• 대한임상검사과학회지
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• 제44권4호
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• pp.229-238
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• 2012

Cyclooxygenase(COX-2) is an inducible enzyme that catalyzes the synthesis of prostaglandins (PGs) from arachidonic acid. Over-expression of COX-2 has been reported to be associated with progressive hepatic fibrosis in chronic hepatic C infection and rat liver fibrosis induced by carbon tetrachloride($CCl_4$). Recently, it is well known that mast cell products can stimulate the proliferation of hepatic stellate cells and key players in liver fibrosis. But little is known regarding their role in $CCl_4$-induced liver fibrosis in rat. Our aim was to investigate the relation between COX-2 expression and mast cells during liver fibrosis after $CCl_4$ treatment. Thirty Wistar rats were divided into five groups (non-treated 0, 2, 4, 6 and 8-week after $CCl_4$-treatment). Reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry were used to assess the expression of ${\alpha}$-smooth muscle actin (${\alpha}$-SMA), collagen-1 and COX-2 in liver tissue from $CCl_4$-treated rats. The density of collagen and mast cells were determined using a computerized image analysis system in liver sections stained with picrosirius red and toluidine blue, respectively. The expression levels of ${\alpha}$-SMA, collagen-1 and COX-2 mRNA were significantly higher at 2 wk in $CCl_4$-treated groups than non-treated group. The number of mast cells in liver tissues increased gradually from 2 wk to 6 wk depending on the fibrosis severity but decreased abruptly at 8 wk. The significant increase of collagen-1 and ${\alpha}$-SMA mRNA expression in $CCl_4$-treated rats was continued until 6 wk while the COX-2 mRNA was significantly decreased at 8 wk. These results suggest that increased mast cells are closely associated with COX-2 over-expression during hepatic fibrogenesis of $CCl_4$-treated rats.

• Biomolecules & Therapeutics
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• 제32권2호
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• pp.224-230
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• 2024

Pinitol (3-O-Methyl-D-chiro-inositol) has been reported to possess insulin-like effects and is known as one of the anti-diabetic agents to improve muscle, liver, and endothelial cells. However, the beneficial effects of pinitol on the skin are not well known. Here, we investigated whether pinitol had effects on human dermal fibroblasts (HDFs), and human dermal equivalents (HDEs) irradiated with ultraviolet A (UVA), which causes various damages including photodamage in the skin. We observed that pinitol enhanced wound healing in UVA-damaged HDFs. We also found that pinitol significantly antagonized the UVA-induced up-regulation of matrix metalloproteinase 1 (MMP1), and the UVA-induced down-regulation of collagen type I and tissue inhibitor of metalloproteinases 1 (TIMP1) in HDEs. Electron microscopy analysis also revealed that pinitol remarkably increased the number of collagen fibrils with regular banding patterns in the dermis of UVA-irradiated human skin equivalents. Pinitol significantly reversed the UVA-induced phosphorylation levels of ERK and JNK but not p38, suggesting that this regulation may be the mechanism underlying the pinitol-mediated effects on UVA-irradiated HDEs. We also observed that pinitol specifically increased Smad3 phosphorylation, which is representative of the TGF-β signaling pathway for collagen synthesis. These data suggest that pinitol exerts several beneficial effects on UVA-induced damaged skin and can be used as a therapeutic agent to improve skin-related diseases.

• Asian-Australasian Journal of Animal Sciences
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• 제20권11호
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• pp.1655-1661
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• 2007

Prolyl 4-hydroxylase (P4H) plays a central role in collagen synthesis by catalyzing the hydroxylation of the proline residue in the X-Pro-Gly amino acid sequence, and controls the biosynthesis of collagen that influences overall meat quality. In order to verify expression level of the catalytic ${\alpha}$ subunit of P4H, a 2.7 kb clone of the ${\alpha}$ subunit gene for P4H was selected from a cDNA library prepared from the muscular tissue of Sancheong berkshire pigs, and the whole gene sequence was determined. As expression level of the ${\alpha}$ subunit of P4H differed between tissues of pigs, we intended to assess more precisely the level of ${\alpha}$-subunit expression between tissues of Sancheong Berkshire pigs by using RT-PCR. Muscular and adipose tissues were taken from each pig grouped by growth stage (weighing 60, 80, and 110 kg) of Yorkshire and Sancheong Berkshire pigs, and the expression levels of the ${\alpha}$-subunit of P4H were examined. Since there were significant differences in the expression level with respect to variation in growth stage (p<0.01), an attempt was made to identify any influences of pig species and tissue variation. The muscular and adipose tissues of pigs weighing 110 kg showed higher expression levels than pigs weighing 60 kg and 80 kg. In general, significantly higher expression levels were found in muscular than in adipose tissue. The expression levels in Sancheong Berkshire were significantly higher than in Yorkshire pigs (p<0.01 or p<0.05). Since expression level of the ${\alpha}$-subunit of P4H affects the activity of P4H and is connected to the biosynthesis of collagen and increased collagen can improve meat texture, this finding may explain why meat quality of the Sancheong Berkshire pig is acclaimed in Korea. Given the higher expression levels of the ${\alpha}$-subunit gene in adipose than in muscular tissue, and also in the heavier pigs, more intensive studies are required to assess the correlation between expression level of the ${\alpha}$ subunit gene and overall meat quality.

• Biomolecules & Therapeutics
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• 제15권4호
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• pp.261-265
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• 2007

Hydroxyproline (HYP) is a post-translational product of proline hydroxylation catalyzed by an enzyme prolyl 4-hydroxylase (P4H) which plays a crucial role in the synthesis of all collagens. Considering the role of collagen and its significance in many clinically important diseases such as liver fibrosis, a great deal of attention has been directed toward the development of an assay at cell-based system. The reason is that cell-based assay system is more efficient than enzyme-based in vitro system and takes much less time than in vivo system. Several assay procedures developed for P4H are laborious, time-consuming and not feasible for the massive-screening. Here, we report the cell-based assay method of prolyl 4-hydroxylase in immortalized rat hepatic stellate HSC-T6 cells. To optimize the cell culture condition to assay for HYP content, various concentrations of reagents were treated for different times in HSC-T6 cells. Our data showed that the treatment with ascorbate in a hypoxic condition for 24 h resulted in the maximal increase of HYP by 1.8 fold. Alternatively, cobalt chloride ($5\;{\mu}M$) and ascorbate ($50\;{\mu}M$) in normoxic states exhibited similar effect on the production of HYP as in a hypoxic condition. Therefore, cobalt chloride can be substituted for a hypoxic condition when an anaerobic chamber is not available. Rosiglitazone and HOE077, known as inhibitors of collagen, synthesis decreased P4H enzyme activity by 32.3% and 15%, respectively, which coincided with previous reports from liver tissues. The level of the smooth muscle ${\alpha}$-actin, a marker of activated stellate cells, was significantly increased under hypoxia, suggesting that our experimental condition could work for screening the anti-fibrotic compounds. The assay procedure took only 3 days after treatment with agents, while assays from the primary stellate cells or liver tissues have taken several weeks. Considering the time and expenses, this assay method could be useful to screen the compounds for the inhibitor of prolyl 4-hydroxylase.

• Archives of Pharmacal Research
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• 제29권9호
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• pp.762-767
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• 2006

Danshen is an herbal medication frequently used in oriental medicine to treat liver or kidney malfunction. In the course of our studies, we observed that compounds purified from Danshen exhibit an inhibitory activity against Discoidin Domain Receptor 2 (DDR2) tyrosine kinase. Through this inhibition, these compounds also inhibited the growth of HSC T6 cells and suppressed the expression of alpha-smooth muscle actin and MMP2, as well as collagen synthesis, all of which are increased in activated liver stellate cells. Given that activation of liver stellate cells is the hallmark of liver fibrosis and that DDR2 plays a critical role in this activation, these results suggest that one of the pharmacological activities of Danshen extract that protects the liver is the inhibition of key cell-signaling kinases, such as DDR2, in liver stellate cells.

• 대한화학회지
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• 제67권2호
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• pp.129-136
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• 2023

Osteoarthritis is to the abnormality of the inflammatory response of joint tissue caused by various causes such as aging, and muscle loss. In this study, the activity in joint inflammation was verified using SKBE, a plant extract, and the expression levels of arthritis-inducing proteins including MMP-1, MMP-3, MMP-13, and collagen type II in vitro were compared and analyzed. Furthermore, we synthesized α-spinasterol, an active ingredient of SKBE, by the previously reported synthesis method and these findings could provide a new starting point for the development of treatments for osteoarthritis.

• Biomolecules & Therapeutics
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• 제28권4호
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• pp.320-327
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• 2020

In current study, we aimed to investigate whether the gentiopicroside (GPS) derived from Gentiana manshurica Kitagawa could block the progression of alcoholic hepatic steatosis to fibrosis induced by chronic ethanol intake. C57BL/6 mice were fed an ethanol-containing Lieber-DeCarli diet for 4 weeks. LX-2 human hepatic stellate cells were treated with GPS 1 h prior to transforming growth factor-β (TGF-β) stimulation, and murine hepatocyte AML12 cells were pretreated by GPS 1 h prior to ethanol treatment. GPS inhibited the expression of type I collagen (collagen I), α-smooth muscle actin (α-SMA) and tissue inhibitor of metal protease 1 in ethanol-fed mouse livers with mild fibrosis. In addition, the imbalanced lipid metabolism induced by chronic ethanol-feeding was ameliorated by GPS pretreatment, characterized by the modulation of lipid accumulation. Consistently, GPS inhibited the expression of collagen I and α-SMA in LX-2 cells stimulated by TGF-β. Inhibition of lipid synthesis and promotion of oxidation by GPS were also confirmed in ethanol-treated AML12 cells. GPS could prevent hepatic steatosis advancing to the inception of a mild fibrosis caused by chronic alcohol exposure, suggesting GPS might be a promising therapy for targeting the early stage of alcoholic liver disease.

• Journal of Periodontal and Implant Science
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• 제36권3호
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• pp.613-625
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• 2006

Periodontal ligament(PDL) cells and human gingival fibroblasts(HGFs) play important roles in development, regeneration, normal function, and pathologic alteration. PDL cells and HGFs have the similarity related with general characteristics of fibroblast such as spindle shaped morphology, the presence of vimentin intermediate filament and the synthesis of interstitial collagens and fibronectin. There were many studies about the differences between PDL cells and HGFs, but they were not about whole gene level. In this study, we tried to explain the differences of gene expression profiles between PDL cells and HGFs, and the differences among three individuals by screening gene expression patterns of PDL cells and HGFs, using cDNA microarray. Although there were some variants among three experiments, a set of genes were consistentely and differentially expressed in one cell type. Among 3,063 genes, 49 genes were more highly expressed in PDL cells and 12 genes were more highly expressed in HGFs. The genes related with cell structure and motility were expressed more highly in PDL cells. These are cofilin 1, proteoglycan 1 secretory granule, collagen type I(${\alpha}$ 1), adducin gamma subunit, collagen type III(${\alpha}$ 1), fibronectin, lumican(keratan sulfate proteoglycan), and ${\alpha}$ -smooth muscle actin. Tissue inhibitor of metalloproteinase known as the enzyme controlling extracellular matrix with matrix metalloproteinase is more highly expressed in PDL cells, osteoprotegerin known as osteoclastogenesis inhibitory factor is more highly expressed in HGFs. We performed northern blot to verify cDNA microarray results on selected genes such as tissue inhibitor of metalloproteinase, fibronectin, osteoprogeterin. The result of northern blot analysis showed that each cell expressed the genes in similar pattern with cDNA microarray result. This result indicates that cDNA microarray is a reliable method in screening of gene expression profiles.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• 제30권4호
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• pp.271-281
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• 2004

Adenoid cystic carcinoma is malignant tumor in salivary gland, and its behavior is very invasive. Of all malignant tumor adenoid cystic carcinoma is occured in frequency of 4.4% in major salivary gland, and 1.29% in minor salivary gland. Histopathologically, adenoid cystic carcinoma is characterized by a cribriform appearance, and tubular form and solid nest type tumor can be seen. The tumor cell structure composed of modified myoepithelial cell, and basaloid cell. Extracellular matrix of this tumor cell contains variable ground substance with basement membrane component. Basement membrane matrix composed of collagen fibers, glycoproteins, proteoglycans, and its function is well known that it participate in differentiation, proliferation, and growth of tumor cell. Basement membrane molecule is essential for invasion of peripheral nerve, blood vessel, skeletal muscle in tumor cell of adenoid cystic carcinoma. In many studies, the tumor cell of adenoid cystic carcinoma containing modified myoepithelial cell participate in synthesis of proteoglycan. In this study, tissue sample of adenoid cystic carcinoma of human salivary gland were obtained from 15 surgical specimen, and all specimen were routinely fixed in 10% formalin and embedded. Serial $4-{\mu}m$ thick sections were cut from paraffin blocks. the histopathologic evaluation was done with light microscopy. And, the immunohistochemical staining, characteristics of glycosaminoglycan were observed. For biochemical analysis of glycosaminoglycan, isolation of crude glycosaminoglycan from tumor tissue and Western bolt analysis were carried out. With transmission electomicroscopy, tumor cell were observed. Biologic behavior of adenoid cystic carcinoma was observed with distribution and expression of basement membrane of glycosaminoglycan in tumor cells, The results obtained were as follows: 1. In immunohistochemical study, chondroitin sulfate is postively stained in tumor cell and interstitial space, dermatan sulfate is weakly stained in ductal cell. But keratan sulfate is negatively stained. 2. In immunohistochemical study, heparan sulfate is strong positive stained in tumor cell and basement membrane, especially in invasion area to peripheral nerve tissue. 3. In transmission electromicroscpic view, the tumor cells are composed modifed myoepithelial cells, and contains many microvilli and rough endoplasmic reticulum. 4. In Western blot analysis, the expression of glycosaminoglycan is expressed mostly in heparan sulfate. From the results obtained in this study, tumor cell of adenoid cystic carcinoma is composed modified myoepithelial cell, and glycosaminoglycan of basement membrane molecule of heparan sulfate and chondroitin sulfate mostly participate in the development and invasiveness of adenoid cystic carcinoma by immunohistochemical study and western blot analysis.