Protective effect of Artemisiae Capillaris Herba water extract on liver injury induced by thioacetamide

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ABSTRACT

Purpose: Thioacetamide (TAA) produces reactive oxygen species (ROS) in the liver, and the generated ROS induces liver injury through inflammatory reactions. The current study was undertaken to investigate the hepatoprotective effect of Artemisiae Capillaris Herba water extract (AC), imparted via its antioxidant activity, in an animal model of TAA-induced liver injury.

Methods: Animal experiments were conducted in 5 groups: normal, control (TAA 200 mg/kg), SM (TAA 200 mg/kg + silymarin 100 mg/kg), ACL (TAA 200 mg/kg + AC 100 mg/kg), ACH (TAA 200 mg/kg + AC 200mg/kg). TAA (intraperitoneal) and treatment compounds (per oral) were administered for 3 days. Serum levels of ammonia concentration and myeloperoxidase (MPO) activity were subsequently measured. Liver tissues were subjected to western blot analysis for measuring the oxidative stress (NADPH oxidase), anti-oxidative activity (Nrf2, heme oxygenase-1 [HO-1], superoxide dismutase [SOD], catalase, and GPx-1/2), tissue inhibitors of metalloproteinase (TIMP)-1, and matrix metalloproteinases (MMPs) protein expressions.

Results: Serum ammonia levels and MPO activity were significantly increased in the TAA- induced control group, whereas groups administered AC treatment showed markedly reduced levels. Western blot analysis revealed significantly increased NOX2 and p22^phox expressions, (oxidative stress-related factors) in the TAA-induced control group. These levels were determined to be significantly decreased after AC exposure. Moreover, antioxidant- related factors including Nrf2, HO-1, SOD, catalase, and GPx-1/2 were significantly decreased in the control group and increased in the AC treated groups. In addition, MMP expressions were significantly suppressed in the AC treatment group due to increased levels of TIMP-1.

Conclusion: Taken together, these data indicate that exposure to AC reduces the oxidative stress by inhibiting the expression of NADPH oxidase (NOX2 and p22^phox) through the Nrf2 signaling pathway. We therefore propose the potential of AC for the prevention and treatment of TAA-induced liver injury.

Keywords: thioacetamide, liver injury, Artemisiae Capillaris, oxidative stress, anti-oxidant

Research Article

Received: Mar 29, 2021 Revised: May 3, 2021 Accepted: May 11, 2021 Correspondence to Seong-Soo Roh

Department of Herbology, College of Korean Medicine, Daegu Haany University, 136 Sincheondong-ro, Suseong-gu, Daegu 42158, Korea.

Tel: +82-53-770-2350 E-mail: [email protected]

creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

ORCID iDs Min Ju Kim

https://orcid.org/0000-0001-5901-3242 Jin A Lee

https://orcid.org/0000-0002-5615-4557 Mi-Rae Shin

https://orcid.org/0000-0002-4365-6988 Hae-Jin Park

https://orcid.org/0000-0002-4283-0809 Seong-Soo Roh

https://orcid.org/0000-0002-4162-6849 Funding

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No.2018R1A5A2025272).

Protective effect of Artemisiae

Capillaris Herba water extract on liver injury induced by thioacetamide

Min Ju Kim ¹, Jin A Lee ¹, Mi-Rae Shin ¹, Hae-Jin Park ², and Seong-Soo Roh ¹

1Department of Herbology, College of Korean Medicine, Daegu Haany University, Daegu 42158, Korea

2DHU Bio Convergence Testing Center, Gyeongsan 38610, Korea

인진호 열수 추출물이 thioacetamide _에

의해 유발된 간손상에 미치는 간보호 효과

김민주 ¹, 이진아 ¹, 신미래 ¹, 박해진 ², 노성수 ¹

1대구한의대학교 한의과대학 본초약리학교실

2대구한의대학교 DHU 바이오융복합시험센터

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Conflict of Interest

There are no financial or other issues that might lead to conflict of interest.

서론

최근과다한음주^,불규칙한식사^,흡연등과같은문제로인하여간기능의손상과심할경우 간질환으로발병하는경우가일어나고있다^[1].이러한다양한원인들로발병되는간질환은 염증반응의반복으로간염이유발되고더욱이간섬유화^,간경변및간암으로진행되어인체

에치명적인영향을주는것으로알려져있다^[2].간질환에대한연구중 thioacetamide (TAA)

로유발된간손상모델에대한연구가많이진행되고있다^.강력한간독성제로알려진^TAA에 의해생성된독성대사산물은간의고분자에결합하여활성산소종 (reactive oxygen species, ROS)을생성하고^,글루타티온을고갈시켜간세포의손상을유도한다고알려져있다^[3].본 연구에서는이러한^TAA를사용하여간손상개선에대한연구를진행하였다^.

국화과 (Asteraceae)에속하는⁴⁰⁰여종의식물중하나인사철쑥 (Artemisia capillaris THUNB) 은다년생식물로우리나라에서는강가또는냇가근처의모래땅에서자라나며^,겨울에도 잘죽지않고후년에줄기에서싹이다시나오는특징을지니고있으며^,이러한사철쑥의지 상부를건조하여사용하는것을인진호 (茵蔯蒿; Artemisiae Capillaris Herba)라고한다^[4,5].

인진호는약간의찬성질을가지고있으며무독하고맛은쓰다^[6].주요성분으로는항산화

와항염증효능이있다고알려진 chlorogenic acid, scopoletin, cineol 및^choline등이있다고알

려져있다^[7,8].인진호에대한연구로는발암물질인 dimethylnitrosamine으로유발된동물모

델에서항간섬유화효과^,항골다공증효과및전뇌허혈 (forebrain ischemia)에대한신경보

호효과등이있으나^{, TAA}로유발된간손상에대한간보호효과에대해서는알려진바가없

다^[9-11].따라서본연구에서는^TAA로유발된간손상동물모델에서인진호열수추출물의

간보호효과에대하여연구를실시하였으며^,이에유의한결과를얻었기에보고하는바이다^.

연구방법

실험재료

본실험에사용된 potassium persulfate, TAA, phenylmethylsulfonyl fluoride (PMSF)는^Sigma Aldrich Co., Ltd. (St. Louis, MO, USA)에서구입하여사용하였으며, protease inhibitor mixture, ethylenediaminetetraacetic acid (EDTA)는 Wako Pure Chemical Industries, Ltd. (Osaka, Japan)에 서구입하였다. Enhanced chemiluminescence (ECL) Western Blotting Detection Reagents는^GE Healthcare (Bloomington, IN, USA)로부터구입하여사용하였다^{. 1}차항체인 β-actin, histone, gp91-phox (NOX2), p22^phox, Nrf2, heme oxygenase-1 (HO-1), superoxide dismutase (SOD), catalase, GPx-1/2, tissue inhibitors of metalloproteinase (TIMP)-1, matrix metalloproteinase (MMP)-1, MMP-2, MMP-8은 Santa Cruz Biotechnology (Dallas, TX, USA)에서구입하였으며^{, 2}차항체인 mouse immunoglobulin G (IgG) antibody와 rabbit IgG antibody는 GeneTex, Inc. (San Antonio,

TX, USA)에서구입하여사용하였다^.

시료추출

본실험에사용된인진호는옹기한약국 (Daegu, Korea)에서구매하였으며^,증류수^{1 L}에인진 호^{100 g}을열탕추출기 (DWT-1800T; Daewoong Pharmaceutical Co., Ltd., Seoul, Korea)에넣고 2시간가열추출하여얻은추출액을농축한후동결건조기 (FD5508; Ilshin Biobase Co., Ltd.,

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Yangju, Korea)를이용하여완전건조시켜가루형태⁽수율^{, 10%)}로만들었으며^,실험에사용

하기전까지^−80°C에서냉동보관하였다^.

실험동물

본실험에사용된수컷^{200 g}내외의⁶주령 Sprague-Dawley rat (DBL Co., Ltd., Eumseong, Korea) 은실험당일까지온도 22 ± 2°C, 습도^{55 ± 5%}가유지되며 conventional system으로명암주기

12시간이잘유지되는사육환경에서물과고형사료 (Zeigler Bros, Inc., PA, USA)를자유롭게

공급받으며 일주일간 사육실 적응기를 가진 후 본 실험에 투입되었다^.또한^,본 실험은 대구한의대학교동물실험윤리위원회 (Institutional Animal Care and Use Committee)에서승인

(DHU2020-082)을받아시행되었으며^,동물관리규정을준수하였다^.

급성간손상동물모델

실험동물은각군당⁸마리씩⁵군으로실험을진행하였다^;정상군 (Normal), 간손상을위한 TAA만을투여한대조군 (Control), silymarin을 100 mg/kg/day로경구투여한^SM군^(SM),인진 호열수추출물을 100 mg/kg/day (성인^{60 kg}일경우, 967.74 mg/60 kg/day)로경구투여한^ACL 군^(ACL),인진호열수추출물을 200 mg/kg/day (성인^{60 kg}일경우, 1,935.48 mg/60 kg/day)로 경구투여한^ACH군^(ACH).모든실험동물은체중을측정하였으며^,정상군을제외한나머지 군은¹일¹회^TAA를 200 mg/kg/day로복강투여하여간손상을유발하였다^{[12]. TAA}와인진 호열수추출물투여를³일간진행한후^{, 4}일째에마취하여복대정맥에서혈액을채취하였으 며^,간조직을적출하였다^.혈액은 4,000 rpm, 4°C으로¹⁰분간원심분리후상층액인혈청을 분리하였으며^,간조직과혈청은실험에사용하기전까지^−80°C에보관하였다^.

혈청내암모니아함량과 myeloperoxidase (MPO) 활성분석

혈청내암모니아함량과^MPO활성은 ammonia assay kit (Abcam, Cambridge, UK)와^MPO colorimetric activity assay kit (BioVision, Milpitas, CA, USA)의프로토콜에따라측정하였다^.

Western blotting

간조직의 western blotting 분석을위해간조직에 buffer A (1.5 M sucrose, pH 7.4/100 mM Tris- HCl, pH 7.5/5 mM Tris–HCl, protease inhibitor cocktail, 0.1 M DTT, 15 mM CaCl2, 2 mM MgCl2) 를넣고조직분쇄기 (BioSpec Product, Bartlesville, OK, USA)를사용하여분쇄하고^ice위에서 30분간정치한다음, 10% NP-40를넣고잘혼합하여 4°C, 12,000 rpm으로²분간원심분리하 여세포질이함유된상층액을분리하였다^.남아있는^pellet을^{10% NP-40}가포함된^{buffer A} 로두번세척한다음 buffer C (0.3 mM NaCl, 50 mM KCl, 10% glycerol, 50 mM HEPES, 0.1 mM EDTA, 1 mM DTT, 0.1 mM PMSF)를넣고¹⁰분간격으로³번^vortex하여 4°C, 12,000 rpm으로 10분간원심분리하여핵을함유하고있는상층액을분리하였다^.분리된세포질과핵시료 는^-80°C에서보관되었다^.세포질내의 β-actin, NOX2, p22^phox, HO-1, SOD, catalase, GPx-1/2, TIMP-1, MMP-1, MMP-2, MMP-8와핵내의 Histone, Nrf2 단백질발현을측정하기위해^{12 µg} 의단백질을 8%–12% SDS-polyacrylamide gel에전기영동후, acrylamide gel을 nitrocellulose membrane으로이동시켰다^.그다음^,각각의¹차항체 (phosphate buffered saline with Tween 20 [PBST]를사용하여^1:1,000으로희석⁾를처리하여^4°C에서 overnight 후^PBST로세척하였 으며^{, 1}차항체에맞는²차항체^(PBST를사용하여^1:3,000으로희석⁾로상온에서²시간반응 후^{, PBST}로세척하였다^.그리고^membrane을^ECL을사용해 Sensi-Q2000 Chemidoc (Lugen

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Sci Co. Ltd, Seoul, Korea)에서감광시켜단백질발현을확인하였다^.또한 ATTO Densitograph Software (ATTO Corporation, Tokyo, Japan) 프로그램으로^band를정량하였으며^,각군들의단 백질수준을정상군의단백질수준⁽정상군의발현비율을¹로기준⁾으로나누어상대적비로 나타내었다^.

병리조직분석

10% neutral buffered formalin에간조직을¹일고정한다음정제된알코올을사용해탈수시키

고^,파라핀을사용해^block을제작하였다^.그후, microtome으로조직을^{5 µm}의두께로절편 하여 hematoxylin & eosin (H&E) 염색을실시하였으며^,광학현미경 (DSCHX50V; Sony, Tokyo,

Japan)을사용하여간조직의병변을확인하였다^.

통계분석

모든수치는평균^±표준편차로표기하였으며^,군간유의수준은 SPSS (version 25.0; IBM, Armonk, NY, USA)에서 one-way analysis of variance test 후, least-significant differences (LSD) test로사후검정을실시하여 p < 0.05에서검증하였다^.

결과

실험동물의체중변화

실험동물의체중변화량 (fold of normal)을측정한결과^,정상군 1.00 ± 0.05에비하여대조군

−2.53 ± 0.30 (p < 0.001)으로유의하게감소하였으며^,간보호유효약물및인진호추출물투

여군은^SM군 −1.92 ± 0.08 (p < 0.01), ACL군 −2.00 ± 0.12 (p < 0.05), ACH군 −1.82 ± 0.10 (p < 0.01) 으로인진호투여군이대조군과비교하여농도의존적으로유의하게증가하는결과를나타 냈다 (Table 1).

혈청내암모니아함량과 MPO 활성분석

혈청내암모니아함량을측정한결과^,정상군에비해대조군에서 50.5% (p < 0.001) 유의하게 증가하였다^{. SM}및인진호추출물투여군들은대조군에비해^SM군 16.3% (p < 0.05), ACL군 28.3% (p < 0.01) 감소하였으며^,특히^ACH군은대조군대비 36.4% (p < 0.001) 감소하여정상

군수준으로나타났다^.혈청내^MPO활성을측정한결과^,대조군은정상군에비해 p < 0.001

Table 1. Initial and final body weight, body weight change

Group Initial body weight (g) Final body weight (g) Body weight change (fold of normal)

Normal 225.30 ± 2.24 237.20 ± 2.08 1.00 ± 0.05

Control 226.95 ± 3.52 196.85 ± 5.71 −2.53 ± 0.30^###

SM 224.58 ± 2.77 201.73 ± 2.93 −1.92 ± 0.08^**

ACL 222.43 ± 2.08 198.64 ± 2.44 −2.00 ± 0.12^*

ACH 225.44 ± 2.12 203.78 ± 2.86 −1.82 ± 0.10^**

All data are expressed as means ± SD (n = 8 rats per group).

Groups are represented as below: Normal, normal rats; Control, TAA-induced rats; SM, TAA-induced + silymarin 100 mg/kg body weight rats; ACL, TAA-induced + Artemisiae Capillaris Herba water extract 100 mg/kg body weight rats; ACH, TAA-induced + Artemisiae Capillaris Herba water extract 200 mg/kg body weight rats.

###p < 0.001 vs. Normal, ^*p < 0.05 and ^**p < 0.01 vs. Control.

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수준으로유의하게증가하였으며^,이러한대조군과비교했을때^SM군은 35.8% (p < 0.01) 감 소하였다^.또한^,인진호투여군인^ACL군과^ACH군은대조군에비하여각각 27.8% (p < 0.05), 51.4% (p < 0.001) 감소함으로농도의존적인경향이나타났다^{(Fig. 1).}

간조직내 NADPH oxidase _발현_분석

간조직내산화적스트레스를측정하기위해 western blotting으로 NADPH oxidase (NOX2, p22^phox)의발현을확인하였다^.그결과^{, NOX2}발현은정상군에비해대조군의발현이^29.0%

(p < 0.001) 유의하게증가하였다^.대조군과비교하여각각^SM군은 11.6% (p < 0.05), ACL군은 19.4% (p < 0.01), ACH군은 22.5% (p < 0.001)로^NOX2의발현이유의하게감소하였으며^,특히

ACH군은정상군수준까지감소하는것을확인할수있었다^{. p22}^phox의발현측정결과^,정상

군과비교해대조군에서 20.0% (p < 0.01) 유의하게증가하였으며^,이러한대조군에비해간 보호약물및추출물투여군들은각각^SM군 11.7% (p < 0.05), ACL군 14.2% (p < 0.01), ACH군 15.0% (p < 0.01)로유의하게감소하였다^{(Fig. 2).}

0 5 10 15 20

Normal Control SM ACL ACH Normal Control SM ACL ACH

Ammonia concentration (nmol/µL) MPO activity (mU/mL)

0 200 100 300 400

###

*

***

** *

###

**

***

A B

Fig. 1. Ammonia concentration and MPO activity in serum. Ammonia concentration (A) and MPO activity (B). TAA (200 mg/kg of body weight, I.P) and drug treatment compounds (P.O) were administered for 3 days. Groups are represented as below: Normal, normal rats; Control, TAA induce rats; SM, TAA-induced + silymarin 100 mg/kg body weight rats; ACL, TAA-induced + Artemisiae Capillaris Herba water extract 100 mg/kg body weight rats; ACH TAA-induced + Artemisiae Capillaris Herba water extract 200 mg/kg body weight rats. All data are expressed as means ± SD (n = 8 rats per group).

MPO, myeloperoxidase.

###p < 0.001 vs. Normal, ^*p < 0.05, ^**p < 0.01, and ^***p < 0.001 vs. Control.

0 0.5 1.0 1.5

Fold of normal Fold of normal

0 0.5 1.0

### 1.5

* ** *** **

##

* **

β-actin p22^phox NOX2

β-actin

A B

Fig. 2. Expressions of NOX2 and p22^phox protein in liver tissue. The protein expression level of NOX2 (A) and p22^phox (B) were detected by western blotting. TAA (200 mg/kg of body weight, I.P) and drug treatment compounds (P.O) were administered for 3 days. Groups are represented as below: Normal, normal rats; Control, TAA induce rats; SM, TAA- induced + silymarin 100 mg/kg body weight rats; ACL, TAA-induced + Artemisiae Capillaris Herba water extract 100 mg/kg body weight rats; ACH TAA-induced + Artemisiae Capillaris Herba water extract 200 mg/kg body weight rats.

All data are expressed as means ± SD (n = 8 rats per group).

##p < 0.01 and ^###p < 0.001 vs. Normal, ^*p < 0.05, ^**p < 0.01, and ^***p < 0.001 vs. Control.

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간조직내항산화관련인자발현분석

간조직내항산화관련전사인자로알려진^Nrf2의발현을측정한결과^,정상군에비해대조

군에서 22.0% (p < 0.001) 유의하게감소하였으며^,대조군과비교해^SM군 15.4% (p < 0.05), ACL군 16.7% (p < 0.05)로증가했으면서^{, ACH}군은증가율은 29.5% (p < 0.001)로정상군수준 까지증가하였다. HO-1, SOD, Catalase 및^GPx-1/2발현측정결과^,모든인자에서정상군대비 대조군이유의하게감소하였으며 (p < 0.001), 간보호약물및추출물투여군들은대조군에 비해증가하는경향을보였다^.특히^ACH군은^GPx-1/2에서대조군보다 46.4% (p < 0.001) 증

가하여정상군수준으로나타났다^{(Fig. 3).}

간조직내 TIMP-1_과 MMPs _발현_분석

간조직내^MMPs발현을측정한결과^,모두정상군에비하여대조군이유의하게증가하는것

을확인하였으며 (p < 0.001), MMP-2와^MMP-8의발현이^ACH군에서대조군에비해정상군

수준까지유의하게감소하는것을확인할수있었다 (p < 0.001). 이러한^MMPs를억제하는

TIMP-1의발현측정결과^,정상군에비해대조군에서^45.0%유의하게감소하였으며^,대조군

과비교해^SM군은 30.9% (p < 0.01), ACL군은 38.2% (p < 0.001), ACH군은 58.2% (p < 0.001) 증

가하는것을확인할수있었다^{(Fig. 4).}

Normal Control SM ACL ACH

Fold of normal Fold of normal Fold of normal

0 0.4 0.8 1.2

0 0.4 0.8

** 1.2

### *

**

###

** **

###

***

**

Catalase β-actin

GPx-1/2 β-actin SOD

β-actin

C D E

0 0.4 0.8 1.2

### * ***

* **

###

* **

β-actin HO-1 Nrf2

Histone

A B

Fig. 3. Expressions of anti-oxidant protein in liver tissue. The protein expression level of Nrf2 (A), HO-1 (B), SOD (C), Catalase (D), and GPx-1/2 (E) were detected by western blotting. TAA (200 mg/kg of body weight, I.P) and drug treatment compounds (P.O) were administered for 3 days. Groups are represented as below:

Normal, normal rats; Control, TAA induce rats; SM, TAA-induced + silymarin 100 mg/kg body weight rats; ACL, TAA-induced + Artemisiae Capillaris Herba water extract 100 mg/kg body weight rats; ACH TAA-induced + Artemisiae Capillaris Herba water extract 200 mg/kg body weight rats. All data are expressed as means

± SD (n = 8 rats per group).

SOD, superoxide dismutase.

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간조직 H&E 염색

간조직을^H&E염색하여관찰한결과^,정상군에비해^TAA를주입한대조군에서중심정맥주

변을기준으로간손상병변과염증세포의침윤을관찰할수있었다^.대조군과비교하여인 진호추출물투여군에서염증세포의침윤이줄어든것을확인할수있었다^{(Fig. 5).}

고찰

간은우리몸에서대사기능에매우중요한역할을하는기관으로^,전세계적으로다양한형태

의간질환의이환율과사망율이증가하고있는추세이다^[13].간에서해독작용이일어날때

많은양의활성산소종^(ROS)이생성되는데^,과도한^ROS로인한산화적스트레스는간에서 염증반응을발생시켜간염을일으키게되고^,간염이만성적으로지속되게되면^,간섬유화와

간경변증및간암등으로진행되어심하면사망까지이르게되는것으로알려져있다^[14].최

근에는이러한산화적스트레스를감소시키는항산화물질에대한연구가많이진행되고있 다^.그렇기에본연구에서는^TAA의산화적스트레스에의한간손상동물모델에서인진호열 수추출물의간보호효과를알아보았다^.

0 0.5 1.0 1.5

### ** *** ***

*

###

*** *

β-actin MMP-1 TIMP-1

β-actin

A B

0 0.5 1.0 1.5

###

*** ** *** **

###

* ***

β-actin MMP-8 MMP-2

β-actin

C D

Fig. 4. Expressions of TIMP-1 and MMPs protein in liver tissue. The protein expression level of TIMP-1 (A), MMP-1 (B), MMP-2 (C), and MMP-8 (D) were detected by western blotting. TAA (200 mg/kg of body weight, I.P) and drug treatment compounds (P.O) were administered for 3 days. Groups are represented as below: Normal, normal rats; Control, TAA induce rats; SM, TAA-induced + silymarin 100 mg/kg body weight rats; ACL, TAA-induced + Artemisiae Capillaris Herba water extract 100 mg/kg body weight rats; ACH TAA-induced + Artemisiae Capillaris Herba water extract 200 mg/kg body weight rats. All data are expressed as means ± SD (n = 8 rats per group).

TIMP, tissue inhibitors of metalloproteinase; MMP, matrix metalloproteinase.

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동물실험후^,혈중암모니아함량과^MPO활성을측정하였다^.간세포효소 (transaminase)에 의해장과간사이의혈관인간문맥을지나간으로이동한암모니아는^TAA를주입할경우질

소화합물인요소^(urea)에의해원할한대사활동을하지못하게되어혈중암모니아수치가

올라가게된다^{[15]. MPO}는호중구과립구에많이있는혈단백질로많은연구에서호중구침

윤의지표로많이사용되며^{, TAA}에의한간손상동물모델에서^MPO활성이증가한다고알려

져있다^[3].이와같은암모니아함량과^MPO활성을혈청내에서측정한결과^,정상군에비

해^TAA로간손상이유발된대조군에서유의하게증가하였으며^,대조군과비교해인진호열 수추출물투여시농도의존적으로유의하게감소시키는것을확인할수있었다^.

인진호열수추출물이산화적스트레스에미치는영향에대해알아보기위해간조직내에서 산화적스트레스와관련된인자의발현을측정하였다. NADPH oxidase (NOX)는여러자극들

에의해반응하여^ROS를생성하는효소이며^[16,17],간내에서^NOX시스템에의하여발생하

는산화적스트레스는간손상등의여러간질환들의진행에중요한역할을한다고알려져있

다^[18].간조직내에서^NOX2와^p22^phox의발현을측정한결과^,정상군에비해대조군에서유

의하게발현이증가한것을확인하였으며^,인진호열수추출물에서대조군보다유의하게감 소하는것을확인하였다^.

우리몸안에서산화적스트레스가일어나게되면세포는산화^-환원반응의항상성을유지하

기위해서항산화작용을하는^Nrf2와기타스트레스반응경로를활성화시킨다^[19].활성화

된^Nrf2는 antioxidant response element와결합하여주요항산화효소들을활성화시키게된다

[20]. 본연구에서는간조직내항산화와관련된 Nrf2, HO-1, SOD, Catalase 및^GPx-1/2의단백 질발현을측정하였으며^,정상군과비교해^TAA로인해산화적스트레스가일어난대조군에 서유의하게감소하였고대조군대비인진호열수투여군에서농도의존적으로유의하게증

A B C

E D

Fig. 5. Pathological observation (hematoxylin & eosin) in liver tissue. Normal (A), Control (B), SM (C), ACL (D), and ACH (E). TAA (200 mg/kg of body weight, I.P) and drug treatment compounds (P.O) were administered for 3 days.

Groups are represented as below: Normal, normal rats; Control, TAA induce rats; SM, TAA-induced + silymarin 100 mg/kg body weight rats; ACL, TAA-induced + Artemisiae Capillaris Herba water extract 100 mg/kg body weight rats; ACH TAA-induced + Artemisiae Capillaris Herba water extract 200 mg/kg body weight rats (×200, scale bar 100 µm).

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가하였다^.이를통해인진호열수추출물이항산화작용을통해^TAA로유발된간손상모델에 서산화적스트레스를일으키는^ROS의생성을억제할것으로판단된다^.

MMPs는세포외기질 (extracellular matrix)의대부분의구성요소를분해할수있는효소로

MMPs의활성은^TIMPs에의해조절된다^[21,22].또한^{, MMPs}는간염^,지방간및간섬유증등 의간질환에서간의재생과정에관여하며^,급성간손상에서산화적스트레스에의해발현이 증가한다고알려져있다^[23].그렇기에본연구에서는간조직내에서^MMPs와^TIMP-1의단 백질발현을측정하였다^.그결과^{, MMPs}는대조군대비인진호열수추출물투여군에서유 의하게발현이감소하였으며^{, TIMP-1}은대조군과비교해인진호열수추출물투여군에서유 의하게발현이증가한것을확인할수있었다^.

종합적으로본연구에서는위와같은결과를통해^TAA로유발된간손상에서인진호열수추 출물이항산화작용을통해산화적스트레스를억제함으로써간보호에효과적인것으로판 단된다^.

요약

본연구는^TAA복강투여로유발된간손상동물모델에서인진호열수추출물의간보호효

능을평가하였으며다음과같은결론을얻었다^{. TAA}로인해줄어드는체중은인진호열수 추출물을투여한군에서유의하게증가하였으며^,간손상에의해증가한혈중암모니아함

량과^MPO활성은인진호열수추출물투여군에서유의하게감소하였다^.간조직의^western

blotting 결과^,인진호열수추출물투여가산화적스트레스관련인자들의발현을유의적으

로감소시키고^,항산화관련인자들의발현을유의하게증가시켰으며^{, MMPs}의발현은감소

시키고^TIMP-1의발현은증가시킴을확인할수있었다^.따라서인진호열수추출물은^TAA로

유발된간손상동물모델에서항산화작용을통해산화적스트레스를억제하여간보호효과 를보이는것으로판단된다^.

REFERENCES

1. Park JY, Park CM, Kim JJ, Song YS. Hepatoprotective activity of Dandelion (Taraxacum officinale) water extract against D-galactosamine-induced hepatitis in rats. J Korean Soc Food Sci Nutr 2008; 37(2): 177-183.

CROSSREF

2. Lee UE, Friedman SL. Mechanisms of hepatic fibrogenesis. Best Pract Res Clin Gastroenterol 2011; 25(2):

195-206.

PUBMED | CROSSREF

3. Özdemir-Kumral ZN, Erkek BE, Karakuş B, Almacı M, Fathi R, Yüksel M, et al. Potential effect of 1,25 Dihydroxyvitamin D3 on thioacetamide-induced hepatotoxicity in rats. J Surg Res 2019; 243: 165-172.

4. Han JM, Kim HG, Choi MK, Lee JS, Lee JS, Wang JH, et al. Artemisia capillaris extract protects against bile duct ligation-induced liver fibrosis in rats. Exp Toxicol Pathol 2013; 65(6): 837-844.

5. Kim JS, Kim KL. Anti-oxidative and anti-inflammatory effects of Artemisiae Capillaris Extract. Korean J Aesthet Cosmetol 2015; 13(6): 805-812.

6. Ham I, Jung SW, Lee KJ, Park KH, Choi HY. Effect of the aerial part of Artemisia capillaris, and A.

iwayomogi on the hyperlipidemia of rats induced by Triton WR-1339. Korea J Herbol 2005; 20(1): 45-52.

(10)

7. Kim HT, Kim JW, Lim MK, Jin TW, Yeo SG, Jang KH, et al. Cytotoxic effect of Artemisia capillaris extracts on the cancer cells on in vitro. J Vet Clin 2007; 24(3): 367-371.

8. Yu F, Qian H, Zhang J, Sun J, Ma Z. Simultaneous quantification of eight organic acid components in Artemisia capillaris Thunb (Yinchen) extract using high-performance liquid chromatography coupled with diode array detection and high-resolution mass spectrometry. J Food Drug Anal 2018; 26(2): 788-795.

9. Kim KS, Yang HJ, Lee JY, Na YC, Kwon SY, Kim YC, et al. Effects of β-sitosterol derived from Artemisia capillaris on the activated human hepatic stellate cells and dimethylnitrosamine-induced mouse liver fibrosis. BMC Complement Altern Med 2014; 14(1): 363-372.

10. Lee SH, Lee JY, Kwon YI, Jang HD. Anti-osteoclastic activity of Artemisia capillaris Thunb. extract depends upon attenuation of osteoclast differentiation and bone tesorption-associated acidification due to chlorogenic acid, hyperoside, and scoparone. Int J Mol Sci 2017; 18(2): 322-335.

CROSSREF

11. Kwon H, Jung JW, Lee YC, Ryu JH, Kim DH. Neuroprotective effect of the ethanol extract of Artemisia capillaris on transient forebrain ischemia in mice via nicotinic cholinergic receptor. Chin J Nat Med 2018;

16(6): 428-435.

12. Luo M, Dong L, Li J, Wang Y, Shang B. Protective effects of pentoxifylline on acute liver injury induced by thioacetamide in rats. Int J Clin Exp Pathol 2015; 8(8): 8990-8996.

PUBMED

13. El Awdan SA, Amin MM, Hassan A. Cilostazol attenuates indices of liver damage induced by

thioacetamide in albino rats through regulating inflammatory cytokines and apoptotic biomarkers. Eur J Pharmacol 2018; 822: 168-176.

14. Kim KJ, Shin MR, Kim SH, Kim SJ, Lee AR, Kwon O, et al. Protective effect of Tongyuhwalhyeol-tang on liver injury in thioacetamide-induced rat. Korea J Herbol 2018; 33(1): 37-46.

15. Brusilow SW. Hyperammonemic encephalopathy. Medicine (Baltimore) 2002; 81(3): 240-249.

16. De Minicis S, Bataller R, Brenner DA. NADPH oxidase in the liver: defensive, offensive, or fibrogenic?

Gastroenterology 2006; 131(1): 272-275.

17. Cheng G, Cao Z, Xu X, van Meir EG, Lambeth JD. Homologs of gp91phox: cloning and tissue expression of Nox3, Nox4, and Nox5. Gene 2001; 269(1-2): 131-140.

18. Paik YH, Kim J, Aoyama T, De Minicis S, Bataller R, Brenner DA. Role of NADPH oxidases in liver fibrosis.

Antioxid Redox Signal 2014; 20(17): 2854-2872.

19. Kasai S, Shimizu S, Tatara Y, Mimura J, Itoh K. Regulation of Nrf2 by mitochondrial reactive oxygen species in physiology and pathology. Biomolecules 2020; 10(2): 320-340.

20. Lee JA, Park HJ, Kim SH, Kim MJ, Kim KJ, Shin MR, et al. Evaluation of Evodiae Fructus extract on the chronic acid reflux esophagitis in rats. Korea J Herbol 2019; 34(2): 15-23.

21. Kessenbrock K, Plaks V, Werb Z. Matrix metalloproteinases: regulators of the tumor microenvironment.

Cell 2010; 141(1): 52-67.

22. Visse R, Nagase H. Matrix metalloproteinases and tissue inhibitors of metalloproteinases: structure, function, and biochemistry. Circ Res 2003; 92(8): 827-839.

23. Naim A, Pan Q, Baig MS. Matrix metalloproteinases (MMPs) in liver diseases. J Clin Exp Hepatol 2017;

7(4): 367-372.

Protective effect of Artemisiae Capillaris Herba water extract on liver injury induced by thioacetamide

Protective effect of Artemisiae

Capillaris Herba water extract on liver injury induced by thioacetamide

인진호 열수 추출물이 thioacetamide 에

의해 유발된 간손상에 미치는 간보호 효과

인진호 열수 추출물이 thioacetamide _에