• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.33 no.6
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• pp.636-642
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• 2007

Background: CpG DNA plays an important role in immune cell function. This study examined whether the temporal control of toll-like receptor (TLR)9 by CpG DNA can regulate the expression of matrix metalloproteinase-9(MMP-9). Methods and materials: Macrophages were cultured in the presence of 10% FBS. For the various MMP genes analysis, RT-PCR and real-time PCR were performed. In addition, zymography assay performed for the MMP activity. The phosphorylation assay did for the ERK1/2 and NF${\kappa}B$ activation, and luciferase promoter assay was for the NF${\kappa}B$ activity. Results: CpG DNA induced the mRNA expression of MMP-2, MMP-9, and MMP-13, but not of MMP-7, MMP-8, and MMP-12, in a time-dependent manner. Especially, the mRNA expression of MMP-9 was strongly induced by CpG DNA using real-time RT-PCR. The TLR9 inhibitor, chloroquine, suppressed CpG DNA-induced MMP-9 expression and its activity. Moreover, CpG DNA induced the phosphorylation of ERK and the inhibition of ERK by U0126 suppressed CpG DNA-induced MMP-9 expression and its activity. CpG DNA stimulated $I{\kappa}B-{\alpha}$ degradation and luciferase activity. In addition, pretreatment of SN-50, the inhibitor of NF${\kappa}B$, strongly blocked the CpG DNA-induced MMP-9 expression and activity. Conclusion: These observations suggest that CpG DNA may play important roles in the activation of macrophages by regulating the production of MMP-9 via the sequential TLR9-ERK-NF${\kappa}B$ signaling pathway.

• Journal of Microbiology and Biotechnology
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• v.12 no.4
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• pp.664-668
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• 2002

Antioxidants either scavenge superoxide and free radicals or stimulate the detoxification mechanisms within cells, resulting in increased detoxification of free radicals formation. Hyperin, isolated from the stem of Uncaria rhynchophylla, prevented oxygen radical formation and inhibited lipid oxidation. The effective concentrations were 31.3 $\mu$M for a radical scavenging assay and 2.2 $\mu$M for a microsome assay. cDNA microarray analysis to determine which genes were modulated by hyperin found that 50 genes were upregulated and 37 genes were downregulated in SNU-668 human gastric cancer cells. Among these genes, thirteen genes that were significantly affected by hyperin were verified by RT-PCR for their effect of genetic reprogramming.

• The Journal of Korean Medicine Ophthalmology and Otolaryngology and Dermatology
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• v.20 no.2 s.33
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• pp.36-46
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• 2007

Objective : In this study, the effect of Atractylodes japonica against LPS induced inflammation in mouse microglia BV2 cells was investigated. Method : Microglia BV2 Cells viability was determined using the MTT assay. We used water, ethanol extract from Atractylodes japonica and studied on the anti-inflammatory effect of lipopolysaccharide-induced inflammation using reverse transcription polymerase chain reaction (RT-PCR), western blot, and nitric oxide detection on mouse microglia BV2 cells. Result : The MTT assay revealed that it's extract has no significant cytotoxicity in the microglia BV2 cell. Various solvent extract from Atractylodes japonica inhibited nitrite production, iNOS protein and mRNA expression levels. And also it's extracts significantly reduced lipopolysaccharide-induced COX-2 activation in RT-PCR and western blot in lipopolysaccharide-induced microglia BV2 cells Conclusion : In this study, it's extracts was shown to suppress NO production by inhibiting iNOS expression and COX-2 activity. With this effects of anti-inflammation, we suggests that, it's extracts may be a useful candidate for the development of a drug on the related inflammatory diseases in brain.

• Journal of Acupuncture Research
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• v.20 no.3
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• pp.63-74
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• 2003

목적 : 한의학에서 관절염이나 진통치료에 사용되어 왔던 봉독약침액이 인간 골육종 세포주인 MG-63 세포에서 항종양효과가 있는지 연구하고자 한다. 특히 본 실험에서는 이러한 봉독의 종양발생 억제작용이 세포사멸과 관련이 있는지, 그리고 프로스타글란딘 합성 효소인 cyclooxygenase(COX)-2의 억제와 관련이 있는지를 연구하고자 한다. 방법 : 인간 골육종 세포주에서 세포사멸의 변화를 관찰하기 위해서 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium brimide(MTT) assay, 4,6-diamidino-2-phenylindole (DAPI), DNA fragmentation assay 및 reverse transcription-polymerase chain reaction(RT-PCR) 방법을 이용하였다. 결과 : 세포독성 검사에서 봉독은 MG-63 세포에서 농도-의존적으로 세포독성을 나타내었다. 이러한 봉독의 세포독성이 세포사멸로 인한 것인지를 여러 가지 형태로 검사한 결과 봉독에 의한 세포독성은 TUNEL 검사와 DAPI 염색시 세포사멸의 특징적인 소견들을 나타내었고, flow cytometric 분석에서도 세포사멸을 의미하는 세포주기의 변화들을 나타내었다. 봉독이 COX-2의 발현에 미치는 영향을 RT-PCR로 실험한 결과 봉독은 COX-2 mRNA의 발현을 선택적으로 억제하였다. 결론 : 본 실험의 결과 봉독은 COX-2 mRNA의 발현을 억제함으로써 골육종 세포에서 세포사멸을 유발하고 그 결과 항종양효과를 나타내는 것으로 보여진다.

• Natural Product Sciences
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• v.23 no.3
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• pp.175-182
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• 2017

This study evaluated the anti-Helicobacter and anti-inflammatory effects of Sohamhyungtang (SHHT). The minimum inhibitory concentration (MIC) of SHHT against Helicobacter pylori (H. pylori) was determined by the agar dilution method. Expression of the H. pylori cagA gene in the presence of SHHT was determined by quantitative real-time polymerase chain reaction (qRT-PCR). Inhibition of H. pylori urease by SHHT was determined by the phenol-hypochlorite assay. Antiadhesion activity of SHHT was measured by urea-phenol red reagent. Inhibition of nitric oxide (NO) production in AGS cells was measured with Griess reagent. Inducible nitric oxide synthase (iNOS) and IL-8 mRNA expression in AGS cells which were infected with H. pylori was determined by qRT-PCR. IL-8 level was measured by enzyme-linked immunosorbent assay (ELISA). The MIC of SHHT was $100{\mu}g/mL$ and the expression of cagA gene was decreased about 25 folds in the presence of SHHT. H. pylori urease was inhibited 90% by SHHT. SHHT inhibited H. pylori adhesion on AGS cell in a concentration dependent manner. mRNA expression of iNOS and IL-8 and the production of NO and IL-8 were significantly decreased in the presence of SHHT. In conclusion, SHHT showed anti-Helicobacter activity and has potent anti-inflammatory effect on H. pylori-induced inflammation in human gastric epithelial AGS cells.

• Journal of Ginseng Research
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• v.30 no.2
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• pp.64-69
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• 2006

Gap junctional channels, allowing rapid intercellular communication and synchronization of coupled cell activities, play crucial roles in many signaling processes, including a variety of cell activities. Consequently, a modulation of the gap junctional intercellular communication (GJIC) should be a potential pharmacological target. In the present, the GJIC of a epithelial-derived rat mammary cells (BICR-M1Rk) was assessed in the presence of ginseng saponin, by using an established method of scrape-loading dye transfer assay. The transfer of Lucifer yellow (diameter: 1.2 nm) among the neighboring BICR-M1Rk cells, in which connexin43 (Cx43) is a major gap junction channel-forming protein, was significantly retarded at a concentration of $10{\mu}g/ml$ ginseng saponin. By using both methods of RT-PCR and Western blotting, it was demonstrated that ginseng saponin modulated neither the mRNA synthesis of Cx43 nor the translational process of Cx43. This ginseng saponin-induced modification of GJIC was a similar phenomenon observed under the $\beta$-glycyrrhetinic acid treatment, a well-known gap junction channel blocker. Taken together, it is reasonable to conclude that the ginseng saponin inhibits GJIC only by modulating the gating property of gap junction channels.

• Journal of Veterinary Clinics
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• v.38 no.6
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• pp.261-268
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• 2021

The study was conducted to evaluate the effects of carboxymethyl chitosan (CMC) on proliferation, migration, and lipopolysaccharide (LPS)-induced inflammatory response in canine bone marrow-derived mesenchymal stem cells (BMSCs). The proliferation and migration of BMSCs were examined after treatment with CMC. The effect of CMC on the mRNA expression of inflammatory cytokines, such as interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, IL-10, and transforming growth factor (TGF)-β, was also evaluated by reverse transcription polymerase chain reaction (RT-PCR). In the proliferation assay, no significant changes were found at all CMC concentrations compared with controls. The migration assay showed that CMC dose-dependently stimulated the migration of BMSCs in normal and LPS-treated conditions. RT-PCR showed that TNF-α and IL-10 expressions were suppressed in the BMSCs after CMC treatment. However, other genes were not affected. Taken together, CMC promoted BMSC migration and inhibited TNF-α and IL-10. Therefore, CMC may be possible to regulate wound healing when mesenchymal stem cells are applied in inflammatory diseases.

• Asian-Australasian Journal of Animal Sciences
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• v.18 no.11
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• pp.1552-1556
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• 2005

The UCPs are members of the mitochondrial inner membrane transporter family, present in the mitochondrial inner membrane. Their main function is increasing the energy expenditure via diminishing the resulting production of ATP from mitochondrial oxidative phosphorylation instead of yielding dissipative heat. They are associated with the metabolism of fat and regulation of energy expenditure. The UCP gene can be viewed as the candidate gene for chicken fatness. In the present study, RT-PCR and Northern Blot methods were developed to investigate the expression of the UCP gene in ten tissues including heart, liver, spleen, lung, kidney, gizzard, intestine, brain, breast muscle and abdominal fat of chicken. The results of both RT-PCR and Northern Blot methods showed that the UCP gene expressed specific in breast muscle. The expression levels of UCP gene in breast muscles from egg-type and meat-type chickens of hatching, 2, 4, 6 and 8 wk of age were detected by RT-PCR assay and results showed that the expression levels of UCP gene were related to breeds. Expression level of UCP gene in layers was higher than that in broilers at various weeks of age except at 6 wk. The UCP gene's expression was higher at 6 wk and had no significant difference among other weeks of age in broilers; in layers the expression level of UCP gene had no significant difference among weeks of age. The experiment results also showed that insulin could increase the expression level of UCP gene by 40% compared with control group.

• Journal of Animal Reproduction and Biotechnology
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• v.34 no.4
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• pp.280-291
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• 2019

Potential utility of 14 candidate housekeeping genes as normalization reference for RT-qPCR analysis with developmental samples (fertilized eggs to late veliger larvae) in Pacific abalone Haliotis discus hannai was evaluated using four different statistical algorithms (geNorm, NormFinder, BestKeeper and comparative ΔCT method). Different algorithms identified different genes as the best candidates, and geometric mean-based final ranking from the most to the least stable expression was as follow: RPL5, RPL4, RPS18, RPL8, RPL7, UBE2, RPL7A, GAPDH, RPL36, PPIB, EF1A, ACTB and B-TU. The findings were further validated via relative quantification of metallothionein (MT) transcripts using the stable and unstable reference genes, and expression levels of MT were greatly influenced according to the choice of reference genes. In overall, our data suggest that RPL5 and RPS18, either singly or in combination, are appropriate for normalizing gene expression in developmental samples of this abalone species, whereas ACTB, B-TU and EF1A are less stable and not recommended. In addition, our findings propose that standard deviations in geometric ranking as well as geometric mean itself should also be taken into account for the final selection of reference gene(s). This study could be a useful basis to facilitate the generation of accurate and reliable RT-qPCR data with developmental samples in this abalone species.

• Parasites, Hosts and Diseases
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• v.50 no.3
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• pp.199-205
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• 2012

Toxoplasmic encephalitis is caused by reactivation of bradyzoites to rapidly dividing tachyzoites of the apicomplexan parasite Toxoplasma gondii in immunocompromised hosts. Diagnosis of this life-threatening disease is problematic, because it is difficult to discriminate between these 2 stages. Toxoplasma PCR assays using gDNA as a template have been unable to discriminate between an increase or decrease in SAG1 and BAG1 expression between the active tachyzoite stage and the latent bradyzoite stage. In the present study, real-time RT-PCR assay was used to detect the expression of bradyzoite (BAG1)- and tachyzoite-specific genes (SAG1) during bradyzoite/tachyzoite stage conversion in mice infected with T. gondii Tehran strain after dexamethasone sodium phosphate (DXM) administration. The conversion reaction was observed in the lungs and brain tissues of experimental mice, indicated by SAG1 expression at day 6 after DXM administration, and continued until day 14. Bradyzoites were also detected in both organs throughout the study; however, it decreased at day 14 significantly. It is suggested that during the reactivation period, bradyzoites not only escape from the cysts and reinvade neighboring cells as tachyzoites, but also converted to new bradyzoites. In summary, the real-time RT-PCR assay provided a reliable, fast, and quantitative way of detecting T. gondii reactivation in an animal model. Thus, this method may be useful for diagnosing stage conversion in clinical specimens of immunocompromised patients (HIV or transplant patients) for early identification of tachyzoite-bradyzoite stage conversion.