• Nutrition Research and Practice
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• v.16 no.5
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• pp.549-564
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• 2022

BACKGROUND/OBJECTIVES: Oxidative stress is caused by an imbalance between harmful free radicals and antioxidants. Long-term oxidative stress can lead to an "exhausted" status of antioxidant defense system triggering development of metabolic syndrome and chronic inflammation. Green perilla (Perilla frutescens) is commonly used in Asian cuisines and traditional medicine in southeast Asia. Green perilla possesses numerous beneficial effects including anti-inflammatory and antioxidant functions. To investigate the potentials of green perilla leaf extract (PE) on oxidative stress, we induced oxidative stress by high-fat diet (HFD) in aging mice. MATERIALS/METHODS: C57BL/6J male mice were fed HFD continuously for 53 weeks. Then, mice were divided into three groups for 12 weeks: a normal diet fed reference group (NDcon), high-fat diet fed group (HDcon), and high-fat diet PE treated group (HDPE, 400 mg/kg of body weight). Biochemical analyses of serum and liver tissues were performed to assess metabolic and inflammatory damage and oxidative status. Hepatic gene expression of oxidative stress and inflammation related enzymes were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: PE improved hepatopathology. PE also improved the lipid profiles and antioxidant enzymes, including hepatic glutathione peroxidase (GPx) and superoxide dismutase (SOD) and catalase (CAT) in serum and liver. Hepatic gene expressions of antioxidant and anti-inflammatory related enzymes, such as SOD-1, CAT, interleukin 4 (IL-4) and nuclear factor erythroid 2-related factor (Nrf2) were significantly enhanced by PE. PE also reduced the levels of hydrogen peroxide (H₂O₂) and malondialdehyde (MDA) in the serum and liver; moreover, PE suppressed hepatic gene expression involved in pro-inflammatory response; Cyclooxygenase-2 (COX-2), nitric oxide synthase (NOS), interleukin 1 beta (IL-1β), and interleukin 6 (IL-6). CONCLUSIONS: This research opens opportunities for further investigations of PE as a functional food and possible anti-aging agent due to its attenuative effects against oxidative stress, resulting from HFD and aging in the future.

• Animal Bioscience
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• v.36 no.7
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• pp.1022-1033
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• 2023

Objective: p66Shc, a 66 kDa protein isoform encoded by the proto-oncogene SHC, is an essential intracellular redox homeostasis regulatory enzyme that is involved in the regulation of cellular oxidative stress, apoptosis induction and the occurrence of multiple age-related diseases. This study investigated the expression profile and functional characteristics of p66Shc during preimplantation embryo development in sheep. Methods: The expression pattern of p66Shc during preimplantation embryo development in sheep at the mRNA and protein levels were studied by quantitative real-time polymerase chain reaction (RT-qPCR) and immunofluorescence staining. The effect of p66Shc knockdown on the developmental potential were evaluated by cleavage rate, morula rate and blastocyst rate. The effect of p66Shc deficiency on reactive oxygen species (ROS) production, DNA oxidative damage and the expression of antioxidant enzymes (e.g., catalase and manganese superoxide dismutase [MnSOD]) were also investigated by immunofluorescence staining. Results: Our results showed that p66Shc mRNA and protein were expressed in all stages of sheep early embryos and that p66Shc mRNA was significantly downregulated in the 4-to 8-cell stage (p<0.05) and significantly upregulated in the morula and blastocyst stages after embryonic genome activation (EGA) (p<0.05). Immunofluorescence staining showed that the p66Shc protein was mainly located in the peripheral region of the blastomere cytoplasm at different stages of preimplantation embryonic development. Notably, serine (Ser36)-phosphorylated p66Shc localized only in the cytoplasm during the 2- to 8-cell stage prior to EGA, while phosphorylated (Ser36) p66Shc localized not only in the cytoplasm but also predominantly in the nucleus after EGA. RNAi-mediated silencing of p66Shc via microinjection of p66Shc siRNA into sheep zygotes resulted in significant decreases in p66Shc mRNA and protein levels (p<0.05). Knockdown of p66Shc resulted in significant declines in the levels of intracellular ROS (p<0.05) and the DNA damage marker 8-hydroxy2'-deoxyguanosine (p<0.05), markedly increased MnSOD levels (p<0.05) and resulted in a tendency to develop to the morula stage. Conclusion: These results indicate that p66Shc is involved in the metabolic regulation of ROS production and DNA oxidative damage during sheep early embryonic development.

• Animal Bioscience
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• v.37 no.2
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• pp.193-202
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• 2024

Objective: Oxidative stress (OS) is a pathological process arising from the excessive production of free radicals in the body. It has the potential to alter animal gene expression and cause damage to the jejunum. However, there have been few reports of changes in the expression of long noncoding RNAs (lncRNAs) in the jejunum in piglets under OS. The purpose of this research was to examine how lncRNAs in piglet jejunum change under OS. Methods: The abdominal cavities of piglets were injected with diquat (DQ) to produce OS. Raw reads were downloaded from the SRA database. RNA-seq was utilized to study the expression of lncRNAs in piglets under OS. Additionally, six randomly selected lncRNAs were verified using quantitative real-time polymerase chain reaction (qRT-PCR) to examine the mechanism of oxidative damage. Results: A total of 79 lncRNAs were differentially expressed (DE) in the treatment group compared to the negative control group. The target genes of DE lncRNAs were enriched in gene ontology (GO) terms and Kyoto encyclopedia of genes and genomes (KEGG) signaling pathways. Chemical carcinogenesis-reactive oxygen species, the Foxo signaling pathway, colorectal cancer, and the AMPK signaling pathway were all linked to OS. Conclusion: Our results demonstrated that DQ-induced OS causes differential expression of lncRNAs, laying the groundwork for future research into the processes involved in the jejunum's response to OS.

• Biomolecules & Therapeutics
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• v.32 no.1
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• pp.154-161
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• 2024

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disorder that causes progressive paralysis. L-Citrulline is a nonessential neutral amino acid produced by L-arginine via nitric oxide synthase (NOS). According to previous studies, the pathogenesis of ALS entails glutamate toxicity, oxidative stress, protein misfolding, and neurofilament disruption. In addition, L-citrulline prevents neuronal cell death in brain ischemia; therefore, we investigated the change in the transport of L-citrulline under various pathological conditions in a cell line model of ALS. We examined the uptake of [¹⁴C]L-citrulline in wild-type (hSOD1wt/WT) and mutant NSC-34/ SOD1^G93A (MT) cell lines. The cell viability was determined via MTT assay. A transport study was performed to determine the uptake of [¹⁴C]L-citrulline. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis was performed to determine the expression levels of rat large neutral amino acid transported 1 (rLAT1) in ALS cell lines. Nitric oxide (NO) assay was performed using Griess reagent. L-Citrulline had a restorative effect on glutamate induced cell death, and increased [¹⁴C]L-citrulline uptake and mRNA levels of the large neutral amino acid transporter (LAT1) in the glutamate-treated ALS disease model (MT). NO levels increased significantly when MT cells were pretreated with glutamate for 24 h and restored by co-treatment with L-citrulline. Co-treatment of MT cells with L-arginine, an NO donor, increased NO levels. NSC-34 cells exposed to high glucose conditions showed a significant increase in [¹⁴C]L-citrulline uptake and LAT1 mRNA expression levels, which were restored to normal levels upon co-treatment with unlabeled L-citrulline. In contrast, exposure of the MT cell line to tumor necrosis factor alpha, lipopolysaccharides, and hypertonic condition decreased the uptake significantly which was restored to the normal level by co-treating with unlabeled L-citrulline. L-Citrulline can restore NO levels and cellular uptake in ALS-affected cells with glutamate cytotoxicity, pro-inflammatory cytokines, or other pathological states, suggesting that L-citrulline supplementation in ALS may play a key role in providing neuroprotection.

• Animal Bioscience
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• v.37 no.3
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• pp.509-521
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• 2024

Objective: It was shown that microRNAs (miRNAs) play an important role in milk protein synthesis. However, the post-transcriptional regulation of casein expression by exogenous miRNA (xeno-miRNAs) in ruminants remains unclear. This study explores the regulatory roles of alfalfa xeno-miR162 on casein synthesis in bovine mammary epithelial cells (bMECs). Methods: The effects of alfalfa xenomiR-162 and G protein subunit gamma 11 (GNG11) on proliferation and milk protein metabolism of bMECs were detected by 5-Ethynyl-2'-Deoxyuridine (EdU) staining, flow cytometry, cell counting kit-8 (CCK-8), enzyme-linked immunosorbent assay, quantitative real-time polymerase chain reaction (qRT-PCR), and Western blot. Dual-luciferase reporter assay was used to verify the targeting relationship between GNG11 and xenomiR-162. Results: Results showed that over-expression of xenomiR-162 inhibited cell proliferation but promoted apoptosis, which also up-regulated the expression of several casein coding genes, including CSN1S1, CSN1S2, and CSN3, while decreasing the expression of CSN2. Furthermore, the targeting relationship between GNG11 and xenomiR-162 was determined, and it was confirmed that GNG11 silencing also inhibited cell proliferation but promoted apoptosis and reduced the expression of casein coding genes and genes related to the mammalian target of rapamycin (mTOR) pathway. Conclusion: Alfalfa xenomiR-162 appears to regulate bMECs proliferation and milk protein synthesis via GNG11 in the mTOR pathway, suggesting that this xeno-miRNA could be harnessed to modulate CSN3 expression in dairy cows, and increase κ-casein contents in milk.

• Journal of The Korean Society of Inherited Metabolic disease
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• v.15 no.3
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• pp.127-137
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• 2015

Purpose: The main aim of this study was to compare and analyze expression profiles of microRNAs (miRNAs) to establish miRNA signature of Fabry nephropathy related epithelial mesenchymal transition (EMT). Methods: Expression profiles of miRNAs in kidney tissue samples and cell lines from normal and Fabry disease mouse model were examined by miRNA expression microarray analysis followed by quantitative real-time polymerase chain reaction analysis (qRT-PCR). Results: In the miRNA expression microarray analysis of Fabry mouse kidney tissues compared to wild type mouse, 5 and 3 miRNAs among 1,247 miRNAs examined were up- and down-regulated, respectively. Among them, miR-149-5p was down-regulated about 2-fold in Fabry kidney samples. The down-regulations of miR-149-5p were observed in kidney tissues of under 35 week-old-Fabry mice. However, this down-regulation was not observed in kidney tissues of 42 week-old Fabry mice. In SV40 MES 13 cells, mouse mesangial cells, treated with globotriaosylsphingosine (lyso-Gb3), miR-149-5p was also downregulated. The down-regulation of miR-149-5p induced up-regulation of its target genes related to EMT. Conclusion: The miRNA expression array and qRT-PCR results show that miR-149-5p expression was decreased in kidney tissues of Fabry mice compared to wild type mice under 35 weeks of age. Along with the observation of miR-149-5p expression in Fabry disease cell models, these results indicate that the down-regulated miR-149-5p were related to the biological response of mesangial cells to lyso-Gb3 and also have influence to the transcriptional up-regulation of its target genes. These results suggest miR-149-5p might play important roles in the Fabry nephropathy.

• Journal of Life Science
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• v.31 no.8
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• pp.710-718
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• 2021

Placenta-derived mesenchymal stem cells (PD-MSCs) are promising candidates for cell-based therapy in regenerative medicine. The migration and homing potential of PD-MSCs to injured sites is a critical property of MSC engraftment. MicroRNAs (miRNAs) have recently been shown to regulate the critical functions of MSCs, such as proliferation, survival, and migration. The objective of the present study was to identify the miRNA and target genes involved in PD-MSCs homing in a bile duct ligation (BDL) rat model. We selected candidate miRNAs targeting genes for PD-MSCs homing based on microarray analysis. PD-MSC engraftment in BDL-injured rat liver was identified by immunofluorescence assay and human-specific Alu gene expression by quantitative real-time polymerase chain reaction (qRT-PCR) one week after transplantation. Compared with migrated naïve PD-MSCs under hypoxic and normoxic conditions (Hyp/Nor), the transplanted group with PD-MSCs (Tx) showed distinct differences in miRNA expressions in BDL-injured rat liver. We also validated the miRNAs and their target genes for PD-MSCs homing. The expressions of integrin α4 (ITGA4) and integrin α5 (ITGA5) target genes for miR-199a-5p and miR-148a-3p were significantly upregulated in the Tx group (p<0.05). In addition, integrin β1 (ITGB1) and integrin β8 (ITGB8) were upregulated by suppressing miR-183-5p and miR-145-5p, respectively. These results demonstrated that PD-MSCs regulate miRNA expression related to the integrin family for their homing effects on the BDL-injured rat liver. The findings further suggest that miRNA-mediated regulation of the integrin family contributes to the therapeutic efficacy of PD-MSCs in the rat hepatic fibrosis model by BDL.

• Journal of Life Science
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• v.33 no.11
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• pp.905-914
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• 2023

To evaluate the effectiveness of the skin barrier improvement of lactic acid (LA) and gluconolactone (GL), the expression of filaggrin, loricrin, hyaluronic acid (HA), hyaluronan syhthase-2 (HAS2), and aquaporine-3 (AQP3) in keratinocytes, and the moisture content and transepidermal water loss (TEWL) by clinical trials were evaluated. The expression levels of filaggrin and locricrin, which are the main factors affecting the proper functioning of skin barrier function, and HA, HAS2, and AQP3, which are skin moisturizing-related proteins measured by quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting. The results showed that the expression levels of the factors that decreased by H2O2 treatment were significantly increased by LA, GL, and a mixture of LA and GL at the mRNA and protein levels (p<0.05). The nanoemulsion containing a mixture of LA and GL was prepared using the emulsion inversion method, and the average particle size was 299.9 ± 0.287 nm. After measuring the TEWL of nanoemulsion using Vapometer, it was found that TEWL significantly decreased by 15.53% and 26.73% after two weeks and four weeks of product use, respectively, compared to TEWL before product use (p<0.001). Similarly, the skin moisture content of the nanoemulsion significantly increased by 15.40% and 26.59% after two weeks and four weeks of product use, respectively, compared to skin moisture content before product use (p<0.001). Therefore, the skin barrier function and moisturizing effect of a mixture of LA and GL are shown by increasing the moisture content and decreasing the TEWL by increasing the expression of filaggrin, loricrin, HA, HAS2, and AQP3. This suggests the possibility for the development of functional cosmetic ingredients in the future.

• Journal of Life Science
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• v.26 no.9
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• pp.1056-1062
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• 2016

Lung cancer is currently the most common malignant disease and the leading cause of mortality in the world and non-small cell lung cancer (NSCLC) accounts for 75-80% of lung cancer cases. miR-155 gene was found to be over expressed in several solid tumors, such as thyroid carcinoma, breast cancer, colon cancer, cervical cancer, pancreatic ductal adenocarcinoma (PDAC) and lung cancer. The aims of this study were to define the expression of miR-155 in lung cancer and its associated clinic-pathologic characteristics. Total RNA was purified from formalin-fixed, paraffin-embedded NSCLC tissues and benign lung tissues. Expression of miR-155 in human lung cancer tissues were evaluated as mean fold changes of miR-155 in cancer tissues compared to benign lung tissues by quantitative real-time reverse transcriptase polymerase chain reaction (real-time qRT-PCR) and associations of miR-155 expression with clinic-pathologic findings of cancer. Compared with the benign control group, miR-155 expression was significantly overexpressed in NSCLCs (p=<0.001). miR-155 was more overexpressed in squamous cell carcinoma than in adenocarcinoma. Poorly differentiated tumors showed significantly overexpression of miR-155 than well-differentiated tumors (p=<0.001). Overexpression of miR-155 was significantly associated with lymph node metastasis (p=<0.05). In survival analysis for all NSCLC patients, high miR-155 expression was significantly correlated with worse overall survival (p=<0.05). These results suggested that miR-155 might play an important role in lung cancer progression and metastasis.