• The Korean Journal of Physiology and Pharmacology
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• 제28권3호
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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.

• International Journal of Stem Cells
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• 제16권1호
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• pp.93-107
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• 2023

Background and Objectives: Chronic periodontitis can lead to alveolar bone resorption and eventually tooth loss. Stem cells from exfoliated deciduous teeth (SHED) are appropriate bone regeneration seed cells. To track the survival, migration, and differentiation of the transplanted SHED, we used super paramagnetic iron oxide particles (SPIO) Molday ION Rhodamine-B (MIRB) to label and monitor the transplanted cells while repairing periodontal bone defects. Methods and Results: We determined an appropriate dose of MIRB for labeling SHED by examining the growth and osteogenic differentiation of labeled SHED. Finally, SHED was labeled with 25 ㎍ Fe/ml MIRB before being transplanted into rats. Magnetic resonance imaging was used to track SHED survival and migration in vivo due to a low-intensity signal artifact caused by MIRB. HE and immunohistochemical analyses revealed that both MIRB-labeled and unlabeled SHED could promote periodontal bone regeneration. The colocalization of hNUC and MIRB demonstrated that SHED transplanted into rats could survive in vivo. Furthermore, some MIRB-positive cells expressed the osteoblast and osteocyte markers OCN and DMP1, respectively. Enzyme-linked immunosorbent assay revealed that SHED could secrete protein factors, such as IGF-1, OCN, ALP, IL-4, VEGF, and bFGF, which promote bone regeneration. Immunofluorescence staining revealed that the transplanted SHED was surrounded by a large number of host-derived Runx2- and Col II-positive cells that played important roles in the bone healing process. Conclusions: SHED could promote periodontal bone regeneration in rats, and the survival of SHED could be tracked in vivo by labeling them with MIRB. SHED are likely to promote bone healing through both direct differentiation and paracrine mechanisms.

• BMB Reports
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• 제40권4호
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• pp.517-524
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• 2007

There were many different families of zinc finger proteins that contained multiple cysteine and/or histidine residues and used zinc to stabilize their folds. The classical C2H2 zinc finger proteins were the founding members of this superfamily and were among the most abundant proteins in eukaryotic genomes. C2H2 proteins typically contained several C2H2 fingers that made tandem contacts along the DNA. Here we reported a novel C2H2 type zinc finger gene, ZNF438, which encoded 828 amino acids that formed five zinc finger domains. Bioinformatics analysis revealed that the ZNF438 was mapped to human chromosome 10p11.2 and shared 62% identity with rat and mouse homologues. RT-PCR analysis indicated that it was ubiquitously expressed in 18 human adult tissues. With immunofluorescence assay, it was shown that the exogenous Flag-tagged ZNF438 was located in nucleus of COS-7 cells. To further explore the function of ZNF438, we examined the transcriptional activity of ZNF438 protein by transfecting recombinant pM-ZNF438 into mammalian cells. The subsequent analysis based on the duel luciferase assay system showed that ZNF438 was a transcriptional repressor.

• Biomolecules & Therapeutics
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• 제28권3호
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• pp.259-266
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• 2020

The present research work primarily investigated whether spinosin has the potential of improving the pathogenesis of Alzheimer's disease (AD) driven by β-amyloid (Aβ) overproduction through impacting the procession of amyloid precursor protein (APP). Wild type mouse Neuro-2a cells (N2a/WT) and N2a stably expressing human APP695 (N2a/APP695) cells were treated with spinosin for 24 h. The levels of APP protein and secreted enzymes closely related to APP procession were examined by western blot analysis. Oxidative stress related proteins, such as nuclear factor-erythroid 2-related factor 2 (Nrf2), and heme oxygenase-1 (HO-1) were detected by immunofluorescence assay and western blot analysis, respectively. The intracellular reactive oxygen species (ROS) level was analyzed by flow cytometry, the levels of Aβ_1-42 were determined by ELISA kit, and Thioflavin T (ThT) assay was used to detect the effect of spinosin on Aβ_1-42 aggregation. The results showed that ROS induced the expression of ADAM10 and reduced the expression of BACE1, while spinosin inhibited ROS production by activating Nrf2 and up-regulating the expression of HO-1. Additionally, spinosin reduced Aβ_1-42 production by impacting the procession of APP. In addition, spinosin inhibited the aggregation of Aβ_1-42. In conclusion, spinosin reduced Aβ_1-42 production by activating the Nrf2/HO-1 pathway in N2a/WT and N2a/APP695 cells. Therefore, spinosin is expected to be a promising treatment of AD.

• 한국동물위생학회지
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• 제18권1호
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• pp.1-21
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• 1995

To elucidate pathogenesis of bovine leukemia virus(BLV) in Korean native goats, the goats experimentally infected with BLV were studied especially for the aspects of infectivity and hematological changes. The experimental goats were examined for 27 months by agar-gel immunodiffusion(AGID) test and syncytium formation assay. During this period, changes of total leucocyte, absolute Iymphocyte and atypical Iymphocyte were examined, and the distribution of surface immunoglobulin ( sIg ) -bearing cells and rosette forming cell (RFC) in the peripheral Iymphocyte were also investigated. By indirect immunofluorescence (IFA) and complement dependent antibody cytotoxicity (CDAC) assay using monoclonal antibody(Mab) against bovine leukosis tumor-associated anti-gen(BL-TAA), changes of BL-TAA positive Iymphocyte in peripheral blood were measured. The results obtained through the experiment were summarized as follows. 1. Antibody titers were measured by AGID using gP51 and P24 antigens. The animals were serologically converted at 2 months post-inoculation(pi) in gP51 antigen, whereas sero-converted at 4 months pi in P24 antigen. In comparison with antibody titers for gP51, P24 antigen showed lower titers throughout the trial period. 2. The peripheral lymphocytes from all of the infected goats, as co-cultivated with F8l cells manifested syncytial formation at 4 months pi. 3. On counting total leucocyte, Iymphocyte and atypical Iymphocyte, two out of four infected goats showed normal distribution, while No 2 of the remaining two revealed temporal and No 3, Persistant increasing number of the cells. 4. The optimal condition of rosette formation of the peripheral Iymphocyte of normal Korean native goats was shown in the sheep erythrocyte treated with 0.1M AET for 30 nun at $37^{\circ}C$. When the Iymphocytes were treated in nylon wool column, the number of sIg-bear-ing cell were increased in the nylon wool adherent cells, but RFC was increased in the non-adherent cells. Of the infected goats, No 2 and No 3 showed significantly increasing number of sIg-bearing cells at 18 months pi. 5. The Iymphocytes of No 2 and No 3 goats reacted positively in IFA using Mab against BL-TAA at 12 months pi and 18 months pi, respectively. In CDAC test, all of four infected goats revealed positive reaction at 24 months pi. The higher positive rates were observed in No 2 and No 3 as compared with the remainders.

• 대한수의학회지
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• 제30권2호
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• pp.177-186
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• 1990

To investigate the etiology, pathogenicity and virological properties of NYJ-1-87 strain of Aujeszky's disease virus (ADV) that was isolated from the diseased piglet in Korea, the virus at $10^{6.0}TCID_{50}/0.1ml$ was inoculated intranasally and subcutaneously into 30 to 35 days-old piglets. Results obtained through the experiments were summarized as follows. 1. Ten of the infected piglets were clinically observed for 15 days. On the 2nd day post-inoculation(pi), the signs of pyrexia, anorexia and convulsion were noted. On the 4th to 7th days pi, nervous signs of incoordination and intermittent spasm were shown in the most of piglets, and one out of 5 piglets infected intranasally was died with severe nervous signs at the 7th day pi. The signs became relieved on the 8th day pi and all of remainder were completely recovered on the 13th to 14th days pi. 2. In hematological study, prominent decrease in the number of total leukocyte and lymphocyte was shown in the ADV-infected piglets on the 6th day pi. On the 8th day pi, the cell numbers were slightly increased and returned to normal level on the 10th day pi. 3. Viral excretion of the ADV-inoculated piglets was examined by swabbing of nasal and oral cavities, and rectal feces. During the periods of the 3rd to 11th days pi, the virus was excreted intermittently from nasal and oral cavities, and rectal feces. The nasal excretions were shown the highest virus concentration of $10^{5.2}TCID_{50}/0.1ml$ at the 5th day pi. 4. Recovery of the inoculated virus from various organs of the piglets that were died or experimentally slaughtered was attempted, and the virus was isolated from the tissues of brain and tonsil by the cultured cell-inoculation method. The highest recovery rate was noted in the tonsil. By indirect immunofluorescence antibody assay using ADV-monoclonal antibody, the viral antigens were detected in tissues of spleen and liver as well as brain and tonsil on the 7th to 9th days pi. The virus was not isolated from blood and the tissues of lung and kidney throughout the experiments. 5. Titers of virus neutralizing antibody in the piglets experimentally infected with ADV became increased after the 6th to 9th days pi in both of intranasal and subcutaneous inoculation showing the highest titers of 64 to 128 on the 29th day pi. When the antibody levels were measured by radial immunodiffusion enzyme assay, the reactive diameter was enlarged to be positive after the 4th to 6th days pi in both of intranasal and subcutaneous inoculation showing the largest diameter of 13 to 14mm on the 29th day pi.

• Journal of Ginseng Research
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• 제42권4호
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• pp.562-570
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• 2018

Background: Lung cancer is the leading cause of cancer-related death worldwide. In this study, we used a bioactivity-guided isolation technique to identify constituents of Korean Red Ginseng (KRG) with antiproliferative activity against human lung adenocarcinoma cells. Methods: Bioactivity-guided fractionation and preparative/semipreparative HPLC purification were used with LC/MS analysis to separate the bioactive constituents. Cell viability and apoptosis in human lung cancer cell lines (A549, H1264, H1299, and Calu-6) after treatment with KRG extract fractions and constituents thereof were assessed using the water-soluble tetrazolium salt (WST-1) assay and terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining, respectively. Caspase activation was assessed by detecting its surrogate marker, cleaved poly adenosine diphosphate (ADP-ribose) polymerase, using an immunoblot assay. The expression and subcellular localization of apoptosis-inducing factor were assessed using immunoblotting and immunofluorescence, respectively. Results and conclusion: Bioactivity-guided fractionation of the KRG extract revealed that its ethyl acetate-soluble fraction exerts significant cytotoxic activity against all human lung cancer cell lines tested by inducing apoptosis. Chemical investigation of the ethyl acetatesoluble fraction led to the isolation of six ginsenosides, including ginsenoside Rb1 (1), ginsenoside Rb2 (2), ginsenoside Rc (3), ginsenoside Rd (4), ginsenoside Rg1 (5), and ginsenoside Rg3 (6). Among the isolated ginsenosides, ginsenoside Rg3 exhibited the most cytotoxic activity against all human lung cancer cell lines examined, with $IC_{50}$ values ranging from $161.1{\mu}M$ to $264.6{\mu}M$. The cytotoxicity of ginsenoside Rg3 was found to be mediated by induction of apoptosis in a caspase-independent manner. These findings provide experimental evidence for a novel biological activity of ginsenoside Rg3 against human lung cancer cells.

• Journal of Ginseng Research
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• 제44권5호
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• pp.704-716
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• 2020

Background: Ginsenoside Rh2 (GRh2) is a characterized component in red ginseng widely used in Korea and China. GRh2 exhibits a wide range of pharmacological activities, such as anti-inflammatory, antioxidant, and anticancer properties. However, its effects on Toxoplasma gondii (T. gondii) infection have not been clarified yet. Methods: The effect of GRh2 against T. gondii was assessed under in vitro and in vivo experiments. The BV2 cells were infected with tachyzoites of T. gondii RH strain, and the effects of GRh2 were evaluated by MTT assay, morphological observations, immunofluorescence staining, a trypan blue exclusion assay, reverse transcription PCR, and Western blot analyses. The in vivo experiment was conducted with BALB/c mice inoculated with lethal amounts of tachyzoites with or without GRh2 treatment. Results and conclusion: The GRh2 treatment significantly inhibited the proliferation of T. gondii under in vitro and in vivo studies. Furthermore, GRh2 blocked the activation of microglia and specifically decreased the release of inflammatory mediators in response to T. gondii infection through TLR4/NF-κB signaling pathway. In mice, GRh2 conferred modest protection from a lethal dose of T. gondii. After the treatment, the proliferation of tachyzoites in the peritoneal cavity of infected mice markedly decreased. Moreover, GRh2 also significantly decreased the T. gondii burden in mouse brain tissues. These findings indicate that GRh2 exhibits an antieT. gondii effect and inhibits the microglial activation through TLR4/NF-κB signaling pathway, providing the basic pharmacological basis for the development of new drugs to treat toxoplasmic encephalitis.

• Journal of Ginseng Research
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• 제41권3호
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• pp.411-418
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• 2017

Background: Recently, protein from ginseng was studied and used for the treatment of several kinds of diseases. However, the effect of ginseng total protein (GTP) on proliferation and wound healing in fibroblast cells remains unclear. Methods: In this study, cell viability was analyzed using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. Cell cycle distribution was analyzed by flow cytometer. The levels of transforming growth factor ${\beta}1$, vascular endothelial growth factor, and collagens were analyzed by enzyme-linked immunosorbent assay and immunofluorescence staining. The expressions of cyclin A, phosphorylation of extracellular signal-related kinase (p-ERK1/2), and ERK1/2 were analyzed by Western blotting. Results: Our results showed that GTP promoted cell proliferation and increased the percentage of cells in S phase through the upregulation of cyclin A in NIH/3T3 cells. We also found that GTP induced the secretion of type I collagen, and promoted the expression of other factors that regulate the synthesis of collagen such as transforming growth factor ${\beta}1$ and vascular endothelial growth factor. In addition, the phosphorylation of ERK1/2 at Thr202/Tyr204 was also increased by GTP. Conclusion: Our studies suggest that GTP promoted proliferation and secretion of collagen in NIH/3T3 cells by activating the ERK signal pathway, which shed light on a potential function of GTP in promoting wound healing.

• 한국안광학회지
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• 제13권4호
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• pp.161-169
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• 2008

목적: 알칼리 화상 후 초기 임상적 손상반응의 진행과 치료를 위한 각막 재생의 이해를 높이기 위하여, 화학적 손상 후 동반하는 다양한 인자에 대한 면역조직화학적 변화를 조사하였다. 방법: 알칼리 화상을 입은 각막의 자가치유과정을 면역형광염색법과 H-E 염색, 그리고 TUNEL assay를 통해 면역조직화학적 측면에서 관찰하였다. 결과: 화상 후 각막의 치유는 진행되었지만 각막기질(stroma)과 내피세포의 세포사는 지속적으로 관찰되었다. 각막가장자리의 혈관신생과 손상된 각막의 ${\alpha}$-SMA의 발현은 알칼리 화상 3일 후부터 나타났으며, 각막기질에서의 콜라젠 III(collagen III)의 형성과 콘드로이친황산(chondroitin sulfate)의 발현은 ${\alpha}$-smooth muscle actin(${\alpha}$-SMA)와 transforming growth factor-${\beta}$(TGF-${\beta}$)의 발현증가와 일치하는 결과를 얻었다. 결론: 각막혼탁을 막기 위해서는 알칼리 화상 후 3일 이내에 혈관신생, 콜라젠 및 콘드로이친황산의 형성을 억제하는데 주력하는 치료가 효과적일 것이라 사료된다. 이 연구는 알칼리 화상을 입은 각막의 치유과정에 있어서의 면역조직화학적 지식을 제공함으로써, 각막의 재생을 촉진하는 치료제의 개발과 이용에 초석이 되리라 사료된다.