• The Journal of Pediatrics of Korean Medicine
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• v.21 no.1
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• pp.117-137
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• 2007

Objectives : In the present study, the author intended to investigate Gagam-jeonggitang(GJT), Gami-hwajeongjeon(GHJ) and Gami-tonggyutang(GTT) significantly affect in vivo and in vitro mucin secretion from airway epithelial cells. Methods : In vivo experiment, the author induced hypersecretion of airway mucin, hyperplasia of tracheal goblet cells and the increase in intraepithelial mucosubstances by exposing rats to SO2 during 3 weeks. Effects of orally-administered GJT, GHJ and GTT during 1 week on in vivo mucin secretion and hyperplasia of tracheal goblet cells were assesed using ELISA and staining goblet cells with alcian blue. For in vitro experiment, confluent HTSE cells were metabolically radiolabeled with 3H-glucosamine for 24 hrs and chased for 30 min in the presence of each agent to assess the effects of each agent on 3H-mucin secretion. Possible cytotoxicities of each agent were assessed by measuring lactate dehydrogenase release. Also, the effects of each agent on contractility of isolated tracheal smooth muscle and effects of each agent on MUC5AC gene expression in cultured HTSE cells were investigated. Results : GJT, GHJ and GTI inhibited hypersecretion of in vivo mucin: GJT and GHJ inhibited the increase of number of goblet cells. However, GTT did not affect the increase of number of goblet cells; GJT and GTT significantly increased mucin secretion from cultured HTSE cells, without significant cytotoxicity. GHJ increased mucin secretion and showed mild cytotoxicity at the highest concentration: GJT, GHJ and GTT chiefly affected the 'mucin' secretion; GJT, GHJ and GTT did not affect Ach-induced contraction of isolated tracheal smooth muscle; GTT did not significantly affect the expression levels of MUC5AC gene. However, GJT significantly. inhibit the expression levels of MUC5AC gene and GHJ significantly increased the expression levels of MUC5AC gene. These results suggest that GJT, GHJ and GTI can increase mucin secretion during short-term treatment(in vitro), whereas it can inihibit hypersecretion of mucin during long-term treatment(in vivo) and GJT and GHJ can not only affect the secretion of mucin but also affect the expression of mucin gene. Conclusions : The author suggests that the effects GJT, GHJ and GTT with their components should be further investigated and it is valuable to find, from oriental medical prescriptions, novel agents which might regulate hypersecretion of mucin from airway epithelial cells.

• The Journal of Pediatrics of Korean Medicine
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• v.21 no.3
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• pp.33-55
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• 2007

Objectives In the present study, the author intended to investigate whether bojung-ikgitang-gamibang(BJGB) and seonbang-paedoktang(SBPT) significantly affect in vivo and in vitro mucin secretion from airway epithelial cells. Methods In vivo experiment, mice's mucin which is on a hypersecretion of airway mucin, mice's tracheal goblet cells in hyperplasia and mice's intraepithelial mucosubstances were exposed with SO2for3weeks. Effects of orally-administered BJGB and SBPT during 1 week on vivo mucin secretion and hyperplasia of tracheal goblet cells were assessed by using both enzyme-linked immunosorbent assay(ELISA) and staining goblet cells with alcian blue. In vitro experiment, confluent hamster tracheal surface epithelial(HTSE) cells were metabolically radiolabeled with 3H-glucosamine for 24hrs and chased for 30 min in the presence of each agent to figure out the effectiveness of 3H-mucin secretion. Total elution profiles of control spent media and treatment sample through Sepharose CL-4B column were analyzed. The effects of each agent on contractility of isolated tracheal smooth muscle and effects of each agent on MUC5AC gene expression in cultured HTSE cells were investigated. Also, possible cytotoxicities of each agent were assessed by measuring lactate dehydrogenase(LDH) release. Additionally, effects of BJGB and SBPT on both MUC5AC gene expression in cultured HTSE cells and TNF- or EGF-induced MUC5AC gene expression in human airway epithelial cells (NCI-H292) were investigated. Results (1) BJGB and SBPT inhibited hypersecretion of in vivo mucin. SBPT also inhibited the increase the number of goblet cells. However, BJGB did not affect the increase of number of goblet cells; (2) BJGB significantly increased mucin secretion from cultured HTSE cells, without significant cytotoxicity, and chiefly affected the 'mucin' secretion; (3) SBPT did not affect mucin secretion from cultured HTSE cells without significant cytotoxicity, and also did not affect the secretion of the other releseable glycoproteins; (4) BJGB and SBPT did not affect Ach-induced contraction of isolated tracheal smooth muscle; (5) SBPT significantly inhibit the expression levels of MUC5AC gene and BJGB significantly increased the expression levels of MUC5AC gene in both HTSE cells and NCI-H292 cells. Conclusions BJGB and SBPT can not only affect the secretion of mucin but also affect the expression of mucin gene. The author suggests that the effects BJGB and SBPT with their components should be further investigated and it is highly desirable to find from oriental medical prescriptions, novel agents which might regulate hypersecretion of mucin from airway epithelial cells.

• Biomolecules & Therapeutics
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• v.18 no.4
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• pp.396-401
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• 2010

We conducted this study to investigate whether baicalin, baicalein or schizandrin significantly affect MUC5AC mucin production induced by epidermal growth factor (EGF) or phorbol ester (PMA) in human airway epithelial cells. Confluent NCI-H292 cells were pretreated with varying concentrations of baicalin, baicalein or schizandrin for 30 min and then stimulated with EGF or PMA for 24 h, respectively. MUC5AC mucin protein production was measured by ELISA. The results were as follows: (1) Baicalin was found to inhibit the production of MUC5AC mucin protein induced by both EGF and PMA. (2) Baicalein, the aglycone of baicalin, also inhibited MUC5AC mucin production. (3) Schizandrin, derived from Schizandrae Fructus, inhibited MUC5AC mucin production by the same inducers. These results suggest that baicalin, baicalein and schizandrin can regulate the production of mucin protein by directly acting on human airway epithelial cells.

• Tuberculosis and Respiratory Diseases
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• v.70 no.3
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• pp.218-223
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• 2011

Background: We investigated whether curcumin and genistein affect the MUC5AC mucin production from human airway epithelial cells that is induced by phorbol 12-myristate 13-acetate (PMA) or tumor necrosis factor-${\alpha}$ (TNF-${\alpha}$). Methods: Confluent NCI-H292 cells were pretreated with each agent for 30 min and then stimulated with PMA or TNF-${\alpha}$ for 24 hours. MUC5AC mucin production was measured by an ELISA. Results: (1) Curcumin dose-dependently inhibited the production of MUC5AC mucin that was induced by PMA or TNF-${\alpha}$; (2) Genistein inhibited PMA-induced MUC5AC mucin production. However, it did not decrease TNF-${\alpha}$-induced MUC5AC mucin production. Conclusion: These results suggest that curcumin and genistein inhibit the production of airway mucin induced by PMA.

• Tuberculosis and Respiratory Diseases
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• v.49 no.1
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• pp.82-92
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• 2000

Background : In chronic airway disease, mucus secretion is increased, but extraction of mucin, which is the main component of mucus secretion, is a very complicated and limited in clinical use. Recently, monoclonal antibody for mucin was developed for possible clinical use. In this study, cellular analysis and quantification of respiratory mucin in sputum of patients with chronic airway diseases were performed. Method : Sputum was collected from patients with asthma(n=33), bronchiectasis(n=8) or chronic bronchitis (n=13) by spontaneous expectoration or by hypertonic saline induction. Collected sputums was treated by 0.1% dithiotreitol to dissociate the disulfide bond of the mucus and filtered through a nylon gauze. Total cell count, viability and differential count were measured. For detection of mucin, collected samples were treated with sodium dodecyl sulfate polyacrylamide gel electrophoresis and then with monoclonal antibody(HMO2), as the primary antibody, and PAS stain. The amount of mucin was measured with ELISA by HMO2. Correlation with clinical information, cellular analysis, and amount of measured mucin were analyzed. Results : Total cell counts of sputum were significantly increased in patients with bronchiectasis but viability remained the same. Eosinophils were significantly increased in patients with asthma, neutrophils in bronchiectasis chronic bronchitis, respectively (p<0.05). The results of Western blotting and PAS staining confirmed the presence of glycoproteins and matched? with mucin. The amounts of mucin measured by ELISA were not significantly different among the disease groups. Significant correlation was identified between the amount of mucin and viability(r=-0.482, p<0.05). Conclusion : Inflammatory cells in the sputum of those with chronic airway disease were different for each disease type. Measurement of mucin by ELISA via monoclonal antibodies may be a simple method for the evaluation of chronic airway disease.

• The Journal of Pediatrics of Korean Medicine
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• v.21 no.3
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• pp.109-124
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• 2007

Objectives In the present study, the author intended to investigate whether Gamitonggyu-tang (GTT) significantly affects (since the subject is GTT, you need an 's') in vivo and in vitro mucin secretion from airway epithelial cells. Methods In vivo experiment, mice's mucin which is on a hypersecretion of an airway, mice's tracheal goblet cells in hyperplasia and mice's intraepithelial mucosubstances were exposed with SO2 for 3 weeks. Effects of orally-administered GTT for 1 week on in vivo mucin secretion and hyperplasia of tracheal goblet cells were assessed by using enzyme-linked immunosorbent assay (ELISA) and staining goblet cells with alcian blue. In vitro experiment, confluent hamster tracheal surface epithelial (HTSE) cells were metabolically radiolabeled with 3H-glucosamine for 24 hrs and chased for 30 min in the presence of GTT to figure out the effectiveness of 3H-mucin secretion. Total elution profiles of control spent media and treatment sample through Sepharose CL-4B column were analyzed.Possible cytotoxicities of each agent were assessed by measuring lactate dehydrogenase (LDH) release. Also, the effect of GTT on contractility of isolated tracheal smooth muscle was investigated. Results (1) GTT inhibited hypersecretion of in vivo mucin. However, it did not affect the increase the number of goblet cells (2) GTT significantly increased mucin release from cultured HTSE cells, without significant cytotoxicity (3) GTT chiefly affected the 'mucin' secretion and did not affect the secretion of the other releasable glycoproteins with less molecular weight than mucin (4) GTT did not affect Ach-induced contraction of isolated tracheal smooth muscle.Conclusions This result suggests that GTT can increase mucin secretion during short-term treatment (in vitro) whereas it can inihibit hypersecretion of mucin during long-term treatment (in vivo). The author suggests that the effect GTT with their components should be further investigated and it is valuable to find from oriental medical prescriptions, novel agents which might regulate mucin secretion from airway epithelial cells.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1996.04a
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• pp.219-219
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• 1996

Mucin release from hamster tracheal surface epithelial(HTSE) cells can be stimulated by extracellular ATP via activation of P$_2$ purinoceptors located on the cell surface which appears to be coupled to phospholipase C via G proteins. However, our preliminary data indicate that the ATP-induced mucin release involves, in part, activation of PKC, but not an increase in the intracellular Ca++ level, suggesting the presence of another pathway which is separate from the PLC-PKC pathway, In this study, we intended to confirm the previous observation and subsequently identify an additional mechanism. Confluent HTSE cells were metabolically labeled with either $^3$H-glucosamine or $^3$H-arachidonic acid(AA), and release of either $^3$H-mucin or $^3$H-AA was quantified following various treatments. $^3$H-mucin was assayed using the sepharose CL-4B gel-filtration method, whereas $^3$H-AA liberation was measured by counting $^3$H-radioactivity in the chase medium. We found that: (1)Desensitization of PKC by pretreatment with PMA completely abolished the mucin releasing effect of PMA but partially inhibited the ATP-induced mucin release; (2) ATP increases release of $^3$H-AA in a dose-dependent fashion; (3) mepacrine, an inhibitor of PLA$_2$, attenuates ATP-induced mucin release in a dose-dependent fashion. These results confirm our previous notion that the PLC-PKC pathway is responsible, in part, for ATP-induced mucin release. Furthermore, activation of PLA$_2$ appears to be an additional pathway which is involved in ATP-induced mucin release.

• Natural Product Sciences
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• v.22 no.4
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• pp.275-281
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• 2016

Perilla frutescens was empirically used for controlling airway inflammatory diseases in folk medicine. We investigated whether caffeic acid, myristicin and rosemarinic acid derived from Perilla frutescens significantly affect the gene expression and production of mucin from airway epithelial cells. Confluent NCI-H292 cells were pretreated with caffeic acid, myristicin or rosemarinic acid for 30 min and then stimulated with phorbol 12-myristate 13-acetate (PMA) for 24 h. The MUC5AC mucin gene expression and production were measured by reverse transcription - polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Additionally, we examined whether caffeic acid, myristicin or rosemarinic acid affects MUC5AC mucin production indued by epidermal growth factor (EGF) and tumor necrosis $factor-{\alpha}$ ($TNF-{\alpha}$), the other two stimulators of production of airway mucin. The results were as follows: (1) Caffeic acid, myristicin and rosemarinic acid inhibited the gene expression and production of MUC5AC mucin induced by PMA from NCI-H292 cells, respectively; (2) Among the three compounds derived from Perilla frutescens, only rosemarinic acid inhibited the production of MUC5AC mucin induced by EGF or $TNF-{\alpha}$, the other two stimulators of production of airway mucin. These results suggest that rosemarinic acid derived from Perilla frutescens can regulate the production and gene expression of mucin, by directly acting on airway epithelial cells and, at least in part, explains the traditional use of Perilla frutescens as remedies for diverse inflammatory pulmonary diseases.

• Archives of Pharmacal Research
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• v.21 no.3
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• pp.253-259
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• 1998

In this study, we developed immunoassay methods for the more convenient and effective detection of rat tracheal mucin and the results were compared with those of [$3^H$]glucosamine based gel-filtratioh method. A monoclonal anti-rat tracheal mucin antibody, mAbRT03, which specifically recognizes rat tracheal mucins, was used throughout in this study. To induce mucin secretion, varying concentrations of ATP (0-2 mM) were applied to the primary rat tracheal surface epithelial (RTSE) cell culture which had been metabolically radiolabeled with [$3^H$]glucosamine and the secretion of mucin was analyzed both by the immunoassay and the gel-filtration chromatography methods. For the immunoassay, the following two procedures were employed. 1) Simple ELISA; the culture spent media were directly coated onto the assay plate and the immunoreactivity with mAbRT03 was assessed from the standard curve generated with the purified rat mucin. 2) Inhibition ELISA; A known amount of the purified rat mucin was coated onto the assay plate and then ATP-stimulated culture spent media were added to inhibit the immunorelitivity with mAbRT03. The contents of mucin in the sample were calculated from the standard inhibition curve which was generated with the purified rat mucin. The assay results obtained from the immunoassays were identical with those from the gel-filtration methods. The present result indicates that ELISA can be substituted for the laborious, time-consuming gel-filtration assay in studying the regulation of airway mucin release from cultured airway epithelial cells.

• The Korean Journal of Physiology and Pharmacology
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• v.10 no.6
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• pp.317-321
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• 2006

In this study, we investigated whether glycyrrhizin, prunetin and morroniside affect mucin secretion from cultured airway epithelial cells and compared the possible activities of these agents with the inhibitory action on mucin secretion by poly-L-lysine (PLL) and the stimulatory action by adenosine triphosphate (ATP). Confluent primary hamster tracheal surface epithelial (HTSE) cells were metabolically radiolabeled using $^{3}H-glucosamine$ for 24 h and chased for 30 min in the presence of varying concentrations of each agent to assess the effects on $^{3}H-mucin$ secretion. The results were as follows: 1) glycyrrhizin and morroniside increased basal mucin secretion from airway; 2) prunetin did not affect basal mucin secretion; 3) glycyrrhizin did not inhibit ATP-induced mucin secretion. We conclude that glycyrrhizin and morroniside can increase basal mucin secretion, by directly acting on airway mucin-secreting cells and suggest that two compounds be further investigated for the possible use as mild expectorants during the treatment of inflammatory airway diseases.