In Vitro Cellular & Developmental Biology. Animal最新文献

Renal ischemia‒reperfusion (I/R) injury is the main cause of acute kidney injury, and its pathological features are manifested primarily by renal tubular epithelial cell injury. The underlying mechanism involves ferroptosis of renal tubular epithelial cells. Atorvastatin (ATO) regulates ferroptosis, and this study explored its role in I/R-induced ferroptosis of renal tubular epithelial cells. We constructed a renal I/R rat model with bilateral renal pedicles using noninvasive arterial clips and placed HK-2 cells in hypoxia/reoxygenation (H/R) incubators to construct the cell model. The damage to rat kidney tissues and HK-2 cells was assessed using enzyme-linked immunosorbent assay (ELISA), hematoxylin and eosin (H&E) staining, and flow cytometry, and the presence of associated proteins was identified through western blotting. Administering ATO markedly lessened the acute kidney damage caused by I/R, decreased the levels of blood urea nitrogen (BUN) and creatinine (CRE), and prevented apoptosis in renal tubular epithelial cells. Treatment with ATO additionally suppressed the production of inflammatory cytokines (TNF-α, IL-1β, and IL-6) and markers linked to ferroptosis (Fe²⁺, ROS, MDA, ACSL4, and COX2), thereby reducing acute kidney damage associated with I/R. The expression of PGE2 in renal I/R injury is related to the degree of renal injury, and it mainly regulates ferroptosis by binding to EP4. ATO effectively inhibited the expression of PGE2 and EP4. Overall, this study revealed that ATO inhibited ferroptosis of renal tubular epithelial cells by blocking the PGE2/EP4 signaling pathway, thereby alleviating I/R-induced kidney injury.

Here, we successfully grew the SNCF (Schizothorax niger caudal fin) cell line from the caudal fin explants of S. niger, an important cold-water fish of the Himalayas. The cells were successfully grown up to 22 passages by planting explant tissues in DMEM medium supplemented with FBS. We observed optimal cell growth at a concentration of 18% FBS. We observed the steady generation of cells from explants from days 2 to 5 of seeding, and obtained a complete monolayer at days 7-10. We tested various temperatures, including 10 °C, 13 °C, 16 °C, 19 °C, 22 °C, 25 °C, and 28 °C, and found that 22 °C was the optimal temperature for cell growth. We examined the response to various doses of epidermal growth factor (EGF) and fibroblast growth factor (FGF) (0, 2, 4, 6, 8, and 10 ng/mL) on cell colony growth at an optimal temperature of 22 °C. We characterized the cell line using karyotyping at the 14th and 20th passages. The cell line showed epithelial cell-like growth by morphology, which was confirmed by immunotyping. We further used the cell line to study the impact of three pesticides (chlorpyrifos, dimethoate, and endosulfan), and a fungicide (mancozeb) and bacterial extracellular product (ECP). The DAPI stain assay and MTT assay confirmed the pesticides toxic effects on the cells, revealing disintegration of the cell nuclei by the formation of micronuclei and LC₅₀ concentrations. ECP treatment showed disruption of the monolayer within 0-36 hrs.

Rainbow trout epithelial cell lines from the gill, RTgill-W1, and gut, RTgutGC, were exposed to NH₄Cl at 18-21 °C in L15 (basal medium) with fetal bovine serum and were found to undergo cytoplasmic vacuolization and cell death, depending on NH₄Cl concentration and exposure time. Vacuolization arose within 24 h of cultures being exposed to 10-100 mM NH₄Cl, and vacuoles disappeared over 24 h after NH₄Cl-exposed cultures were returned to just L15/FBS. RTgill-W1 appeared more sensitive to vacuolization, with one indicator being the maximal proportion of vacuolated cells in a culture, which approached 100% in 50 mM NH₄Cl for 72 h. RTgill-W1 also were more sensitive to NH₄Cl-induced cell killing. For 7-d exposures, the inhibitory concentrations (IC50s) for the 50% loss of cell viability as evaluated with Alamar Blue were 30 mM NH₄Cl for RTgill-W1 and 80 mM for RTgutGC. In a wound-healing assay, RTgutGC cells in 0.1 and 1 mM NH₄Cl were able to migrate and cover a 500-μm gap in 5 d, like the control, but in 50 mM NH₄Cl healing was blocked. In 10 mM NH₄Cl, repair was slowed but by 14 d the gap was covered with cells and many of these were vacuolated. Overall, the results provide a foundation for using these two cell lines to study the physiology and toxicology of ammonia in fish.

Mycotoxins in aquatic feeds and their effects on fish are becoming more important in aquaculture, as fishmeal and fish oil in feeds are being replaced with more sustainable plant protein. Here, we investigated the potential of the mycotoxin, beauvericin (BEA), to impact the rainbow trout (RT) intestine by using cultures of the epithelial cell line, RTgutGC. BEA was dosed in different ways and exposed at temperatures ranging from 4 to 26 °C before being evaluated for cell viability by the metabolic reduction of Alamar Blue, by the accumulation of Neutral Red (lysosomal activity), cytotoxicity (CellTox Green), and for wound healing. BEA induces cell death in RTgutGC cells. The lysosomes are the main target (Neutral Red assay is the most sensitive) while cytotoxicity and plasma membrane rupture (CellTox Green) occur at considerably higher concentrations. BEA caused a dose-dependent decline in Neutral Red reading at all tested temperatures but Alamar Blue readings did not decline at 4 °C. Under these conditions, BEA appears to impair only lysosomal activity. Wound healing was reduced at 4, 10, and 26 °C compared to 18 °C. Also BEA treatment, at non-cytotoxic concentrations, reduced wound healing, but the temperature had little influence on this. Different carrier vehicles (methanol, DMSO) and exposure methods (passive or active dispersal) for BEA exposure were also studied. Here, methanol and passive dispersal gave comparable results to exposure with DMSO and active dispersal. In contrast, when DMSO was dosed with passive dispersal, immediate cytotoxicity in combination with BEA was induced.

The Warburg effect, a common feature of solid tumors, rewires the metabolism and promotes growth, survival, proliferation, and long-term maintenance in gastric cancer (GC). We performed in vitro and in vivo studies of the pathogenesis of GC to investigate the effects and mechanism of LINC01224 in this cancer. qRT-PCR was used to measure the expression of LINC01224 or miR-486-5p in GC cells, and the expression of LINC01224 in GC tissues by FISH (Fluorescence in situ hybridization) analysis was evaluated. Bioinformatics predicted the target gene of LINC01224, Western blotting was used to measure the protein expression of genes in the PI3K/AKT/mTOR/HIF-1α axis and Warburg effect in GC cells. The function of LINC01224 in GC cells was determined using measurements of EDU assay, colony formation, apoptosis, cell migration, and cell invasion. Glucose metabolism was evaluated using a glucose uptake assay and measurements of lactate. A tumor xenograft model was used to examine the effect of LINC01224 on GC growth in vivo. We found that upregulation of LINC01224 in GC cells activated the miR-486-5p/PI3K axis and promoted aerobic glycolysis, thereby increasing cell viability, proliferation, migration, invasion and anti-apoptosis. LINC01224 downregulation had the opposite effect. LINC01224 expression promoted the in vitro and in vivo pathogenesis of GC by promoting aerobic glycolysis. LINC01224 is a promising target in the treatment of GC.

Acute pancreatitis (AP) is a serious inflammatory disease with high incidence rate and mortality. It was confirmed that overactivation of autophagy in acinar cells can increase the risk of AP. Nevertheless, the regulatory mechanism of autophagy in AP is unclear. The role of ubiquitin-specific peptidase 7 (USP7) in controlling autophagy during AP development was examined in this study. AR42J cells were subjected to caerulein to establish a cell model of AP. ELISA utilized to assess IL-6, IL-1β, and TNF-α secretion levels. Cell viability and death were detected using CCK8 assay and flow cytometry, respectively. The interaction between USP7 and ATF4 was analyzed by Co-IP assay. USP7 and ATF4 were abnormally overexpressed in AP patients and cellular models. Loss of function of USP7 increased cell viability, but alleviated cell death and secretions of inflammatory cytokines including IL-6, IL-1β, and TNF-α in AP cellular models. Importantly, autophagy level was activated in AP cells, and could be repressed after USP7 knockdown, and rapamycin treatment greatly diminished the beneficial functions mediated by USP7 downregulation in AP cells. Mechanically, ATF4, an activator of stress autophagy in AP, was proved to be a deubiquitination modification target downstream of USP7, and its protein stability was weakened after USP7 reduction. ATF4 upregulation abolished the protective effect of USP7 silencing on caerulein-induced autophagy, inflammation, and cell injury in AR42J cells. USP7 knockdown reduced inflammation and cell injury during AP progression by inhibiting ATF4-mediated autophagy activation.

The objective of this study is to explore how adipose-derived stem cells (ASCs) regulate mitochondrial structure and function and the impact of this regulation on slowing cellular senescence. HFF-1 cells were induced by H₂O₂ to establish a cellular senescence model, and ASCs or Mdivi-1 (mitochondrial fission inhibitor) was added. MTT examined the cell proliferation; flow cytometry detected mitochondrial membrane potential as well as apoptosis and cell cycle; kit measured ATP production; ELISA analyzed the levels of interleukin-6 (IL-6), interleukin 1 beta (IL-1β), tumor necrosis factor alpha-like (TNF-α), glutathione (GSH), malondialdehyde (MDA), and superoxide dismutase (SOD); Western blotting and qRT-PCR detected the expression of protein and mRNA levels; and β-galactosidase staining observed the degree of cellular senescence. Compared to normal HFF-1 cells, senescent HFF-1 cells exhibited weaker proliferative capacity, marked apoptosis, and G0-G1 cell cycle arrest. These cells also showed lower mitochondrial membrane potential and ATP production, higher expression of inflammatory factors, oxidative damage, and increased levels of senescence. Treatment with Mdivi-1 or ASCs enhanced HFF-1 cell proliferation, reduced apoptosis and cell cycle arrest, increased mitochondrial membrane potential and ATP production, decreased the expression of inflammatory factors, and mitigated oxidative stress, thereby reducing the degree of cellular senescence. Concurrent intervention with Mdivi-1 and ASCs further diminishes the impacts of cellular senescence. In conclusion, ASCs regulate mitochondrial dynamics (promoting mitochondrial fusion and inhibiting mitochondrial fission), enhance ATP production, and upregulate mitochondrial membrane potential, thereby alleviating cell cycle arrest, apoptosis, inflammatory responses, and oxidative stress induced by senescence in HFF-1 cells.

Cleft lip and palate (CL/P) are prevalent congenital anomalies with complex genetic causes. The G874A mutation of T-box transcription factor 22 (TBX-22) gene is notably associated with CL/P, while the underlying mechanism remains to be clarified. Studies have shown that the restriction of epithelial-mesenchymal transformation (EMT) process in medial edge epithelial cells (MEEs) is crucial for CL/P development. In our current research, primary MEEs, isolated and cultured from mouse embryos, were genetically introduced the TBX-22 G874A mutation and subsequently treated with TGF-β1. They were then utilized to test the hypothesis that the G874A mutation in TBX22 plays a role in the regulation of the EMT in MEEs. Our findings reveal that TBX22 reduces miR140-5p transcription by binding to its promoter, while miR140-5p downregulates TGFBR1 protein expression by targeting its mRNA 3'-UTR. In other words, TBX22 could indirectly positively regulates TGFBR1 expression post-transcriptionally. Functional cellular assays showed that the G874A mutation of TBX-22 counteracted TGF-β1-induced decrease in proliferation and migration. Western blotting results showed that the G874A mutation of TBX-22 inhibited EMT protein expression (α-SMA and Vimentin) and promoted E-cadherin in TGF-β1-induced MEEs. To summarize, our research reveals that the G874A mutation of TBX22 impedes the progression of EMT in MEEs through the upregulation of miR140-5p and the downregulation of TGFBR1. This highlights TGFBR1 as a viable target for the prevention of CL/P.

The overall goal of this research was to develop an embryonic stem cell (ESC) line from the Pacific white shrimp, Litopenaeus vannamei, to support production of cell-based cultivated seafood products towards meeting a growing global demand for sustainable seafood. It was hypothesized that characteristics of ESCs, such as high proliferation and pluripotency, would facilitate development of a continuous cell line that could be triggered to differentiate into a muscle cell phenotype. The targeted approach was based on collection of ESCs from fertilized shrimp eggs at the blastomere stage. Various media, supplements, growth factors, and plate coatings were tested to achieve growth of the shrimp ESCs. Although successful in early culture, this manuscript describes substantial challenges encountered as cultures grew over time. The cell cultures were initially dominated by shrimp as indicated by 18S rDNA community analysis, but after multiple passages, thraustochytrids, a common contaminant of invertebrate cell culture, became the predominant cell type. Presence of shrimp cells was confirmed through species-specific primers for the cytochrome C oxidase subunit 1 gene. Presence of thraustochytrids was also confirmed using species-specific primers, morphological features, growth properties, and acriflavine staining. Unsuccessful attempts to eradicate thraustochytrid contamination prevented shrimp cells from thriving. The future of shrimp cell culture depends on eliminating culture contaminants while encouraging growth of shrimp ESCs.

Tilapia parvovirus (TiPV) is an emerging viral pathogen and responsible for severe economic loss in tilapia culture production. Lethargic, cutaneous haemorrhages; ocular lesions; discolouration of gill and cloudy eye and exophthalmia are common symptoms of TiPV. The TiPV-suspected tilapia fish were collected from grow-out ponds situated in different parts of Tamil Nadu, India, and screened for TiPV by PCR. The results showed the presence of TiPV in disease-suspected fish which was further confirmed by PCR using different primer sets specific to different genomic regions of TiPV. Sequence analysis of 534 bp of genomic region of TiPV showed 100% similarity with the sequence of TiPV strain of Thailand and India. TiPV was found in different organs including eggs of infected fish and showed the possibility of systemic infection and vertical transmission. Snakehead kidney (CSK), snubnose pompano fin (SPF) and tilapia heart (TH) cell lines showed susceptibility to TiPV. The viral replication in cell lines was confirmed by PCR, TiPV-specific cytopathic effect of Cowdry A inclusion bodies with clear halo surrounding them and infectivity experiment. The disease was reproduced in normal fish by intramuscular route using viral inoculum from TiPV-infected fish or virus multiplied in susceptible cell lines to satisfy Koch's postulates.