pRAX-EGFP) as a key biomarker of RPCs. Considering that the pRAX-hESC line showed significant pluripotency capacity and differentiation potential towards RPCs, this cell line can provide a valuable biological tool for optimization and introduction of more suitable in vitro culture conditions in order to ensure the long-term maintenance of the derived RPCs. This cell line might also be considered a reliable cellular source for monitoring the differentiation process towards RPCs in preclinical studies in the future.

Pterygium syndrome is a common eye disease that often leads to vision loss and even blindness. There is increasing evidence that miRNAs play a key role in the progression of pterygium, but the function of miR-381-3p in pterygium has not been studied. Therefore, this study aimed to investigate the effect of miR-381-3p on the progression of pterygium and to elucidate its potential molecular mechanisms. Human pterygium fibroblasts (HPFs) were isolated from clinical pterygium tissues. The expression of key genes and proteins was detected via RT-qPCR and western blotting. Cell proliferation was detected by CCK-8 and scratch assay, while cell invasion was examined by Transwell assay. Protein interactions were investigated by coimmunoprecipitation. First, we found that the expression level of miR-381-3p was significantly reduced in pterygium tissues. Second, we found that the overexpression of miR-381-3p in HPFs inhibited the proliferation, migration, and invasion abilities of HPFs while inducing cell apoptosis. In addition, in pterygium tissue, the expression of MCPIP1 was downregulated, and the expression of HACE1 and TRIP12 was upregulated. Importantly, MCPIP1 interference partially attenuated the positive effects of miR-381-3p overexpression described above, and miR-381-3p could target HACE1, while HACE1 could bind to TRIP12. Mechanistic studies revealed that miR-381-3p inhibited the binding of HACE1 to TRIP12 through the inhibition of HACE1 expression, thereby inhibiting the ubiquitination and degradation of MCPIP1 and improving the progression of pterygium. Our study highlights the powerful potential of miR-381-3p in improving the progression of pterygium, laying the foundation for the development of new intervention targets for related diseases.

Mosquito-borne diseases represent a growing global health crisis, exacerbated by climate change and insecticide resistance. RNA interference (RNAi), a natural mechanism of gene silencing, offers a promising, target-specific alternative for mosquito control. This review explores the potential of RNAi to disrupt critical physiological processes, such as reproduction and disease transmission, thereby reducing vector populations and competence. We examine the mechanisms of RNAi, its application in combatting insecticide resistance, and recent advancements in delivery systems, including nanobody- and chitosan-based nanoparticles, which enhance the stability and uptake of double-stranded RNA (dsRNA) molecules. However, significant challenges remain, such as optimizing field-effective delivery methods and assessing potential off-target effects on non-target organisms. Continued innovation in RNAi technology is pivotal for developing sustainable and environmentally sound vector control strategies. This review synthesizes current research, highlighting the molecular insights, practical applications, and future directions for integrating RNAi into modern public health initiatives.

Cyclic adenosine monophosphate (cAMP), an intracellular messenger, regulates granulosa cell (GC) proliferation, differentiation, and apoptosis via downstream effectors like PKA. Previous studies focused on cAMP promoting GC proliferation and its unidirectional transfer from GC to oocytes through gap junctions to sustain meiotic arrest. This study hypothesized cAMP bidirectionally interacts between GC and oocytes: partially entering oocytes to maintain meiotic arrest, while regulating GC apoptosis in a concentration-dependent manner. Immunofluorescence confirmed intracellular cAMP signaling in GC. Treatment with 0-10 μmol/L cAMP for 48 h revealed dual effects: low concentrations (2-4 μmol/L) significantly suppressed apoptosis and enhanced viability (CCK-8 assay), whereas concentrations > 4 μmol/L increased apoptosis and inhibited proliferation. This study provides new insights into the direct regulatory role of cAMP in sheep GC, emphasizes its dual role in cell survival and apoptosis, and deepens our understanding of the mechanism of follicular development in sheep.

Fish cell lines are indispensable tools for virology, biotechnology, and toxicology research. This study established a new marine fish cell line, designated EfE, from the eye tissue of the brown-marbled grouper (Epinephelus fuscoguttatus). The EfE cell line has been stably subcultured for over 70 passages in vitro for more than 300 days. It proliferated optimally in Leibovitz's L-15 medium supplemented with 15% fetal bovine serum at 28°C. Species origin was confirmed by molecular analysis of the mitochondrial CO1 gene. Chromosome analysis revealed a diploid count of 48, which is consistent with the karyotype of E. fuscoguttatus. The cell line demonstrated high transfection efficiency (25.6%) with a pEGFP-N1 plasmid, indicating its potential for genetic manipulation. In virus susceptibility tests, EfE cells were highly permissive to red-spotted grouper nervous necrosis virus (RGNNV), developing severe cytopathic effects (CPE), including extensive vacuolation, cell rounding, and detachment. Viral replication was further confirmed by semi-quantitative RT-PCR and the observation of virus particles via transmission electron microscopy (TEM). In conclusion, the novel EfE cell line provides a valuable in vitro model for virus isolation, propagation, investigation of pathogenic mechanisms, and genetic studies.

Canine distemper virus (CDV), a highly contagious member of the Morbillivirus genus in the Paramyxoviridae family, continues to threaten the health of domestic and wild carnivores worldwide. However, isolation of field strains is often limited by the low susceptibility of conventional cell lines lacking essential viral entry receptors. In this study, a recombinant Vero cell line stably expressing the canine signaling lymphocyte activation molecule (SLAM/CD150) receptor was established to improve CDV isolation efficiency. The SLAM gene was amplified from canine peripheral blood mononuclear cells (PBMCs), cloned into the pTargeT mammalian expression vector, and transfected into Vero cells. Functional expression of SLAM was validated by inoculation with a field isolate (CDV34388), which induced pronounced cytopathic effects and extensive syncytium formation in Vero-dogSLAM cells, whereas no syncytia were observed in non-transfected controls. Quantitative analyses revealed a mean 2.8-fold increase in minimum effective dilution (MED) values and an average area-under-the-curve (ΔAUC) of + 98 units in SLAM-expressing cells compared with normal Vero cells (p < 0.001), indicating markedly enhanced viral replication and cytopathic activity. These findings demonstrate that canine SLAM expression significantly improves CDV detection and isolation from field samples, establishing a robust in vitro model for future diagnostic and vaccine development studies.

The abnormal proliferation, migration, and angiogenesis of retinal microvascular endothelial cells (RMECs) are key pathological mechanisms involved in diabetic retinopathy (DR). This study aims to investigate the regulatory role of PAX interacting protein 1 (PTIP) in modulating proliferation, angiogenesis, and inflammatory responses in RMECs under high-glucose conditions. The levels of PTIP, VEGF, MDA, and SOD were measured in RMECs cultured under both normal and high-glucose conditions. A PTIP overexpression vector and a PTIP interference vector were constructed and transfected into RMECs exposed to high glucose. Cell proliferation was assessed using the CCK-8 assay, cell migration capacity was evaluated through wound healing assays, and tube formation ability was analyzed using Matrigel-based assays. Intracellular MDA and SOD levels were determined biochemically, while TNF-α and IL-6 concentrations in the culture supernatants were quantified by ELISA. The expression levels of EGR3, VEGF, MMP3, and MMP9 were detected using Western blotting and immunofluorescence techniques. The results showed that the expressions of PTIP and SOD were down-regulated in RMECs exposed to high glucose, whereas the levels of VEGF and MDA were up-regulated. Overexpression of PTIP in high-glucose-treated RMECs significantly suppressed cell proliferation, tube formation, and migration abilities. Additionally, it markedly reduced the levels of MDA, IL-6, TNF-α, EGR3, VEGF, MMP3, and MMP9 while increasing the level of SOD. Conversely, PTIP knockdown in RMECs under high-glucose conditions elicited opposite effects. Thus, overexpression of PTIP mitigated the impairment of proliferation, migration, and tube formation abilities, as well as reduced the inflammatory response induced by high glucose in RMECs.

Saikogenin F (SGF) is a metabolite of Saikosaponin A (SSA) in vivo. However, in comparison to SSA, the neuroprotective efficacy and mechanisms of SGF remain uncertain in depression. The objective of this study was to explore the neuroprotective effects and mechanisms of SGF in corticosterone (CORT)-induced PC12 cells. Initially, analyses using MTT assays and flow cytometry demonstrated that SGF enhanced cell viability, inhibited cell death, and reduced levels of reactive oxygen species, lactate dehydrogenase and mitochondrial membrane potential. Furthermore, metabolomic analysis revealed that metabolic disorders were occurring in CORT-induced PC12 cells. SGF significantly reversed alterations in 13 metabolites and influenced 5 metabolic pathways. Of the five metabolic pathways, the regulation of purine metabolism is the most significantly affected by SGF. This study subsequently examined the regulatory impact of SGF on the P2X7R-NLRP3 and cAMP-PKA signaling pathways associated with purine metabolism, aiming to elucidate its neuroprotective mechanism. Enzyme-linked immunoassays and western blot analyses indicated that SGF significantly modulated the expression of proteins involved in these two pathways. These results show for the first time that SGF protected PC12 cells from damage caused by CORT through the regulation of the P2X7R-NLRP3 and cAMP-PKA signaling pathways in this study.

This study aims to elucidate the role of mixed lineage leukemia 1 (MLL1) in regulating the invasion of fibroblast-like synoviocytes (FLSs) in rheumatoid arthritis (RA). RA-FLSs and HC-FLSs were isolated and cultured from synovial tissues of RA patients and healthy controls (HC). MLL1 knockdown was achieved in RA-FLSs using shRNA transfection. The expression of MLL1, Krüppel-like factor 7 (KLF7), and ubiquitin carboxyl-terminal hydrolase 7 (USP7) was assessed via quantitative real-time polymerase chain reaction (qRT-PCR) or Western blot. Cell proliferation and invasion were evaluated using cell counting kit-8 (CCK-8) and Transwell assays. Chromatin immunoprecipitation (ChIP) assays were conducted to determine the enrichment of MLL1 and histone H3 lysine 4 trimethylation (H3K4me3) at the KLF7 promoter and the binding of KLF7 to the USP7 promoter. A dual-luciferase assay was used to validate the transcriptional activation of USP7 by KLF7. Results demonstrated that MLL1 was significantly overexpressed in RA-FLSs, and its inhibition suppressed FLS proliferation and invasion. Mechanistically, MLL1 promoted KLF7 transcription through H3K4me3 modification, and KLF7 subsequently upregulated USP7 expression. These findings reveal a novel MLL1/KLF7/USP7 regulatory axis that facilitates RA-FLS invasion and may represent a potential therapeutic target in RA.