• Animal Bioscience
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• v.36 no.9
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• pp.1367-1375
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• 2023

Objective: Pigment production and distribution are controlled through multiple proteins, resulting in different coat color phenotypes of sheep. Methods: The expression distribution of vimentin (VIM) and transthyretin (TTR) in white and black sheep skins was detected by liquid chromatography-electrospray ionization tandem MS (LC-ESI-MS/MS), gene ontology (GO) statistics, immunohistochemistry, Western blot, and quantitative real time polymerase chain reaction (qRT-PCR) to evaluate their role in the coat color formation of sheep. Results: LC-ESI-MS/MS results showed VIM and TTR proteins in white and black skin tissues of sheep. Meanwhile, GO functional annotation analysis suggested that VIM and TTR proteins were mainly concentrated in cellular components and biological process, respectively. Further research confirmed that VIM and TTR proteins were expressed at significantly higher levels in black sheep skins than in white sheep skins by Western blot, respectively. Immunohistochemistry notably detected VIM and TTR in hair follicle, dermal papilla, and outer root sheath of white and black sheep skins. qRT-PCR results also revealed that the expression of VIM and TTR mRNAs was higher in black sheep skins than in white sheep skins. Conclusion: The expression of VIM and TTR were higher in black sheep skins than in white sheep skins and the transcription and translation were unanimous in this study. VIM and TTR proteins were expressed in hair follicles of white and black sheep skins. These results suggested that VIM and TTR were involved in the coat color formation of sheep.

• Animal Bioscience
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• v.36 no.9
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• pp.1403-1413
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• 2023

Objective: Intestinal alkaline phosphatase (IAP) maintains intestinal homeostasis by detoxifying bacterial endotoxins and regulating gut microbiota, and lipid absorption. Antibiotics administered to animals can cause gut dysbiosis and barrier disruption affecting animal health. Therefore, the present study sought to investigate the role of IAP in the intestinal environment in dysbiosis. Methods: Young male mice aged 9 weeks were administered a high dose of antibiotics to induce dysbiosis. They were then sacrificed after 4 weeks to collect the serum and intestinal organs. The IAP activity in the ileum and the level of cytokines in the serum samples were measured. Quantitative real-time polymerase chain reaction analysis of RNA from the intestinal samples was performed using primers for tight junction proteins (TJPs) and proinflammatory cytokines. The relative intensity of IAP and toll-like receptor 4 (TLR4) in intestinal samples was evaluated by western blotting. Results: The IAP activity was significantly lower in the ileum samples of the dysbiosis-induced group compared to the control. The interleukin-1 beta, interleukin-6, and tumor necrosis factor-alpha concentrations were significantly higher in the ileum samples of the dysbiosis-induced group. The RNA expression levels of TJP2, claudin-3, and claudin-11 showed significantly lower values in the intestinal samples from the dysbiosis-induced mice. Results from western blotting revealed that the intensity of IAP expression was significantly lower in the ileum samples of the dysbiosis-induced group, while the intensity of TLR4 expression was significantly higher compared to that of the control group without dysbiosis. Conclusion: The IAP activity and relative mRNA expression of the TJPs decreased, while the levels of proinflammatory cytokines increased, which can affect intestinal integrity and the function of the intestinal epithelial cells. This suggests that IAP is involved in mediating the intestinal environment in dysbiosis induced by antibiotics and is an enzyme that can potentially be used to maintain the intestinal environment in animal health care.

• The Korean Journal of Physiology and Pharmacology
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• v.27 no.4
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• pp.375-381
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• 2023

Numerous studies have revealed the importance of tumor-derived exosomes in rectal cancer (RC). This study aims to explore the influence of tumor-derived exosomal integrin beta-1 (ITGB1) on lung fibroblasts in RC along with underlying mechanisms. Exosome morphology was observed using a transmission electron microscope. Protein levels of CD63, CD9, ITGB1, p-p65 and p65 were detected using Western blot. To determine ITGB1's mRNA expression, quantitative real-time polymerase chain reaction was used. Moreover, levels of interleukin (IL)-8, IL-1β, and IL-6 in cell culture supernatant were measured via commercial ELISA kits. ITGB1 expression was increased in exosomes from RC cells. The ratio of p-p65/p65 as well as levels of interleukins in lung fibroblasts was raised by exosomes derived from RC cells, while was reduced after down-regulation of exosomal ITGB1. The increased ratio of p-p65/p65 as well as levels of pro-inflammatory cytokines caused by exosomes from RC cells was reversed by the addition of nuclear factor kappa B (NF-κB) inhibitor. We concluded that the knockdown of RC cells-derived exosomal ITGB1 repressed activation of lung fibroblasts and the NF-κB pathway in vitro.

• Animal Bioscience
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• v.36 no.11
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• pp.1693-1699
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• 2023

Objective: Cows that are nursing get around 80% of their glucose from liver gluconeogenesis. Propionate, a significant precursor of liver gluconeogenesis, can regulate the key genes involved in hepatic gluconeogenesis expression, but its precise effects on the activity of enzymes have not yet been fully elucidated. Therefore, the aim of this study was to investigate the effects of propionate on the activity, gene expression, and protein abundance of the key enzymes involved in the gluconeogenesis of dairy cow hepatocytes. Methods: The hepatocytes were cultured and treated with various concentrations of sodium propionate (0, 1.25, 2.50, 3.75, and 5.00 mM) for 12 h. Glucose content in the culture media was determined by an enzymatic coloring method. The activities of gluconeogenesis related enzymes were determined by enzyme linked immunosorbent assay kits, and the levels of gene expression and protein abundance of the enzymes were detected by real-time quantitative polymerase chain reaction and Western blot, respectively. Results: Propionate supplementation considerably increased the amount of glucose in the culture medium compared to the control (p<0.05); while there was no discernible difference among the various treatment concentrations (p>0.05). The activities of cytoplasmic phosphoenolpyruvate carboxylase (PEPCK1), mitochondrial phosphoenolpyruvate carboxylase (PEPCK2), pyruvate carboxylase (PC), and glucose-6-phosphatase (G6PC) were increased with the addition of 2.50 and 3.75 mM propionate; the gene expressions and protein abundances of PEPCK1, PEPCK2, PC, and G6PC were increased by 3.75 mM propionate addition. Conclusion: Propionate encouraged glucose synthesis in bovine hepatocytes, and 3.75 mM propionate directly increased the activities, gene expressions and protein abundances of PC, PEPCK1, PEPCK2, and G6PC in bovine hepatocytes, providing a theoretical basis of propionate-regulating gluconeogenesis in bovine hepatocytes.

• Parasites, Hosts and Diseases
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• v.61 no.4
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• pp.418-427
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• 2023

Toxoplasma gondii infections are primarily diagnosed by serological assays, whereas molecular and fluorescence-based techniques are garnering attention for their high sensitivity in detecting these infections. Nevertheless, each detection method has its limitations. The toxoplasmosis detection capabilities of most of the currently available methods have not been evaluated under identical experimental conditions. This study aimed to assess the diagnostic potential of enzyme-linked immunosorbent assay (ELISA), real-time polymerase chain reaction (RT-PCR), immunohistochemistry (IHC), and immunofluorescence (IF) in BALB/c mice experimentally infected with various doses of T. gondii ME49. The detection of toxoplasmosis from sera and brain tissues was markedly enhanced in mice subjected to high infection doses (200 and 300 cysts) compared to those subjected to lower doses (10 and 50 cysts) for all the detection methods. Additionally, increased B1 gene expression levels and cyst sizes were observed in the brain tissues of the mice. Importantly, IHC, IF, and ELISA, but not RT-PCR, successfully detected T. gondii infections at the lowest infection dose (10 cysts) in the brain. These findings may prove beneficial while designing experimental methodologies for detecting T. gondii infections in mice.

• Journal of Veterinary Science
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• v.24 no.5
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• pp.55.1-55.12
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• 2023

Background: Peste des petits ruminants (PPR), caused by the PPR virus (PPRV), is an acute and fatal contagious disease that mainly infects goats, sheep, and other artiodactyls. Peripheral blood mononuclear cells (PBMCs) are considered the primary innate immune cells. Objectives: PBMCs derived from goats were infected with PPRV and analyzed to detect the relationship between PPRV replication and apoptosis or the inflammatory response. Methods: Quantitative real-time polymerase chain reaction was used to identify PPRV replication and cytokines expression. Flow cytometry was conducted to detect apoptosis and the differentiation of CD4⁺ and CD8⁺ T cells after PPRV infection. Results: PPRV stimulated the differentiation of CD4⁺ and CD8⁺ T cells. In addition, PPRV induced apoptosis in goat PBMCs. Furthermore, apoptosis and the inflammatory response induced by PPRV could be suppressed by Z-VAD-FMK and Z-YVAD-FMK, respectively. Moreover, the virus titer of PPRV was attenuated by inhibiting caspase-1-dependent apoptosis and inflammation. Conclusions: This study showed that apoptosis and the inflammatory response play an essential role in PPR viral replication in vitro, providing a new mechanism related to the cell host response.

• Journal of Veterinary Science
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• v.24 no.5
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• pp.69.1-69.11
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• 2023

Background: Kalkitoxin (KT) is an active lipopeptide isolated from the cyanobacterium Lyngbya majuscula found in the bed of the coral reef. Although KT suppresses cell division and inflammation, KT's mechanism of action in vascular smooth muscle cells (VSMCs) is unidentified. Therefore, our main aim was to investigate the impact of KT on vascular calcification for the treatment of cardiovascular disease. Objectives: Using diverse calcification media, we studied the effect of KT on VSMC calcification and the underlying mechanism of this effect. Methods: VSMC was isolated from the 6 weeks ICR mice. Then VSMCs were treated with different concentrations of KT to check the cell viability. Alizarin red and von Kossa staining were carried out to examine the calcium deposition on VSMC. Thoracic aorta of 6 weeks mice were taken and treated with different concentrations of KT, and H and E staining was performed. Real-time polymerase chain reaction and western blot were performed to examine KT's effect on VSMC mineralization. Calcium deposition on VSMC was examined with a calcium deposition quantification kit. Results: Calcium deposition, Alizarin red, and von Kossa staining revealed that KT reduced inorganic phosphate-induced calcification phenotypes. KT also reduced Ca⁺⁺-induced calcification by inhibiting genes that regulate osteoblast differentiation, such as runtrelated transcription factor 2 (RUNX-2), SMAD family member 4, osterix, collagen 1α, and osteopontin. Also, KT repressed Ca²⁺-induced bone morphogenetic protein 2, RUNX-2, collagen 1α, osteoprotegerin, and smooth muscle actin protein expression. Likewise, Alizarin red and von Kossa staining showed that KT markedly decreased the calcification of ex vivo ring formation in the mouse thoracic aorta. Conclusions: This experiment demonstrated that KT decreases vascular calcification and may be developed as a new therapeutic treatment for vascular calcification and arteriosclerosis.

• Animal Bioscience
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• v.36 no.12
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• pp.1796-1805
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• 2023

Objective: This study aims to identify heat shock protein70-2 (HSP70-2) and protamine-1 (PRM1) mRNA and protein in Madura bull sperm and demonstrate their relation as bull fertility biomarkers. Methods: The Madura bull fertility rates were grouped based on the percentage of first service conception rate (%FSCR) as high fertility (HF) (79.04%; n = 4), and low fertility (LF) (65.84%; n = 4). mRNA of HSP70-2 and PRM1 with peptidylprolyl isomerase A (PPIA) as a housekeeping gene were determined by quantitative real-time polymerase chain reaction, while enzyme-linked immunoassay was used to measure protein abundance. In the post-thawed semen samples, sperm motility, viability, acrosome integrity, and sperm DNA fragmentation index were analyzed. Data analysis was performed on the measured parameters of semen quality, relative mRNA expression, and protein abundance of HSP70-2 and PRM1, among the bulls with various fertility levels (HF and LF) in a one-way analysis of variance analysis. The Pearson correlation was used to analyze the relationship between semen quality, mRNA, proteins, and fertility rate. Results: Relative mRNA expression and protein abundance of HSP70-2 and PRM1 were detected and were found to be highly expressed in bulls with HF (p<0.05) and were associated with several parameters of semen quality. Conclusion: HSP70-2 and PRM1 mRNA and protein molecules have great potential to serve as molecular markers for determining bull fertility.

• Animal Bioscience
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• v.36 no.12
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• pp.1905-1917
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• 2023

Objective: Nanog homeobox (NANOG) is a core transcription factor that contributes to pluripotency along with octamer binding transcription factor-4 (OCT4) and sex determining region-Y box-2 (SOX2). It is an epiblast lineage marker in mammalian pre-implantation embryos and exhibits a species-specific expression pattern. Therefore, it is important to understand the lineage of NANOG, the trophectoderm, and the primitive endoderm in the pig embryo. Methods: A loss- and gain-of-function analysis was done to determine the role of NANOG in lineage specification in parthenogenetic porcine blastocysts. We analyzed the relationship between NANOG and pluripotent core transcription factors and other lineage makers. Results: In NANOG-null late blastocysts, OCT4-, SOX2-, and SOX17-positive cells were decreased, whereas GATA binding protein 6 (GATA6)-positive cells were increased. Quantitative real-time polymerase chain reaction revealed that the expression of SOX2 was decreased in NANOG-null blastocysts, whereas that of primitive endoderm makers, except SOX17, was increased. In NANOG-overexpressing blastocysts, caudal type homeobox 2 (CDX2-), SOX17-, and GATA6-positive cells were decreased. The results indicated that the expression of primitive endoderm markers and trophectoderm-related genes was decreased. Conclusion: Taken together, the results demonstrate that NANOG is involved in the epiblast and primitive endoderm differentiation and is essential for maintaining pluripotency within the epiblast.

• Nutrition Research and Practice
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• v.17 no.6
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• pp.1128-1142
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• 2023

BACKGROUND/OBJECTIVES: Inonotus obliquus has been used as antidiabetic herb around the world, especially in the Russian and Scandinavian countries. Diabetes is widely believed to be a key factor in Alzheimer's disease (AD), which is widely considered to be type III diabetes. To investigate whether I. obliquus can also ameliorate AD, it would be interesting to identify new clues for AD treatment. We tested the anti-AD effects of raw Inonotus obliquus polysaccharide (IOP) in a mouse model of AD (3×Tg-AD transgenic mice). MATERIALS/METHODS: SPF-grade 3×Tg-AD mice were randomly divided into three groups (Control, Metformin, and raw IOP groups, n = 5 per group). β-Amyloid deposition in the brain was analyzed using immunohistochemistry for AD characterization. Gene and protein expression of pertinent factors of the ubiquitin-proteasome system (UPS) was determined using real-time quantitative polymerase chain reaction and Western blotting. RESULTS: Raw IOP significantly reduced the accumulation of amyloid aggregates and facilitated UPS activity, resulting in a significant reduction in AD-related symptoms in an AD mouse model. The presence of raw IOP significantly enhanced the expression of ubiquitin, E1, and Parkin (E3) at both the mRNA and protein levels in the mouse hippocampus. The mRNA level of ubiquitin carboxyl-terminal hydrolase isozyme L1, a key factor involved in UPS activation, also increased by approximately 50%. CONCLUSIONS: Raw IOP could contribute to AD amelioration via the UPS pathway, which could be considered as a new potential strategy for AD treatment, although we could not exclude other mechanisms involved in counteracting AD processing.