• Herbal Formula Science
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• v.29 no.2
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• pp.71-80
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• 2021

Objectives : This study investigated cellular-protective effects of Nardotidis seu Sulculii Concha water extract (NSCE) against oxidative stress induced by arachidonic acid (AA)+iron or tert-butylhydroperoxide (tBHP). Methods : In vitro, MTT assay was assessed for cell viability, and immunoblotting analysis was performed to detect expression of AMP-activated kinase (AMPK) signaling pathway and autophagy related proteins. In vivo, mice were orally administrated with the aqueous extract of NSCE of 500 mg/kg for 3 days, and then injected with CCl₄ 0.5 mg/kg body weight to induce acute damage. The level of liver damage was measured by serum aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) analysis. Results : Treatment with NSCE inhibited cell death induced by AA+iron and tBHP. NSCE induced the phosphorylation of AMPK, and this compound also induced the phosphorylation of LKB1, an upstream kinase of AMPK, and Acetyl-CoA carboxylase (ACC), a primary downstream target of AMPK. NSCE increased the protein levels of autophagic markers (LC3II and beclin-1) and decreased the phosphorylation of mammalian target of rapamycin (mTOR) and simultaneously increased the phosphorylation of unc-51-like kinase-1 (ULK-1) in time-dependent manner. Conclusions : NSCE has the ability 1) to protect cells against oxidative stress induced by AA+iron or tBHP. NSCE 2) to activate AMP-activated protein kinase (AMPK), and 3) to regulate autophagy, an important regulator in cell survival.

• Journal of Korean Medicine Rehabilitation
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• v.32 no.2
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• pp.19-36
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• 2022

Objectives This study is to investigate the effects and mechanisms of Imyo-san (IMS) on the obese mice model induced by high-fat diet. Methods Antioxidative capacity was measured by in vitro method. C57BL/6 mice were randomly assigned into 5 groups (n=7). Normal group was fed general diet (Normal). The other 4 groups were fed high fat diet (HFD) with water (Control), with Garcinia gummi-gutta (GG, Garcinia gummi-gutta 200 mg/kg), with low-dose IMS (IMSL, Imyo-san 0.54 g/kg) and with high-dose IMS (IMSH, Imyo-san 1.08 g/kg). Results IMS showed high radical scavenging activity. After 6 week experiment, body weight, food intake, food efficiency ratio (FER), epididymal fat and liver weight, triglyceride (TG), total cholesterol (TC), high density lipoprotein (HDL) cholesterol, low density lipoprotein (LDL) cholesterol, very low density lipoprotein (VLDL) cholesterol, sterol regulatory element-binding protein-1 (SREBP-1), phospho-acetyl-CoA carboxylase (p-ACC), fatty acid synthase (FAS), stearoyl-CoA desaturase-1 (SCD-1), SREBP-2, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), phospho-liver kinase B1 (p-LKB1), phospho-AMP-activated protein kinase (p-AMPK), peroxisome proliferator-activated receptor 𝛼 (PPAR𝛼), peroxisome proliferator-activated receptor 𝛾 coactivator-1𝛼 (PGC-1𝛼), uncoupling protein-2 (UCP-2), carnitine palmitoyltransferase 1A (CPT-1A), and histology of liver and epididymal fat were measured and analysed. Body weight gain, FER, liver and epididymal fat weight of IMS groups were significantly decreased. There were significant improvements in blood lipids with less TG, TC, LDL-cholesterol, VLDL-cholesterol and more HDL-cholesterol. Proteins associated with lipid synthesis (SREBP-1, p-ACC, FAS, SCD-1) and cholesterol (SREBP-2, HMGCR) was improved. Factors regulating lipid synthesis and lipid catabolism (p-LKBI, p-AMPK, PPARα, PGC-1α, UCP-2, CPT-1A) were increased. In histological examinations, IMS group had smaller fat droplets than control group. All results increased depending on concentration. Conclusions It can be suggested that IMS has anti-obesity effects with improving lipid metabolism.

• Herbal Formula Science
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• v.32 no.1
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• pp.51-61
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• 2024

Objectives : This study induced oxidative stress in HepG2 cells by treating them with AA+iron and investigated the effects of forsythia suspensa extract on this stress, as well as elucidated the molecular mechanisms underlying its hepatoprotective effects. Methods : To confirm the antioxidative effects of FSE, HepG2 cells were induced with AA+iron to induce oxidative stress, followed by MTT assay. Additionally, the effect of FSE in reducing the increased ROS levels and mitochondrial damage induced by AA+iron in HepG2 cells was confirmed using FACS. Furthermore, western blot analysis were conducted to investigate the molecular mechanisms underlying the hepatoprotective effects of FSE. Results : FSE increased the decreased cell viability induced by AA+iron. Additionally, FSE normalized the expression of apoptosis-related proteins induced by AA+iron. The elevated ROS levels in HepG2 cells induced by AA+iron were reduced by FSE, and the increase in Rh123-negative cells induced by AA+iron was attenuated by FSE. Moreover, FSE activated the protein expression of AMPK and its related phosphorylating enzymes, LKB1 and ACC. Furthermore, FSE activated YAP and its upstream phosphorylating enzyme, LATS1. Conclusions : These results demonstrate that FSE has an inhibitory effect on oxidative stress induced by AA+iron and may have potential hepatoprotective effects.

• Applied Microscopy
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• v.24 no.2
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• pp.78-92
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• 1994

Lysozyme has been reported to be present in the secretory granules of the Paneth cell, and lysozyme immunoreactivity has been detected by immunogold method in Paneth cells of the intestine of human, mouse and rat. The present study was aimed at clarifying the intracellular distribution and changes of the lysozyme immunoreactivity in rabbit Paneth cell after common bile duct ligation of rabbit, using the electron microscope immunogold technique. Healthy adult rabbits weighing about 2kg body weight were divided into normal and bile duct ligated groups. Common bile duct ligation was performed aseptically under ether anesthesia. Experimental animals were sacrificed on the 1st, the 3rd, the 5th, the 7th and the 14th day after the operation. Mucosal specimens from the intestinal gland of ileum were fixed in 2.5% glutaraldehyde-1.5% paraformaldehyde, followed by 1% osmium tetroxide, embedded in araldite mixture, cut with LKB-V ultratome. Ultrathin sections were placed on parlodion coated nickel grids (200mesh). The section-bearing grids were floated upside down on the added substance in a moist chamber at room temperature except for the primary antibody step, which was at $4^{\circ}C$. Sections were etched with a saturated solution of sodium m-periodate for 60min. After etching, sections were pretreated with 0.02M tris buffered saline (TBS), pH 8.4, with 1% bovine serum albumin (BSA, Sigma) for 60min, then treated polyclonal rabbit anti-human lysozyme (Dakipatts) diluted 1 : 50 in TBS with 0.1% BSA for 20hr. Subsequently, grids were incubated 60min in biotinylated goat anti rabbit IgG (Amersham) diluted 1 : 100 in TBS with 0.1% BSA. After this, sections were incubated 60min on streptavidin gold G10 (Amersham) diluted 1 : 50 in TBS with 0.1% BSA. After each step, the grids were briefly rinsed with TBS with 0.1% BSA. After the strepavidin gold step, the sections were jet washed with distilled water. Counterstain of the sections performed by uranyl acetate and lead citrate, and observed with JEM 100 CX II electron microscope. Observed results were as follow; 1. Secretory granules of mouse Paneth cells have a lysozyme immunoreactivity and also eosinophil leucocyte of rabbit applied for the positive-control stain, are well labeld with gold particles. 2. Normal rabbit Paneth cells have a lysozyme immunoreactivity restricted on the secretory granules. 3. Amount lysosomes containing myelin figures in the Paneth cells were significantly increased from 5th day after the common bile duct ligation. 4. Immunoreactivity of Paneth cell secretory granules were more activated on the 3rd day after the common bile duct ligation as compared with those of the normal animal. But the lysozyme immunoreactivity were decreased from the 5th day after the common bile duct ligation. 5. Considering the above finding, lysozyme contained Paneth cell are affected following of common bile duct ligation, whereas lysosomes containing myelin-figure do not exhibit any immunoreactive relationship with those of secretory granules.

• Applied Microscopy
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• v.15 no.2
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• pp.31-68
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• 1985

Authors are trying to unveil the ultrastructural organization of olfactory bulb, which has been summerized under light microscopic level or communicated only in some detail in different view point until now. For the critical point of view, since the phylogenetical approach will give the ultimate value in the correlative study between structural and functional bases (Brodal, 1969), the present study was carried out light and electron microscopic analyses of the structures of the neurons and synaptic organizations in olfactory bulbs from different animals in phylogenetical scale. We selected each one species from five animal classes: the house rabbit(Oryctolagus cuniculus var. domesticus [Gmelin]) from Mammalia, the domestic fowl (Gallus gallus domesticus Brisson) from Aves, the viper (Agkistrodon hylys [G.P. Pallas]) from Reptilia, a frog (Bombiana orientalis Boulenger) from Amphibia and the crussian carp (Carassius carassius [Linne]) from Pisces. For light microscopic study, samples were fixed in 10% formalin and paraffin sections were stained with hematoxylin-eosin. For the electron microscopic study, the tissues were fixed by perfusion through the heart or immersion with 1% paraform-aldehyde-glutaraldehyde mixture (phosphate buffer, pH 7.4), and final tissue block trimmed under dissecting microscope were osmicated (1% OsO4), they were embedded in Araldite or Epon 812, and ultrathin sections were made by LKB-V ultratome following the inspection of semi-thin sections stained with toluidine blue-borax solution. Ultra-thin sections contrasted with uranyl acetate and lead citrate were observed with JEM 100CX electron microscope. We have summerized our morphological analyses as follows: 1. The olfactory bulb of rabbit, viper and frog shows the eight layers of fila olfactoria, glomerular, external granular, external plexiform, mitral cell, internal plexiform, internal granular, medullary but domestic fowl shows the five layers of glomerular, fibrillar, mitral, granular and medullary and the three layers of fibrilla, glomerular and medullary in crussian carp. The sharpness of demarcation between the layers shows deferential tendency according to phylogenetical order. 2. Mitral cells of vertebrate have large triangular or oval shape with spherical nuclei which contain not so much chromatin. The cytoplasm contains numerous cell organelles, of which Nissl's bodies or granular endoplasmic reticula arranged as parallel strands. Development of granular endoplasmic reticula were declined as the phylogentical grade is going lower. 3. Tufted cells of all animal are mostly spindle or polygonal contour and contain oval nuclei which located in periphery of cytoplasm. The nuclei of rabbit, fowl, viper and frog has relatively space chromatin, but a nucleus of crussian carp contain irregularly aggregated chromatin in karyoplasm. Their cytoplasmic volume and cell organelle contents are in between those of mitral cell and granular cell. They contain moderate amount of mitochondria, granular endoplasmic reticula, a few Golgi complex, polysomes, lysosome, etc. 4. Granule of cells of all the vertebrate amimals studied exhibit similar features; cells and their dense nuclei show spherical or oval contour, and they have the thin rim of cytoplasm which contain only a few cell organelles. 5. In rabbit, the soma of mitral cells were in contact with boutons with two types of synaptic vesicles, that is, round and flat vesicles, especially flat vesicles in boutons were showing reciprocal synapses. However, in domestic fowls, vipers, frogs and crussian carps, there were found boutons showing only spherical synaptic vesicles. 6. The boutons containing round synaptic vesicles were made contact with the some of tufted cell of olfactory bulb in the rabbits, fowls, vipers and frogs, but no synaptic boutons were observed in soma of tufted cells in crussian carps. In the frogs, there were observed dendrites were contact with the soma of tufted cells. 7. In the neuropils of plexiform, granular and glomerular layers olfactory bulbs in the vertebrate, the synapses were axo-large dendrites, axo-median and small dendrites, dendrodendritic, and axo-axonal contacts. However, in the neuropil of crussian carps, synapses were observed only in glomerular layer.