In Vitro Cellular & Developmental Biology. Animal最新文献

Lipocalin-2 (LCN-2) has a variety of biological functions and produces various effects on adipocytes, such as promoting cell apoptosis, inhibiting preadipocyte differentiation, and weakening insulin signaling. Tumor necrosis factor-α (TNF-α) is one of the first secreted products discovered in adipocytes, which plays an important role in the regulation of adipose metabolism. This experiment was conducted to investigate the regulatory effect of LCN-2 on TNF-α induced inflammatory response in porcine intramuscular adipocytes. Porcine intramuscular adipocytes were cultured in vitro and treated with LCN-2 overexpression or silencing plasmids. After TNF-α treatment, the expressions of LCN-2, interleukin 6 (IL-6), IL-8, and IL-1β were detected by enzyme-linked immunosorbent assay (ELISA) and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). Western blot analysis was used to detect the expression of phosphatidylinositol 3-kinase (PI3K), total protein kinase B (Akt), and phosphorylated Akt (pAkt) proteins. After the action of the PI3K/Akt pathway inhibitor LY294002, the effects of LCN-2 overexpression on IL-6, IL-8, and IL-1β were evaluated. The results showed that TNF-α induced LCN-2 expression in a dose-/time-dependent manner. Overexpression or silencing of LCN-2 had an impact on TNF-α induced IL-6, IL-8, and IL-1β in porcine intramuscular adipocytes. Overexpression of LCN-2 significantly promoted inflammatory factors IL-6, IL-8, and IL-1β secretion while silencing of LCN-2 inhibited the secretion of these inflammatory factors (P < 0.01). Overexpression of LCN-2 significantly increased the expression of pAkt protein in cells, while silencing of LCN-2 decreased pAkt protein expression (P < 0.01). After blocking the PI3K/Akt signaling pathway, compared with the control group, overexpression of LCN-2 affected IL-6, IL-8, and IL-1β secretion, but the impact was not significant (P > 0.05). This study suggests that LCN-2 regulates TNF-ɑ induced IL-6, IL-8, and IL-1β secretion in porcine intramuscular adipocytes by targeting the PI3K/Akt pathway, which provides a theoretical basis for LCN-2 regulating the inflammatory response of porcine intramuscular adipocytes.

Sperm capacitation, a prerequisite for fertilization, is regulated not only by intrinsic signaling but also by paracrine factors within the female tract. Analysis of previously published RNA-seq datasets identified the ectodysplasin-A2 receptor (EDA2R), an X-linked member of the TNF-receptor superfamily, as a candidate regulator of this process. This study was conducted to test the hypothesis that the EDA-A2/EDA2R axis is a regulator that directly regulates sperm capacitation during fertilization process. Western blotting and immunofluorescence showed that EDA2R was localized in late spermatogenic cells and in the midpiece of epididymal sperm. Incubation of mouse sperm in HTF medium containing the corresponding ligand EDA-A2 (0-1 µg/mL) resulted in a dose-dependent improvement in the amplitude of lateral head displacement and curvilinear velocities. Ligand exposure promoted the appearance of capacitation hallmarks: tyrosine phosphorylation level was elevated within 30 min and the proportion of FITC-PNA positive, acrosome-reacted cells increased at 30 and 60 min (p < 0.05). The EDA-A2 treated sperm yielded a higher cleavage rate (78.5% vs. 48.3%) and a higher blastocyst formation rate (97.6% vs. 88.4%) after in vitro fertilization. qPCR in hormonally synchronized females revealed transient ovarian and prolonged oviductal Eda-a2 upregulation surrounding ovulation, suggesting that the ligand is present at the site of sperm-oocytes fertilization. These results clarify that EDA-A2/EDA2R is a rapid physiological driver of sperm capacitation. This provides a tractable cytokine axis for optimizing assisted reproduction.

Studies in rodents have shown that systemic inflammation induced by prenatal exposure to the viral mimetic polyinosinic:polycytidylic acid (Poly I:C) triggers maternal immune activation. Cytokines released by the maternal immune system can cross the placenta and enter fetal circulation. In the fetal brain, embryonic microglia may produce additional cytokines and other inflammatory mediators in response to maternally derived cytokines. This resulting cytokine imbalance is suggested to impair neurogenesis and brain development, potentially contributing to the onset of neuropsychiatric disorders in offspring. To investigate microglial involvement in neurogenesis under pathological conditions, we used the spontaneously immortalized microglial cell line (SIM-A9), and confirmed the expression of Iba1 and CD68 via immunocytochemistry. Additionally, SIM-A9 cells expressed CX3CR1, Ki67, and isolectin. Upon Poly I:C stimulation, SIM-A9 cells released the cytokines interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α), as well as nitric oxide (NO), as determined by ELISA and Griess assay, respectively. After confirming SIM-A9 cell activation by Poly I:C, we co-cultured these cells with neural stem/progenitor cells (NSPCs) from embryonic mouse neocortex using a transwell system. We examined how chronically activated microglia influence NSPC differentiation and characterized the resulting cell phenotypes using immunocytochemistry. Our results demonstrate that SIM-A9 cells support NSPC differentiation into neurons as early as three days in culture. However, the number of neurons decreased with prolonged culture. Furthermore, Poly I:C in the NSPC culture media, as well as cytokines secreted by Poly I:C-activated SIM-A9 cells, showed a supportive effect on astrocyte differentiation.

Beef is primarily made up of skeletal muscle tissue. Therefore, the cultivation of bovine muscle stem cells (MSCs) to provide a consistent supply of muscle cells would enhance the sustainability of the cultured beef industry. Here, we report a high-yield, simple, economic, and convenient protocol for the isolation of active MSCs from bovine skeletal muscle tissue. We optimized the enzymatic tissue dissociation protocol and the composition of the medium used for differential plating (DP) to enhance the purity of active MSCs isolated from primary cells derived from the tissue. In addition, the optimal source of bovine muscle tissue for the isolation of active MSCs was determined. The yield of active MSCs was maximized by incubating round area-derived skeletal muscle tissue for 30 min in 0.2% (w/v) collagenase type II in high-glucose DMEM (HG-DMEM), followed by 1% (w/v) pronase in HG-DMEM for 5 min, and conducting DP of the enzymatically dissociated skeletal muscle tissue-derived primary cells in HG-DMEM supplemented with 10% (v/v) FBS and 5 ng/mL bFGF. In conclusion, we established a simple, convenient, and inexpensive protocol for the high-yield isolation of active MSCs from bovine skeletal muscle tissue. This protocol could overcome the technical challenges that hamper the large-scale production of bovine muscle cells, thereby enabling the commercialization of cultured beef.

Epsilon toxin produced by Clostridium perfringens type D is the third most potent clostridial toxin. It causes enterotoxemia in sheep and lambs. The clostridial vaccine has been used against clostridial disease, and its efficacy is evaluated using the serum neutralization (SN) assay as a gold standard. Researchers are concerned about replacing in vivo tests with in vitro tests. Our study aimed to evaluate the cell culture assay to measure the neutralizing antibodies against Clostridium perfringens epsilon toxin as an alternative SN assay. Madin-Darby canine kidney (MDCK) and African green monkey kidney (Vero) cell lines were used to monitor the cell line response after treatment with purified epsilon toxin through microscopic examination and 3-[4,5-imethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) staining. Antibodies were calculated in cell culture assays, and SN results were analyzed using Pearson's correlation. Based on our results, only MDCK was sensitive to the epsilon toxin. The cytopathic effect in this cell culture was rounded. The relationship between toxin concentration and cell viability showed that increasing toxin concentrations significantly decreased cell viability. Good correlation coefficients were obtained between SN and the in vitro assay (r = 0.987) (p < 0.01). The antibody titers obtained by SN were within the range of the cytotoxicity assay and had high reproducibility. Therefore, cell culture may be a suitable alternative for SN assays. Cell culture is one of the tools used in toxicity testing, resulting in consistent and reproducible results.

Breast cancer (BC) refers to a malignant neoplasm that takes place in the epithelial tissue of the breast. α-Cyperone (α-CYP) is one of the principal active components of Cyperus rotundus. However, research on the role of α-CYP in the development of BC is still lacking. This study investigates the effect and underlying mechanism of α-CYP in the progression of BC. Our findings revealed that both low-dose and high-dose α-CYP inhibited the colony formation ability of MCF-7 and BT474 cells, accompanied by the decrease in Ki67 expression and the obstruction of the cell cycle. Moreover, α-CYP treatment increased the activity of caspase-3, which leads to an increase in apoptosis. Moreover, the combination of α-CYP and cisplatin (DDP) remarkably suppressed cell viability and further facilitated apoptosis, indicating that α-CYP could enhance the sensitivity of chemotherapeutic agents in BC cells. Further, α-CYP treatment decreased TRIM24 expression through the ubiquitin-proteasome pathway. Notably, α-CYP counteracted the robust proliferation of BC cells triggered by TRIM24 overexpression. Taken together, this study confirmed that α-CYP is an effective anticancer component for BC treatment. α-CYP inhibits proliferation and induces apoptosis of BC cells via the modulation of TRIM24.

Background: Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are devastating acute pulmonary conditions with high mortality rates and limited effective treatment options. This study aimed to investigate the therapeutic potential of XBJ on ALI and its potential mechanism.

Methods: We developed an in vitro model of lipopolysaccharide (LPS)-induced ALI and evaluated the effects of XBJ pre-treatment on oxidative stress, inflammatory responses, and the polarization state of alveolar macrophages.

Results: LPS exposure significantly elevated the levels of reactive oxygen species (ROS) and oxidants 8-hydroxy-2'-deoxyguanosine (8-OHDG) and malondialdehyde (MDA) in alveolar macrophages. It also elevated the concentrations of pro-inflammatory cytokines, including tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and IL-23. XBJ and quercetin significantly mitigated the increase in these indicators. Moreover, XBJ and quercetin both downregulated the expression of key proteins in the NLRP3 inflammasome pathway in the ALI model. Similar to the ROS inhibitor N-acetylcysteine (NAC), XBJ and quercetin significantly decreased M1 polarization markers like CD86 and inducible nitric oxide synthase (iNOS), while increasing M2 polarization markers such as CD206 and arginase-1 (Arg-1). Notably, the overexpression of NLRP3 was able to reverse the inhibitory effect of XBJ on macrophage M1 polarization.

Conclusion: XBJ inhibits the M1 polarization of alveolar macrophages by targeting ROS-mediated NLRP3 inflammasome signaling, thereby reducing the inflammatory response. These results indicate that XBJ may offer a novel therapeutic strategy for ALI/ARDS by modulating macrophage polarization and inflammation.

5-Methoxytryptophan (5-MTP), a candidate biomarker for chronic kidney disease (CKD), has an undefined role in cerebrovascular pathophysiology. To investigate this, we employed a folic acid (FA)-induced CKD to simulate cerebrovascular complications in vivo. Additionally, in vitro models of cerebral ischemia and cerebrovascular endothelial cell injury were established. 5-MTP was administered to rats and cells, along with nuclear factor-κB (NF-κB) expression. The pathological characteristics of kidney and brain tissue were observed by histological staining. Cell proliferation was assessed using the Cell Counting Kit 8, while tube formation and migration were examined using tube formation and wound healing assays. Cell apoptosis was detected using both TdT-mediated dUTP-biotin nick end labeling and flow cytometry. Levels of renal injury markers, blood biomarkers of cerebrovascular disease, and inflammatory cytokines were measured using biochemical assays. Quantitative real-time PCR and Western blot were used to detect the mRNA and protein expression, respectively. Key findings revealed that FA successfully induced CKD in rats, which subsequently exacerbated cerebrovascular dysfunction. 5-MTP reduced the levels of proteinuria, N-acetyl-beta-D-glucosaminidase, nephrin, endothelin-1, von Willebrand factor, and thrombomodulin; improved the degree of renal fibrosis and structural damage to the brain tissue; and inhibited cell apoptosis in rats. In vitro, 5-MTP promoted cell proliferation, tube formation, migration, and the upregulation of B-cell lymphoma-2 and caspase-3 expression. This treatment also led to an increase in interleukin (IL)-10 levels while suppressing cell apoptosis, Bcl-2-associated X protein (Bax), and cleaved caspase-3 expression. Furthermore, it reduced the IL-6 and tumor necrosis factor-alpha levels. NF-κB overexpression reversed the effects of 5-MTP in vitro and in vivo. Our results demonstrate that 5-MTP ameliorated CKD-induced cerebrovascular injury through the NF-κB pathway, indicating its potential as an innovative and efficacious therapeutic target for CKD-induced cerebrovascular dysfunction.

Nonexudative age-related macular degeneration (dry AMD) is characterized by the progressive degeneration of retinal pigment epithelial (RPE) cells and photoreceptors, resulting in central vision loss. The disease is primarily marked by the accumulation of drusen and RPE atrophy. Given the emerging role of miR-21-5p in various ocular diseases, including diabetic retinopathy, glaucoma, pterygium, and choroidal neovascularization, we hypothesized that miR-21-5p may also impact RPE cell integrity in AMD. To test this hypothesis, we employed a rat model of dry AMD induced by sodium iodate (NaIO3) and evaluated the effects of miR-21-5p modulation via intravitreal injections of miR-21-5p agomir or antagomir. Comprehensive assessments were performed using optical coherence tomography (OCT), fundus imaging, histopathology, and biochemical markers. Our results demonstrated an upregulation of miR-21-5p in response to NaIO3 treatment. Administration of miR-21-5p agomir exacerbated RPE damage, while pretreatment with miR-21-5p antagomir mitigated these detrimental effects. Furthermore, in vitro experiments revealed that miR-21-5p regulates necroptosis in CoCl2-treated RPE cells by targeting Pellino1 (Peli1) via its 3' untranslated region, thereby inhibiting Peli1 expression. Overexpression of Peli1 effectively counteracted the necroptotic effects induced by CoCl2. These findings highlight the potential of miR-21-5p as a therapeutic target in dry AMD, expanding our understanding of miRNA-mediated regulation of RPE cells and suggesting new avenues for treatment strategies.