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1 대한한방내과학회지제 27 권 1 호 (26 년 3 월 ) Korean J.Orient.Int. Med. 26:27(1): Studies on the Effects of Selected Oriental Herbal Medicines on Inhibitory Activity of Airway Mucus Secretion Joon-myoung Kim, Chung-jae Lee *, Yang-chun Park In the present study, the author intended to investigate whether three oriental medical prescriptions named Jeongcheonhwadam-tang(JHT), Haengso-tang(HST), Socheungryong-tang(SCRT) significantly affect mucin release from cultured hamster tracheal surface epithelial (HTSE) cells. The results were as follows : (1) JHT significantly inhibited mucin release from cultured HTSE cells, without significant cytotoxicity : (2) HST significantly inhibited mucin release from cultured HTSE cells, without significant cytotoxicity : (3) SCRT significantly inhibited mucin release from cultured HTSE cells, without significant cytotoxicity : (4) JHT, HST chiefly inhibited the mucin release and did not significantly affect the release of the other releasable glycoproteins with less molecular weight than mucin. These results suggest that the three herbal prescriptions specifically inhibit the release of mucin : (5) JHT significantly inhibited the expression levels of MUC 5AC mrna. This result suggests that JHT affects the synthesis of mucin at gene level in cultured HTSE cells. All agents showed no significant cytotoxicity. In view of these results, further investigation of the effects of JHT and HST are likely to be instrumental in yielding novel agents from oriental medical prescriptions which have inhibitory effects or expectorative effects on airway mucus secretion. 1,,, , (Tel , Fax omdpyc@dju.ac.kr) 1,,,, 6,7. 8,,,,,,,,,,,, 7,8.

2 (mucus) ,,,,, 12,13.,, 14-9, 2, 21.,, (hamster tracheal surface epithelial, HTSE), (lactate dehydrogenase, LDH),. 1) (hamster tracheal surface epithelial cells) 81 Golden Syrian. 2),, 1. (g) Citri Pericarpium Pinelliae Rhizoma Arisaematis Rhizoma Ansu Semen Schizandrae Fructus Glycyrrhizae Radix Farearae Flos Ginsengs Radix Zingiberis Rhizoma Total Amount 45. (g) Total Amount Ansu Semen Perillae Folium Mori Cortex Citri Pericarpium Pinelliae Rhizoma Fritikkariae Royeli Bulbus Atractylis Rhizoma Schizandrae Fructus Glycyrrhizae Radix Zingiberis Rhizoma

3 (g) Total Amount Ephedrae Herba Paeoniae Radix Alba Schizandrae Fructus Pinelliae Rhizoma Asari Herba cum Radice Zingiberis Rhizoma Cinnamonni Ramulus Glycyrrhizae Radix ) (JHT), (HST), (SCRT), , 13, 8.,.22 filter, 4. 1) Kim, Wu, Lee incubator 32 incubator., 1, 3, 5, 7. 2) (radiolabeling) Kim, Lee 23-5,, (24 well plate, cells/well), 1Ci/ [6-3 H] glucosamine (39.2 Ci/m, New England neuclear) [insulin(5/), transferrin(5/), epidermal growth factor(12.5ng/), hydrocortisone(.1 M), sodium selenite(.1m), fetal bovine serum (5%, V/V, FBS), retinoic acid(.1m), penicillin G(1U/), streptomycin(1/), gentamicin(5 /) Dulbecco s Modified Eagle s Medium(DME)Medium 199(M199)1:1 ]well23224 (metabolic radiolabeling). 3) 24 (pretreatment sample, PT), 28PBS 2 well treatment sample( T sample). sample, 5 (LDH activity assay) ) Hyaluronidase, sepharose CL-4B columnexcludeglycoconjugate, Lee ) (LDH activity assay), (24 well plate, cells/well), 1Ci/[6-3 H] glucosamine(39.2 Ci/m, Amersham) well23224,, 28 PBS 2 well (T sample), 5

4 (LDH activity assay). LDH commercial kit(sigma, LD-L 1) ) Sepharose CL-4B column T sample1. 5 sepharose CL-4B column..35void volume included volumetotal bed volume. scintillation cocktaillsc,. sample, ) RT-PCR RNA, HTSE MUC 5AC mrna(), total RNA GIBCO BRL TRIZOL reagent(total RNA isolation reagent)., HTSE,, 4, 4, 8 2 well, 32 24, PBS2. Matrix collagen.2% collagenase type well 4, 37 2., total RNA isolation reagentlysis, 5. 5, microtube chloroform, 15 vortexing234, 12, rpm (Hanil centrifuge, MICRO 17R)2 microtube. isopropanol 1 4, 12,rpm 15 RNA. diethyl pyrocarbonate (DEPC) 8% ethanol 4, 7,5 rpm 5. RNA, RNase-free water, spectrophotometer (Beckman, DU-65) 26nm RNA, (1.A 26 =single strand RNA 4/) 28. Primer MUC 5AC sense primer 5'-CACCGGCCTCACCCGAC GCCCACC-3', antisense primer5'-gat GGGGCCGGCCTCCCGGAGAGC-3', primer PCR 44bp. actin sense primer 5'-TGGAGAAGAGCTATGAGCTGCCTG-3', antisense primer 5'-GTGCCACCAGACAG CACTGTGTTG-3' primer DNA 29bp. RT-PCR total RNA, (reverse transcription; RT) cdna, (polymerase chain reaction; PCR). total RNA 5755denaturation, 4 5 RT buffer (25mM Tris-HCl/pH 8.3, 375mM KCl, 15mM MgCl 2, 5mM Dithiothreitol), 2.51mM dntp, 1oligo-dT15 (25p), 1 (M-MLV RT; 2 U/) cDNA. cdna MUC 5AC (PCR), cdna 5, 41 PCR buffer (1mM Tris/pH 8., 3mM MgCl 2, 2.5/ BSA), 42.5mM dntp, 1MUC 5AC

5 sense, antisense primer (1pmol/), 1 Taq DNA polymerase (.2U/) 42., PCR (PCR thermal cycler; Takara MP-3, Japan)3, denaturation941, annealing6 1, extension RNA cdna MUC 5AC mrna ()., PCR (2)1 gel loading buffer (.25 bromphenol blue,.25 xylene cyanol FF, 5 glycerol), Tris-acetate- EDTA buffer (4 mm Tris-acetate, 1mM EDTA) 1/ ethidium bromide 1.2 agarose gel. Gel DNA band(ultraviolet transilluminator),. 8) mean±s.e.m.,. unpaired Student s t-test, p< Cont Confluent HTSE cells were metabolically radiolabeled with 3 H-glucosamine for 24 hrs and chased for 3 min in the presence of varying concentrations of JHT extract and the amount of 3 H-mucins in the spent media was measured. Each bar represents a mean± S.E.M. from 4 culture wells. *: significantly different from control(p<.5). 2) (HST) 4/PBS 2 (Fig. 2) Cont ) (JHT)4/PBS 2 (Fig. 1). Confluent HTSE cells were metabolically radiolabeled with 3H-glucosamine for 24 hrs and chased for 3 min in the presence of varying concentrations of HST extract and the amount of 3H-mucins in the spent media was measured. Each bar represents a mean±s.e.m. from 4 culture wells. *: significantly different from control(p<.5). 3) (SCRT), 4

6 8/PBS 2 3% (Fig. 3). 15 2) (HST) LDH (Fig. 5) Cont Cont Confluent HTSE cells were metabolically radiolabeled with 3 H-glucosamine for 24 hrs and chased for 3 min in the presence of varying concentrations of SCRT extract and the amount of 3 H-mucins in the spent media was measured. Each bar represents a mean±s.e.m. from 4 culture wells. *: significantly different from control(p<.5). 1) (JHT) LDH (Fig. 4). Confluent HTSE cells were treated with varying concentrations of HST extract for 3 min and supernatants were collected for LDH activity assay at the end of the treatment. Each bar represents a mean±s.e.m. from 4 culture wells. 3) (SCRT) LDH (Fig. 6) Cont Cont Confluent HTSE cells were treated with varying concentrations of JHT extract for 3 min and supernatants were collected for LDH activity assay at the end of the treatment. Each bar represents a mean±s.e.m. from 4 culture wells. Confluent HTSE cells were treated with varying concentrations of SCRT extract for 3 min and supernatants were collected for LDH activity assay at the end of the treatment. Each bar represents a mean±s.e.m. from 4 culture wells.

7 1) (JHT) 4/PBS 2, JHT, (Fig. 7). 2) (HST) 4/PBS 2, HST, (Fig. 8). 3) (SCRT) 8/PBS 2, SCRT, (Fig. 9) Confluent HTSE cells were metabolically radiolabeled with 3 H-glucosamine for 24 hrs and chased for 3 min in the presence of JHT 4 μl / PBS 2 μl and total elution profiles of control spent media and treatment sample through Sepharose CL-4B column were analysed, as described in Materials and Methods Confluent HTSE cells were metabolically radiolabeled with 3 H-glucosamine for 24 hrs and chased for 3 min in the presence of HST 4 μl /PBS 2 μl and total elution profiles of control spent media and treatment sample through Sepharose CL-4B column were analysed, as described in Materials and Methods.

8 Confluent HTSE cells were metabolically radiolabeled with 3 H-glucosamine for 24 hrs and chased for 3 min in the presence of SCRT 8 μl / PBS 2 μl and total elution profiles of control spent media and treatment sample through Sepharose CL-4B column were analysed, as described in Materials and Methods. RT-PCR, 3 (4/PBS 2), (4/PBS 2) (8 /PBS 2) 24 MUC 5AC mrna. (),. M MUC5AC (44 bp) -actin (29 bp) M : 1kb marker 1: Control 2: JHT 3: HST 4: SCRT HTSE cells were incubated with the indicated concentration (JHT, 4 μl /PBS 2 μl, HST 4 μl /PBS 2 μl, SCRT 8 μl /PBS 2 μl ) of each agent for 24 hrs. Total RNA was isolated and MUC 5AC mrna levels were analyzed by RT-PCR. The PCR products were separated on 1.2% agarose gel and stained with ethidium bromide, as described in Materials and Methods., 7.,,,,,,,,, 7,29. (mucus) 6-8 (mucin, )

9 .,, 9,1,3. 1,,, 2-4,,,,,,,,, 14, ,,,,,,,,,,, 6,7,15,31. 8,,,,,, 8,31.,,,,,,,,,, 7,8. glucocorticoid 32 poly-l-lysine (PLL) 23..,. 2,21, 7, (mucin) (HTSE),,. 3% (Fig. 1), 3% (Fig. 2), 8/PBS 2 5% (Fig. 3).,,,,.,,,,,,,,,,

10 .,. LDH. LDH 33,34. (Fig. 46)..,,,,. Gel filtration chromatographyresinloading, loadingvoid volme total volume,,, 24,35. HTSE 3H-glucosamine, glucosamine,,, 3 H- 24,25., sepharose CL-4B column loading, 3 H-dalton, 3 H- glucosamine (elution profile).,, (total elution profile), 3H-. (Fig. 79), fraction void volume fraction, included volume total volume fraction.,,.,,., HTSE 3,,, (production) MUC 5AC mrna., 24 MUC 5AC mrna. (Fig. 1), 24, MUC 5AC mrna.,. mrna,, RT-PCR

11 ,, real-time PCR northern blot analysis.,,,,, ( ). (HTSE),,,, (LDH) ,,. 3.,,,,. 4.,.,,, : ; 1999, p.469, : ; 1975, p : ; 198, p : : 2, p : ; 199, p : ; 1984, p : ; 22, p.52-5, 12-14, 144-9, 191, 542, ,,,,.. : ; 199, p : ; 1997, p : ; 199, p Rubin BK. Physiology of airway mucus clearance. Respir Care. 22;47(7): Frigas E, Loegering DA, Solley GO, Farrow GM, Gleich GJ. Elevated levels of the eosinophil granule major basic protein in the sputum of patients with bronchial asthma. Mayo Clin Proc. 1981;56:

12 13. Gleich GJ. The eosinophil and bronchial asthma. Current understanding. J Allergy Clin Immuno. 199;85: ,, ;5(2): ,, ;11: ,,,,,,. IgE T.. 22;23(1): ,,,. Helper T cell.. 22;16(4): ,,,,,. IL-4, IL-5, IFN-.. 22; 23(2): ,,,. B IgEcytokine.. 23;24(2): ,,... 24;18(3): ,,,,,,... 25;26(1): : ; 1992, p.15-5, Lee CJ. Specificity in the inhibition of mucin release from airway goblet cells by polycationic peptides. Appl. Pharmacol. 21;9(3): : ; Kim KC, Rearick JI, Nettesheim P, Jetten AM. Biochemical characterization of mucous glyco- proteins synthesized and secreted by hamster tracheal epithelial cells in primary culture. Biol. Chem. 1985;26: Kim KC, Brody JS. Use of primary cell culture to study regulation of airway surface epithelial mucus secretion. Symp Soc Exp Biol. 1989;43: Wu R, Smith D. Continuous multiplication of rabbit tracheal epithelial cells in a defined, homone-supplemented medium. In Vitro. 1982;18: Karlinsey J, Stamatoyannopoulos G, Enver T. Simultaneous purification of DNA and RNA from small numbers of eukaryotic cells. Anal Biochem. 1989;18(2): ,, ;1(1): Rubin BK. Physiology of airway mucus clearance. Respir Care. 22;47(7): : ; 2, p.113-6, 121, 124-5, 165, 334, 347, 448, 45, 453, 458, 463, 478, 479, 481-4, 531, 536, 54, 542, 581, Mutschler E, Derendorf H. Drug actions. Boca Raton, Florida: CRC press; 1995, p Freshny. Measurement of viability and cytotoxicity. In: Culture of animal cells(3rd edn). Willey- Liss; 1994, p Yu XY, Schofield BH, Croxton T, Takahashi N, Gabrielson EW, Spannhake EW. Physiologic modulation of bronchial epithelial cell barrier function by polycationic exposure. Am J Respir Cell Mol Biol. 1994;11(2): Cheng PW, Sherman JM, Boat TE, Bruce M. Quantitation of radiolabeled mucous glycoproteins secreted by tracheal explants. Anal. Biochem. 1981;117:31-6.

15.hwp

15.hwp airway, mucin, Mahwangyunpye-tang (MYT), MUC5AC mrna LDH release (% control) 150 120 90 60 30 0 cont 10 20 40 80 Concentrations (µ l / 200µ l PBS) Absorbance (% control) 150 100 50 0 cont 0.2 Concentrations