• The Journal of Pediatrics of Korean Medicine
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• v.28 no.2
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• pp.56-71
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• 2014

Objectives In this study, the author tried to investigate whether piryongbang-gamgil-tang (PGGT) significantly affect in vitro airway mucin secretion, PMA- or EGF- or TNF-${\alpha}$-induced MUC5AC mucin production / gene expression from human airway epithelial cells and increase in airway epithelial mucosubstances and hyperplasia of tracheal goblet cells of rats. Materials and Methods For in vitro experiment, confluent RTSE cells were chased for 30 min in the presence of PGGT to assess the effect of PGGT on mucin secretion by enzyme-linked immunosorbent assay (ELISA). Also, effect of PGGT on PMA- or EGFor TNF-${\alpha}$-induced MUC5AC mucin production and gene expression from human airway epithelial cells (NCI-H292) were investigated. Confluent NCI-H292 cells were pretreated for 30 min in the presence of PGGT and treated with PMA (10 ng/ml) or EGF (25 ng/ml) or TNF-${\alpha}$ (0.2 nM) for 24 hrs, to assess both effect of PGGT on PMA- or EGF- or TNF-${\alpha}$-induced MUC5AC mucin production by ELISA and gene expression by reverse transcription-polymerase chain reaction (RT-PCR). For in vivo experiment, the author induced hypersecretion of airway mucus and goblet cell hyperplasia by exposure of rats to $SO_2$ during 3 weeks. Effect of orally-administered PGGT during 2 weeks on increase in airway epithelial mucosubstances from tracheal goblet cells of rats and hyperplasia of goblet cells were assesed by using histopathological analysis after staining the epithelial tissue with alcian blue. Possible cytotoxicities of PGGT in vitro were assessed by examining LDH release from RTSE cells and the rate of survival and proliferation of NCI-H292 cells. In vivo liver and kidney toxicities of PGGT were evaluated by measuring serum GOT/GPT activities and serum BUN/creatinine concentrations of rats after administering PGGT orally. Results (1) PGGT did not affect in vitro mucin secretion from cultured RTSE cells. (2) PGGT significantly inhibited PMA-, EGF-, and TNF-${\alpha}$-induced MUC5AC mucin productions and the expression levels of MUC5AC mRNA from NCI-H292 cells. (3) PGGT decreased the amount of intraepithelial mucosubstances and showed the tendency of expectorating airway mucus already produced. (4) PGGT increased LDH release from RTSE cells. However, PGGT did not show in vivo liver and kidney toxicities and cytotoxicity to NCI-H292 cells. Conclusion The result from this study suggests that PGGT can regulate the production and gene expression of airway mucin observed in diverse respiratory diseases accompanied by mucus hypersecretion and do not show in vivo toxicity to liver and kidney functions after oral administration. Effect of PGGT with their components should be further studied using animal experimental models that reflect the diverse pathophysiology of respiratory diseases through future investigations.

• Natural Product Sciences
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• v.20 no.3
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• pp.196-201
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• 2014

Pyunkang-hwan (Pyunkang-tang) extract (PGT) is a traditional folk medicine for controlling diverse pulmonary diseases including bronchitis, tonsiltis and pneumonitis. We investigated whether PGT significantly affects secretion, production and gene expression of airway mucin using in vivo and in vitro experimental models reflecting the hypersecretion and/or hyperproduction of mucus observed in inflammatory pulmonary diseases. For in vivo experiment, effect of PGT was checked on hypersecretion of pulmonary mucin in sulfur dioxide-induced bronchitis in rats. For in vitro experiment, confluent NCI-H292 cells were pretreated with PGT for 30 min and then stimulated with EGF (epidermal growth factor), PMA (phorbol 12-myristate 13-acetate) or TNF-${\alpha}$ (tumor necrosis factor-${\alpha}$) for 24 h. The MUC5AC mucin gene expression and mucin protein production were measured by RT-PCR and ELISA. The results were as follows: (1) PGT inhibited the expression of MUC5AC mucin gene induced by EGF, PMA or TNF-${\alpha}$ from NCI-H292 cells, respectively; (2) PGT also inhibited the production of MUC5AC mucin protein induced by the same inducers from NCI-H292 cells, respectively; (3) PGT inhibited secretion of mucin in sulfur dioxide-induced bronchitis rat model. This result suggests that PGT can regulate secretion, production and gene expression of airway mucin.

• The Journal of Pediatrics of Korean Medicine
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• v.31 no.2
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• pp.1-13
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• 2017

Objectives In this study, the author intended to investigate whether Gami-cheongpetang (GCP), Gagam-jeongkitang (GJG), Gami-samooltang (GSM) and Gami-ijoongtang (GIJ) significantly affect in vivo (animal model) and in vitro (cultured cells) mucin secretion and MUC5AC gene expression in airway epithelial cells. Methods For in vivo experiment, the author induced hypersecretion of airway mucin in rats by introducing SO2 for 3 weeks. Enzyme-linked immunosorbent assay (ELISA) was used to assess the effects of orally-administered GCP, GJG, GSM and GIJ in vivo mucin secretion from tracheal goblet cells of rats after 1 week. Also, the effects of the agents on TNF- or EGF-induced MUC5AC gene expression in human airway epithelial cells (NCI-H292) were investigated. Possible cytotoxicities of the agents were assessed by examining the rate of survival and proliferation of NCI-H292 cells. Results (1) GCP and GJG significantly inhibited hypersecretion of in vivo mucin, although GSM and GIJ did not affect hypersecretion of in vivo mucin; (2) GCP and GJG significantly increased in vitro mucin secretion from cultured HTSE cells. However, GSM and GIJ did not affect in vitro mucin secretion from HTSE cells; (3) GCP and GJG significantly inhibited the expression levels of EGF-induced MUC5AC gene in NCI-H292 cells. However, GSM and GIJ increased the expression levels of EGF-induced MUC 5AC gene in NCI-H292 cells; (4) GCP, GJG, GSM and GIJ did not significantly inhibit the survival and proliferation of NCI-H292 cells. Conclusions These results suggest that GCP, GJG, GSM and GIJ can not only affect the secretion of mucin but also affect the expression of mucin gene. The author suggests that the effects of GCP, GJG, GSM and GIJ with their components should be further investigated by using animal experimental models that simulate the diverse pathophysiology of pulmonary diseases.

• Journal of Physiology & Pathology in Korean Medicine
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• v.22 no.6
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• pp.1500-1508
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• 2008

The author investigated whether Chwiyeon-tang(PC), Haengso-tang(PH), Jawanchihyo-san(PJ) and Gamisocheongryong-tang(PS) significantly affect both PMA-induced mucin production and MUC5AC gene expression in airway epithelial cells and sulfur-dioxide-induced airway goblet cell hyperplasia and mucus hypersecretion animal model using rat. Possible cytotoxicity of each herbal medicine was assessed by measuring the survival and proliferation rate of NCI-H292 cells. Confluent NCI-H292 cells were pretreated for 30 min in the presence of PC, PH, PJ and PS, respectively, and treated with PMA(10 $ng/m{\ell}$), to assess the effect of each herbal medicine on PMA-induced mucin production by enzyme-linked immunosorbent assay(ELISA). Effects of each herbal medicine on PMA-induced MUC5AC gene expression from the same cells were investigated. Also, hypersecretion of airway mucus and goblet cell hyperplasia were induced by exposure of rats to $SO_2$ during 3 weeks. Effects of orally-administered PC, PH, PJ and PS during 1 week on intraepithelial mucosubstances and hyperplasia of goblet cells were examined using histological analysis after staining the epithelial tissue with PAS-alcian blue. (1) PC, PJ, PS and PH did not show significant effects on the survival and proliferation of NCI-H292 cells ; (2) PC, PJ and PS significantly decreased PMA-induced mucin production from NCI-H292 cells ; (3) PC, PJ and PS significantly inhibit the expression levels of PMA-induced MUC5AC gene in NCI-H292 cells ; (4) Among PC, PJ, PS and PH, only PS decreased $SO_2$-induced hyperplasia of airway goblet cells and intraepithelial mucosubstances. This result suggests that PC, PJ and PS can not only affect the production of mucin but also affect the expression of mucin gene and this can explain, at least in part, the traditional use of PC, PJ and PS for controlling airway diseases showing hypersecretion of mucus in oriental medicine.

• Journal of the Korean Society of Tobacco Science
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• v.32 no.1
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• pp.19-27
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• 2010

The aim of this study is to compare the sensitivity of both two NCI-H292 and A549 cell types to acute inflammatory responses induced by cigarette smoke. For this, we treated two kinds of smoke fractions derived from 2R4F reference cigarettes: total particulate matter(TPM) collected onto a Cambridge filter pad and gas/vapor phase(GVP) prepared by bubbling through in buffer solution. When we measured cellular cytotoxicity by neutral red uptake assay after treatment for 24 hours, TPM and GVP induced cytotoxic effect in a dose-dependent manner in the range of 10-$100{\mu}g$/mL and 60-$300 {\mu}g$/mL., respectively, in both cell types without any cellular difference. Additionally, when we examined acute inflammatory responses by analyzing cytokines secreted into culture media including tumor necrosis factor-$\alpha$ (TNF-$\alpha$), interleukin-8(IL-8), and transforming growth factor-$\alpha$(TGF-$\alpha$) as well as matrix metalloproteinase-1(MMP-1), the treatment with smoke fractions increased those marker proteins in a dose-dependent manner in NCI-H292. Meanwhile, in A549 cells only MMP-1 was observed to be increased in a dose-dependent fashion. Collectively, our data indicate that NCI-H292 cell type is more sensitive to cigarette smoke-induced inflammatory response than A549 cells. This suggests that NCI-H292 could be useful as an in vitro evaluation tool to assess harmful effects of cigarette smoke.

• The Journal of Korean Medicine
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• v.29 no.3
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• pp.76-87
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• 2008

Objectives: In this study, the author investigated whether Haengso-tang (HST) and Chwiyeon-tang (CHT) affect both in vitro mucin secretion and MUC5AC gene expression in airway epithelial cells and in vivo mucin secretion from animal model for airway mucus hypersecretion. Materials and Methods: Confluent HTSE cells (non-labeled) were chased for 30 min in the presence of HST and CHT to assess the effects of the agents on mucin secretion by enzyme-linked immunosorbent assay (ELISA), with removal of oriental herbal medicine extract from each agent-treated sample by centrifuge microfilter. Also, the effects of the agents on TNF- or EGF-induced MUC5AC gene expression in human airway epithelial cells (NCI-H292) were investigated. The author also induced hypersecretion of airway mucus by exposure of rats to SO2 for 3 weeks. Effects of orally-administered HST and CHT during 1 week on in vivo mucin secretion from tracheal goblet cells of rats were assessed using ELISA. Results: (1) HST significantly decreased in vitro mucin secretion from cultured HTSE cells. However, CHT did not affect in vitro mucin secretion from HTSE cells; (2) CHT significantly inhibited the expression levels of EGF- or TNF-alpha-induced MUC5AC gene in NCI-H292 cells. However, HST did not affect the expression levels of EGF- or TNF-alpha-induced MUC5AC gene in NCI-H292 cells; (3) CHT significantly inhibited hypersecretion of in vivo mucin. However, HST did not affect hypersecretion of in vivo mucin. Conclusion: These results suggest that CHT can not only affect the secretion of mucin but also the expression of the mucin gene and could be helpful for treating pulmonary disease caused by secretion of mucin.

• The Journal of Internal Korean Medicine
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• v.28 no.4
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• pp.797-807
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• 2007

Objectives : The purpose of this study was to investigate whether Mahwangyunpye-tang(MYT) significantly affects mucin secretion from airway epithelial cells. Methods : 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 MYT to assess the effect of the agent on 3H-mucin secretion. Total elution profiles of control spent media and treatment sample through Sepharose CL-4B column were analyzed. Effect of MYT on contractility of isolated tracheal smooth muscle was investigated; also investigated was effect of the agent on MUC5AC gene expression in cultured NCI-H292 cells. Possible cytotoxicities of the agent were assessed both by measuring lactate dehydrogenase (LDH) release from HTSE cells and examining the rate of survival and proliferation of NCI-H292 cells. Results : MYT significantly increased mucin secretion from cultured HTSE cells, without significant cytotoxicity. MYT chiefly affected the 'mucin' secretion. MYT inhibited Acetylcholine-induced contraction of isolated tracheal smooth muscle. MYT did not significantly affect the expression levels of MUC 5AC gene in cultured NCI-H292 cells. Conclusions : Based on the above results, it is suggested that MYT increased mucin secretion from cultured HTSE cells without significant cytotoxicity and inhibited contraction of isolated tracheal smooth muscle.

• Biomolecules & Therapeutics
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• v.19 no.1
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• pp.65-69
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• 2011

In this study, we investigated whether ambroxol significantly affects secretion, production and gene expression of mucin from cultured airway epithelial cells. Confluent primary rat tracheal surface epithelial (RTSE) cells were pretreated with adenosine triphosphate (ATP) for 5 min and then treated for 30 min with ambroxol to assess the effect on mucin secretion using ELISA. Additionally, confluent NCI-H292 cells were pretreated with ambroxol for 30 min and then stimulated with EGF or PMA for 24 h. The MUC5AC mucin gene expression and mucin protein production were measured by RT-PCR and ELISA. The results were as follows: (1) ambroxol did not significantly affect ATP-induced mucin secretion from cultured RTSE cells; (2) ambroxol inhibited the production of MUC5AC mucin protein induced by EGF and PMA in NCI-H292 cells; (3) ambroxol also inhibited the expression of MUC5AC mucin gene induced by EGF and PMA in NCI-H292 cells. This result suggests that ambroxol can inhibit the production and gene expression of MUC5AC mucin, by directly acting on human airway epithelial cells.

• The Journal of Internal Korean Medicine
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• v.29 no.2
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• pp.318-333
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• 2008

Objectives : In this study, the author tried to examine whether Cheogjogupye-tang (淸燥救肺湯, CGPT) and Yieum-jeon (理陰煎, YEJ) significantly affect in vitro and in vivo mucin secretion, MUC5AC gene expression in airway epithelial cells and contractility of isolated tracheal smooth muscle of rabbit. Materials and Methods : For in vitro experiment, confluent hamster tracheal surface epithelial (HTSE) cells were chased for 30 minutes in the presence of CGPT and YEJ to assess the effects of the agents on mucin secretion by enzyme-linked immunosorbent assay (ELISA), with removal of oriental herbal medicine extract from each agent-treated sample by centrifuge microfilter. Also, the effects of the agents on TNF-alpha or EGF-induced MUC5AC gene expression in human airway epithelial cells (NCI-H292) were investigated. Possible cytotoxicities of the agent were assessed by examining both LDH release from HTSE cells and the rate of survival and proliferation of NCI-H292 cells. For in vivo experiment, hypersecretion of airway mucin and goblet cell hyperplasia was induced by exposure of rats to $SO_2$ over 3 weeks. Effects of CGPT and YEJ orally administered for 1 week on in vivo mucin secretion from tracheal goblet cells of rats and hyperplasia of goblet cells were assessed using ELISA and histological analysis after staining the epithelial tissue with alcian blue, respectively. Also, the effects of CGPT and YEJ on contractility of isolated tracheal smooth muscle were investigated. Results : (1) CGPT significantly inhibited in vitro mucin secretion from cultured HTSE cells. However, YEJ did not affect in vitro mucin secretion; (2) CGPT and YEJ did not affect hypersecretion of in vivo mucin and hyperplasia of tracheal goblet cells; (3) CGPT and YEJ slightly increased the expression levels of TNF-alpha or EGF-induced MUC5AC gene in NCI-H292 cells; (4) CGPT and YEJ inhibited acetylcholine-induced contraction of isolated tracheal smooth muscle of rabbit; (5) CGPT and YEJ did not affect LDH release from HTSE cells and the survival and proliferation of NCI-H292 cells. Conclusion : The results from the present study suggest that CGPT and YEJ mainly affect the expression of mucin gene rather than secretion of mucin and do not show remarkable cytotoxicity to respiratory epithelial cells.

• Biomolecules & Therapeutics
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• v.22 no.6
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• pp.525-531
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• 2014

In the present study, we investigated whether apigenin significantly affects tumor necrosis factor-${\alpha}$ (TNF-${\alpha}$)-induced production and gene expression of MUC5AC mucin in airway epithelial cells. Confluent NCI-H292 cells were pretreated with apigenin for 30 min and then stimulated with TNF-${\alpha}$ for 24 h or the indicated periods. The MUC5AC mucin gene expression and mucin protein production were measured by reverse transcription - polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Apigenin significantly inhibited MUC5AC mucin production and down-regulated MUC5AC gene expression induced by TNF-${\alpha}$ in NCI-H292 cells. To elucidate the action mechanism of apigenin, effect of apigenin on TNF-${\alpha}$-induced nuclear factor kappa B (NF-${\kappa}B$) signaling pathway was also investigated by western blot analysis. Apigenin inhibited NF-${\kappa}B$ activation induced by TNF-${\alpha}$. Inhibition of inhibitory kappa B kinase (IKK) by apigenin led to the suppression of inhibitory kappa B alpha ($I{\kappa}B{\alpha}$) phosphorylation and degradation, p65 nuclear translocation. This, in turn, led to the down-regulation of MUC5AC protein production in NCI-H292 cells. Apigenin also has an influence on upstream signaling of IKK because it inhibited the expression of adaptor protein, receptor interacting protein 1 (RIP1). These results suggest that apigenin can regulate the production and gene expression of mucin through regulating NF-${\kappa}B$ signaling pathway in airway epithelial cells.