In Vitro Cellular & Developmental Biology. Animal最新文献

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.

This study aimed to evaluate the effects of conditioned medium derived from murine skeletal muscle (SMCM) on oxidative stress and testicular morphology in vitro. Initially, Wistar rats underwent treadmill familiarization and a maximal incremental test (MIT). Animals were then submitted to a single exercise session at 60% of the maximum speed established by MIT. In Experiment 1, femoral muscles from trained animals were cultured in αMEM supplemented with 1.25 mg/mL BSA to produce SMCM. In Experiment 2, testes from sedentary rats were fragmented and cultured for 24 h in αMEM alone or αMEM added to irisin at 100 ng/mL or αMEM added to SMCM at 25, 50, 75, or 100%. HPLC confirmed the presence of irisin in SMCM. Oxidative stress analyses demonstrated catalase activity was higher in irisin and 75% of SMCM treatments, while glutathione peroxidase (GPX) activity was significantly higher in the irisin when compared to fresh control. It is important to highlight that 25% of SMCM was similar to fresh control in GPX activity and thiol content. Histological assessment revealed structural alterations in cultured testes, although overall tubular organization was preserved. These findings suggest that skeletal muscle SMCM modulates oxidative balance and testicular structure, with the 25% concentration yielding the most favorable antioxidant profile.

This study aimed to extract and isolate endometrial stromal cells from Arabian mares and investigate their growth and differentiation potential. Endometrial biopsies were obtained from three healthy 6-year-old Arabian mares using a standardized, minimally invasive protocol. The isolated cells were characterized using flow cytometry and differentiation analysis. Flow cytometry revealed mesenchymal markers CD90 (95.2%) and CD105 (97.4%) and hematopoietic markers CD34 (1.17%) and CD45 (0.339%). The cells exhibited differentiation potential into adipocytes, osteoblasts, and chondrocytes. The findings suggest that endometrial cells from Arabian mares represent a promising autologous source of MSCs, particularly suited for regenerative applications in musculoskeletal disorders.

Research shows that transplanted bone marrow mesenchymal stem cells (BMSCs) have been shown to improve functional outcomes in mice with spinal cord injury (SCI). Many experimental centers have demonstrated that systemic delivery of MSCs in mice can treat neurological diseases, but whether or how it works in acute spinal cord injury is not understood. Various methods such as Basso, Beattie, and Bresnahan (BBB) locomotor rating scale score and biological detection of inflammatory factors were used to test the changes in inflammatory factors of spinal cord injury at different time points in 24 h, 7 d, and 14 d. Twenty-four hours after injury, the functional measurement results of the injured group were significantly weakened compared with the control rats. The functional results of the BMSCs injection injured group were also significantly weakened compared with the control rats. There was no statistical difference between the injured group and the BMSCs injection group. However, the injury group had the highest mortality rate (p < 0.05). Biochemical results showed that compared with the control group, the expression of high-mobility group box 1 (HMGB1) and receptor for advanced glycation end-products (RAGE) and related inflammatory factors in the injury group and BMSCs injection group increased significantly at 7 d after the experiment in Western blot. Similarly, the expression of HMGB1 and RAGE in the injury group was also greater than that in the injection group, and there was a statistical difference in immunohistochemical assessment. Injecting BMSCs into rats with acute spinal cord injury could reduce rat mortality and improve prognostic functional measurements after SCI. BMSCs may promote spinal cord re-repair by inhibiting the HMGB1/RAGE signaling pathway after acute contusive spinal cord injury.

The skin is the largest organ of the human body, capable of protecting it from external harm. However, due to trauma, paralysis, and other external factors, skin damage can occur, and scars may form. Exosomes have regenerative functions and, as a cell-free therapy, show great potential for wound healing. In this study, we aimed to investigate whether thrombin-preconditioned umbilical cord mesenchymal stem cells (T-pre-UCMSCs) increase the production of exosomes. Different umbilical cord mesenchymal stem cell exosomes can accelerate the healing of skin. In our study, umbilical cord mesenchymal stem cells (UCMCs) were cultured in DMEM/F12 medium without fetal bovine serum (FBS) for 72 h with 200U/ml thrombin. Exosomes were isolated from the supernatant by ultracentrifugation. There are two kinds of exosomes: UCMCs culture supernatant-derived exosomes (UCMSCs-Exos) and T-pre-UCMSCs culture supernatant-derived exosomes (T-UCMSCs-Exos). The skin injury cell model was constructed by treating HaCats with a tip. Additionally, the wound healing capacity of exosomes was evaluated in vivo using a mouse skin injury model. Compared to UCMSCs-Exos, T-UCMSCs-Exos significantly promoted cell proliferation and migration of cells. In vivo experiments demonstrated that T-UCMSCs-Exos can accelerate wound closure and enhance collagen maturation, promoting angiogenesis in the vascularized wound area. These results indicate that T-UCMSCs-Exos have good potential for accelerating wound healing and minimizing scar formation. Our research indicates that thrombin pre-UCMSCs significantly increased the production of exosomes. These findings demonstrate that T-UCMSCs-Exos for skin wounds are a promising cell-free therapy that can be applied in the treatment of skin injuries.

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.

Obesity resulting from chronic overnutrition and physical inactivity promotes the development of metabolic disorders by disrupting physiological processes in metabolically active organs, including skeletal muscles. To investigate whether skeletal muscle stem cells (satellite cells, SCs) are affected by systemic metabolic stress, we established primary SC cultures from male mice fed a high-fat diet (HFD) for 8 wk, and from control mice fed a standard chow (CTL). This model allowed us to assess diet-induced obesity (DIO)-related changes in SC-specific molecular and cellular signatures. Although body weight, body fat composition, and adipose tissue-associated macrophages differed significantly between DIO and CTL ex vivo, we observed no differences in the in vitro behaviour of primary SC-derived myoblasts from either group. Parameters such as proliferation and differentiation following serum deprivation were comparable. Expression levels and distribution patterns of myogenic regulatory factors (MRF), SC-specific markers (Pax7, CD56, Itga7), and hallmarks for senescence (GLB1), autophagy (p62, LC3B), and oxidative stress (ALDH1A1, ALDH1A3) remained unchanged. Thus, potential differences in the signatures of SC-derived myoblasts after 8 wk of a high-fat diet cannot be depicted in vitro. However, future experiments should address whether prolonged and metabolically more susceptible diets will exert long-term effects on myogenesis in vitro or not. Overall, we propose that primary SC cultures are better suited for acute in vitro testing regarding the molecular and cellular plasticity in metabolic shifts as induced by pharmacological treatments or genetical modifications, rather than for modeling long-term dietary effects.