• Animal Bioscience
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• 제37권5호
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• pp.929-943
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• 2024

Objective: The present study was executed to explore the molecular mechanism of fibroblast growth factor 10 (FGF10) gene in bovine adipogenesis. Methods: The bovine FGF10 gene was overexpressed through Ad-FGF10 or inhibited through siFGF10 and their negative control (NC) in bovine adipocytes, and the multiplicity of infection, transfection efficiency, interference efficiency were evaluated through quantitative real-time polymerase chain reaction, western blotting and fluorescence microscopy. The lipid droplets, triglycerides (TG) content and the expression levels of adipogenic marker genes were measured during preadipocytes differentiation. The differentially expressed genes were explored through deep RNA sequencing. Results: The highest mRNA level was found in omasum, subcutaneous fat, and intramuscular fat. Moreover, the highest mRNA level was found in adipocytes at day 4 of differentiation. The results of red-oil o staining showed that overexpression (Ad-FGF10) of the FGF10 gene significantly (p<0.05) reduced the lipid droplets and TG content, and their down-regulation (siFGF10) increased the measurement of lipid droplets and TG in differentiated bovine adipocytes. Furthermore, the overexpression of the FGF10 gene down regulated the mRNA levels of adipogenic marker genes such as CCAAT enhancer binding protein alpha (C/EBPα), fatty acid binding protein (FABP4), peroxisome proliferator-activated receptor-γ (PPARγ), lipoprotein lipase (LPL), and Fas cell surface death receptor (FAS), similarly, down-regulation of the FGF10 gene enriched the mRNA levels of C/EBPα, PPARγ, FABP4, and LPL genes (p<0.01). Additionally, the protein levels of PPARγ and FABP4 were reduced (p<0.05) in adipocytes infected with Ad-FGF10 gene and enriched in adipocytes transfected with siFGF10. Moreover, a total of 1,774 differentially expressed genes (DEGs) including 157 up regulated and 1,617 down regulated genes were explored in adipocytes infected with Ad-FGF10 or Ad-NC through deep RNA-sequencing. The top Kyoto encyclopedia of genes and genomes pathways regulated through DEGs were the PPAR signaling pathway, cell cycle, base excision repair, DNA replication, apoptosis, and regulation of lipolysis in adipocytes. Conclusion: Therefore, we can conclude that the FGF10 gene is a negative regulator of bovine adipogenesis and could be used as a candidate gene in marker-assisted selection.

• 대한화장품학회지
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• 제50권2호
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• pp.171-178
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• 2024

본 연구에서는 목서(Osmanthus fragrans, O. fragrans) 꽃 추출물을 화장품 소재로 활용하기 위해 피부에 대한 다양한 효능을 확인하고자 하였다. 이를 위해 제주산 목서 꽃 추출물(O. fragrans flower extract, OFFE)을 제조하여 실험에 이용하였다. 실험은 실시간 중합효소 연쇄반응인 qRT-PCR과 lipid droplet 염색법으로 평가하였다. 먼저 OFFE는 표피 각질세포에서 poly I:C로 유도된 3 종의 대표적인 전염증성 사이토카인 (IL-8, IL-6, IL-1α)과 염증 관련 효소인 PTGS2의 유전자 발현을 감소시켰다. 또한 OFFE는 진피 섬유아세포에서 콜라겐(COL1A1)과 엘라스틴(ELN) 유전자 발현을 증가시켰다. 더 나아가 OFFE는 피지세포에서 linoleic acid에 의해 유도된 피지 생성을 억제하는 효능 보여주었다. 따라서 본 연구를 통해 OFFE은 항염, 항노화, 그리고 피지억제 효능을 위한 천연 화장품 소재로써 적용이 가능할 것으로 기대된다.

• Animal Bioscience
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• 제37권8호
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• pp.1345-1354
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• 2024

Objective: The objective of this study was to identify candidate genes that play important roles in skeletal muscle development in ducks. Methods: In this study, we investigated the transcriptional sequencing of embryonic pectoral muscles from two specialized lines: Liancheng white ducks (female) and Cherry valley ducks (male) hybrid Line A (LCA) and Line C (LCC) ducks. In addition, prediction of target genes for the differentially expressed mRNAs was conducted and the enriched gene ontology (GO) terms and Kyoto encyclopedia of genes and genomes signaling pathways were further analyzed. Finally, a protein-to-protein interaction network was analyzed by using the target genes to gain insights into their potential functional association. Results: A total of 1,428 differentially expressed genes (DEGs) with 762 being up-regulated genes and 666 being down-regulated genes in pectoral muscle of LCA and LCC ducks identified by RNA-seq (p<0.05). Meanwhile, 23 GO terms in the down-regulated genes and 75 GO terms in up-regulated genes were significantly enriched (p<0.05). Furthermore, the top 5 most enriched pathways were ECM-receptor interaction, fatty acid degradation, pyruvate degradation, PPAR signaling pathway, and glycolysis/gluconeogenesis. Finally, the candidate genes including integrin b3 (Itgb3), pyruvate kinase M1/2 (Pkm), insulin-like growth factor 1 (Igf1), glucose-6-phosphate isomerase (Gpi), GABA type A receptor-associated protein-like 1 (Gabarapl1), and thyroid hormone receptor beta (Thrb) showed the most expression difference, and then were selected to verification by quantitative real-time polymerase chain reaction (qRT-PCR). The result of qRT-PCR was consistent with that of transcriptome sequencing. Conclusion: This study provided information of molecular mechanisms underlying the developmental differences in skeletal muscles between specialized duck lines.

• Animal Bioscience
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• 제37권7호
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• pp.1156-1167
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• 2024

Objective: MicroRNAs (miRNAs) are endogenous non-coding RNAs that can play a role in the post-transcriptional regulation of mammalian preadipocyte differentiation. However, the precise functional mechanism of its regulation of fat metabolism is not fully understood. Methods: We identified bta-miR-365-3p, which specifically targets the 3' untranslated region (3'UTR) of the FK506-binding protein 5 (FKBP5), and verified its mechanisms for regulating expression and involvement in adipogenesis. Results: In this study, we found that the overexpression of bta-miR-365-3p significantly decreased the lipid accumulation and triglyceride content in the adipocytes. Compared to inhibiting bta-miR-36 5-3p group, overexpression of bta-miR-365-3p can inhibit the expression of adipocyte differentiation-related genes C/EBPα and PPARγ. The dual-luciferase reporter system further validated the targeting relationship between bta-miR-365-3p and FKBP5. FKBP5 mRNA and protein expression were detected by quantitative real-time polymerase chain reaction and Western blot. Overexpression of bta-miR-365-3p significantly down-regulated FKBP5 expression, while inhibition of bta-miR-365-3p showed the opposite, indicating that bta-miR-365-3p negatively regulates FKBP5. Adenosine 5'-monophosphate (AMP)-activated protein kinase/mammalian target of rapamycin (AMPK/mTOR) signaling pathway is closely related to the regulation of cell growth and is involved in the development of bovine adipocytes. In this study, overexpression of bta-miR-365-3p significantly inhibited mRNA and protein expression of AMPK, mTOR, and SREBP1 genes, while the inhibition of bta-miR-365-3p expression was contrary to these results. Overexpression of FKBP5 significantly upregulated AMPK, mTOR, and SREBP1 gene expression, while inhibition of FKBP5 expression was contrary to the above experimental results. Conclusion: In conclusion, these results indicate that bta-miR-365-3p may be involved in the AMPK/mTOR signaling pathway in regulating Yanbian yellow cattle preadipocytes differentiation by targeting the FKBP5 gene.

• Animal Bioscience
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• 제37권2호
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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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• 제32권1호
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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.

• Journal of Animal Science and Technology
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• 제66권4호
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• pp.663-681
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• 2024

Co-infection with porcine reproductive and respiratory syndrome virus (PRRSV) and Haemophilus parasuis (HPS) has severely restricted the healthy development of pig breeding. Exploring disease resistance of non-coding RNAs in pigs co-infected with PRRSV and HPS is therefore critical to complement and elucidate the molecular mechanisms of disease resistance in Kele piglets and to innovate the use of local pig germplasm resources in China. RNA-seq of lungs from Kele piglets with single-infection of PRRSV or HPS and co-infection of both pathogens was performed. Two hundred and twenty-five differentially expressed long non-coding RNAs (DElncRNAs) and 30 DEmicroRNAs (DEmiRNAs) were identified and characterized in the PRRSV and HPS co-infection (PRRSV-HPS) group. Compared with the single-infection groups, 146 unique DElncRNAs, 17 unique DEmiRNAs, and 206 target differentially expressed genes (DEGs) were identified in the PRRSV-HPS group. The expression patterns of 20 DEmiRNAs and DElncRNAs confirmed by real-time quantitative polymerase chain reaction (RT-qPCR) were consistent with those determined by high-throughput sequencing. In the PRRSV-HPS group, the target DEGs were enriched in eight immune Gene Ontology terms relating to two unique DEmiRNAs and 16 DElncRNAs, and the unique target DEGs participated the host immune response to pathogens infection by affecting 15 immune-related Kyoto Encyclopedia of Genes and Genomes enrichment pathways. Notably, competitive endogenous RNA (ceRNA) networks of different groups were constructed, and the ssc-miR-671-5p miRNA was validated as a potential regulatory factor to regulate DTX4 and AEBP1 genes to achieve innate antiviral effects and inhibit pulmonary fibrosis by dual-luciferase reporter assays. These results provided insight into further study on the molecular mechanisms of resistance to PRRSV and HPS co-infection in Kele piglets.

• 한국가금학회지
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• 제43권2호
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• pp.77-88
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• 2016

본 연구에서는 국산종계 개발을 위하여 5계통의 토종닭 순계를 이용하여 $5{\times}5$ 이면교배조합 시험을 수행하고, 공시된 25계통에 대한 스트레스 반응 정도를 비교 분석하였다. 스트레스 반응정도 표지로 텔로미어 함량 및 감축율, 열 스트레스 단백질 유전자(HSPs)로 HSP-70, $HSP-90{\alpha}$, $HSP-90{\beta}$의 발현율 및 DNA 손상율을 이용하여 분석하였다. 텔로미어 함량은 양적 형광접합보인법을 이용하고, HSPs 발현율은 quantitative real-time PCR 기법으로, DNA 손상율은 Comet assay법을 적용하였다. 분석 결과, 교배조합 25계통 간 텔로미어 함량과 감축율, HSPs 유전자 발현율 및 DNA 손상율 모두에서 유의한 차이를 보였고, 더불어 계통 간 체중 및 생존율에도 유의한 차이가 나타났다. 텔로미어 감축율, HSPs 유전자 발현율 및 DNA 손상율 모든 분석 값에서 W 및 Y 계통과의 교잡구가 다른 교잡구에 비해 유의하게 낮은 값을 보였고, 반면 G계통의 순계구가 가장 높은 값을 나타내었다. 생존율과 스트레스 표지 값들 간의 상관계수를 추정한 결과, 텔로미어 함량과는 저도의 정(+)의 상관, HSP-70, $HSP-90{\alpha}$, $HSP-90{\beta}$ 및 DNA 손상율과는 중도 및 저도의 부(-)의 상관을 나타내었다. 또한 폐사 개체와 생존 개체 간 HSP-70, $HSP-90{\alpha}$ 및 $HSP-90{\beta}$의 발현율을 비교 분석한 결과, 폐사 개체의 HSPs 발현율이 생존 개체에 비해 유의하게 높은 발현율을 보였다. 이상 스트레스 표지 값의 분석 결과를 종합할 때, 교잡계들이 순계에 비해 스트레스 저항성이 높은 것으로 판단되고, 더불어 W, Y와 같은 경량종 계통의 교잡계들이 외적 스트레스에 강한 반면, G, H, F와 같은 고체중 계통의 교잡계들은 스트레스에 민감한 것으로 사료된다.

• 한국가금학회지
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• 제41권2호
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• pp.115-125
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• 2014

본 연구에서는 닭의 품종에 따른 개체의 스트레스 반응정도를 알아보고자 한국 재래닭과 단관 백색 레그혼종을 공시하고, 고밀도 사육에 따른 생리적 스트레스 표지 값을 비교 분석하였다. 스트레스 반응 정도는 혈액과 각 조직별 세포들에 대한 텔로미어 함량, DNA 손상율 및 열 스트레스 단백질(HSPs) 유전자의 발현율을 분석하고 비교하였다. 텔로미어 함량 및 감축율은 양적 형광 접합 보인법으로 분석하였고, DNA 손상율은 Comet assay로 분석하였다. 열 스트레스 단백질 유전자 발현율은 HSP70, HSP90-${\alpha}$, HSP90-${\beta}$ 및 HMGCR을 표적으로 하여 real-time PCR로 분석하였다. 분석 결과, 한국 재래닭과 단관 백색 레그혼 간 품종에 따른 체중, 증체량, 텔로미어 감축율 및 DNA 손상율의 차이는 없는 것으로 나타났다. 그러나 고밀도 사육과 같은 스트레스 사양 관리는 품종에 상관없이 닭의 성장을 저해하고, 텔로미어 감축 및 DNA 손상을 촉진시키는 것으로 나타났다. 한편, HMGCR을 제외한 HSPs 유전자 발현율의 경우, 밀사 사육에 따른 요인뿐만 아니라, 품종 간에도 유의적 차이를 보였다. HSPs의 분석에 따른 스트레스 정도는 단관 백색 레그혼종이 한국 재래닭에 비해 보다 민감하게 반응하는 것으로 나타났다. 따라서 품종과 관계없이 닭에 있어 고밀도 사육이 강한 환경적 스트레스 요인임을 시사하고, 품종 간 스트레스의 반응 정도는 레그혼종이 한국 재래닭에 비해 다소 높은 것으로 사료된다.

• Asian-Australasian Journal of Animal Sciences
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• 제30권11호
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• pp.1620-1632
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• 2017

Objective: This study evaluated the effects of a traditional Chinese medicine formula (TCMF) on muscle fiber characteristics in finishing pigs and the effects of the formula's extract (distilled water, ethyl acetate and petroleum ether extraction) on porcine cell proliferation and isoforms of myosin heavy chain (MyHC) gene expression in myocytes. Methods: In a completely randomized design, ninety pigs were assigned to three diets with five replications per treatment and six pigs per pen. The diets included the basal diet (control group), TCMF1 (basal diet+2.5 g/kg TCMF) and TCMF2 (basal diet+5 g/kg TCMF). The psoas major muscle was obtained from pigs at the end of the experiment. Muscle fiber characteristics in the psoas major muscle were analyzed using myosin ATPase staining. Cell proliferation was measured using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) dye and cytometry. Isoforms of MyHC gene expression were detected by real-time quantitative polymerase chain reaction. Results: The final body weight and carcass weight of finishing pigs were increased by TCMF1 (p<0.05), while the psoas major muscle cross-sectional area was increased by TCMF (p<0.05). The cross-sectional area and diameter of psoas major muscle fiber Ι, IIA, and IIB were increased by TCMF2 (p<0.05). The cross-sectional area and fiber diameter of psoas major muscle fiber IIA and IIB were increased by diet supplementation with TCMF1 (p<0.05). Psoas major muscle fiber IIA and IIB fiber density from the pigs fed the TCMF1 diet and the type IIB fiber density from the pigs fed the TCMF2 diet were lower compared to pigs fed the control diet (p<0.05). Pigs fed TCMF2 had a higher composition of type Ι fiber and a lower percentage of type IIB fiber in the psoas major muscle (p<0.05). The expression levels of MyHC Ι, MyHC IIa, and MyHC IIx mRNA increased and the amount of MyHC IIb mRNA decreased in the psoas major muscle from TCMF2, whereas MyHC Ι and MyHC IIx mRNA increased in the psoas major muscle from TCMF1 (p<0.05). Peroxisome proliferator-activated receptor ${\gamma}$ $coactivator-1{\alpha}$ and CaN mRNA expression in the psoas major muscle were up-regulated by TCMF (p<0.05). Porcine skeletal muscle satellite cell proliferation was promoted by $4{\mu}g/mL$ and $20{\mu}g/mL$ TCMF water extraction (p<0.05). Both $1{\mu}g/mL$ and $5{\mu}g/mL$ of TCMF water extraction increased MyHC IIa, MyHC IIb, and MyHC IIx mRNA expression in porcine myocytes (p<0.05), while MyHC Ι mRNA expression in porcine myocytes was decreased by $5{\mu}g/mL$ TCMF water extraction (p<0.05). Porcine myocyte MyHC Ι and MyHC IIx mRNA expression were increased, and MyHC IIa and MyHC IIb mRNA expression were down-regulated by $5{\mu}g/mL$ TCMF ethyl acetate extraction (p<0.05). MyHC Ι and MyHC IIa mRNA expression in porcine myocytes were increased, and the MyHC IIb mRNA expression was decreased by $1{\mu}g/mL$ TCMF ethyl acetate extraction (p<0.05). Four isoforms of MyHC mRNA expression in porcine myocytes were reduced by $5{\mu}g/mL$ TCMF petroleum ether extraction (p<0.05). MyHC IIa mRNA expression in porcine myocytes increased and MyHC IIb mRNA expression decreased by $1{\mu}g/mL$ in a TCMF petroleum ether extraction (p<0.05). Conclusion: These results indicated that TCMF amplified the psoas major muscle cross-sectional area through changing muscle fiber characteristics in finishing pigs. This effect was confirmed as TCMF extraction promoted porcine cell proliferation and affected isoforms of MyHC gene expression in myocytes.