• Journal of Ginseng Research
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• v.47 no.2
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• pp.218-227
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• 2023

Background: Myocardial fibrosis (MF) is an advanced pathological manifestation of many cardiovascular diseases, which can induce heart failure and malignant arrhythmias. However, the current treatment of MF lacks specific drugs. Ginsenoside Re has anti-MF effect in rat, but its mechanism is still not clear. Therefore, we investigated the anti-MF effect of ginsenoside Re by constructing mouse acute myocardial infarction (AMI) model and AngII induced cardiac fibroblasts (CFs) model. Methods: The anti-MF effect of miR-489 was investigated by transfection of miR-489 mimic and inhibitor in CFs. Effect of ginsenoside Re on MF and its related mechanisms were investigated by ultrasonographic, ELISA, histopathologic staining, transwell test, immunofluorescence, Western blot and qPCR in the mouse model of AMI and the AngII-induced CFs model. Results: MiR-489 decreased the expression of α-SMA, collagenI, collagen III and myd88, and inhibited the phosphorylation of NF-κB p65 in normal CFs and CFs treated with AngII. Ginsenoside Re could improve cardiac function, inhibit collagen deposition and CFs migration, promote the transcription of miR-489, and reduce the expression of myd88 and the phosphorylation of NF-κB p65. Conclusion: MiR-489 can effectively inhibit the pathological process of MF, and the mechanism is at least partly related to the regulation of myd88/NF-κB pathway. Ginsenoside Re can ameliorate AMI and AngII induced MF, and the mechanism is at least partially related to the regulation of miR-489/myd88/NF-κB signaling pathway. Therefore, miR-489 may be a potential target of anti-MF and ginsenoside Re may be an effective drug for the treatment of MF.

• Journal of Microbiology and Biotechnology
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• v.33 no.3
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• pp.398-409
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• 2023

LncRNAs play crucial roles in the progression of colon adenocarcinoma (COAD), but the role of LINC01232 in COAD has not received much attention. The present study was designed to explore the related mechanisms of LINC01232 in the progression of COAD. LINC01232, miR-181a-5p, p53, c-myc, Bcl-2, cyclin D1, p16, Bax, VEGF, E-cadherin, vimentin, N-cadherin and SDAD1 expressions were determined by western blot and qRT-PCR. CCK-8, tubule formation, and Transwell assays were employed to detect proliferation, angiogenesis, and migration/invasion of COAD cells, respectively. The relationship between LINC01232 and miR-181a-5p was predicted by LncBase Predicted v.2, and then verified through dual luciferase reporter gene assay. According to the results, LINC01232 was highly expressed in COAD cells and enhanced proliferation, angiogenesis, migration, and invasion of COAD cells. Downregulated LINC01232 promoted expression of p53 and p16, and inhibited c-myc, Bcl-2 and cyclin D1 expressions in COAD cells, while upregulation of LINC01232 generated the opposite effects. LINC01232 was negatively correlated with miR-181a-5p while downregulated miR181a-5p could reverse the effects of siLINC01232 on cell proliferation, angiogenesis, migration, and invasion. Similarly, miR-181a-5p mimic could also offset the effect of LINC01232 overexpression. SiLINC01232 increased the expressions of Bax and E-cadherin, and decreased the expressions of VEGF, vimentin, N-cadherin and SDAD1, which were partially attenuated by miR-181a-5p inhibitor. Collectively, LINC01232 enhances the proliferation, migration, invasion, and angiogenesis of COAD cells by decreasing miR-181a-5p expression.

• Journal of Ginseng Research
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• v.47 no.3
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• pp.440-447
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• 2023

Background: The human hair follicle undergoes cyclic phases-anagen, catagen, and telogen-throughout its lifetime. This cyclic transition has been studied as a target for treating hair loss. Recently, correlation between the inhibition of autophagy and acceleration of the catagen phase in human hair follicles was investigated. However, the role of autophagy in human dermal papilla cells (hDPCs), which is involved in the development and growth of hair follicles, is not known. We hypothesized that acceleration of hair catagen phase upon inhibition of autophagy is due to the downregulation of Wnt/β-catenin signaling in hDPCs, and that components of Panax ginseng extract can increase the autophagic flux in hDPCs. Methods: We generated an autophagy-inhibited condition using 3-methyladenine (3-MA), a specific autophagy inhibitor, and investigated the regulation of Wnt/β-catenin signaling using the luciferase reporter assay, qRT-PCR, and western blot analysis. In addition, cells were cotreated with ginsenoside Re and 3-MA and their roles in inhibiting autophagosome formation were investigated. Results: We found that the unstimulated anagen phase dermal papilla region expressed the autophagy marker, LC3. Transcription of Wnt-related genes and nuclear translocation of β-catenin were reduced after treatment of hDPCs with 3-MA. In addition, treatment with the combination of ginsenoside Re and 3-MA changed the Wnt activity and hair cycle by restoring autophagy. Conclusions: Our results suggest that autophagy inhibition in hDPCs accelerates the catagen phase by downregulating Wnt/β-catenin signaling. Furthermore, ginsenoside Re, which increased autophagy in hDPCs, could be useful for reducing hair loss caused by abnormal inhibition of autophagy.

• Journal of Animal Reproduction and Biotechnology
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• v.38 no.3
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• pp.99-108
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• 2023

Background: Efficient gene editing technology is needed for successful knock-in. Homologous recombination (HR) is a major double-strand break repair pathway that can be utilized for accurately inserting foreign genes into the genome. HR occurs during the S/G2 phase, and the DNA mismatch repair (MMR) pathway is inextricably linked to HR to maintain HR fidelity. This study was conducted to investigate the effect of inhibiting MMR-related genes using CdCl₂, an MMR-related gene inhibitor, on HR efficiency in HC11 cells. Methods: The mRNA and protein expression levels of MMR-related genes (Msh2, Msh3, Msh6, Mlh1, Pms2), the HR-related gene Rad51, and the NHEJ-related gene DNA Ligase IV were assessed in HC11 cells treated with 10 μM of CdCl₂ for 48 hours. In addition, HC11 cells were transfected with a CRISPR/sgRNA expression vector and a knock-in vector targeting Exon3 of the mouse-beta casein locus, and treated with 10 μM cadmium for 48 hours. The knock-in efficiency was monitored through PCR. Results: The treatment of HC11 cells with a high-dose of CdCl₂ decreased the mRNA expression of the HR-related gene Rad51 in HC11 cells. In addition, the inhibition of MMR-related genes through CdCl₂ treatment did not lead to an increase in knock-in efficiency. Conclusions: The inhibition of MMR-related gene expression through high-dose CdCl₂ treatment reduces the expression of the HR-related gene Rad51, which is active during recombination. Therefore, it was determined that CdCl₂ is an inappropriate compound for improving HR efficiency.

• The Korean Journal of Physiology and Pharmacology
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• v.28 no.3
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• pp.239-252
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• 2024

Dexmedetomidine displays multiple mechanisms of neuroprotection in ameliorating ischemic brain injury. In this study, we explored the beneficial effects of dexmedetomidine on blood-brain barrier (BBB) integrity and neuroinflammation in cerebral ischemia/reperfusion injury. Sprague-Dawley rats were subjected to middle cerebral artery occlusion (MCAO) for 1.5 h and reperfusion for 24 h to establish a rat model of cerebral ischemia/reperfusion injury. Dexmedetomidine (9 ㎍/kg) was administered to rats 30 min after MCAO through intravenous injection, and SB203580 (a p38 MAPK inhibitor, 200 ㎍/kg) was injected intraperitoneally 30 min before MCAO. Brain damages were evaluated by 2,3,5-triphenyltetrazolium chloride staining, hematoxylin-eosin staining, Nissl staining, and brain water content assessment. BBB permeability was examined by Evans blue staining. Expression levels of claudin-5, zonula occludens-1, occludin, and matrix metalloproteinase-9 (MMP-9) as well as M1/M2 phenotypes-associated markers were assessed using immunofluorescence, RT-qPCR, Western blotting, and gelatin zymography. Enzyme-linked immunosorbent assay was used to examine inflammatory cytokine levels. We found that dexmedetomidine or SB203580 attenuated infarct volume, brain edema, BBB permeability, and neuroinflammation, and promoted M2 microglial polarization after cerebral ischemia/reperfusion injury. Increased MMP-9 activity by ischemia/reperfusion injury was inhibited by dexmedetomidine or SB203580. Dexmedetomidine inhibited the activation of the ERK, JNK, and p38 MAPK pathways. Moreover, activation of JNK or p38 MAPK reversed the protective effects of dexmedetomidine against ischemic brain injury. Overall, dexmedetomidine ameliorated brain injury by alleviating BBB permeability and promoting M2 polarization in experimental cerebral ischemia/reperfusion injury model by inhibiting the activation of JNK and p38 MAPK pathways.

• KSBB Journal
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• v.23 no.3
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• pp.231-238
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• 2008

The second family member of tissue inhibitors of matrix metalloproteinases, TIMP-2, is a 21kDa protein which inhibits matrix metalloproteinases 2 (MMP-2). Expression of mammalian proteins in E. coli often forms inclusion bodies that are made up of mis-folded or insoluble protein aggregates. The requirement for the formation of 6 disulfide bonds in the process of the TIMP-2 folding is likely to be incompatible with the reducing environment of E. coli. However, this incompatibility can be often overcome by introducing a mutagenesis that could lead to enhancement of the protein solubility. In this reason, we have attempted to express the soluble TIMP-2 in E. coli by applying a modified staggered extension process (StEP), one of the in vitro PCR-based recombinant mutagenesis methods, and error-prone PCR. C-terminally located CAT fusion protein with respect to mutated TIMP-2 proteins enables us to differentiate the soluble TIMP-2 from the insoluble in E. coli by virtue of chloramphenicol resistance. According to this scheme, E. coli harboring properly-folded CAT fused to TIMP-2 protein was selected, and some of the resulting colonies exhibited an enhanced, soluble expression of TIMP-2 compared to the wild type, implying (i) the StEP technique is successfully employed to enhance the proper folding thereby increasing the solubility of TIMP-2, and (ii) the CAT dependent screening may be a simple and effective method to differentiate the soluble protein expression in E. coli.

• Journal of Chest Surgery
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• v.42 no.5
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• pp.576-587
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• 2009

Background: The up-regulation of the nitric oxide (NO)-cGMP pathway might be involved in the change of vascular reactivity in rats 3 days after they suffer acute myocardial infarction. However, the underlying mechanism for this has not been clarified. Material and Method: Acute myocardial infarction (AMI) was induced by occluding the left anterior descending coronary artery (LAD) for 30 min (Group AMI), whereas the sham-operated control rats were treated similarly without LAD occlusion (Group SHAM), The concentration-response relationships for phenylephrine (PE), KCl, acetylcholine (Ach) and sodium nitroprusside (SNP) were determined in the endothelium intact E(+) and endothelium denuded E(-) thoracic aortic rings from the rats 3 days after AMI or a SHAM operation. The concentration-response relationships of PE in the E(+) rings from the AMI rats were compared with those relationships in the rings pretreated with nitric oxide synthase (NOS) inhibitor $N{\omega}$-nitro-L-arginine methyl ester (L-NAME) or the cyclooxygenase inhibitor indomethacin. The plasma nitrite/nitrate concentrations were checked via a Griess reaction. The cyclic GMP content in the thoracic aortic rings was measured by radioimmunoassay and the endothelial nitric oxide synthase (eNOS) mRNA expression was assessed by real time PCR. Result: The mean infarct size (%) in the rats with AMI was $21.3{\pm}0.62%$. The heart rate and the systolic and diastolic blood pressure were not significantly changed in the AMI rats. The sensitivity of the contractile response to PE and KCl was significantly decreased in both the E(+) and E(-) aortic rings of the AMI group (p<0.05). L-NAME completely reversed these contractile responses whereas indomethacin did not (p<0.05). Moreover, the sensitivity of the relaxation response to Ach was also significantly decreased in the AMI group (p<0.05). The plasma nitrite and nitrate content (p<0.05), the basal cGMP content (p<0.05) and the eNOS mRNA expression (p=0.056) in the AMI rats were increased as compared with the SHAM group. Conclusion: Our findings indicate that the increased eNOS activity and the up-regulation of the NO-cGMP pathway can be attributed to the decreased contractile or relaxation response in the rat thoracic aorta 3 days after AMI.

• Radiation Oncology Journal
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• v.18 no.4
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• pp.321-328
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• 2000

Purpose :NOS2 induce NO Production and NO activate TGF-${\beta}$. The TGF-${\beta}$ is a inhibitor of NOS2. If this negative feedback mechanism operating in radiation pneumonitis model, NOS2 inhibitor may play a role in TGF-${\beta}$ suppression. We planned this study to evaluate the expression patterns of NO, NOS2 and TGF-${\beta}$ in vivo radiation pneumonitis model. Materials and Methods : Sixty sprague-Dawley rat were irradiated 5 Gy or 20 Gy. They were sacrificed 3, 7, 14, 28 and 56 days after irradiation. During sacrifice, we peformed broncho-alveolar lavage (BAL). The BAL fluids were centrifuged and supernatents were used for measure NO and TGF-${\beta}$, and the cells were used for RT-PCR. Results : After 5 Gy of radiation, NO in BAL fluid increased at 28 days in both lung and TGF-${\beta}$ in left lung at 56 days. NO increased in BAL fluid at 28 days in both lung after irradiation and TGF-${\beta}$ in right lung at 28-56 days after 20 Gy of radiation. After 5 Gy of radiation, NOS2 expression was increased in right lung at 14 days, in both lung at 28 days and in left lung at 56 days. TGF-${\beta}$ expression was reduced in both lung at 28 days and increased in left lung at 56 days. Conclusions :The Proposed feedback mechanism of NO, NOS2 and TGF-${\beta}$ was operated in vivo radiation pneumonitis model. At 56 days, however, NOS2 and TGF-${\beta}$ expressed concurrently in left lung after 5 Gy and in both lung after 20 Gy of radiation.

• The Korean Journal of Nuclear Medicine
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• v.38 no.1
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• pp.99-108
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• 2004

Purpose: The ability to noninvasively track the migration of neural progenitor cells would have significant clinical and research implications. We generated stably transfected F3 human neural progenitor cells with human sodium/iodide symporter (hNIS) for noninvasively tracking F3. In this study, the expression patterns of hNIS gene in F3-NIS were examined according to the cultured time and the epigenetic modulation. Materials and Methods: F3 human neural stem cells had been obtained from Dr. Seung U. Kim (Ajou University, Suwon, Korea). hNIS and hygromycin resistance gene were linked with IRES (Internal Ribosome Entry Site) under control of CMV promoter. This construct was transfected to F3 with Liposome. To investigate the restoration of hNIS gene expression in F3-NIS, cells were treated with demethylating agent (5-Azacytidine) and Histone deacetylase inhibitor (Trichostatin A: TSA). The expression of hNIS was measured by I-125 uptake assay and RT-PCR analysis. Results: The iodide uptake of the F3-NIS was higher 12.86 times than F3 cell line. According to the cell passage number, hNIS expression in F3-NIS gradually diminished. After treatment of 5-Azacytidine and TSA with serial doses (up to $20{\mu}M$, up to 62.5nM, respectively) for 24 hours, I-125 uptake and mRNA of hNIS in F3-NIS were increased. Conclusion: These results suggest that hNIS transfected F3 might undergo a change in its biological characters by cell passage. Therefore, the gene ex[ressopm of exogenous gene transferred human stem cell might be affected to the epigenetic modulation such as promoter methylation and Histone deacetylation and to the cell culture conditions.

• Radiation Oncology Journal
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• v.19 no.3
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• pp.245-251
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• 2001

Prupose : The genes involved on the suppression or radiation-induced apoptosis by genistein in K562 leukemia cell line was investigated. Materials and methods : K562 cells in exponential growth phase were irradiated with a linear accelerator at room temperature. For X-ray irradiation and drug treatment, cultures were prepared at $2\times10^5\;cells/mL$. The cells were irradiated with 10 Gy (Clinac 1800C, Varian, USA), Stock solutions of herbimycin A (HMA, Calbiochem, UK) and genistein (Calbiochem, UK) were prepared in dimethylsulfoxide (DMSO, Sigma, UK). After incubation at $37^{\circ}C$ for 24 h, PCR-select cDNA subtractive hybridization, dot hybridization, DNA sequencing and Northern hybridization were examined. Results : Smad6 gene was identified from the differentially expressed genes in K562 cells incubated with genistein which had been selected by PCR-select cDNA subtractive hybridization. The mRNA expression of Smad6 in K562 cells incubated with genistein was also higher than control group by Northern hybridization analysis. Conclusion : We have shown that Smad6 involved on the suppression of radiation-induced apoptosis by genistein in K562 leukemia cell line. It is plausible that the relationship between Smad6 and the suppression of radiation-induced apoptosis is essential for treatment development based on molecular targeting designed to modify radiation-induced apoptosis.