In Vitro Cellular & Developmental Biology. Animal最新文献

Improving the diagnosis and treatment of gastric cancer is a significant challenge worldwide. Circular RNAs (circRNAs), a recently identified class of endogenous non-coding RNAs with covalently closed-loop structures, have emerged as key regulators in tumorigenesis. CircFoxo3 has been studied in various cancer types, while its functional role in GC remains poorly understood. In this study, we found that circFoxo3 is significantly upregulated in GC tissues and cell lines compared to paired normal controls. Functional analyses demonstrated that knockdown of circFoxo3 markedly inhibited GC cell proliferation and migration, whereas overexpression of circFoxo3 produced the opposite effects. Mechanistically, circFoxo3 knockdown reduced forkhead box (Fox) transcription factors FOXO3 mRNA and protein levels. FOXO3a is involved in regulating cancer cell proliferation. Bioinformatic analysis revealed high expression of FOXO3 in GC tumor samples, a finding confirmed in both GC tissues and cell lines. A tumor xenograft model was used to examine the effect of circFoxo3 on tumor growth in vivo. The low circFoxo3 expression reduced the volume of the tumor and decreased its proliferation. Collectively, our findings identify circFoxo3 as an oncogenic factor in GC progression.

Diabetic nephropathy (DN) is a critical complication of diabetes mellitus. Icariside II, a bioactive compound from epimedium, is known for its anti-hyperglycemic properties, but its mechanism in DN remains unclear. Our study aimed to explore Icariside II's protective effects against high-glucose (HG) induced podocytes injury using an in vitro model. We assessed cell viability and proliferation using the CCK8 assay after treating cells with Icariside II. qPCR and Western blot analysis were used to measure the mRNA and protein expressions of DNMT1, α-SMA, fibronectin and collagen IV. Molecular docking studies were performed using DNMT1's 3D structure from the Protein Data Bank. DNMT1 overexpression levels were quantified via qRT-PCR and western blot. Immunofluorescence staining and ELISA assays evaluated TGF-β1, inflammatory cytokines, respectively. GSH, MDA, and intracellular Fe²⁺ were measured using biochemical assay kits and FerroOrange probes, respectively. Western blot analysis was used to measure the protein expressions of GPX4, SLC7A11, ACSL4 and TFR1. Results showed Icariside II inhibits HG induced proliferation, inflammation and extracellular matrix (ECM) accumulation in MPC-5 cells. Besides, Icariside II also reduced inflammation, ECM accumulation and ferroptosis by downregulating DNMT1. However, the intervention treatment with Ferrostatin-1 could effectively counteract this effect. Icariside II mitigated HG-induced inflammation and ECM accumulation by down-regulating DNMT1 and ferroptosis.

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.

Non-alcoholic steatohepatitis (NASH) is a progressive form of non-alcoholic fatty liver disease (NAFLD), characterized by hepatic steatosis, inflammation, and fibrosis, which may advance to cirrhosis and hepatocellular carcinoma. Treatment options remain limited, underscoring the need to elucidate its mechanisms and develop effective therapeutics. Salidroside (Sal), a primary active compound of Rhodiola rosea, has shown potential in alleviating NASH, yet its underlying mechanisms are not fully understood. This study investigates whether Sal mitigates palmitic acid (PA)-induced hepatocyte injury by regulating the leukocyte immunoglobulin-like receptor B2 (LILRB2)-mediated autophagy pathway. In vitro NASH model were established by inducing AML-12 cells with PA. Cells were divided into control, PA, and PA + Sal groups. To validate the role of LILRB2, an LILRB2 overexpression group was included. Cell proliferation, apoptosis, inflammatory factors (TNF-α, IL-1β, IL-6), and autophagy-related proteins were detected. Autophagic flux was evaluated using mCherry-GFP-LC3B transfection. PA treatment significantly suppressed proliferation, promoted apoptosis and inflammation, and inhibited autophagy, indicated by decreased LC3B-II/Beclin-1 and accumulated p62. Sal reversed these effects. Mechanistically, Sal downregulated LILRB2 expression, which was upregulated by PA. Overexpressing LILRB2 counteracted Sal's beneficial effects. These findings reveal that Sal attenuates PA-induced injury by inhibiting LILRB2, enhancing autophagy, and reducing apoptosis and inflammation, suggesting LILRB2 as a potential therapeutic target for NASH.

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.

Polycystic ovary syndrome (PCOS), a common endocrine disorder affecting over 10% of women, is characterized by hyperandrogenism and ovarian dysfunction. While linked to chronic inflammation and granulosa cell apoptosis, its molecular mechanisms remain unclear. In this study, we elucidated the novel role of Krüppel-like factor 5 (KLF5) in the pathogenesis of PCOS and its regulatory role with thioredoxin interacting protein (TXNIP). In a mouse model of PCOS induced by dehydroepiandrosterone (DHEA), KLF5 expression was significantly elevated in ovarian tissues (up-regulated 2.62-fold, P < 0.001), correlating with hyperandrogenism (testosterone: up-regulated 2.83-fold, P < 0.001) and cystic follicle formation. The proliferative capacity of testosterone-treated KGN cells was reduced to 59% after KLF5 was knocked down (P < 0.01), attenuated apoptosis by inhibiting the increase of Bax and Cleaved-caspase 3 proteins and thus attenuated inflammation by down-regulating NLRP3 and Interleukin- (IL-) 1β. Most importantly, dual luciferase assay showed that KLF5 transcriptionally activated TXNIP, resulting in a 3.04-fold enhancement of its promoter activity (P < 0.001). Meanwhile, overexpression of TXNIP reversed the silencing effect of KLF5, resulting in a significant increase in apoptosis and secretion of inflammatory factors. These results reveal a previously unrecognized KLF5/TXNIP axis driving granulosa cell (GC) dysfunction in polycystic ovary syndrome, in which KLF5 transcriptionally upregulates TXNIP to promote apoptosis and NLRP3 inflammasome activation. Our findings provide the first evidence linking KLF5 to the pathogenesis of PCOS and establish this pathway as a potential therapeutic target, bridging a significant gap in understanding the molecular basis of the disease and providing compelling evidence for clinical drug development.

Cisplatin-based chemotherapy is the first-line treatment for lung cancer. However, cisplatin resistance (CR) remains a major challenge, leading to treatment failure. A key driver of CR is enhanced DNA damage repair. Although males absent on the first (MOF) participate in DNA repair, their specific role in mediating CR remains unclear. In this study, CR models were established in PC9 and A549 lung cancer cell lines. Our results showed that high expression of Williams syndrome transcription factor (WSTF) in lung cancer cells was associated with CR. WSTF knockdown inhibited proliferation and promoted apoptosis, DNA damage, and γ-H2AX levels in CR cells. Moreover, MOF was highly expressed in lung cancer cells and regulated by WSTF acetylation. Furthermore, MOF knockdown downregulated H4K16ac levels in CR cells. MOF overexpression significantly upregulated H4K16ac levels, enhanced proliferation, and suppressed apoptosis in CS cells, concomitant with DNA damage repair and reduced γ-H2AX expression. Notably, transfection with the K46R attenuated these MOF-mediated effects in CS cells. Collectively, our study demonstrates that MOF promotes DNA damage repair and enhances CR in lung cancer cells via H4K16ac-mediated WSTF acetylation. These findings provide valuable insights for overcoming chemoresistance and improving patient outcomes.