대한한방내과학회지제 27 권 1 호 (26 년 3 월 ) Korean J.Orient.Int. Med. 26:27(1):126-137 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,,, 2-4. 5 26. 2. 1. 26. 2. 27., 173-9 (Tel. 43-229-374, Fax. 43-253-8757 E-mail : omdpyc@dju.ac.kr) 1,,,, 6,7. 8,,,,,,,,,,,, 7,8.
(mucus) 9-11..,,,,, 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 8. 6. 6. 8. 3.2 3.2 2.8 2.8 5. 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 2. 5. 39.
(g) Total Amount Ephedrae Herba Paeoniae Radix Alba Schizandrae Fructus Pinelliae Rhizoma Asari Herba cum Radice Zingiberis Rhizoma Cinnamonni Ramulus Glycyrrhizae Radix 6. 6. 6. 6. 4. 3) (JHT), (HST), (SCRT), 22. 81, 13, 8.,.22 filter, 4. 1) Kim, Wu, Lee 23-7. 1337 incubator 32 incubator., 1, 3, 5, 7. 2) (radiolabeling) Kim, Lee 23-5,, (24 well plate, 5 1 5 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 32 3. 3 treatment sample( T sample). sample, 5 (LDH activity assay) -7 23-5. 4) Hyaluronidase, sepharose CL-4B columnexcludeglycoconjugate, Lee 23-5. 5) (LDH activity assay), (24 well plate, 5 1 5 cells/well), 1Ci/[6-3 H] glucosamine(39.2 Ci/m, Amersham) well23224,, 28 PBS 2 well 32 3. 3 (T sample), 5
(LDH activity assay). LDH commercial kit(sigma, LD-L 1) 23-5. 6) Sepharose CL-4B column T sample1. 5 sepharose CL-4B column..35void volume included volumetotal bed volume. scintillation cocktaillsc,. sample, 23-5. 7) 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/) 2237 1cDNA. cdna 94 5. MUC 5AC (PCR), cdna 5, 41 PCR buffer (1mM Tris/pH 8., 3mM MgCl 2, 2.5/ BSA), 42.5mM dntp, 1MUC 5AC
sense, antisense primer (1pmol/), 1 Taq DNA polymerase (.2U/) 42., PCR (PCR thermal cycler; Takara MP-3, Japan)3, denaturation941, annealing6 1, extension 72 2. 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<.5. 15 1 5 Cont 1 2 4 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). 15 1 5 Cont 1 2 4 1) (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
8/PBS 2 3% (Fig. 3). 15 2) (HST) LDH (Fig. 5). 1 15 5 1 Cont 1 2 4 8 5 Cont 1 2 4 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). 15 15 1 1 5 5 Cont 1 2 4 Cont 1 2 4 8 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.
1) (JHT) 4/PBS 2, JHT, (Fig. 7). 2) (HST) 4/PBS 2, HST, (Fig. 8). 3) (SCRT) 8/PBS 2, SCRT, (Fig. 9). 5 4 3 2 1 1 2 3 4 5 6 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. 6 5 4 3 2 1 1 2 3 4 5 6 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.
6 5 4 3 2 1 1 2 3 4 5 6 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 1 2 3 4 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, )
.,, 9,1,3. 1,,, 2-4,,,,,,,,, 14,31. 5 1,,,,,,,,,,, 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).,,,,.,,,,,,,,,,
.,. 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
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