• Journal of Veterinary Science
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• 제19권6호
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• pp.735-743
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• 2018

We investigated the effect of transforming growth factor beta 1 ($TGF-{\beta}1$) on equine hyaluronan synthase 2 (HAS2) gene expression and hyaluronan (HA) synthesis in culture models of articular chondrocytes. Equine chondrocytes were treated with $TGF-{\beta}1$ at different concentrations and times in monolayer cultures. In three-dimensional cultures, chondrocyte-seeded gelatin scaffolds were cultured in chondrogenic media containing 10 ng/mL of $TGF-{\beta}1$. The amounts of HA in conditioned media and in scaffolds were determined by enzyme-linked immunosorbent assays. HAS2 mRNA expression was analyzed by semi-quantitative reverse transcription polymerase chain reaction. The uronic acid content and DNA content of the scaffolds were measured by using colorimetric and Hoechst 33258 assays, respectively. Cell proliferation was evaluated by using the alamarBlue assay. Scanning electron microscopy (SEM), histology, and immunohistochemistry were used for microscopic analysis of the samples. The upregulation of HAS2 mRNA levels by $TGF-{\beta}1$ stimulation was dose and time dependent. $TGF-{\beta}1$ was shown to enhance HA and uronic acid content in the scaffolds. Cell proliferation and DNA content were significantly lower in $TGF-{\beta}1$ treatments. SEM and histological results revealed the formation of a cartilaginous-like extracellular matrix in the $TGF-{\beta}1$-treated scaffolds. Together, our results suggest that $TGF-{\beta}1$ has a stimulatory effect on equine chondrocytes, enhancing HA synthesis and promoting cartilage matrix generation.

• Journal of Animal Science and Technology
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• 제63권2호
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• pp.426-439
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• 2021

It is already well known that castration improves marbling quality but exact timing of castration is still highly debated in beef cattle production industry. After castration, blood hormonal changes occur in steer and objective of this study was to investigate the effects of growth hormone (GH) levels on adipocyte differentiation in stromal vascular cells (SVCs) and transdifferentiation into adipocytes in C2C12 myoblasts. Total GH concentrations were measured via enzyme-linked immunosorbent assay (ELISA) in 24 male calves and 4 female calves. Cell proliferation, cellular triglyceride (TG) accumulation, and the cell's lipolytic capability were measured in C2C12 myoblasts and SVCs. Myogenic, adipogenic, and brown adipocyte-specific gene expression was measured via real-time polymerase chain reaction (PCR) using SYBR green. Serum GH levels were the highest in late-castrated calves. Treatment with 5 ng/mL GH resulted in greater TG accumulation as well as increased CCAAT-enhancer-binding protein (C/EBP)α and peroxisome proliferator-activated receptor (PPAR)γ expression compared to that after treatment with 15 ng/mL GH. Treatment with 5 ng/mL GH also resulted in lower myogenin (myo)G and myoD expression compared to that after treatment with 15 ng/mL GH. The expression of bone morphogenetic protein (BMP) 7 after treatment with 5 ng/mL GH was higher than that after treatment with 15 ng/mL GH. But carcass characteristics data showed no significant difference between early and late castrated steers. Therefore, our results indicate that castration timing does not seem to be inevitable determinate of carcass qualities, particularly carcass weight and marbling score in Hanwoo beef cattle.

• The Korean Journal of Physiology and Pharmacology
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• 제26권3호
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• pp.165-174
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• 2022

As the mechanism underlying glucose metabolism regulation by oxymatrine is unclear, this study investigated the effects of oxymatrine on pyroptosis in INS-1 cells. Flow cytometry was employed to examine cell pyroptosis and reactive oxygen species (ROS) production. Cell pyroptosis was also investigated via transmission electron microscopy and lactate dehydrogenase (LDH) release. Protein levels were detected using western blotting and interleukin (IL)-1β and IL-18 secretion by enzyme-linked immunosorbent assay. The caspase-1 activity and DNA-binding activity of nuclear factor kappa B (NF-κB) and nuclear factor (erythroid-derived 2)-like 2 protein (Nrf2) were also assessed. In the high glucose and high fat-treated INS-1 cells (HG + PA), the caspase-1 activity and LDH content, as well as Nod-like receptor family pyrin domain containing 3, Gsdmd-N, caspase-1, apoptosis-associated speck-like protein containing a CARD, IL-1β, and IL-18 levels were increased. Moreover, P65 protein levels increased in the nucleus but decreased in the cytoplasm. Oxymatrine attenuated these effects and suppressed high glucose and high fat-induced ROS production. The increased levels of nuclear Nrf2 and heme oxygenase-1 (HO-1) in the HG + PA cells were further elevated after oxymatrine treatment, whereas cytoplasmic Nrf2 and Keleh-like ECH-associated protein levels decreased. Additionally, the elevated transcriptional activity of p65 in HG + PA cells was reduced by oxymatrine, whereas that of Nrf2 increased. The results indicate that the inhibition of pyroptosis in INS-1 cells by oxymatrine, a key factor in its glucose metabolism regulation, involves the suppression of the NF-κB pathway and activation of the Nrf2/HO-1 pathway.

• Journal of Veterinary Science
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• 제24권5호
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• pp.70.1-70.14
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• 2023

Background: Mycobacterium avium subspecies paratuberculosis (MAP) causes a chronic and progressive granulomatous enteritis and economic losses in dairy cattle in subclinical stages. Subclinical infection in cattle can be detected using serum MAP antibody enzyme-linked immunosorbent assay (ELISA) and fecal polymerase chain reaction (PCR) tests. Objectives: To investigate the differences in blood parameters, according to the detection of MAP using serum antibody ELISA and fecal PCR tests. Methods: We divided 33 subclinically infected adult cattle into three groups: seronegative and fecal-positive (SNFP, n = 5), seropositive and fecal-negative (SPFN, n = 10), and seropositive and fecal-positive (SPFP, n = 18). Hematological and serum biochemical analyses were performed. Results: Although the cows were clinically healthy without any manifestations, the SNFP and SPFP groups had higher platelet counts, mean platelet volumes, plateletcrit, lactate dehydrogenase levels, lactate levels, and calcium levels but lower mean corpuscular volume concentration than the SPFN group (p < 0.017). The red blood cell count, hematocrit, monocyte count, glucose level, and calprotectin level were different according to the detection method (p < 0.05). The SNFP and SPFP groups had higher red blood cell counts, hematocrit and calprotectin levels, but lower monocyte counts and glucose levels than the SPFN group, although there were no significant differences (p > 0.017). Conclusions: The cows with fecal-positive MAP status had different blood parameters from those with fecal-negative MAP status, although they were subclinically infected. These findings provide new insights into understanding the mechanism of MAP infection in subclinically infected cattle.

• Clinical and Experimental Vaccine Research
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• 제12권1호
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• pp.47-59
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• 2023

Purpose: The development and study of hepatitis C virus (HCV) vaccine candidates' individualized responses are of great importance. Here we report on an HCV DNA vaccine candidate based on selected envelope (E1/E2) epitopes. Besides, we assessed its expression and processing in human peripheral blood mononuclear cells (PBMCs) and in vivo cellular response in mice. Materials and Methods: HCV E1/E2 DNA construct (EC) was designed. The antigen expression of EC was assayed in PBMCs of five HCV-uninfected donors via a real-time quantitative polymerase chain reaction. Serum samples from 20 HCV antibody-positive patients were used to detect each individual PBMCs expressed antigens via enzyme-linked immunosorbent assay. Two groups, five Swiss albino mice each, were immunized with the EC or a control construct. The absolute count of lymph nodes' CD4⁺ and CD8⁺ T-lymphocytes was assessed. Results: Donors' PBMCs showed different levels of EC expression, ranging between 0.83-2.61-fold in four donors, while donor-3 showed 34.53-fold expression. The antigens expressed in PBMCs were significantly reactive to the 20 HCV antibody repertoire (all p=0.0001). All showed comparable reactivity except for donor-3 showing the lowest reactivity level. The absolute count % of the CD4⁺ T-cell significantly increased in four of the five EC-immunized mice compared to the control group (p=0.03). No significant difference in CD8⁺ T-cells % was observed (p=0.89). Conclusion: The inter-individual variation in antigen expression and processing dominance was evident, showing independence in individuals' antigen expression and reactivity levels to antibodies. The described vaccine candidate might result in a promising natural immune response with a possibility of CD4⁺ T-cell early priming.

• 한국식품과학회지
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• 제42권3호
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• pp.329-334
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• 2010

본 연구에서는 최근 식품이나 농축산물 등에서 빈번하게 검출되고 있는 장관출혈성 대장균인 E. coli O157:H7에 대한 단크론성 항체를 생산하고, 신속 정확하게 검색할 수 있는 간접효소면역분석법을 개발한 후 시료적용가능성을 확인하였다. 먼저 LPS, HKEC 및 FKEC를 E. coli O157:H7의 면역원으로 준비하여 mouse 면역에 사용하였고, 세포융합과 cloning을 실시하여 단클론성 항체를 생산하였으며, 생산된 항체 중 높은 역가를 나타낸 HKEC 4G8-5 항체로 ID-ELISA법을 개발하였다. 개발된 IDELISA법의 최저검출한계는 $10^5\;cell/mL$이었고, S. aureus와 Sal. Typhimurium에 대해서 약간의 반응성을 나타내었지만 E. coli O157:H7에 대해 보다 강한 반응성을 나타내었기 때문에 E. coli O157:H7에 매우 특이적인 분석법인 것으로 확인되었다. 개발된 ID-ELISA법을 이용하여 돼지고기, 소고기, 닭고기 및 새싹채소에 대한 검출률 향상을 위해 각 시료를 증균한 결과, 3종의 육류와 새싹채소는 각각 증균 6시간과 2시간부터 검출이 가능한 것으로 나타나 기존의 증균법보다 신속하게 시료 속 E. coli O157:H7을 분석할 수 있을 것으로 판단된다. 따라서 본 연구에서 개발된 IDELISA법을 적용하여 농축산물 중에 존재하는 E. coli O157:H7을 분석한다면 보다 신속하고 간편하게 분석할 수 있을 것으로 판단된다.

• Asian-Australasian Journal of Animal Sciences
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• 제31권8호
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• pp.1103-1109
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• 2018

Objective: This study aimed to screen and identify the target genes of miR-375 in pig Sertoli (ST) cells and to elucidate the effect of miR-375 on the proliferation of ST cells. Methods: In this study, bioinformatics software was used to predict and verify miR-375 target genes. Quantitative polymerase chain reaction (PCR) was used to detect the relationship between miR-375 and its target genes in ST cells. Enzyme-linked immunosorbent assay (ELISA) of rearranged L-myc fusion (RLF) and hypoxia-induced gene domain protein 1A (HIGD1A) was performed on porcine ST cells, which were transfected with a miR-375 mimics and inhibitor to verify the results. Dual luciferase reporter gene assays were performed to assess the interactions among miR-375, RLF, and HIGD1A. The effect of miR-375 on the proliferation of ST cells was analyzed by CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS). Results: Five possible target genes of miR-375, including RLF, HIGD1A, colorectal cancer associated 2, POU class 3 homeobox 1, and WW domain binding protein 1 like, were found. The results of quantitative PCR suggested that mRNA expression of RLF and HIGD1A had a negative correlation with miR-375, indicating that RLF and HIGD1A are likely the target genes of miR-375. The ELISA results revealed that RLF and HIGD1A were negatively correlated with the miR-375 protein level. The luminescence results for the miR-375 group cotransfected with wild-type RLF and HIGD1A vector were significantly lower than those of the miR-375 group co-transfected with the blank vector or mutant RLF and HIGD1A vectors. The present findings suggest that RLF and HIGD1A are target genes of miR-375 and that miR-375 inhibits ST cell proliferation according to MTS analysis. Conclusion: It was speculated that miR-375 affects cell proliferation through its target genes, which play an important role in the development of testicular tissue.

• 대한한방내과학회지
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• 제31권2호
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• pp.224-241
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• 2010

Objectives : The aim of the current study was to investigate the effects of Sargassum (Sargassum pallidum (TURN.) C. AG.; SP) on the experimental colitis induced by 2,4,6-trinitrobenzene sulfonic acid (TNBS) in mice. Methods : ICR mice were divided into 7 groups (NOR, CON, $SS50\times5$, $SP20\times3$, $SP50\times3$, $SP20\times5$, $SP50\times5$). TNBS processing was intrarectally applied to all experimental groups on the 3rd experiment day, except the normal group (NOR). For investigating the prophylactic effect, SP at doses of 20 mg/kg ($SP20\times5$) and 50 mg/kg ($SP50\times5$) were orally administered for 5 days. The SP at doses of 20 mg/kg ($SP20\times3$) and 50 mg/kg ($SP50\times3$) were orally administered for 3 days after the colitis induction in order to check the effect of treatment. As a positive control group, sulfasalazine 50 mg/kg ($SS50\times5$) was administrated. Macroscopic findings of epithelial tissue on mice were measured by colon length and macroscopic score. Histologic findings were also checked by crypt cell, epithelial cell, inflammatory cell and edema of submucosa. We measured the ability of SP to inhibit lipid peroxidation and myeloperoxidase activity. We also measured levels of the inflammatory markers, interleukin (IL)-$1\beta$ and cyclooxygenase-2 (COX-2), its transcription factor activation, phospho-NF-${\kappa}B$ (pp65), in the colon by enzyme-linked immunosorbent assay and immunoblot analysis. We measured activation of fecal bacterial enzyme, $\beta$-glucuronidase and degradation activation of fecal glycosaminoglycan (GAG), and hyaluronic acid. Results : Oral administration of SP on mice inhibited TNBS-induced colon shortening and myeloperoxidase activity in the colon of mice as well as IL-$1\beta$ and COX-2 expression. SP also inhibited TNBS-induced lipid peroxidation and pp65 activation in the colon of mice. SP inhibited $\beta$-glucuronidase activation and fecal hyaluronic acid degradation activation as well. Conclusions : SP could be a possible herbal candidate and preventive prebiotic agent for treating inflammatory bowel disease (IBD). Further experiments to differentiate effects of SP on IBD, such as other solutions and extracting times, might be promising.

• 한방재활의학과학회지
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• 제31권1호
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• pp.33-46
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• 2021

Objectives The aim of this study is to evaluate anti-inflammatory and anti-arthritic effects of Sogyunghwalhyel-tang-gamibang (SGHHTGB) in cell and animal models and also to suggest one of putative mechanisms underlying its anti-arthritic effects. Methods Enzyme-linked immunosorbent assay was applied to measure the concentrations of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6 and prostaglandin E2 (PGE2) in culture medium and blood serum and nitric oxide (NO) was assayed by Griess reagent. The expressions of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) were analyzed by Western blot method. Results In a cell model using RAW264.7 macrophages stimulated with the endotoxin lipopolysaccharide (LPS), the drug, at its non-cytotoxic concentrations, inhibited the production of the pro-inflammatory cytokine TNF-α, IL-1β and IL-6. In addition, it suppressed the expression of the inflammatory enzyme iNOS and COX-2, and reduced the synthesis of the enzyme product NO (as stable nitrite) and PGE2 in activated macrophages. Meanwhile, in an animal model using rheumatic arthritis (RA) mice induced with injection of type II collagen antibody (CAb) and LPS, the drug improved clinical symptom of arthritis and reduced paw thickness and inflammatory cell infiltration. In blood of RA mice, the drug reduced serum levels of TNF-α, IL-1β, IL-6, nitrite, and PGE2, all inflammatory mediators produced by activated macrophages. Conclusions SGHHTGB may ameliorate CAb and LPS-induced RA in mice, presumably by inactivating macrophages that are capable of initiating joint inflammation by producing pro-inflammatory cytokines and expressing inflammatory enzymes.

• The Journal of Advanced Prosthodontics
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• 제3권3호
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• pp.145-151
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• 2011

PURPOSE. This study was performed to investigate the ability of recombinant human-bone morphogenic protein-2 immobilized on a heparin-grafted bone substrate to enhance the osteoblastic functions. MATERIALS AND METHODS. The Bio-$Oss^{(R)}$, not coated with any material, was used as a control group. In rhBMP-2-Bio-$Oss^{(R)}$ group, rhBMP-2 was coated with Bio-$Oss^{(R)}$ using only deep and dry methods (50 ng/mL, 24 h). In heparinized rhBMP-2-Bio-$Oss^{(R)}$ group, dopamine was anchored to the surface of Bio-$Oss^{(R)}$, and coated with heparin. rhBMP-2 was immobilized onto the heparinized- Bio-$Oss^{(R)}$ surface. The release kinetics of the rhBMP-2-Bio-$Oss^{(R)}$ and heparinized rhBMP-2-Bio-$Oss^{(R)}$ were analyzed using an enzyme-linked immunosorbent assay. The biological activities of the MG63 cells on the three groups were investigated via cytotoxicity assay, cell proliferation assay, alkaline phosphatase (ALP) measurement, and calcium deposition determination. Statistical comparisons were carried out by one-way ANOVA test. Differences were considered statistically significant at $^*$P<.05 and $^{**}$P<.001. RESULTS. The heparinized rhBMP-2-Bio-$Oss^{(R)}$ showed more sustained release compared to the rhBMP-2-Bio-$Oss^{(R)}$ over an extended time. In the measurement of the ALP activity, the heparinized group showed a significantly higher ALP activity when compared with the non-heparinized groups (P<.05). The MG63 cells cultivated in the group with rhBMP-2 showed increased calcium deposition, and the MG63 cells from the heparinized group increased more than those that were cultivated in the non-heparinized groups. CONCLUSION. Heparin increased the rhBMP-2 release amount and made sustained release possible, and heparinized Bio-$Oss^{(R)}$ with rhBMP-2 successfully improved the osteoblastic functions.