In Vitro Cellular & Developmental Biology. Animal最新文献

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.

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.

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.

Immunoglobulin A (IgA) nephropathy (IgAN) is characterized by the deposition of IgA1 in the glomerular mesangium, which induces secondary glomerular and tubulointerstitial inflammation and subsequently leads to podocyte apoptosis and fibrosis. This condition often progresses to end-stage renal disease and lacks effective targeted treatment. Our study aimed to explore the role of M2 macrophage-mediated Ubiquitin C-terminal hydrolase L1 (UCHL1) expression in podocytes and its potential impact on the progression of IgAN. This study established an IgAN cellular model by exposing podocytes to aggregated IgA1 (aIgA1)-treated glomerular mesangial cells supernatants and assessed the impact of M2 macrophage polarization on UCHL1 expression and podocyte apoptosis. Additionally, we utilized siRNA technology and overexpression constructs to investigate the direct effects of UCHL1 modulation on podocyte apoptosis. The supernatant from aIgA1-treated glomerular mesangial cells significantly induced apoptosis in podocytes. Based on this, M2 macrophage polarization was induced using interleukin (IL)-4. The results showed that M2 macrophages (CD163⁺) effectively alleviated podocyte apoptosis by reducing the secretion of inflammatory cytokines IL-6, tumor necrosis factor (TNF)-α, and IL-1β, as well as downregulating the expression of apoptosis-related proteins. Notably, M2 macrophages (CD163⁺) inhibited the expression of UCHL1 in podocytes. Blockade of UCHL1 promoted podocyte proliferation, reduced apoptosis, and downregulated the protein expression of the fibrotic markers vascular endothelial growth factor and collagen type IV. Overexpression of UCHL1 reversed the protective effects of M2 macrophages on podocyte apoptosis. M2 macrophage (CD163⁺)-mediated UCHL1 downregulation in podocytes presents a potential therapeutic approach for IgAN by alleviating apoptosis.

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 reports cyprinid herpesvirus-2 infection in farm-reared goldfish in Tamil Nadu during surveillance between 2022 and 2024. CyHV-2 is a temperature-dependent viral pathogen that causes mortality during temperature fluctuations of 18-24°C (post monsoon). Live goldfish showing hemorrhage, skin ulcers, pale gill color, and high mortality rates were collected from aquarium shops in Kolathur, Chennai District, Tamil Nadu, India. To examine pathogenesis, PCR and RT-PCR assays were performed on disease-suspected samples using primer sets for viral infections, such as CyHV-2, CyHV-3, CEV, VHSV, and SVCV. These results confirmed that the CyHV-2 infection caused mortality. CyHV-2 was confirmed by sequence analysis of the ORF92 of CyHV-2 with the reported CyHV-2 strains worldwide. Sequence analysis results showed 97.1 to 100% similarity with the CyHV-2 sequence reported in GenBank. The snakehead kidney cells (CSK) were susceptible to CyHV-2 and replication was confirmed by virus-specific cytopathic effects, PCR, and bioassays. The CyHV-2 was injected in healthy fish through the IM route using viral inoculum derived from infected fish, and the virus was cultivated in susceptible cell lines. Prominent PCR bands indicated that CyHV-2 demonstrated tissue tropism in all the essential organs. The relative expression level of immune-related genes TLR22, HSP70, IL-1β-1, IL-1β, IFNγ-1, TGF-β, and TNF1 was examined in the kidney and spleen of CyHV-2-infected fish using RT-qPCR. During the early stage of infection (48-72 h post-infection), the expression level of TLR22, HSP70, IL-1β, IL-1β-1, IFNγ-1, and TNF1 was significantly upregulated, whereas they were downregulated at 96 h post-infection onwards. In contrast, the TGF-β gene was consistently downregulated throughout the experimental period.

The current study aimed to explore the effect of Cadmium (Cd) on nucleus pulposus derived mesenchymal stem cells (NPMSCs) and the possible mechanism of IVDD caused by Cd. In this study, cell viability assay, EdU assay, TUNEL staining, flow cytometry assay, mRNA transcriptome sequencing, quantitative real-time polymerase chain reaction (PCR) assay, immunofluorescence assay and western blot assay were used to prove that Cadmium induces apoptosis of NPMSCs. Cd impaired the proliferation of NPMSCs and promoted cell apoptosis, and this effect was time and concentration dependent. Further study also found that the expression levels of senescence-related molecules (P16, P21 and P53) in the Cd group were up-regulated and the expression levels of pro-apoptotic molecules Bax and Caspase-3 in the Cd group were significantly up-regulated, while the expression level of anti-apoptotic molecule Bcl-2 was significantly down-regulated compared with those of the Control group. The MAPK signaling pathway-related proteins were detected, and the results found that the ratios of p-P38/P38 and p-JNK/JNK in the Cd group were significantly increased, while the ratios of p-ERK/ERK was significantly less compared with the control group, and it was in a concentration-dependent relationship. Cd can inhibit the activity and proliferation of NPMSCs in a dose and time-dependent manner, and promote cell aging and apoptosis. Cd may promote the apoptosis of NPMSCs by activating MAPK signaling pathway.

Bergapten (BG), a furanocoumarin compound, has demonstrated diverse pharmacological properties, including the reduction of oxidative stress and inflammation in osteoarthritic chondrocytes. However, the mechanisms underlying BG's effects on chondrocyte differentiation and proliferation remain incompletely understood. This study aimed to elucidate BG's role in chondrocyte differentiation, proliferation, and inflammation prevention using in vitro and in vivo osteoarthritis (OA) models. An OA model was established by treating rabbit primary chondrocytes with sodium nitroprusside (SNP) to induce apoptosis and inflammation. BG treatment significantly upregulated chondrogenic differentiation markers, including type II collagen, SOX9, and aggrecan, and histological staining revealed increased proteoglycan accumulation in OA-induced cells treated with BG. Importantly, BG exhibited pronounced anti-inflammatory effects by modulating the NF-κB pathway: it increased IκBα expression and reduced cyclooxygenase-2 (COX-2) and p50 expression, thereby preventing inflammation in chondrocytes. BG also activated p38 kinase signaling, suggesting its contribution to Chondroprotective effects. Flow cytometry demonstrated that BG reduced SNP-induced apoptosis from 22 to 4%. Western blot analysis confirmed that BG downregulated apoptotic proteins p53 and BAX while upregulating the anti-apoptotic protein Bcl-2. In vivo validation using a zebrafish model showed that BG rescued SNP-induced craniofacial cartilage structural defects. These findings provide novel insights into BG's pharmacological role in promoting chondrocyte differentiation and survival, as well as preventing inflammation, by modulating key inflammatory pathways, apoptotic proteins, and chondrogenic markers. Given its clinical potential, BG may hold promise as a therapeutic agent for OA treatment.

Tissue densification, as a hallmark of development, injury, and fibrosis, alters the mechanical and structural properties of the extracellular matrix (ECM). However, its specific effects on neural cell behavior remain poorly understood. To address this, we developed a 3D in vitro microtissue model composed of collagen and Matrigel, incorporating co-cultures of N2A neuroblastoma and 3T3 fibroblast cells. By modulating fibroblast-driven contraction through scaffold-guided constraint, we generated microtissues with distinct levels of densification with Young's moduli ranging from approximately 0.5 to 1 kPa. Our results demonstrated that increased microtissue densification significantly enhanced N2A migration and aggregate formation, indicating that mechanical compaction facilitates neuronal clustering. Furthermore, higher densification promoted N2A cell proliferation, while apoptosis remained at relatively low baseline levels, suggesting that dense environments support cell expansion without affecting overall viability. Additionally, higher densification suppressed the proportion of neurite-bearing cells without affecting neurite length, implying impaired initiation of neuronal differentiation but not morphological maturation. Together, these findings reveal that ECM densification serves as a critical mechanical cue shaping multiple aspects of neural cell behavior. Compared to conventional hydrogel systems, our collagen-based microtissue model provides a more physiologically relevant in vitro platform for studying neurodevelopmental mechanobiology and guiding biomaterial design for neural tissue engineering.