Journal of Korean Medicine Rehabilitation Vol. 23 No. 3, July 2013 pissn 1229-1854 eissn 2288-4114 Original Article 이수연 박원형 * 차윤엽 * 이은상지대학교보건과학대학제약공학과, 상지대학교한의과대학한방재활의학과교실 * Effects of Ojeoksangamibang Extract on the Recovery of Liver Function in CCl 4 -exposed Rats Soo-Yeon Lee, M.D., Won-Hyung Park, K.M.D.*, Yun-Yeop Cha, K.M.D.*, Eun Lee, Ph.D. Department of Pharmaceutical Engineering of Health Sciences, Sangji University, Department of Korean Rehabilitation Medicine, College of Korean Medicine, Sangji University* RECEIVED June 25, 2013 REVISED July 16, 2013 ACCEPTED July 18, 2013 CORRESPONDING TO Yun-Yeop Cha, Department of Korean Rehabilitation Medicine, College of Korean Medicine, Sangji University, 80, Sangjidae-gil, Wonju 220-717, Korea TEL (033) 741-9261 FAX (033) 732-2124 E-mail omdcha@sangji.ac.kr Objectives The objective of present study was to investigate the effect of Ojeoksangamibang (Wǔjīsǎnjiāwèifāng) extract on recovery of liver function in carbontetrachloride (CCl 4)-exposed rat. Methods Male rats weighing 230±7.21 g fed experimental diet for 1 week and 28 rats were divided into 4 groups. Each of 7 rats was devided into a control group and experimental groups. We fed a control group of rats a basal diet and administered normal saline (100 mg/kg, 1 time/1 day) for 3 weeks. And we fed each experimental group of rats basal diet and administered an extract of Ojeoksangamibang extracts (100 mg/kg, 200 mg/kg, 300 mg/kg, 1 time/1 day) for 3 weeks. We measured lipid of plasma and liver, concentration of proinflammatory cytokines (IL-1β, TNF-α, IL-6 and IL-10). Statistical analysis was done by one-way analysis of variance (ANOVA) and Duncan's multiple range test with significance level at p<0.05. Results Plasma a-fetoprotein (AFP) and total protein concentration showed a tendency to decrease in Ojeoksangamibang extract-treated groups. However, plasma albumin concentration showed no significant differences in all treatment groups. Activity of plasma Aspartate aminotransferase (AST) and Alanine aminotransferase (ALT) in the Ojeoksangamibang extract-treated groups, increased addition amount of Ojeoksangamibang extracts tended to decline. Alkaline phosphatase (ALP), lactate dehydrogenase (LDH) and γ-glutamyl transferase (γ-gt) activities showed a tendency to decrease in Ojeoksangamibang extract-treated groups, increased addition amount of Ojeoksangamibang extracts tended to decline. Concentration of plasma triglyceride and total cholesterol showed a lower value than that of control group. The liver IL-1β, IL-6 and TNF-α concentration were decreased, and IL-10 was increased in Ojeoksangamibang extract groups, compared to control group. Plasma IL-1β, IL-6 and TNF-α concentration were decreased, and IL-10 was increased in Ojeoksangamibang extract groups, compared to control group. Conclusions This study suggested that Ojeoksangamibang may alleviate liver inflammatory reaction induced by liver toxicity. (J Korean Med Rehab 2013;23(3):45-53) Copyright 2013 The Society of Korean Medicine Rehabilitation Key words Ojeoksangamibang extract, Liver function, A-fetoprotein, AST, ALT, Cytokines www.e-jkmr.org 45
이수연 박원형 차윤엽 이은 서론»»» 최근청장년층에서간질환의발병률이높아져간질환의예방과치료에대한관심이고조되고있다. 간질환으로인한간기능의이상은영양소대사에문제를일으켜여러종류의복합적임상증후를나타낸다 1). 따라서손상된간세포의부활, 인체의면역기능및간기능을증진시켜간질환을치료하려는연구가진행되고있으며, 특히천연물이나생약을이용한연구가많이수행되어 2,3), 간질환의치료와예방을위해천연물응용에대한인식이높아졌다. 五積散은 太平惠民和劑局方ㆍ傷寒門 4) 에서처음서술된처방으로蒼朮, 麻黃, 陳皮, 厚朴, 桔梗, 枳殼, 當歸, 乾薑, 白芍藥, 白茯苓, 川芎, 白芷, 半夏, 肉桂, 甘草, 生薑으로구성되어있으며, 주로祛痰, 袪瘀血之劑로많이사용한다. 최근임상에서비만환자등에게五積散加味方으로祛痰, 消食, 利氣, 利水하는薏苡仁, 蘿葍子, 木通, 澤瀉, 車前子, 靑皮등을加味하여사용하던중간기능개선등의효과가있는례가있어일차적으로본실험을하게되었다. 五積散효능에대한이전의연구결과에서는자궁근종세포의사멸및 apoptosis 의촉진과五積散농도에비례한사멸세포수의증가가보고되었으며 5), 해열, 진통, 소염에있어서는아스피린과유사한효과가관찰되었고 6-8), 신장질환에있어서는백서세뇨관의재흡수기능과신사구체여과율의촉진 9,10) 등이보고되었다. 상기의연구를통해서간질환을비롯한생체내염증반응에五積散加味方이긍정적으로작용할것으로판단되었기에, CCl 4 로간중독을유도한쥐에게五積散加味方추출물을급여한후혈액내간기능에관계하는생물학적수치들과전염증성 cytokine 의생산량을확인하여유의한결과를얻었기에보고하는바이다. 재료및방법»»» 1. 시험동물및시험군평균체중이 230±7.21 g의 Sprague-Dawley 계쥐수컷 28두를 1주일간시험식이에적응시킨후, 평균체중이유 사하게대조군 ( 생리식염수 100 mg/kg), 처리1군 ( 五積散加味方추출액 100 mg/kg), 처리2군 ( 五積散加味方추출액 200 mg/kg) 및처리3군 ( 五積散加味方추출액 300 mg/kg) 으로나누어, 처리군당 7두씩임의로배치하였다. 본실험은실험동물윤리에관한규정을준수하였다. 2. 식이및물 식이및물은시험기간 3주동안자유롭게먹게하였다 (Table I). 3. 사염화탄소처리 시험개시기에 Conc. CCl 4 와올리브오일을 1:3으로희석한용액을 2.5 mg/kg 수준으로격일로 3회존대를이용하여경구투여하여간장중독을유도했다. 4. 五積散加味方추출물및급여방법 시중에서구입한五積散加味方 (Table II) 500 g을적량으로나누어수조상에서냉각수환류하에서 5시간씩 3회추출하고, 여과, 감압, 농축하여 EtOH 추출물 97 g을만들어, 냉동실에보관, 공시했다. 급여는매일오후 3시 Table I. Composition of Experimental Diet Ingredients Basal diet (%) Casein 20.0 α-corn starch 35.5 Sucrose 11.0 Lard 4.0 Corn oil 1.0 Mineral mix* 3.5 Vitamin mix 1.0 Cellurose powder 23.7 DL-methione 0.3 *g/kg diet: CaCO 3, 29.29; CaHPO 4 ㆍ2H 2O, 0.43; KH 2PO 4, 34.30; NaCl, 25.06; MgSO 4 ㆍ7H 2O, 9.98; Feric citrate hexahydrate, 0.623; CuSO 4 ㆍ5H 2O, 0.516; MnSO 4 ㆍH 2O, 0.121; ZnCl 2, 0.02; KI, 0.005; (NH 4) 6 MO 7O 24 ㆍ4H 2O, 0.0025. mg/kg diet: Thiamine-HCl, 12; Riboflavin, 40; Pyridoxin- Hcl, 8; Vitamin-B 12, 0.005; Ascorbic acid, 300; D-biotin, 0.2; Menadione, 52; Folic acid, 2; D-calcium pantothenate, 50; P-aminobenzoic acid, 50; Nicotinic acid, 60; Choline chloride, 2,000 (IU/kg diet); Rethinyl acetate, 5,000 (IU/kg diet); Cholecalciferol, 250 (IU/kg diet). 46 J Korean Med Rehab 2013;23(3):45-53
Table II. The Prescription of Ojeoksangamibang Herb medicine Scientific name Amount (g) 蒼朮 Atractylodes japonica 8.0 麻黃 Ephedra sinica 4.0 陳皮 Citrus unshiu Markovich 4.0 厚朴 Machilus thunbergii 3.0 桔梗 Platycodon grandiflorum 3.0 枳殼 Ponicirus trifoliata Rafin 3.0 當歸 Angelica gigas Nakai 3.0 乾薑 Zingiber officinale Roscoe 3.0 白灼藥 Paeonia lactiflora 3.0 白茯苓 Poria cocos Wolf 3.0 川芎 Cnidium officinale 2.8 白芷 Angelica dahurica Benthamet Hooker 2.8 半夏 Pinellia ternata 2.8 肉桂 Cinnamomum cassia Blume 2.8 甘草 Glycyrrhiza uralensis 2.4 生薑 Zingiber officinale 3.0 薏苡仁 Coicis Semen 8.0 蘿葍子 Raphani Semen 4.0 木通 Akebiae Caulis 4.0 澤瀉 Alismatis Rhizoma 4.0 車前子 Plantaginis Semen 4.0 靑皮 Citri Reticulatae viride Pericarpium 4.0 Total amount 85.6 경에존대를이용하여 4주간경구투여했다. 대조군은동일한방법으로생리식염수를투여했다. 7. Cytokine 정량혈장 Cytokine 정량용시료는채혈직후, 혈장을분리하여 -80 o C에냉동보관하였다. 간장 Cytokine 정량용시료는 1 g의간장을채취하여 5 ml의 phosphate buffered saline (PBS, PH 7.4, containing a protease inhibitors cocktail) 과함께혼합하여얼음위에서분쇄 (homogenized) 하였다. 분쇄혼합물을 4 o C, 15,000 rpm 15분간원심분리한후, 상층부를 0.45μm 필터로여과하고, 다시원심분리해서상층부를 -80 o C에냉동보관했다. Cytokine (IL-1β, TNF-α, IL-6 및 IL-10) 정량은시판 kit (Biosource International, USA) 를이용했다. TNF-α 의최적측정농도는 0.7 pg/ml 이며, 다른 Cytokine 들은 3~8 pg/ml 이다. 간장 Cytokine 량은 5 ml의 PBS에생간장 1 g을혼합한조정액으로측정하였으며, pg/mg 단위로나타내었다. 8. 통계처리실험결과는 SPSS 17.0 version 을이용하여 one-way ANOVA 검정을수행하였으며, 각처리군간의유의성검정은 Duncan's multiple range test에의해 p<0.05 수준에서실시했다. 5. 혈액및간장채취혈액채취는시험종료 12시간전에급여사료를중단, 절식한상태에서약 5~6 ml의혈액을심장천자법에의해채혈, 공시했다. 간장채취는혈액채취가끝난직후적출했다. 6. 혈액분석혈청 AFP (α-fetoprotein), Total protein, Albumin, ALP (Alkaline phosphatase), AST (Asparate Aminotransferase), ALT (Alanine Aminotransferase), γ-gt (γ-glutamyl transferase), Triglyceride, Total cholesterol, LDH (Lactate Dehydrogenase) 의활성측정은혈액자동분석기 (Boeheringer Manheim, 독일 ) 에의해측정했다. 결과»»» 1. Plasma a-fetoprotein (AFP), Total protein 및 Albumin 의농도 AFP의농도는전처리군에서 3.17 ng/ml에서 4.09 ng/ml 의변동범위를보여주었으며, 대조군보다五積散加味方추출물처리군들이낮은값을나타내었다. Total protein 농도는대조군과비교하여五積散加味方처리군들이낮은수치를나타내었으며, 五積散加味方추출물첨가량이증가함에따라하락하는경향을보였다. Albumin 의농도는대조군과五積散加味方추출물처리군간에유의한차이를나타내지않았다 (Table III). www.e-jkmr.org 47
이수연 박원형 차윤엽 이은 Table III. Effects of Ojeoksangamibang Extract on Plasma AFP, Total Protein and Albumin Concentration in Rat Injured CCl 4 Solution Treatment AFP (ng/ml) Total protein (g/dl) Albumin (g/dl) I 4.09±0.42 6.95±0.47 3.47±0.25 NS II 3.17±0.39 6.72±0.41 3.83±0.59 NS III 2.88±0.41* 5.81±0.35* 3.96±0.52 NS IV 2.31±0.22* 5.29±0.37* 3.91±0.631 NS NS: Not significantly different (p>0.05), I: Control group (CCl 4), II: CCl 4+100 mg/kg Ojeoksangamibang ext. group, III: CCl 4+200 mg/kg Ojeoksangamibang ext. group, IV: CCl 4+300 mg/kg Ojeoksangamibang ext. group. *,, : Means in the same column with different superscripts are significantly different (p<0.05). Table IV. Effects of Ojeoksangamibang Extract on Plasma AST and ALT in Rat Injured CCl 4 Solution Treatment AST (U/L) ALT (U/L) I 201.44±23.57 187.39±19.54 II 151.39±17.28 172.83±17.69 III 133.27±21.93* 124.11±22.94* IV 115.64±19.68* 121.72±21.65* I: Control group (CCl 4), II: CCl 4+100 mg/kg Ojeoksangamibang ext. group, III: CCl 4+200 mg/kg Ojeoksangamibang ext. group, IV: CCl 4+ 300 mg/kg Ojeoksangamibang ext. group. *,, : Means in the same column with different superscripts are significantly different (p<0.05). Table VI. Effects of Ojeoksangamibang Extract on Plasmid Cholesterol and Triglyceride Concentration in Rat Injured CCl 4 Solution Treatment Total cholesterol (mg/dl) Triglyceride (mg/dl) I 93.51±5.47 121.73±6.88 II 85.65±4.94* 108.35±5.15* III 81.96±5.27* 93.27±6.11* IV 82.73±4.09* 91.53±5.47* I: Control group (CCl 4), II: CCl 4+100 mg/kg Ojeoksangamibang ext. group, III: CCl 4+200 mg/kg Ojeoksangamibang ext. group, IV: CCl 4+300 mg/kg Ojeoksangamibang ext. group. *, : Means in the same column with different superscripts are significantly different (p<0.05). Table V. Effects of Ojeoksangamibang Extract on Plasma ALP, γ-gt and LDH in Rat Injured CCl 4 Solution Treatment ALP (U/L) γ-gt (U/L) LDH (U/L) I 291.72±23.59 2.83±0.21 1,854.39±52.18 II 274.38±19.57 2.56±0.23* 1,539.23±36.47* III 227.41±15.28* 2.47±0.25* 1,427.31±31.46* IV 211.53±19.57* 2.23±0.17* 1,392.58±32.17* I: Control group (CCl 4), II: CCl 4+100 mg/kg Ojeoksangamibang ext. group, III: CCl 4+200 mg/kg Ojeoksangamibang ext. group, IV: CCl 4+300 mg/kg Ojeoksangamibang ext. group. *, : Means in the column row with different superscripts are significantly different (p<0.05). 2. AST, ALT 의효소활성 3. ALP, γ-gt 및 LDH 의생물학적수치 AST의활성치는대조군보다五積散加味方추출물처리군에서현저하게낮은수치를나타내었으며, 五積散加味方추출물첨가량이증가함에따라하락하는경향을보였다. ALT의활성치는대조군과五積散加味方추출물 100 mg 처리군은상호간에유의한차이를나타내지않았으나, 五積散加味方추출물 200 mg 및 300 mg 처리군은대조군보다현저하게하락하였다 (Table IV). ALP는五積散加味方처리군들이대조군보다낮은수치를보였고, γ-gt 는처리군간에유의한차이를나타내지않았다. LDH의효소활성치는 CCl 4 처리에의해상당한수준으로그활성치가증가하였으나, 五積散加味方처리군들은대조군보다낮은수치를나타내었다 (Table V). 48 J Korean Med Rehab 2013;23(3):45-53
Table VII. Effects of Ojeoksangamibang Extract on Liver Cytokines Concentration in CCl 4-exposed Rats Treatment IL-1β (pg/mg) IL-6 (pg/mg) TNF-α (pg/mg) IL-10 (pg/mg) I 31.57±4.89 15.71±1.15 2.11±0.47 NS 1.68±0.47 NS II 27.32±4.52 9.64±0.97 1.71±0.59 NS 1.77±0.39 NS III 21.55±3.81* 7.12±0.83* 1.65±0.35 NS 1.93±0.38 NS IV 18.75±3.29* 7.27±0.91* 1.53±0.41 NS 1.95±0.31 NS NS: Not significantly different (p>0.05), I: Control group (CCl 4), II: CCl 4+100 mg/kg Ojeoksangamibang ext. group, III: CCl 4+200 mg/kg Ojeoksangamibang ext. group, IV: CCl 4+300 mg/kg Ojeoksangamibang ext. group. *,, : Means in the same column with different superscripts are significantly different (p<0.05). Table VIII. Effects of Ojeoksangamibang Extract on Plasma Cytokines Concentration in CCl 4-exposed Rats Treatment IL-1β (pg/mg) IL-6 (pg/mg) TNF-α (pg/mg) IL-10 (pg/mg) I 291.57±21.75 672.39±58.53 803.27±79.38 82.15±7.15* II 247.35±22.53 571.48±63.15* 621.35±49.33* 93.69±9.87* III 185.49±27.31* 488.78±51.45* 639.71±56.28* 87.47±8.11* IV 192.57±31.55* 517.44±72.19* 571.49±51.23* 118.57±8.19 NS: Not significantly different (p>0.05), I: Control group (CCl 4), II: CCl 4+100 mg/kg Ojeoksangamibang ext. group, III: CCl 4+200 mg/kg Ojeoksangamibang ext. group, IV: CCl 4+300 mg/kg Ojeoksangamibang ext. group. *,, : Means in the same column with different superscripts are significantly different (p<0.05). 4. Total Cholesterol량과 Triglyceride량본실험의결과에서 Cholesterol 과 Triglyceride 농도가五積散加味方처리군들이대조군보다낮게나타났다 (Table VI). 5. Liver cytokine 간 IL-1β 및 IL-6 농도 (Table VII) 는五積散加味方추출물처리군들이대조군보다낮은값을나타내어, 五積散加味方추출물에의해간염증상태가개선되었음을알수있다. CCl 4 처리후간장 TNF-α 농도는대조군과五積散加味方추출물처리군들간에유의한차이를나태내지않았으며, 간 IL-10은높은경향을나타냈으나, 유의성은없었다 (Table VII). 6. Plasma cytokine 본연구에서 Plasma IL-1β, IL-6 및 TNF-α농도모두가五積散加味方추출물처리군들에서대조군보다낮은수치를보였다. Plasma IL-10 농도는대조군보다五積散加味方추출물처리군들이유의하게높은값을나타내었다. 고찰»»» 五積散加味方의간기능회복효과를알아보기위하여 CCl 4 로간손상을유도한흰쥐에게五積散加味方추출물을급여한후간장기능에관여하는혈액내생물학적수치와 cytokine 농도를처리군간에비교, 검토하였다. 먼저, 각처리군별 AFP, Total protein 및 Albumin 의농도를알아보았다. AFP의농도는전처리군에서 3.17 ng/ml 에서 4.09 ng/ml 의변동범위를보여주었으며, 대조군보다五積散加味方추출물처리군들이낮은값을나타내었다. Total protein 농도는대조군과비교하여五積散加味方처리군들이낮은수치를나타내었으며, 그중 200 mg/kg 과 300 mg/kg 농도에서는유의성있게낮았다. AFP는간암지표로가장널리사용이되며 11), 간염ㆍ간경변증등으로간세포가파괴되어재생할경우에도 AFP 값이상승한다 12). Total protein 은혈액중의모든단백질의총칭으로혈청단백의대부분은간장에서합성되고파괴되며, 신장에서비슷한양으로배설이이루어지므로항상일정치를유지하게된다. 그러므로혈청단백의양은간과신장의건강유무의지표가될수있고, 따라서혈장 AFP농도및 Total protein 량의증가는간조직의염증 www.e-jkmr.org 49
이수연 박원형 차윤엽 이은 유발이나이상을의미한다 13). 본연구의결과에서五積散加味方추출물처리군에서 AFP농도및 Total protein 량의감소는五積散加味方추출물이손상된간세포의회복에긍정적인영향을준것으로판단된다. Albumin은혈청단백의 50~70% 를차지하며체내단백질대사이상의좋은지표가되며, 교질삼투압유지및생체내화합물과약제를수송하는기능을하고 CCl 4 투여이후 total protein 과 albumin 이유의하게감소하는것으로알려져있다 14). 본연구에서 Albumin 의농도는대조군과五積散加味方추출물처리군간에유의한차이를나타내지않았다. 이는간에서생산되는 Albumin 이간세포의회복이다소진전되어도간기능이완전히복구되지않아 Albumin 생산이정상으로회복되지않았을것으로판단된다 15-18). AST의활성치는대조군보다五積散加味方추출물처리군에서현저하게낮은수치를나타내었으며, 五積散加味方추출물첨가량이증가함에따라하락하는경향을보였다. ALT의활성치는대조군과五積散加味方추출물 100 mg 처리군은상호간에유의한차이를나타내지않았으나, 五積散加味方추출물 200 mg 및 300 mg 처리군은대조군보다현저하게하락하였다. AST 및 ALT는아미노산대사에관여하는전이효소로서 AST는주로심장, 간, 골격및신장순으로많이분포되어있으며이들조직들이파괴되면손상된세포로부터이효소들이방출되어혈액내활성치가높아진다. 일반적으로활성도의상승은간담도질환및심장질환에서일어나고, 특히급성간염의경우에현저하다 19). 또한세포장애정도와상관성이높을뿐만아니라다른혈청효소에비해민감하게변화하여간염및간경변등간질환의지표로널리사용되고있다. ALT는세포막내효소로서절대량은 AST보다적지만심장및근육에비해주로간세포내존재하는효소로간의활성도가높아이들세포의괴사및파괴정도를반영하는것 20) 으로잘알려져있다. 특히 CCl 4 처리등급성간손상시혈중 AST 및 ALT 의활성도가급격하게증가하며, 심한바이러스성간염및독성물질에의한간손상등과같이상당한간괴사가있는경우에혈액중으로유리되어높은활성을나타내게된다 21,22). 본연구에서五積散加味方추출물처리군들에서간질환지표효소들의활성이하락하였으며, 이는 CCl 4 에의해 손상된간조직의복구에五積散加味方추출물이긍정적인영향을준것으로판단된다. 각처리군별 ALP, γ-gt 및 LDH의생물학적수치에서 ALP는五積散加味方처리군들이대조군보다낮은수치를보였고, γ-gt 는처리군간에유의한차이를나타내지않았다. LDH의효소활성치는 CCl 4 처리에의해상당한수준으로그활성치가증가하였으나, 五積散加味方처리군들은대조군보다낮은수치를나타내었다. ALP는담관손상의지표로서종양, 간염, 간경화등의질환일경우에증가한다 23). 간, 신장, 소장, 뼈, 태반등에비교적고농도로존재하여담즙정체시에담즙으로배설된다. 그러나담즙으로의배설장애나담관내압항진으로인해간에서생성량이증가하여혈중유입이증가된다 14). γ-gt 는대부분의간질환에서증가하며, 특히만성알코올성간질환에서현저하게증가한다 24). 본연구에서는처리군간에유의한차이를보이지않았다. LDH는세포내해당작용에관여하는효소로간장기능에이상이있을경우그활성치가증가한다 25). CCl 4 는간세포소포체의약물대사효소계에작용하여 free radical metabolite 를생성시켜간세포의손상을야기하며, 그결과로혈청중의 LDH의활성치가상승하게된다 26). 본연구에서는간손상으로증가된 ALP 및 LDH 두효소의활성치가五積散加味方처리군들에서대조군보다낮은수치를보였다. 이로서五積散加味方이손상된간세포의회복에긍정적인영향을준것으로판단된다. 그리고 Total Cholesterol 및 Triglyceride 량의변동치를알아보았는데, 생체내에서지질대사에직접관여하는장기는간으로간기능에이상이있을경우 Triglyceride 와 Cholesterol 의혈중양이증가한다. 특히담도의이상으로 Bile acid의배출에문제가생길경우에는전구물질인 Cholesterol 량이증가한다 13). CCl 4 는내형질세망의불포화지방산을산화시켜과산화지질을형성하고, 중성지질의축적을유도하고 27), 사염화탄소에의한간조직의손상은간세포기능을저하시킴으로써콜레스테롤과중성지질의함량이증가한다 28,29). 본실험의결과에서 Cholesterol 과 Triglyceride 농도가五積散加味方처리군들이대조군보다낮게나타났다. 즉, 五積散加味方이손상된간장세포의회복을유도하여간장내에서의지질대사를정상적으로회복시켜가고있음을알수있다. 50 J Korean Med Rehab 2013;23(3):45-53
각처리군별간내전염증성 Cytokines 농도를알아본바는다음과같다. 간은생체의염증반응시에각종전염증성 Cytokine 들을생산하는기관인데 30), 본연구에서간 IL-1β 및 IL-6 농도는五積散加味方추출물처리군들이대조군보다낮은값을나타내어, 五積散加味方추출물에의해간내염증상태가개선되었음을알수있다. TNF-α는 LPS shock 에의해간장의 Kuffer cell로부터방출되는전염증성사이토카인이다 31). 또한 TNF-α는단핵구, Kuffer cell 및간세포에서 IL-8 생산을자극하며, 이때분비되는 IL-8은간내중성구의침윤과간장손상을일으키는간경변의예후인자가되는데 32-34), 본연구에서 CCl 4 처리후간장 TNF-α 농도는대조군과五積散加味方추출물처리군들간에유의한차이는없었다. 이러한결과는간에서생산된 TNF-α가혈액으로유출된후소량으로잔류한것에기인한것으로생각된다. 항염증성사이토카인 IL-10은간이주요공급원이며 macrophage, Kupffer cell, T와 B lymphocytes 및 hepatocytes 등에서생성된다 35). 점막의 Tcell 활성화와 metalloproteinase, extracellular matrix의결손을하향조절하여점막손상을억제하고, 활성화된대식세포나단핵구의 TNF-α, IL-1β, IL-6 및 IL-8의합성을억제한다 36-38). 본연구에서五積散加味方추출물처리군모두가대조군보다높은경향을나타내었으며, 이와같은결과가전염증성 cytokine 들의생산에영향을주었을것으로생각된다. Plasma IL-1β의농도는五積散加味方추출물처리군들이대조군보다낮은수치를나타내었다. IL-1β는 monocyte, macrophage, β-cell, dendritic cell, endothelial cell, neutrophil 및 hepatocyte 에서분비되며, TNF-α, IL-2 및 IL-6와함께 pro-inflammatory cytokine 으로서여러면역학적작용들과연관되어있다. 특히 IL-1β는 T-cell 의 activity 를활성화한다 39). 또한 IL-1β와 TNF-α 는여러형태의실험에서상호상가효과를나타낸다는것이밝혀졌으며, 급성기염증반응에서급격히증가한다 40) 고알려져있다. IL-6는 T림프구, B림프구, 대식세포및섬유아세포등의여러세포에서생산되는 Cytokine 으로면역응답, 조혈계와신경계세포의증식과분화및급성반응등에관여 한다. 그러나과잉생산은여러가지면역이상, 염증성질환및림프계종양의발생과깊은관련이있는것으로알려져있다 41-43). TNF-α는대표적인전염증성 cytokine으로여러가지의자극에반응하여 Kuffer cell 및 monocytes 와 macrophage 에의해방출되는 peptide mediator 이며 44), endotoxin의제거효과를가지는가장중요한 mediator 로가정되었으며 45), IL-1β와함께감염및종양생성시나타나는면역반응의 inflammatory cytokine으로작용한다. 그러나다량의 TNF-α 발현은심근수축력감소, 혈압강하, 대사과정의손상을유발하기도한다 46,47). 또한 T cell의수를감소시켜방어체계를무너뜨리며 48), 광범위의 pathogenic 상태를일으키고간장내에서간세포의사멸을일으키는것으로알려져있다 31). 본연구에서 Plasma IL-1β, IL-6 및 TNF-α농도모두가五積散加味方추출물처리군들에서대조군보다낮은수치를보였다. 이러한결과는五積散加味方추출물처리군들의간장내염증상태가대조군보다완화되었음을시사해준다. Plasma IL-10은 lympocytes 와 macrophage 에의해생산되는강력한 pleiotropic anti-inflammatory cytokine이며, IL-1β나 IL-6 및 TNF-α의분비를조절하여면역조절작용에관여하는것으로알려져있으며 38,49), 전염증성 cytokine 들이과량분비되어 IL-10과균형을이루지못할경우, 숙주의생존력에크게영향을미친다 50). 본연구에서는 Plasma IL-10 농도가대조군보다五積散加味方추출물처리군들이유의하게높은값을나타내었다. 이와같은결과는증가된 IL-10이 IL-1β, IL-6 및 TNF-α의분비를조절하여염증반응완화에영향을주었을것으로생각된다. 이상의결과는五積散加味方이간손상회복에긍정적효과를나타내었다. 하지만약물투여의횟수와용량그리고기간등에대한다양한검토가이루어지지않았고동물실험이라는한계를가지고있다. 다만五積散관련처방의간손상후회복에대한기초자료로사용될수있을것으로판단되며, 향후더많은연구가필요할것으로판단된다. www.e-jkmr.org 51
이수연 박원형 차윤엽 이은 결론»»» 五積散加味方의간기능회복효과를알아보기위하여 CCl 4 로간손상을유도한흰쥐에게五積散加味方추출물을급여한후간장기능에관여하는혈액내생물학적수치와 cytokine 농도를처리군간에비교ㆍ검토한결과, AFP와 Total protein 농도, AST, ALT 및 ALP, γ-gt, LDH의효소활성치들은五積散加味方추출물처리군들이대조군보다낮은수치를나타내었으며, 五積散加味方추출물첨가량이증가함에따라하락하는경향을보였다. Total Cholesterol 량과 Triglyceride 량역시五積散加味方추출물처리군들이대조군보다낮은수치를보였다. Albumin 농도는대조군과처리군간에유의한차이를나타내지않았다. 또한五積散加味方추출물은전염증성 cytokine 들로분류되는 IL-1β, IL-6 및 TNF-α의생산을하락시키는경향을보였으며, 반면에 IL-10의생산을촉진하는경향을보였다. 이상의결과를종합해보면五積散加味方추출물은간기능회복에효과를나타내는기능성물질들이내재하고있음을알수있다. 참고문헌»»» 1. Rabson A, Roitt IM, Delves PJ. Really essential medical immunology. Oxford:Blackwell Publishing Ltd. 2005: 1-14. 2. Lee JC, Lee E, Lee YC, Oh H, Yoon HS, Ha TK, Hong EH. Effects of Fructus Foeniculi extract on recovering liver function. Korean J Herbology. 2007;22(4):213-18. 3. 백상철. 말초성안면마비환자군의한약복용이간기능에미치는영향. 원광대학교석사학위논문. 2011. 4. 陳師文. 太平惠民和劑局方. 서울 : 경희대학교출판국. 2006: 27-8. 5. 전미혜, 백승희, 신상우, 이영선. 五積散이자궁근종세포의사멸과 Cell Apoptosis 에미치는영향. 대한한방부인과학회지. 2003;16(2):45-55. 6. 한대희, 이학인. 五積散전탕액이해열및진통에미치는영향. 대한한의학회지. 1984;5(1):72-86. 7. 박종기, 채우석. 五積散엑기스과립의경구투여와수침이동통에미치는영향. 대한침구학회지. 1992;9(1):273-91. 8. 문영희, 박영준. 五積散의소염진통효과에관한연구. 생약학회지. 1994;25(3):258-63. 9. 손숙영, 조동현, 두호경. 가미소창음자, 가미오적산및가 미보중익기탕이 Gentamicin Sulfate 로유발된白鼠腎損傷에미치는영향. 경희한의대논문집. 1990;13:27-59. 10. 신규원, 이선동, 박해모, 전성진, 변진석. 다용한약처방투여가흰쥐의신장기능에미치는영향. 대한예방한의학회지. 2005;9(1):119-33. 11. 김승조, 김병각, 오도연, 손의섭. 암어떻게치료할것인가. 서울 : 건강신문사. 2003:19. 12. 정용재. 간경화암나으려면바보가되세요. 서울 : 건강다이제스트사. 2004:256. 13. 이삼열. 임상병리해석법. 서울 : 연세대학교출판부. 1991:61-3. 14. Kim KS, Park JH. Effect of Artemisia iwayomomi water extract on hepatic injury by carbon tetrachloride in rats II, Effect on serum ALP, LAP activities, total protein, bilirubin content and liver lycogen content. Kor J Res. 1992;32(3):357-64. 15. Berg B, Johansson BG. Effects on parameters of liver function, plasma lipid oncentration and lipoprotein patterns. Acta Medica Scandinavica. 1973;194(552):13-8. 16. DeCarli LM, Lieber CS. Fatty liver in the rat after prolonged intake of ethanol with a nutritionally adequate new liquid diet. J Nutr. 1967;91(3):331-6. 17. Lieber CS. Biochemical and molecular basis of alcohol induced injury to liver and other tissues. N Eng J Med. 1988;319(25):1639-50. 18. Lieber CS, DeCarli LM. An experimental model of alcohol feeding and liver injury in the baboon. J Med Prim. 1974;3(3):153-63. 19. Kim SW, Kim HW, Cho SI, Yun YC. Effects of Sayeoksanhap-pyeongweisan-Gamibang(SPG) on Hepatological Changes in Animals Damared by CCl4. The Journal of Korean Acupuncure & Moxibustion Society. 2007;24(4): 197-207. 20. 고려의학출판부. 간, 담도질환. 서울 : 고려의학. 1989:27. 21. Jeong TC, Kim HJ, Park JI, Ha CS, Park JD, Kim SI, Roh JK. Protective effects of red gineng saponins against carbon tetrachloride-induced hepatotoxicity in Sprague dawley rats. Planta Medica. 1997;63:136-40. 22. Jung ME, Jung CK. Ham SS, Nam SM, Kang IJ, Kim SJ. Biochemical and histological effects of Phellinus inteus methanol extract on liver lipid metabolism of rats CCl4 and high fat. J Korean Soc Food SCI Nutr. 2001; 30:331-7. 23. Sipes IG, McQueen CA, Gandolifi AJ, McCuskey RS, Earnest DL. Hepatic and gastrointestinal toxicology. In comprehensive Toxicology. Didcot:Pergamon. 1997:251-71. 24. Simko V, Connell AM, Banks B. Nutritional status in alcoholics without liver disease. Amer J Clin Nutr. 1982; 35(2):197-203. 25. Mezey E. Alcoholic liver disease: roles of alcohol and malnutrition. Am J Clin Nutr. 1980;33(12):2709-81. 26. Kim SY, Kim HP, Lee MK, Byun SJ, Kim SH, Han HM, 52 J Korean Med Rehab 2013;23(3):45-53
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