• Asian-Australasian Journal of Animal Sciences
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• v.25 no.5
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• pp.621-628
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• 2012

The FAT-1 protein is an n-3 fatty acid desaturase, which can recognize a range of 18- and 20-carbon n-6 substrates and transform n-6 polyunsaturated fatty acids (PUFAs) into n-3 PUFAs while n-3 PUFAs have beneficial effect on human health. Fat1 gene is the coding sequence from Caenorhabditis elegans which might play an important role on lipometabolism. To reveal the function of fat1 gene in bovine fetal fibroblast cells and gain the best cell nuclear donor for transgenic bovines, the codon of fat1 sequence was optimized based on the codon usage frequency preference of bovine muscle protein, and directionally cloned into the eukaryotic expression vector pEF-GFP. After identifying by restrictive enzyme digests with AatII/XbaI and sequencing, the fusion plasmid pEF-GFP-fat1 was identified successfully. The pEF-GFP-fat1 vector was transfected into bovine fetal fibroblast cells mediated by Lipofectamine2000$^{TM}$. The positive bovine fetal fibroblast cells were selected by G418 and detected by RT-PCR. The results showed that a 1,234 bp transcription was amplified by reverse transcription PCR and the positive transgenic fat1 cell line was successfully established. Then the expression level of fat1 gene in positive cells was detected using quantitative PCR, and the catalysis efficiency was detected by gas chromatography. The results demonstrated that the catalysis efficiency of fat1 was significantly high, which can improve the total PUFAs rich in EPA, DHA and DPA. Construction and expression of pEF-GFP-fat1 vector should be helpful for further understanding the mechanism of regulation of fat1 in vitro. It could also be the first step in the production of fat1 transgenic cattle.

• Animal Bioscience
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• v.34 no.10
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• pp.1590-1599
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• 2021

Objective: This study investigates the expression patterns of toll-like receptors (TLRs) and intracellular mediators in horse muscle cells after exercise, and the relationship between TLRS expression in stressed horse muscle cells and immune cell migration toward them. Methods: The expression patterns of the TLRs (TLR2, TLR4, and TLR8) and downstream signaling pathway-related genes (myeloid differentiation primary response 88 [MYD88]; activating transcription factor 3 [ATF3]) are examined in horse tissues, and horse peripheral blood mononuclear cells (PBMCs), polymorphonuclear cells (PMNs) and muscles in response to exercise, using the quantitative reverse transcription-polymerase chain reaction (qPCR). Expressions of chemokine receptor genes, i.e., C-X-C motif chemokine receptor 2 (CXCR2) and C-C motif chemokine receptor 5 (CCR5), are studied in PBMCs and PMNs. A horse muscle cell line is developed by transfecting SV-T antigen into fetal muscle cells, followed by examination of muscle-specific genes. Horse muscle cells are treated with stressors, i.e., cortisol, hydrogen peroxide (H₂O₂), and heat, to mimic stress conditions in vitro, and the expression of TLR4 and TLR8 are examined in stressed muscle cells, in addition to migration activity of PBMCs toward stressed muscle cells. Results: The qPCR revealed that TLR4 message was expressed in cerebrum, cerebellum, thymus, lung, liver, kidney, and muscle, whereas TLR8 expressed in thymus, lung, and kidney, while TLR2 expressed in thymus, lung, and kidney. Expressions of TLRs, i.e., TLR4 and TLR8, and mediators, i.e., MYD88 and ATF3, were upregulated in muscle, PBMCs and PMNs in response to exercise. Expressions of CXCR2 and CCR5 were also upregulated in PBMCs and PMNs after exercise. In the muscle cell line, TLR4 and TLR8 expressions were upregulated when cells were treated with stressors such as cortisol, H₂O₂, and heat. Migration of PBMCs toward stressed muscle cells was increased by exercise and oxidative stresses, and combinations of these. Treatment with methylsulfonylmethane (MSM), an antioxidant on stressed muscle cells, reduced migration of PBMCs toward stressed muscle cells. Conclusion: In this study, we have successfully cultured horse skeletal muscle cells, isolated horse PBMCs, and established an in vitro system for studying stress-related gene expressions and function. Expression of TLR4, TLR8, CXCR2, and CCR5 in horse muscle cells was higher in response to stressors such as cortisol, H₂O₂, and heat, or combinations of these. In addition, migration of PBMCs toward muscle cells was increased when muscle cells were under stress, but inhibition of reactive oxygen species by MSM modulated migratory activity of PBMCs to stressed muscle cells. Further study is necessary to investigate the biological function(s) of the TLR gene family in horse muscle cells.

• Journal of Nutrition and Health
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• v.56 no.5
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• pp.455-468
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• 2023

Purpose: Abeliophyllum distichum (A.distichum) is a plant native to Korea. In this study, we investigated the mechanism of antioxidant and anti-inflammatory effects of the leaf extract of A.distichum. Methods: The antioxidant capacity of the A.distichum leaf extract was determined based on the total polyphenol content, 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay, and the ferric reducing antioxidant power (FRAP) assay. The anti-inflammatory effects of the A.distichum leaf extract were evaluated by measuring the production of nitric oxide (NO) and the expression levels of proinflammatory cytokines including tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 using the enzyme-linked immunosorbent assay (ELISA) and reverse transcription quantitative real-time PCR (RT-qPCR). In addition, the expression of heme oxygenase-1 (HO-1), nuclear transcription factor-erythroid 2 related factor (Nrf2), inducible nitric oxide synthase (iNOS), and cyclooxygenase 2 (COX-2), as well as the activation of nuclear factorkappa B (NF-ĸB) were examined using the western blot analysis. Results: The total polyphenol content of the A.distichum leaf extract was 329.89 ± 30.17 gallic acid equivalents mg/g and the DPPH and ABTS scavenging activities were 55% and 70%, respectively. Additionally, the FRAP value of the extract was 743.68 ± 116.59 mg/mL. After 12-hour treatment with the A.distichum leaf extract, there was a tendency for the Nrf2 expression to increase, and the expression of HO-1 was significantly elevated in the RAW264.7 cells. The A.distichum leaf extract treatment resulted in decreased levels of NO, TNF-α, IL-6, and IL-1β, as well as reduced expression of iNOS and COX-2, along with inhibition of NF-κB activation in lipopolysaccharide-stimulated RAW264.7 cells. Conclusion: These results suggest that the A.distichum leaf extract exerts antioxidative and anti-inflammatory effects by upregulating the expression of HO-1 and downregulating NF-κB activation.

• Animal Bioscience
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• v.36 no.9
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• pp.1336-1349
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• 2023

Objective: The study was conducted to screen differentially expressed miRNAs in sows at early pregnancy by high-throughput sequencing and explore its mechanism of action on embryo implantation. Methods: The blood serum of pregnant and non-pregnant Landrace×Yorkshire sows were collected 14 days after artificial insemination, and exosomal miRNAs were purified for high throughput miRNA sequencing. The expression patterns of 10 differentially expressed (DE) miRNAs were validated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The qRT-PCR quantified the abundance of serum exosomal miR-192 in pregnant and control sows, and the diagnostic power was assessed by receiver operating characteristic (ROC) analysis. The target genes of DE miRNAs were predicted with bioinformatics software, and the functional and pathway enrichment analysis was performed on gene ontology and the Kyoto encyclopedia of genes and genomes terms. Furthermore, a luciferase reporter system was used to identify the target relation between miR-192 and integrin alpha 4 (ITGA4), a gene influencing embryo implantation in pigs. Finally, the expression levels of miRNAs and the target gene ITGA4 were analyzed by qRT-PCR, and western blot, with the proliferation of BeWo cells detected by cell counting kit-8 (CCK-8). Results: A total of 221 known miRNAs were detected in the libraries of the pregnant and non-pregnant sows, of which 55 were up-regulated and 67 were down-regulated in the pregnant individuals compared with the non-pregnant controls. From these, the expression patterns of 10 DE miRNAs were validated. The qRT-PCR analysis further confirmed a significantly higher expression of miR-192 in the serum exosomes extracted from pregnant sows, when compared to controls. The ROC analysis revealed that miR-192 provided excellent diagnostic accuracy for pregnancy (area under the ROC curve [AUC]=0.843; p>0.001). The dual-luciferase reporter assay indicated that miR-192 directly targeted ITGA4. The protein expression of ITGA4 was reduced in cells that overexpressed miR-192. Overexpression of miR-192 resulted in the decreased proliferation of BeWo cells and regulated the expression of cell cycle-related genes. Conclusion: Serum exosomal miR-192 could serve as a potential biomarker for early pregnancy in pigs. miR-192 targeted ITGA4 gene directly, and miR-192 can regulate cellular proliferation.

• Biomedical Science Letters
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• v.8 no.3
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• pp.127-135
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• 2002

Anterior subcapsular cataract was developed by opacification with transdifferentiation and abnormal proliferation of lens epithelial cells (LECs) and pathological accumulation of extracellular matrix (ECM). After-cataract also be caused by a similar transdifferentiation of LECs remaining after surgery and the accompanying increase of ECM deposits. It is blown that prostaglandin E2 and cytokine, such as TGF-$\beta$, bFGF, and IL-1, were associated with abnormal proliferation and transdifferentiation of LECs. The aim of this study was to detect the expression of transforming growth factor-$\alpha$ (TGF-$\alpha$), transforming growth factor-$\beta_1$(TGF-$\beta_1$) and basic fibroblast growth factor (bFGF) in LECs of senile and diabetic cataract. The expressions of these growth factors in lens epithelial cells were determined. The sample for growth factor determination were collected in senile cataract patients without metabolic disorder, especially diabetes mellitus and diabetic cataract patients. The mRNA expression of growth factors was detected by semi-quantitative reverse transcription - polymerase chain reaction (RT-PCR) followed by Southern blot analysis. Statistics were analysed using Wilcoxon rank sum test. Semi-quantitative RT-PCR/southern analysis of RNA obtained from thirty surgical specimens demonstrated that the level of mRNA expression of TGF-$\alpha$, -$\beta_1$ and bFGF was increased in diabetic cataract lens tissues compared with senile cataract specimens but non-significant, bFGF and TGF-$\beta_1$ mRNA expression were detected in most patients, expression level of TGF-$\beta_1$ was most high on the basis of normal ocular concentration. Detection rate of TGF-$\alpha$ in diabetic cataract was 1.5 fold higher than in senile cataract (P=0.098). TGF-$\alpha$, TGF-$\beta_1$, and bFGF mRNA expression of LECs were detected in senile and diabetic cataract. In both patient groups, expression level of TGF-$\beta_1$, mRNA was high, so We suggest TGF-$\beta_1$ strong influence in development of senile cataract and of diabetic cataract also. TGF-$\alpha$ expression level was similar but more frequently detected in diabetic cataract than in senile cataract. In conclusion, TGF-$\alpha$ may be associated with early development of diabetic cataract.

• Development and Reproduction
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• v.15 no.3
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• pp.265-272
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• 2011

It is well known that adipose tissue or body fat has been proved as a crucial component of brain-peripheral axis which can modulate the activities of reproductive hormonal axis in female mammals including rodents and human. Concerning the male reproduction, however, the role of adipose tissue has not been thoroughly studied. The present study was carried out to elucidate the effect of a high-fat (HF) diet on the reproductive system of postpubertal male rats. The HF diet (45% energy from fat, HF group) was applied to male rats from week 8 after birth for 4 weeks. The blood glucose levels, body and tissue weights were measured. Histological studies were performed to assess the structural alterations in the reproductive tissues. To determine the transcriptional changes of reproductive hormone-related genes in hypothalamus and pituitary, total RNAs were extracted and applied to the semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). Body weights (p<0.01) and blood glucose levels (p<0.01) of HF group were significantly higher than those of control animals. Similarly, the weights of epididymis (p<0.05), prostate (p<0.01), seminal vesicle (p<0.01) in HF group were higher than control levels. The weights of testis were not changed. The weights of kidney (p<0.001) and spleen (p<0.01) were significantly higher than control levels while the adrenal and pancreas weights were not changed. There were only slight alterations in the microstructures of accessory sex organs; the shape of luminal epithelial cells in epididymis from HF group were relatively thicker and bigger than those from control animals. In the semi-quantitative RT-PCR studies, the mRNA levels of hypothalamic GnRH (p<0.05) in HF group were significantly higher than those from the control animals. The mRNA levels of kisspeptin in HF group tend to be higher than control levels, the difference was not significant. Unlike the hypothalamic GnRH expression, the mRNA levels of pituitary $LH{\beta}$ and $FSH{\beta}$ were significantly decreased in HF group (p<0.05). The present study indicated that the 4-weeks feeding HF diet during the postpubertal period can alter the hypothalamus-pituitary (H-P) neuroendocrine reproductive system These results suggest that the increased body fat and the altered leptin input might disturb the H-P reproductive hormonal activities in male rats, and the changed activities seem to be responsible for the changes of tissue weights in accessory sex organs.

• Development and Reproduction
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• v.13 no.3
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• pp.183-190
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• 2009

In mammals, puberty is a dynamic transition process from infertile immature state to fertile adult state. The neuroendocrine aspect of puberty is started with functional activation of hypothalamus-pituitary-gonadal hormone axis. The timing of puberty can be altered by many factors including hormones and/or hormone-like materials, social cues and metabolic signals. For a long time, attainment of a particular body weight or percentage of body fat has been thought as crucial determinant of puberty onset. However, the precise effect of high-fat (HF) diet on the regulation of hypothalamic GnRH neuron during prepubertal period has not been fully elucidated yet. The present study was undertaken to test the effect of a HF diet on the puberty onset and hypothalamic gene expressions in immature female rats. The HF diet (45% energy from fat, HF group) was applied to female rats from weaning to around puberty onset (postnatal days, PND 22-40). Body weight and vaginal opening (VO) were checked daily during the entire feeding period. In the second experiment, all animals were sacrificed on PND 36 to measure the weights of reproductive tissues. Histological studies were performed to assess the effect of HF diet feeding on the structural alterations in the reproductive tissues. To determine the transcriptional changes of reproductive hormone-related genes in hypothalamus, total RNAs were extracted and applied to the semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). Body weights of HF group animals tend to be higher than those of control animals between PND 22 and PND 31, and significant differences were observed PND 32, PND 34, PND 35 and PND 36 (p<0.05). Advanced VO was shown in the HF group (PND $32.8{\pm}0.37$ p<0.001) compared to the control (PND $38.25{\pm}0.25$). The weight of ovaries (p<0.01) and uteri (p<0.05) from HF group animals significantly increased when compared to those from control animals. Corpora lutea were observed in the ovaries from the HF group animals but not in control ovaries. Similarly, hypertrophy of luminal and glandular uterine epithelia was found only in the HF group animals. In the semi-quantitative RT-PCR studies, the transcriptional activities of KiSS-1 in HF group animals were significantly higher than those from the control animals (p<0.001). Likewise, the mRNA levels of GnRH (p<0.05) were significantly elevated in HF group animals. The present study indicated that the feeding HF diet during the post-weaning period activates the upstream modulators of gonadotropin such as GnRH and KiSS-1 in hypothalamus, resulting early onset of puberty in immature female rats.

• Journal of Microbiology and Biotechnology
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• v.27 no.9
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• pp.1566-1575
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• 2017

MicroRNAs (miRNAs) are abundant in bovine milk and milk derived from other livestock, and they have functional roles in infants and in the secretion process of mammary glands. However, few studies have evaluated miRNAs in dairy processes, such as during cheese making and ripening. Thus, we investigated the characteristics of milk-derived miRNAs during the manufacturing and ripening of Camembert cheese as well as the microbiota present using the quantitative reverse transcription polymer chain reaction (RT-qPCR) and 16S rRNA pyrosequencing, respectively. Pyrosequencing showed that the cheese microbiota changed dramatically during cheese processing, including during the pasteurization, starter culture, and ripening stages. Our results indicated that the RNA contents per $200mg/200{\mu}l$ of the sample increased significantly during cheese-making and ripening. The inner cheese fractions had higher RNA contents than the surfaces after 12 and 22 days of ripening in a time-dependent manner (21.9 and 13.2 times higher in the inner and surface fractions than raw milk, respectively). We performed a comparative analysis of the miRNAs in each fraction by RT-qPCR. Large amounts of miRNAs (miR-93, miR-106a, miR-130, miR-155, miR-181a, and miR-223) correlated with immune responses and mammary glands were present in aged cheese, with the exception of miR-223, which was not present on the surface. Considerable amounts of miRNAs were also detected in whey, which is usually disposed of during the cheese-making process. Unexpectedly, there were no significant correlations between immune-related miRNAs and the microbial populations during cheese processing. Taken together, these results show that various functional miRNAs are present in cheese during its manufacture and that they are dramatically increased in amount in ripened Camembert cheese, with differences according to depth.

• Journal of The Korean Society of Integrative Medicine
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• v.9 no.2
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• pp.83-92
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• 2021

Purpose : Keratinocytes are the main cellular components involved in wound healing during re-epithelization and inflammation. Dysfunction of tight junction (TJ) adhesions is a major feature in the pathogenesis of various diseases. The purpose of this study was to identify the various effects of a Sparassis crispa water extract (SC) on HaCaT cells and to investigate whether these effects might be applicable to human skin. Methods : We investigated the effectiveness of SC on cell HaCaT viability using MTS. The antioxidant effect of SC was analyzed by comparing the effectiveness of ABTS to that of the well-known antioxidant resveratrol. Reverse-transcription quantitative polymerase chain reaction (qRT-PCR) is the most widely applied method Quantitative RT-PCR analysis has shown that SC in HaCaT cells affects mRNA expression of tight-junction genes associated with skin moisturization. In addition, Wound healing is one of the most complex processes in the human body. It involves the spatial and temporal synchronization of a variety of cell types with distinct roles in the phases of hemostasis, inflammation, growth, re-epithelialization, and remodeling. wound healing analysis demonstrated altered cell migration in SC-treated HaCaT cells. Results : MTS analysis in HaCaT cells was found to be more cytotoxic in SC at a concentration of 0.5 mg/㎖. Compared to 100 µM resveratrol, 4 mg/㎖ SC exhibited similar or superior antioxidant effects. SC treatment in HaCaT cells reduced levels of claudin 1, claudin 3, claudin 4, claudin 6, claudin 7, claudin 8, ZO-1, ZO-2, JAM-A, occludin, and Tricellulin mRNA expression by about 1.13 times. Wound healing analysis demonstrated altered cell migration in SC-treated HaCaT cells and HaCaT cell migration was also reduced to 73.2 % by SC treatment. Conclusion : SC, which acts as an antioxidant, reduces oxidative stress and prevents aging of the skin. Further research is needed to address the effects of SC on human skin given the observed alteration of mRNA expression of tight-junction genes and the decreased the cell migration of HaCaT cells.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.4
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• pp.2503-2508
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• 2013

Histone deacetylation mediated by histone deacetylases (HDACs) has been reported as one of the epigenetic mechanisms associated with tumorigenesis. The poor responsiveness of anticancer drugs found with cholangiocarcinoma (CCA) leads to short survival rate. We aimed to investigate mRNA expression of HDACs class I and II, and the effect of HDAC inhibitors, suberoylanilide hydroxamic acid (SAHA) and valproic acid (VPA), in CCA in vitro. Expression of HDACs was studied in CCA cell lines (M213, M214 and KKU-100) and an immortal cholangiocyte (MMNK1) by semi-quantitative reverse transcription-PCR. SAHA and VPA, as well as a classical chemotherapeutic drug 5 -fluorouacil (5-FU) were used in this study. Cell proliferation was determined by sulforhodamine assay. $IC_{50}$ and $IC_{20}$ were then analyzed for each agent and cell line. Moreover, synergistic potentional of VPA or SAHA in combination with 5-FU at sub toxic does ($IC_{20}$) of each agent was also evaluated. Statistic difference of HDACs expression or cell proliferation in each experimental condition was analyzed by Student's t-test. The result demonstrated that HDACs were expressed in all studied cell types. Both SAHA and VPA inhibited cell proliferation in a dose-dependent manner. Interestingly, KKU-100 which was less senstitive to classical chemotheraoeutic 5-FU was highly was sensitive to HDAC inhibitors. Simultaneous combination of subtoxic doses of HDAC inhibitors and 5-FU signiicantly inhibited cell proliferation in CCA cell lines compared to single sgent treatment($P{\leq}0.01$), while sequentially combined treatments were less effective. The present study showed inhibitory effects of HDACIs on cell proliferation in CCA cell lines, with synergistic antitumor potential demonstrated by simultaneous combination of VPA or SAHA with 5-FU, suggesting a novel alternative therapeutic strategy in effective treatment of CCA.