Improved anti-fibrotic effects by combined treatments of simvastatin and NS-398 in experimental liver fibrosis models

Background/Aims@#Efficient anti-fibrotic therapies are required for the treatment of liver cirrhosis. Hydroxymethylglutaryl-coenzyme A reductase inhibitors (statins) and cyclooxygenase-2 (COX-2) inhibitors have been reported to have anti-fibrotic effects. Here, we investigated whether combined treatment with a statin and a COX-2 inhibitor has synergistic anti-fibrotic effects. @*Methods@#The effects of treatment strategies incorporating both simvastatin and a COX-2 inhibitor, NS-398, were investigated using an immortalized human hepatic stellate cell line (LX-2) and a hepatic fibrosis mouse model developed using thioacetamide (TAA) in drinking water. Cellular proliferation was investigated via 5-bromo-2-deoxyuridine uptake. Pro- and anti-apoptotic factors were investigated through Western blotting and real-time polymerase chain reaction analysis. @*Results@#The evaluation of the anti-proliferative effects on LX-2 cells showed that the observed effects were more pronounced with combination therapy than with single-drug therapy. Moreover, hepatic fibrosis and collagen deposition decreased significantly in TAA-treated mice in response to the combined treatment strategy. The mechanisms underlying the anti-fibrotic effects of the combination therapy were investigated. The effects of the combination therapy were correlated with increased expression levels of extracellular signal-regulated kinase 1/2 signaling molecules, upregulation of the Bax/Bcl-2 signaling pathway, inhibition of the transforming growth factor-β signaling pathway, and inhibition of tissue inhibitor of matrix metalloproteinases 1 and 2. @*Conclusions@#The combination of simvastatin and NS-398 resulted in a synergistic anti-fibrotic effect through multiple pathways. These findings offer a theoretical insight into the possible clinical application of this strategy for the treatment of advanced liver diseases with hepatic fibrosis.

Transgenic fluorescent zebrafish lines that have revolutionized biomedical research / 한국실험동물학회지

Since its debut in the biomedical research fields in 1981, zebrafish have been used as a vertebrate model organism in more than 40,000 biomedical research studies. Especially useful are zebrafish lines expressing fluorescent proteins in a molecule, intracellular organelle, cell or tissue specific manner because they allow the visualization and tracking of molecules, intracellular organelles, cells or tissues of interest in real time and in vivo. In this review, we summarize representative transgenic fluorescent zebrafish lines that have revolutionized biomedical research on signal transduction, the craniofacial skeletal system, the hematopoietic system, the nervous system, the urogenital system, the digestive system and intracellular organelles.

Transgenic fluorescent zebrafish lines that have revolutionized biomedical research / 한국실험동물학회지

Since its debut in the biomedical research fields in 1981, zebrafish have been used as a vertebrate model organism in more than 40,000 biomedical research studies. Especially useful are zebrafish lines expressing fluorescent proteins in a molecule, intracellular organelle, cell or tissue specific manner because they allow the visualization and tracking of molecules, intracellular organelles, cells or tissues of interest in real time and in vivo. In this review, we summarize representative transgenic fluorescent zebrafish lines that have revolutionized biomedical research on signal transduction, the craniofacial skeletal system, the hematopoietic system, the nervous system, the urogenital system, the digestive system and intracellular organelles.

Notch Signaling Controls Oligodendrocyte Regeneration in the Injured Telencephalon of Adult Zebrafish

The myelination of axons in the vertebrate nervous system through oligodendrocytes promotes efficient axonal conduction, which is required for the normal function of neurons. The central nervous system (CNS) can regenerate damaged myelin sheaths through the process of remyelination, but the failure of remyelination causes neurological disorders such as multiple sclerosis. In mammals, parenchymal oligodendrocyte progenitor cells (OPCs) are known to be the principal cell type responsible for remyelination in demyelinating diseases and traumatic injuries to the adult CNS. However, growing evidence suggests that neural stem cells (NSCs) are implicated in remyelination in animal models of demyelination. We have previously shown that olig2+ + radial glia (RG) have the potential to function as NSCs to produce oligodendrocytes in adult zebrafish. In this study, we developed a zebrafish model of adult telencephalic injury to investigate cellular and molecular mechanisms underlying the regeneration of oligodendrocytes. Using this model, we showed that telencephalic injury induced the proliferation of olig2+ + RG and parenchymal OPCs shortly after injury, which was followed by the regeneration of new oligodendrocytes in the adult zebrafish. We also showed that blocking Notch signaling promoted the proliferation of olig2+ RG and OPCs in the normal and injured telencephalon of adult zebrafish. Taken together, our data suggest that Notch-regulated proliferation of olig2+ RG and parenchymal OPCs is responsible for the regeneration of oligodendrocytes in the injured telencephalon of adult zebrafish.

Notch Signaling Controls Oligodendrocyte Regeneration in the Injured Telencephalon of Adult Zebrafish

The myelination of axons in the vertebrate nervous system through oligodendrocytes promotes efficient axonal conduction, which is required for the normal function of neurons. The central nervous system (CNS) can regenerate damaged myelin sheaths through the process of remyelination, but the failure of remyelination causes neurological disorders such as multiple sclerosis. In mammals, parenchymal oligodendrocyte progenitor cells (OPCs) are known to be the principal cell type responsible for remyelination in demyelinating diseases and traumatic injuries to the adult CNS. However, growing evidence suggests that neural stem cells (NSCs) are implicated in remyelination in animal models of demyelination. We have previously shown that olig2+ + radial glia (RG) have the potential to function as NSCs to produce oligodendrocytes in adult zebrafish. In this study, we developed a zebrafish model of adult telencephalic injury to investigate cellular and molecular mechanisms underlying the regeneration of oligodendrocytes. Using this model, we showed that telencephalic injury induced the proliferation of olig2+ + RG and parenchymal OPCs shortly after injury, which was followed by the regeneration of new oligodendrocytes in the adult zebrafish. We also showed that blocking Notch signaling promoted the proliferation of olig2+ RG and OPCs in the normal and injured telencephalon of adult zebrafish. Taken together, our data suggest that Notch-regulated proliferation of olig2+ RG and parenchymal OPCs is responsible for the regeneration of oligodendrocytes in the injured telencephalon of adult zebrafish.

Pharmacokinetic and pharmacodynamic integration of enrofloxacin against Salmonella Enteritidis after administering to broiler chicken by per-oral and intravenous routes

It is crucial to optimize the dose of fluoroquinolones to avoid antibiotic resistance and to attain clinical success. We undertook this study to optimize the dose of enrofloxacin against Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) in chicken by assessing its pharmacokinetic/pharmacodynamic (PK/PD) indices. The antibacterial activities of enrofloxacin against S. Enteritidis were evaluated. After administering 10 mg/kg body weight (b.w.) of enrofloxacin to broiler chickens of both sexes by intravenous (IV) and peroral (PO) routes, blood samples were drawn at different intervals and enrofloxacin concentrations in plasma were determined. PK/PD indices were calculated by integrating the PK and PD data. The elimination half-lives (T(1/2)), time required to reach peak concentration (T(max)), peak concentration (C(max)), and area under curve (AUC) after administering enrofloxacin by PO and IV routes were 25.84 ± 1.40 h, 0.65 ± 0.12 h, 3.82 ± 0.59 µg/mL, and 20.84 ± 5.0 µg·h/mL, and 12.84 ± 1.4 h, 0.22 ± 0.1 h, 6.74 ± 0.03 µg/mL, and 21.13 ± 0.9 µg.h/mL, respectively. The bioavailability of enrofloxacin was 98.6% ± 8.9% after PO administration. The MICs of enrofloxacin were 0.0625–1 µg/mL against S. Enteritidis strains, and the MIC₅₀ was 0.50 µg/mL. The C(max)/MIC₅₀ were 7.64 ± 0.2 and 13.48 ± 0.7 and the 24 h AUC/MIC₅₀ were 41.68 ± 0.1 and 42.26 ± 0.3 after administering the drug through PO and IV routes, respectively. The data in this study indicate that the application of 50 mg/kg b.w. of enrofloxacin to chicken through PO and IV routes with a dosing interval of 24 h can effectively cure S. Enteritidis infection, indicating the need for a 5-fold increase in the recommended dosage of enrofloxacin in chicken.

Development of high-performance liquid chromatography methods for the anticoccidials: toltrazuril and diclazuril

This study was undertaken to develop new analytical methods for assessment of anticoccidials. High-performance liquid chromatography (HPLC) was found to be a fast, reliable, and practical method. The anticoccidials used in this experiment were toltrazuril and diclazuril, and the analysis factors were specificity, linearity, accuracy, repeatability, and intermediate precision. The linearity of each anticoccidial was better than 0.99, and the accuracies were 99.5% and 99.1% with relative SD of 0.5 and 0.4, respectively. To assess whether the developed HPLC method could be effectively applied, toltrazuril and diclazuril post-market veterinary products (five products) that are currently sold were tested. The results revealed no non-compliant items and the method was applied successfully. Therefore, the newly developed HPLC method for anticoccidial assessment described in this study may be useful as a reference method in the Korean Standards of Veterinary Pharmaceuticals for the analysis of toltrazuril and diclazuril.

Morphologic Changes of Zebrafish Melanophore after Intense Pulsed Light and Q-Switched Nd:YAG Laser Irradiation

BACKGROUND: Recently, the pulse-in-pulse mode of intense pulsed light (IPL) has been used increasingly for the treatment of melasma. OBJECTIVE: To observe the morphologic changes in the melanophore in adult zebrafish after irradiation with conventional and pulse-in-pulse IPL and Q-switched Nd:YAG (QSNY) laser. METHODS: Adult zebrafish were irradiated with conventional and pulse-in-pulse mode of IPL. The conditions for conventional IPL were 3 mJ/cm², 560 nm filter, and pulse widths of 7, 20, and 35 msec. The pulse-in-pulse conditions were 3 mJ/cm² and on-time 1/off-time 2. The QSNY laser was used with the settings of 1,064 nm, 0.4 J/cm², a 7 mm spot size, and one shot. Specimens were observed using a light microscope, a transmission electron microscope (TEM), a scanning electron microscope (SEM) and a confocal microscope. RESULTS: After conventional IPL irradiation with a 7 msec pulse width, melanophore breakage was observed using light microscopy. Under TEM, irradiation with conventional IPL for 7 msec and pulse-in-pulse IPL induced melanophore thermolysis with vacuolization. However, changes in the melanophore were not observed with 35 msec IPL. Under SEM, unlike the control and QSNY groups, IPL-irradiated zebrafish showed finger-like fusion in the protein structure of scales. Specimens examined by a confocal microscope after conventional IPL irradiation showed a larger green-stained area on TUNEL staining than that after pulse-in-pulse mode IPL irradiation. CONCLUSION: Zebrafish irradiated with long pulse-IPL showed no morphologic changes using light microscopy, while morphological changes in melanophores were evident with use of TEM. Pulse-in-pulse mode IPL caused less damage than conventional IPL.

Influence of Pulse Type on Subcellular Selective Photothermolysis of Melanosomes in Adult Zebrafish Skin Following 1,064-nm, Q-switched, Nd:YAG Laser Irradiation: A Pilot Study

No abstract available.

Protective Role of Trimetazidine Against Neomycin-induced Hair Cell Damage in Zebrafish

OBJECTIVES: Trimetazidine (TMZ) is known to reduce the generation of oxygen-derived free radicals. The objective of the present study was to evaluate the effects of TMZ on neomycin-induced ototoxicity in transgenic zebrafish (Brn3C: EGFP). METHODS: Five-day, postfertilization zebrafish larvae were exposed to 125 microM neomycin and one of the following TMZ concentrations for 1 hour: 10 microM, 100 microM, 500 microM, 1,000 microM, 1,500 microM, or 2,000 microM. Hair cells within the neuromasts of the supraorbital (SO1 and SO2), otic (O1), and occipital (OC1) lateral lines were analyzed using fluorescence microscopy and confocal microscopy (n=10). Hair cell survival was calculated as a percentage of hair cells in the control group that were not exposed to neomycin. Ultrastructural changes were evaluated using scanning electron microscopy. RESULTS: TMZ protected against neomycin-induced hair cell loss in the neuromasts (TMZ 1,000 microM, 11.2+/-0.4 cells; 125 microM neomycin only, 4.2+/-0.5 cells; n=10; P<0.05) and decreased the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) reaction. In the ultrastructural analysis, structures of mitochondria and hair cells within the neuromasts were preserved in zebrafish exposed to 125 microM neomycin and 1,000 microM TMZ. CONCLUSION: TMZ attenuated neomycin-induced hair cell loss in zebrafish. The results of this study suggest that neomycin induces apoptosis, and that apoptotic cell death can be prevented by treatment with tremetazidine.

Microarray Screening for Genes Involved in Oligodendrocyte Differentiation in the Zebrafish CNS

Within the vertebrate nervous system, myelination is required for the normal function of neurons by facilitating the rapid conduction of action potentials along axons. Oligodendrocytes are glial cells which myelinate axons in the central nervous system. Disruption of myelination and remyelination failure can cause human diseases such as multiple sclerosis. Despite the importance of myelination, the molecular basis of oligodendrocyte differentiation and myelination are still poorly understood. To understand the molecular mechanisms which regulate oligodendrocyte differentiation and myelination, novel genes were identified using a microarray analysis. The analysis used oligodendrocyte lineage cells isolated from transgenic zebrafish expressing fluorescent proteins in the oligodendrocyte lineage cells. Seven genes not previously known to be involved in oligodendrocyte differentiation were identified, and their expression during oligodendrocyte development was validated.

Modulation of Glial and Neuronal Migration by Lipocalin-2 in Zebrafish

BACKGROUND: Glial cells are involved in immune and inflammatory responses in the central nervous system (CNS). Glial cells such as microglia and astrocytes also provide structural and functional support for neurons. Migration and morphological changes of CNS cells are associated with their physiological as well as pathological functions. The secreted protein lipocalin-2 (LCN2) has been previously implicated in regulation of diverse cellular processes of glia and neurons, including cell migration and morphology. METHODS: Here, we employed a zebrafish model to analyze the role of LCN2 in CNS cell migration and morphology in vivo. In the first part of this study, we examined the indirect effect of LCN2 on cell migration and morphology of microglia, astrocytes, and neurons cultured in vitro. RESULTS: Conditioned media collected from LCN2-treated astrocytes augmented migration of glia and neurons in the Boyden chamber assay. The conditioned media also increased the number of neuronal processes. Next, in order to further understand the role of LCN2 in the CNS in vivo, LCN2 was ectopically expressed in the zebrafish spinal cord. Expression of exogenous LCN2 modulated neuronal cell migration in the spinal cord of zebrafish embryos, supporting the role of LCN2 as a cell migration regulator in the CNS. CONCLUSION: Thus, LCN2 proteins secreted under diverse conditions may play an important role in CNS immune and inflammatory responses by controlling cell migration and morphology.