活體即時性肝炎報導平台之建立

第二章文獻探討

2.5 活體即時性肝炎報導平台之建立

傳統上，抗慢性肝炎藥物在活體的研究，大多都是利用動物解剖

終點組織病理實驗以及血清生化指標加以評估，但這種方式需要長達

數週至數個月的時間，而且需要犧牲相當數量的動物才能達到客觀的

標準。除了活體模式 (in vivo) 外，以疾病分生為標的之細胞模式也

被應用於抗肝炎藥物的篩選。雖然以疾病分生標的之細胞模式進行搜

尋，確實可以加快藥物的評估，但是體外試驗 (in vitro) 證實有效的

藥物，進入活體試驗時的失敗率相當高。此外，有許多方法可以直接

以活體試驗偵測肝炎分生標的的活性，例如：免疫組織化學染色法

(immunohistochemistry stain; IHC) 、原位雜交反應 (in situ

9 hybridization) 等，但這些方法都無法即時性地反應動物體內肝臟發炎的現象。目前，非侵入性 (noninvasive) 、即時性 (real-time) 分子影像技術已經被廣泛地應用在分子、細胞和組織階層的影像化及量化，

例如：顯微電腦斷層 (micro-computed tomography; micro-CT) 可應用於當作肝硬化程度的評估工具 (66) ；同步遠紅外線 (Synchrotron Infrared) 及飛行時間型二次離子質譜儀結合光譜儀 (ToF-SIMS Microspectroscopies) 可用來評估脂肪肝切片 (67) ；磁振造影 (magnetic resonance image; MRI) 可使用在臨床預測肝纖維化及藥物治療效果的評估 (68) 。近年來，靈敏度相當高的新興生物冷光影像 (bioluminescence imaging; BLI) 已被運用於偵測及定量小鼠體肝部位生長激素訊息傳遞及肝腫瘤的成像 (69, 70) ，然而在肝纖維化及肝發炎偵測卻尚還未被應用。

2.6 Microarray 分析平台的建立

Microarray 的雛形是由 Augenlicht 等發展的(71)。他們將人類大腸癌細胞 HT-29 的 mRNA 合成約 4000 種 cDNA，再將所有 cDNA 點到狀似小方格的硝化纖維濾紙上。之後抽取自大腸癌早期及晚期病人活體組織樣品的 mRNA，合成放射線標定的 cDNA 探針，與這些濾紙進行雜合，再分析放射線的強度，觀察基因表現的波動。真正出現 microarray 並加入自動化系統技術，是由史丹福大學的 Schena 等（1995）及 Shalon 等（1996）發展的。他們將阿拉伯芥

（Arabidopsis thaliana）約 50 種 cDNA 利用聚合酶連鎖反應增殖後，

點到玻璃玻片上，再將取材自野生型及 HAT4 基因轉殖後阿拉伯芥

的 mRNA，分別合成有螢光標定及 lisaamine 標定的 cDNA，進一

步將這兩種 cDNA 與玻片進行競爭性的雜合反應。結果發現，在

10 HAT4 基因的位置，lissamine 的強度較螢光多 50 倍，顯示利用不同

標定方式，可以在同一時間在同一個雜合玻片上，分析到不同處理

組基因表現的變化 (72)。這套系統被開發後，隨即被應用於監測不

同狀態下細胞內所有基因表現的波動，例如酵母菌的細胞週期、果

蠅在變態時的發育、細胞不同代謝狀態的基因表現變化(73-76)，另

外也應用於分析腫瘤轉移、癌症、其他疾病的基因表現圖譜及疾病

病程的預測和疾病的診斷(77-84)。因此，microarray 的技術也常被

用來研究中藥及其組成物的作用機轉。此研究中我使用活體分子影

像來進行肝炎的即時監控，搭配 microarray 為分析工具所形成的一

個新型平台，我們將它命名為「分子影像導引轉錄學平台」

(bioluminescent imaging-guided transcriptomic) 。我們利用分子影像

進行中藥作用位置的活性偵測，再利用 microarray 進行 gene

ontology、pathway 及 clustering 分析，這樣的模式在中醫藥作用機

轉的偵測提供非常多的方便性。

11 第三章材料與方法

3.1 材料

CCl 4 購自昭和化學工業株式會社 (Showa chemical industry co.,ltd) 。橄欖油購自Sigma (St. Louis, MO) 。天狼星紅Sirius red購自 Sigma (St. Louis, MO) 。picric acid 購自Panreac (Panreac, Barcelona, Spain) 。水飛薊素 (Silymarin) 購自Sigma (St. Louis, MO) ，並溶於無菌水 (混懸) 最終濃度20 mg/ml 冷凍在-20℃保存。D-Luciferin 購自 Xenogen (Hopkinton, MA) ，溶於PBS。茵陳蒿湯、茵陳蒿、大黃、山梔子科學化中藥購自順天堂。 Genipin購自Wako (Osaka, Japan) 。 Lipopolysaccharide (LPS) 及 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazoliumbromide (MTT) 購自Sigma (St. Louis, MO) ，並分別溶於無菌水及磷酸鹽緩衝液 (phosphate-buffered saline; PBS) (137 mM NaCl, 1.4 mM KH ₂ PO ₄ , 4.3 mM Na ₂ HPO ₄ , 2.7 mM KCl, pH 7.2) 。單株抗體試劑luciferase、IL-1β、TNF-α、TGF-β1和α-SMA購自Santa Cruz (Santa Cruz, CA) 及NF-κB p65 購自Chemicon (Temecula, CA) 。

3.2 NF-κB基因轉殖鼠的構築

實驗室先前探討中藥的抗發炎作用，大多利用 NF-κB/luciferase 之重組細胞株來進行中藥及主要活性物質的測試。為了使報導發炎疾病研究模式更接近真實發炎情況，我們將此細胞平台進一步的推進到 NF-κB/luciferase 基因轉殖鼠之發炎疾病動物模式，以利中醫藥在抗發炎藥物上的探勘，在本研究中主要以研究茵陳蒿湯在抗肝炎上的效果與細部機轉。

我們首先構築 NF-κB 冷光報導的基因轉殖小鼠。構築載體的策

略是將 NF-κB response element 接在 TATA-BOX 上游，下游報導基因

採用

3. 2 NF-κ

14 老鼠以腹腔注射 0.5 ml/kg 10% (v/v) 溶在橄欖油的 CCl ₄ 每週 2 次，

持續 8 週，並在第 5 週開始同時以胃管餵食投予依照每天人體劑量的茵陳蒿湯、茵陳蒿、大黃、梔子 150 mg/kg 每天一次到第 8 週。

3.4 體內及器官內冷光酵素活性偵測

將每公斤體重 150 mg 之冷光素 (luciferin) 以腹腔注射方式打入基因轉殖鼠體內，五分鐘後以異氟烷 (isoflurane) 進行麻醉，將基因轉殖鼠置於密封不透光裝有 25 mm 鏡頭的 CCD 相機和影像增強器結合構成的影像系統 (IVIS Imaging Systems 100 Series, Xenogen Corporation) 中，進行全身性活體影像分析，並利用 Living Images software (Xenogen) 測定冷光值，單位以 photons/sec/cm ² /steradian (photons/sec/cm ² /sr) 表示。

進一步進行器官剖檢，冷光素以腹腔注射方式打入基因轉殖鼠體內，五分鐘後將基因轉殖鼠犧牲，並取其全身實質臟器進行冷光值偵測，單位以 photons/sec 表示。

3.5 肝纖維化的定量分析

為了偵測肝臟纖維化，我們將肝臟切片以溶解在飽合苦味酸

(picric acid) 的 0.1% (w/v) 天狼星紅 (Sirius red；Sigma, St Louis, MO)

進行染色。經染色一小時後，切片以 0.5% (v/v) 的醋酸水潤濕，並

換過兩次新的醋酸水。接著經過 100%酒精脫水乾燥後，再以阿拉伯

膠進行封片後以顯微鏡觀察。我們利用 Image-Pro Plus (Media

Cybernetics, Bethesda, MD) 這套軟體進行分析天狼星紅陽性 (紅色

絲狀) 的區域。天狼星紅陽性區域代表肝纖維化程度，利用軟體分

析結果，將肝纖維化的區域除以觀察總區域所得即為肝纖維化比

15 例。

3.6 免疫組織化學染色

將肝臟組織從小鼠體內取出後浸泡於 10% 福馬林 (phosphatebuffered formalin solution) 中二天進行固定，然後，以生理食鹽水(saline)沖洗，再將組織塊進行石蠟包埋。石臘包埋的肝臟組織我們將其切成5-μm的切片並且做蘇木紫-伊紅染色法 (hematoxylin and eosin stain; H&E stain) 的染色。為了進行免疫組織化學染色 (immunohistochemistry stain; IHC) ，切片先以二甲苯 (xylene) 除掉石蠟，然後以一系列濃度梯度之酒精 (99.5%、95%、75% alcohols) 進行覆水作用 (rehydration) 。接著，將切片浸泡在3%雙氧水 (hydrogen peroxide) 中作用十五分鐘以清除組織中的過氧酵素 (endogenous peroxidase)，再以1% 胎牛血清白蛋白 (bovine serum albumin) 作用一小時以阻斷非特異的蛋白質。接著切片再與1:50稀釋的一級抗體於4°C 下作用 16-24小時。然後，再以接有biotin的二級抗體 (biotinylated secondary antibody) (Zymed Laboratories, South San Francisco, CA) 作用二十分鐘。最後，切片以生物素 -過氧化酵素複合物 (avidin-biotin complex; ABC) 的試劑作用二十分鐘，然後再以3,3’-diaminobenzidine (DAB) 染色。染色後的切片於光學顯微鏡下進行判讀。

3.7 RNA 萃取

我們依照 RNeasy Mini kit (Qiagen, Valencia, CA) 的操作步驟萃

取組織的 total RNA。隨後再利用 Beckman DU800 分光光度計

(Beckman Coulter, Fullerton, CA) 進行 total RNA 的定量。若樣品的

O.D. 值 A260/A280 比值大於 1.8，則進一步利用 Aglient 2100

16 bioanalyzer (Agilent Technologies, Santa Clara, CA) 評估其 total RNA 的品質。當樣品的 RNA integrity number (RIN) 高於 8.0 時，樣品才會進入下述的 DNA 微陣列實驗分析。

3.8 DNA 微陣列實驗分析

DNA微陣列 (microarray) 實驗分析方法如Cheng等報告中所述 (86) 。簡述之，將5 μg total RNA利用MessageAmp ^TM aRNA kit (Ambion, Austin, TX) ，經由試管外轉錄 (in vitro transcription) 的步驟增量樣品。

增量後的 RNA (amplified RNA ，簡稱 aRNA) 再和螢光物質 Cy5

(Amersham Pharmacia, Piscataway, NJ) 進行標定，當aRNA被Cy5染劑標

定上，將使得aRNA成為帶有螢光標定的標的物 (target) 。接著，利用

蓋玻片和 Phalanx 公司所提供的雜合反應緩衝劑 (hybridization

buffer)，將螢光標定的標的物與 Mouse Whole Genome OneArray ^TM

(Phalanx Biotech Group, Hsinchu, Taiwan) 進行雜合反應

(hybridization) 。於50°C下，經隔夜 (overnight) 的雜合反應之後，藉

由後續清洗處理步驟將非專一性結合的標的物從晶片上清除。接著將

晶片以離心的方式乾燥，並利用掃描器Axon 4000 Scanner (Molecular

Devices, Sunnyvale, CA) 進行螢光強度的掃描。掃描的圖檔利用

Genepix (Molecular Devices Co., PA) 分析軟體 (Genepix 4.1 版) 進行

螢光強度進行分析。每一點的訊號經由扣除周圍背景值的方式校正其

強度。我們刪除作為內在控制的探針 (probe) 或是訊雜比

(signal-to-noise ratio) 小於零的點。通過這些門檻的點藉由R程式的

limma package進行normalization (87) 。經過normalized 資料我們利用

Gene Expression Pattern Analysis Suite v3.1 計算有顯著差異的基因

(88) 。我們選擇大於等於 2 倍或小於等於 2 倍變化的基因，利用

17 WebGestalt (http://bioinfo.vanderbilt.edu/webgestalt/login.php) 網頁分析基因群關聯的訊息路徑。另外，這些表現有差異的基因再藉由TIGR Multiexperiment Viewer (89) (http://www.tm4.org/index.html) 進行階層式叢集分析 (hierarchical clustering analysis) 用以展現它們標的器官的表現形式。接著，將表現有差異的基因表單送到Gene Ontology Tree Machine 網頁 (http://bioinfo.vanderbilt.edu/gotm/) 進行Gene Ontology (GO)分析，以找出這些受調控的基因所影響的細胞行為模組。Gene Ontology Tree Machine是一個在網路上使用，以基因群組 (gene set) 為分析基礎的資料探勘 (data mining) 工具 (90) 。最後，我們利用 BiblioSphere Pathway Edition 軟體 (Genomatix Applications, http://www.genomatix.de/index.html) 建構表現有差異基因之間的交互作用網路 (interaction network) 。BiblioSphere Pathway Edition 軟體以知識庫分析為基礎，整合了文獻探勘 (literature mining) 、基因註解分析和啟動子 (promoter) 序列分析來建構基因交互作用網路 (91) 。本實驗的重複數為三次。

3.9 即時螢光定量聚合酶連鎖反應 (qPCR)

我們利用qPCR的方式定量cytochrome c oxidase 基因 (Cox6a2,

Cox7a1, and Cox8b) 、 interferon inducible GTPase 1 (Iigp1) 及

interferon-inducible gene (Ifi202) 的表現量。 RNA 樣品利用 High

Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster

City, CA, USA) 經2小時 37℃反轉錄的反應轉換成cDNA。接著利用1

μl 的 cDNA, 2× SYBR Green PCR Master Mix (Applied Biosystems) ，

以及200 nM的正股引子 (forward) 跟反股引子 (reverse) 進行聚合酶

反應，反應條件為 95℃ 10分鐘；接著95℃ 15秒，60℃ 1分鐘 40

18 次循環。這些分析我們利用Applied Biosystems 7300 Real-Time PCR 系統且三重複。基因表現的倍率變化我們利用相對CT 值的方法做定量計算。本研究所用引子如下： Cox6a2 forward, 5'-CAGAGAAGGACAGTGCCATTC-3'; Cox6a2 reverse, 5'-GAAGAGCCAGCACAAAGGTC-3'; Cox7a1 forward, 5'-CAATGACCTCCCAGTACACTTG-3'; Cox7a1 reverse, 5'-CCAAGCAGTATAAGCAGTAGGC-3'; Cox8b forward, 5'-TCCCAAAGCCCATGTCTCTG-3'; Cox8b reverse, 5'-CATCCTGCTGGAACCATGAAG-3'; Iigp1 forward, 5'-CTTGACATGGTGACTGAGGATG-3'; Iigp1 reverse, 5'-AGGTGGATAAAGCCCGAATAAC-3'; Ifi202 forward, 5'-AAGGCTGGTTGATGGAGAG-3'; Ifi202 reverse, 5'-GTCAATTCAAAGCAGACAAGTC-3'; glyceraldahyde-3-phosphate

dehydrogenase (GAPDH) forward, 5′-TCACCCACACTGTGCCCATCTATGA-3′; GAPDH reverse,

5′-GAGGAAGAGGATGCGGCAGTGG-3′.

3.10 細胞培養及Lipopolysaccharide (LPS) 的處理

重組HepG2/NF-κB細胞株帶有NF-κB-responsive elements可驅使 luciferase表現，構築詳細內文參照我們過往發表的文獻 (62) ，以含有10%胎牛血清 (Fetal bovine serum) (HyClone, Logan, Utah) 和400 ng/ml 建那黴素 (Geneticin; G418) 的 Dulbecco’s modified Eagle medium (DMEM) (Life Technologies, Gaithersburg, MD) 於37°C培養箱中培養。將HepG2/NF-κB培養於96孔盤，24小時後，將培養液置換成DMEM繼續培養24小時，然後再處理不同濃度的LPS作用16小時。

3.11 細胞培養及genipin的處理

19 重組細胞株HepG2/NF-κB以含有10%胎牛血清 (HyClone, Logan, Utah) 和400 ng/ml建那黴素 (Geneticin; G418) 的Dulbecco′s modified Eagle′s medium (DMEM) (Life Technologies, Gaithersburg, MD) 於 37°C培養箱中培養。將HepG2/NF-κB細胞培養於96孔盤，24小時後，

再處理100 ng/ml LPS及不同濃度的genipin作用24小時。

3.12 NF-κB活性測試

為了測試發炎誘發物 LPS 是否會誘發細胞中 NF-κB 的活性， HepG2/NF-κB處理不同濃度的LPS，16小時之後，以 350 μl Triton lysis buffer (50 mM Tris-HCl, 1% Triton X-100, 1 mM dithiothreitol, pH 7.8) 使細胞溶解，以12,000 xg的轉速，在4°C下離心2分鐘。將20 μl的細胞溶解液與20 μl 冷光試劑 (470 μM luciferin, 33.3 mM dithiothreitol, 270 μM coenzyme A, 530 μM ATP, 20 mM Tricine, 1.07 mM (MgCO 3 ) 4 , Mg(OH) 2 , 2.67 mM MgSO 4 , 0.1 mM EDTA, pH 7.8) 混合，利用冷光儀 (luminometer, FB15, Zylux Corp., Maryville, TN) 測定冷光值，單位以 relative luciferase unit (RLU) 表示。活化倍率的計算方式是以LPS處理細胞的RLU除以未受LPS處理細胞的RLU。

3.13 MTT 分析法

為了測試發炎反應誘發物LPS是否會毒殺細胞，HepG2/NF-κB處理不同濃度的LPS處理24小時，利用MTT比色分析法偵測細胞存活率。

細胞存活率(%)的計算公式為：(處理LPS之細胞OD值/未處理LPS之細胞OD值)×100%。

3.14 Genipin動物實驗

所使用的基因轉殖鼠請參照材料與方法3.1及3.2。我們將25頭基

20 因轉殖鼠 (母鼠6-8週大) 隨機分成5組，每組5頭： (1) mock組，沒有任何的處理； (2) LPS組，腹腔注射4 mg/kg劑量的LPS； (3) LPS加 genipin 1 mg/kg組，腹腔注射4 mg/kg劑量的LPS後10分鐘，接著腹腔注射genipin 1 mg/kg； (4) LPS加genipin 10 mg/kg組，腹腔注射4 mg/kg 劑量的LPS後10分鐘，接著腹腔注射genipin 10 mg/kg； (5) LPS加 genipin 100 mg/kg組，腹腔注射4 mg/kg劑量的LPS經過10分鐘後，接著腹腔注射genipin 100 mg/kg。經過4小時後，基因轉殖鼠以IVIS照影，

分析其體內冷光活性，之後解剖摘取實質器官進行冷光照影、組織取樣提供RNA萃取及免疫組織化學染色。

3.15 細胞激素的酵素連結免疫吸附法

我們利用酵素連結免疫吸附法套組 (Enzyme-linked immunosorbent assay, ELISA) OptEIA ^TM mouse IL-1β 和 TNF-α sets (Pharmingen, San Diego, CA) 進行IL-1β 和 TNF-α的定量。基因轉殖鼠犧牲後，取其血液。於4 °C環境下，以轉速3000 rpm、離心10分鐘，

之後，取其血清。血清以每一格200 μl的體積加入作用盤中與盤底材料進行室溫接合反應2小時，接著使用IL-1β或 TNF-α單株抗體進與血清中標的接合2小時，經3次 PBS含0.05% Tween 20洗滌後，以biotin 接合的2級抗體進行反應1.5個小時，而後以過氧化氫酶接合avidin的試劑進行催化作用呈色，再以O.D. 450 nm吸收光分析。

3.16 統計分析

實驗所得資料數據我們以平均值±標準差來呈現。大於3個組別的

數據，我們利用PASW Statistics (SPSS) 軟體以one way ANOVA 及事後檢

定LSD 進行分析當p value小於0.05為顯著差異。

21 第四章結果

4.1 NF-κB活體即時性肝炎報導平台之建立

4.1.1 Silymarin穩定下降CCl 4 誘發的肝臟NF-κB活性

基因轉殖鼠經過投予CCl 4 或 silymarin，在第4 週、第6週、第8 週、第12週，經過分子影像照影偵測NF-κB活性，結果呈現如圖4.1。

與Mock組對照起來，CCl 4 很顯著的誘發小鼠腹腔區域冷光值。經過解剖觀察實質器官後，發現CCl 4 的確很專一的誘發肝臟冷光，圖4.2。

這個結果顯示， CCl 4 誘發小鼠腹腔區域生物冷光訊號是來自於肝臟。

以上結果證實CCl 4 可以專一的誘發肝臟NF-κB活化，另外silymarin 也顯示可以依投予時間穩定的下降CCl 4 在肝臟所誘發的NF-κB活性。

4.1.2 Silymarin 降低肝臟 NF-κB 活性與改善肝纖維化之間的關聯性為了評估肝病變和肝纖維化的程度，我們利用H&E染色和天狼星紅進行的肝臟切片染色。肝。天狼星紅是一種很強的陰離子染料，已經在膠原蛋白的專一染色與在組織切片纖維化的量化上(92, 93) 。纖維化主要是由於被積累在肝臟組織的膠原蛋白所誘發 (94) 。因此，

天狼星紅陽性粉紅絲狀面積可以直接用來標記肝纖維化的程度。如圖 4.3所示， H&E染色顯示四氯化碳的攻擊使肝臟組織結構受損，這是由免疫細胞的浸潤、出血、肝細胞形成空泡變性所造成，如圖中箭頭所示。天狼星紅在mock組的肝門靜脈周圍出現陽性區域而非在肝臟實質，天狼星紅染色區域很清晰地出現在肝臟組織上呈網狀結構，經過定量統計肝纖維化面積的比例為3.86±0.54%。圖中CCl 4 與silymarin 組相較之下， silymarin明顯的改善CCl 4 誘導的肝組織病理變化。此外，

silymarin組與CCl 4 組相較之下，天狼星紅染色區域明顯的減少，經過

定量統計經silymarin治療後肝纖維化區域的比例為1.94±0.29%，有顯

22 著下降的現象。這些結果顯示，silymarin可改善CCl ₄ 誘導的肝纖維化。

我們進一步利用免疫組織化學染色去偵測NF-κB活性與肝纖維化的相關性。我們利用α-SMA抗體進行肝切片的免疫染色，以檢測肌纖維母細胞所產生膠原蛋白是否存在 (95) 。另外，我們利用TGF-β1抗體做免疫組織化學染色，TGF-β1在肝纖維化中扮演著很重要的一種細胞因子 (96) 。如圖4.4所示，在四氯化碳組的肝臟呈現出許多褐色的TGF-β1陽性細胞和α-SMA陽性的肌纖維母細胞。然而，silymarin 口服組明顯的降低了肝臟的褐色細胞的數量。這些發現顯示， silymarin明顯改善四氯化碳誘導的肝纖維化，這些結果與上述H&E和天狼星紅組織染色的結果吻合。進一步，我們利用p65 (NF-κB) 抗體進行免疫組織化學染色，結果顯示經過四氯化碳誘導的肝細胞有許多褐色的p65陽性細胞。然而，silymarin明顯下降在肝臟中p65的陽性細胞的數量。這些結果顯示，silymarin可能透過抑制NF-κB、TGF-β1及 α-SMA進而改善四氯化碳所誘發的肝纖維化。此外，我們也發現 NF-κB的活性與肝纖維化在基因轉殖鼠上所發出的冷光影像關係成正比，因此證實NF-κB活體即時性影像可以當作抗肝纖維化藥物療效評估的可行性。

4.1.3 Silymarin 治療四氯化碳誘發肝纖維化的基因表現圖譜分析

我們進一步利用 microarray 分析了 silymarin 治療組的肝基因反應

圖譜，以確定 silymarin 的新穎作用機轉。CCl ₄ 組基因反應圖譜與 Mock

組相比，在 CCl ₄ 誘發下有 420 個基因上調 439 個基因下調超過 2 倍

以上。而 silymarin 基因反應圖譜在與 CCl ₄ 組比較下，silymarin 處理

之下有 67 個基因變化大於等於 2.0 倍或小於等於-2.0 倍，其中包括 2

23 個基因上調和 65 個基因下調。進一步，我們將這些基因進行 pathway 的分析。分析結果 (表 4.1) 顯示由 silymarin 顯著改變的 34 個 pathway (p <0.01) ，在這 34 個 pathway 當中，有一半以上與代謝途徑相關，

而另外有一些 pathway 與細胞生存過程的調控和信號轉導有關。而 TGF-β 相關的 pathway，包括 TGF-βsignaling pathway，TGF-β-induced apoptosis 和 TGF-β-mediated pathway 都有明顯的受 silymarin 調控。

因為 TGF-β1 與 TGF-β pathway 的調控在肝纖維化的過程中扮演很重要的角色跟作用，而 silymarin 調控這個分子進而調控 pathway 可能有助於改善 CCl ₄ 所誘發的肝纖維化。在四氯化碳的攻擊之下 silymarin 一共下調了 65 個基因的表現，這些基因倍率變化小於等於 -4.0 倍如表 4.2 顯示。有一半被 silymarin 所下調的基因是與細胞骨架組成和肌肉收縮有關，而其中有 3 個基因，包括 Cox6a2、Cox7a2 和 Cox8b，

均與粒腺體電子傳遞鏈有關聯。這些結果顯示，silymarin 可能改善 CCl ₄ 誘導的肝纖維化，主要是透過調控參與細胞骨架組織和電子傳遞鏈的基因表現。

4.1.4 qPCR 確認受 silymarin 調控的新穎基因表現程度

Microarray結果顯示，與粒腺體呼吸鏈相關的基因，包括Cox6a2, Cox7a1 和 Cox8b基因都受到silymarin的下調。我們進一步利用定量 PCR來驗證這些基因的轉錄表現程度。如表4.3所示，Cox6a2、Cox7a1 和Cox8b基因在 CCl ₄ 組的表現程度都高於Mock組，倍率分別496.21，

21.36和240.38倍。然而，Silymarin組相較於Mock組，Cox6a2，Cox7a1，

Cox8b基因的表現倍率分別為9.84，0.72，0.7倍，此結果證明CCl ₄ 所

上調的基因表現都被silymarin給抑制了。

)

(B)

4.1 小鼠肝 Cl 4 或 sily

身上的冷色位為 photo 定量統計圖示

p<0.00

肝炎即時 ymarin，並

色系到暖 on/sec，圖圖。冷光值 1；與 CC

時性活體 N 並且在各個暖色系從藍

圖中每個組值以平均 Cl ₄ 比較*表

24 NF-κB/冷光個指定時間藍到紅的顏

組別皆有均值 ± 標準

表示

p<0.0

光報導。基間內照影顏色，分別

8 重複。

準差，組別 05，**表

基因轉殖鼠

。 (A) 活別代表冷光 (B) 全身別與 mock 示

p<0.01

殖鼠經過投活體影像光表現強度身影像冷光

k 比較，

1。

投予

，小度，

光值

###

(A)

(A)

圖

28 表 4.1 Silymarin 所調控的 pathway 分析。

Pathway p value

Regulation of cellular process/ cell cycle and death

TGF-β signaling pathway 2.75×10 ^-7

p53-mediated pathway 0.00171

Tight junction 0.00014

TGF-β-induced apoptosis 0.00261

Adherens junction 0.00494

TGF-β-mediated pathway 0.00976

Metabolism Urea cycle and metabolism of amino groups 2.49×10 ^-5

Citrate cycle 2.45×10 ^-6

Arginine and proline metabolism 0.00016

Galactose metabolism 0.00034

Biosynthesis of steroids 0.00049

Glycine, serine and threonine metabolism 0.00070

Glycolysis / Gluconeogenesis 0.00100

Butanoate metabolism 0.00098

Folate biosynthesis 0.00218

Pyruvate metabolism 0.00223

Fatty acid metabolism 0.00273

Bile acid biosynthesis 0.00273

Alanine and aspartate metabolism 0.00442

Glutathione metabolism 0.00544

Starch and sucrose metabolism 0.00601

Glycosaminoglycan degradation 0.00799

Glutamate metabolism 0.00921

Signal tansduction

Adipocytokine signaling pathway 8.26×10 ^-5

IL6 signaling pathway 0.00016

PPAR signaling pathway 0.00039

Insulin signaling pathway 0.00047

Vitamin D3 signaling pathway 0.00067

RANKL signaling pathway 0.00548

TNF signaling pathway 0.00629

IGF signaling pathway 0.00655

Chemokine signaling pathway 0.00709

EGF signaling pathway 0.00770

PTH/PTHrP signaling pathway 0.00840

a p value 的數據是利用 WebGestalt 網頁藉由 hypergeometric test 所計算.

29 表 4. 2 silymarin 下調 CCl ₄ 所誘發的肝臟基因表現倍率變化。

Gene symbol Description Fold changes

Acta1 Actin, alpha 1, skeletal muscle -90.21±0.001

Myl1 Myosin, light polypeptide 1 -77.05±0.001

Tnni2 Troponin I, skeletal, fast 2 -49.39±0.001

Atp2a1 ATPase, Ca ⁺² transporting, cardiac muscle, fast twitch 1 -48.46±0.001 Mylpf Myosin light chain, phosphorylatable, fast skeletal muscle -41.90±0.001

Mb Myoglobin -35.39±0.002

Cox6a2 Cytochrome c oxidase, subunit VI a, polypeptide 2 -28.43±0.003

Cox8b Cytochrome c oxidase, subunit VIII b -18.60±0.004

Eno3 Enolase 3, beta muscle -8.17±0.009

Tnnt1 Troponin T1, skeletal, slow -7.60±0.011

Tnnc1 Troponin C, cardiac/slow skeletal -7.56±0.010

Cox7a1 Cytochrome c oxidase, subunit VIIa 1 -6.67±0.015

Eef1a2 Eukaryotic translation elongation factor 1 alpha 2 -4.64±0.022 EG433229 Predicted gene, EG433229, transcript variant 7 -4.06±0.016

a 倍率變化表示為平均值±標準差 (n=3) 。

30

表4.3 qPCR分析Cox6a2、Cox7a1、和 Cox8b 基因表現程度。

Sample Average C

of target Average C

of GAPDH ΔC

ΔΔC

Relative to mock Cox6a2

Mock 35.82 ± 0.40 19.71 ± 0.03 16.11 ± 0.40 0.00 ± 0.40 1.00

CCl

25.80 ± 0.05 18.64 ± 0.05 7.16 ± 0.07 -8.96 ± 0.07 496.21 Silymarin 31.17 ± 0.09 18.36 ± 0.01 12.81 ± 0.09 -3.30 ± 0.09 9.84 Cox7a1

Mock 29.98 ± 0.10 19.71 ± 0.03 10.27 ± 0.11 0.00 ± 0.11 1.00

CCl

24.49 ± 0.04 18.64 ± 0.05 5.85 ± 0.07 -4.42 ± 0.07 21.36 Silymarin 29.10 ±0 .11 18.36 ± 0.01 10.74 ± 0.11 0.47 ± 0.11 0.72 Cox8b

Mock 32.81 ± 0.11 19.71 ± 0.03 13.09 ± 0.12 0.00 ± 0.12 1.00

CCl

23.83 ± 0.05 18.64 ± 0.05 5.18 ± 0.07 -7.91 ± 0.07 240.38 Silymarin 31.97 ± 0.29 18.36 ± 0.01 13.61 ± 0.29 0.52 ± 0.29 0.70

The ΔC

value is determined by subtracting the average GAPDH C

value from the average target gene C

value. The standard deviation of the difference is calculated from the standard deviations of the target gene and GAPDH.

The calculation of ΔΔC

involves subtraction by the ΔC

calibrator value. This is a subtraction of an arbitrary constant, so the standard deviation ofΔΔC

is the same as the standard deviation of ΔC

value.

30

31

4.2 茵陳蒿湯及其組成在肝炎之療效分析

4.2.1 茵陳蒿湯中的大黃與梔子在肝臟明顯抑制CCl ₄ 所誘發的NF-κB 活性

基因轉殖鼠經過投予CCl ₄ 或 silymarin，在第4 週與第8週經過分子影像照影偵測NF-κB活性，結果呈現如圖4.5，經過CCl ₄ 處理後與 Mock組對照起來，很顯著的誘發小鼠腹腔區域NF-κB活性。經過解剖後，實質器官經影像照影顯示，CCl ₄ 的確很專一的誘發肝臟冷光 (圖 4.6) 。茵陳蒿湯複方及其組成單方包括茵陳蒿、大黃和梔子以口服方式投予，結果發現大黃與梔子在第8週時有顯著的抑制小鼠腹腔CCl ₄ 所誘發的冷光強度。進一步器官剖檢進行冷光影像照影，發現口服大黃及梔子組確實抑制肝臟CCl ₄ 所誘發的冷光。這些結果顯示，大黃與梔子都可以明顯的抑制肝臟CCl 4 所誘發的NF-κB活性。

4.2.2 大黃與梔子下降肝臟 NF-κB 活性與改善肝纖維化之間的關係為了評估肝病理改變和肝纖維化程度，肝臟切片我們利用H&E 染色和天狼星紅進行染色， H&E可以用來觀查肝臟組織受到破壞的程度，而天狼星紅陽性粉紅絲狀面積可以直接用來標記肝纖維化的程度。

H&E染色很明顯可以看見，CCl ₄ 組的肝臟小葉結構嚴重受損，這是由

免疫細胞的浸潤、出血、肝細胞形成空泡變性所造成，反而在大黃或

梔子組有明顯的緩解下來。天狼星紅染色區域很清晰地出現在肝臟小

葉上呈網狀結構，經過定量統計 CCl ₄ 組的肝纖維化面積的比例為

3.43±0.1% 。與 CCl 4 組相較之下，肝臟纖維化程度在大黃組為

1.58±0.08%與梔子組為0.92±0.05%各有顯著下降。這些結果顯示，大

黃與梔子可能是茵陳蒿湯裡主要的肝纖維化療效組成 (圖4.7) 。

32 4.2.3 梔子主要化合物 genipin 抗肝炎效果

基因轉殖鼠經過投予 CCl ₄ 或 genipin，在第4 週時開始投於

genipin, 直到第8週經過分子影像照影偵測NF-κB活性，結果呈現如圖

4.8。經過CCl ₄ 處理後與Mock組對照起來，很顯著的誘發小鼠腹腔區

域的NF-κB活性。而genipin在三個濃度分別為1、10、100 mg/kg，其

中以10 mg/kg的抗肝臟發炎最具效果，另外100 mg/kg，我們推測該濃

度過高，可能導致肝毒性。以上結果顯示Genipin 可以在一定的劑量

下大約10 mg/kg，在小鼠體內使肝臟下降CCl ₄ 所誘發的NF-κB活性。

4.5 茵陳投予 CCl ₄ ) 活體影像冷光表現強身影像冷光 ock 比較,

0.01。

陳蒿湯處理

4 或茵陳蒿

像，小鼠身強度，單位光值的定

###表示

理後小鼠活蒿湯及其組

身上的冷位為 phot 定量統計圖示

p<0.001

33 活體 NF-κ 組成單方冷色系到暖

ton/sec，圖圖。冷光值 1；與 CC

κB 冷光的

，並且在各暖色系從藍

圖中每個組值以平均值 Cl ₄ 比較*表

的效應。基各個指定時藍到紅的顏

組別皆有值 ± 標準

表示

p<0

基因轉殖鼠時間內照顏色，分別

5 重複。

準差，組別 0.05，**表

鼠經影。

別代

(B)

別與

表示

4.6 基因

35 (A

37

4.3 Genipin抗發炎分析

4.3.1 Genipin在體外(in vitro) 和體內 (in vivo) 影響的NF-κB活性為了要評估genipin對LPS所誘發的NF-κB活性的影響，我們首先將LPS和不同濃度的genipin處理到HepG2/NF-κB的重組細胞，LPS很明顯誘發NF-κB的活性有1.8倍，而genipin顯著抑制NF-κB的活性呈現劑量反應(圖4.9)。而且在genipin處理過程中並無觀察到細胞毒性，這些結果顯示，genipin在體外實驗確實可以抑制NF-κB的活性。

接著，我們利用LPS及不同濃度的genipin處理到基因殖鼠，並且在4小時後進行偵測經NF-κB活性，如圖4.10所示。比較未處理LPS組，

LPS組在腹部區域發出的冷光強度增加約14.7倍。然而，genipin在體內可顯著抑制LPS所誘發的冷光，且抑制效果呈也現出劑量反應 (1、

10和100 mg/kg) 。這些結果暗示，genipin在體內抑制了NF-κB活性，

因此我們選擇了100 mg/kg這個濃度進行更進一步的實驗。由於我們先前研究已經證明NF-κB所驅使的冷光與發炎反應有密切關係，這些結果也顯示，genipin在體內抑制了急性全身性發炎反應。

4.3.2 Genipin 在基因轉殖鼠各個實質臟器的 NF-κB 活性

我們進一步進行器官剖檢的影像照影，用以評估genipin的主要作

用標的器官，如圖4.11所示。結果發現處理LPS的基因轉殖鼠其各個

器官的冷光強度比對照組高，尤其是肝臟、脾臟、胃及小腸等器官冷

光強度明顯的增加。而經過投予genipin後，在腦、心臟、肝臟及腎臟

的LPS所誘發冷光都有明顯的下降。我們發現genipin在腦、心臟、肝

臟及腎臟有各個不同程度顯著差異。因此，我們接著利用cytokine

ELISA、免疫組織化學染色進行分析，進一步我們萃取這些器官的的

RNA利用DNA microarray分析genipin的作用基因。

38 4.3.3. Genipin 抑制IL-1β 和 TNF-α表現

IL-1β和TNF-α是急性發炎反應的細胞因子，大量的產生表示開始急性的發炎並導致後端的發炎反應 (97) 。我們進一步利用ELISA和免疫組織化學染色分析IL-1β和TNF-α，以驗證genipin是否抑制全身性發炎反應。如圖4.12所示，ELISA分析結果，LPS組的小鼠血清中IL-1β 和TNF-α的濃度分別為969 ± 47 pg/ml和339 ± 25 pg/ml。然而，genipin 的投予組降低了IL-1β和TNF-α的表現量。利用IL-1β和TNF-α抗體進行免疫組織化學染色結果顯示，與mock組相比，在LPS組的器官有大量褐色的IL-1β或TNF-α活性細胞，而且在LPS組棕色區域的比例均有顯著增加 (圖4.13) 。然而，genipin這些器官IL-1β和TNF-α活性區的比例顯著下降。所以Genipin組的IL-1β和TNF-α在血清和器官表現量都有明顯下降，這結果顯示，genipin會透過抑制cytokine的產生進而抑制發現反應。

4.3.4 Microarray分析genipin調控的基因表現圖譜

Microarray 結果先利用 Gene Expression Pattern Analysis Suite

v3.1 進行分析腦、心臟、肝臟和腎臟受到LPS和/或genipin處理後具

有差異的基因表現變化。在microarray上的全部30968個基因中，在LPS

組及LPS/Genipin組有79個基因呈現有差異的倍率變化。我們更進一

步利用WebGestalt工具將這些基因進行定義，並且經過分析獲得

genipin在這些器官中的細胞生理狀態變化的總覽。Gene ontology類別

(GO categories) 只要是它們包含至少兩個基因且其p值小於0.01就會

被認定為該GO類別成立。如圖4.14所示，大部份的顯著表現差異的

基因被歸類到免疫相關 (immunity-related) 的GO類別裡，例如抗原生

成及抗原呈現 (antigen processing and presentation) 、B細胞分化的調

39 控、發炎反應以及急性期發炎反應等類別。只有少數基因被歸納到代謝類別裡。

這些基因反應圖譜，我們進一步利用 TIGR Multiexperiment Viewer 進行hierarchical clustering分析。如圖4.15所示，有趣的是，從結果中發現到在這些器官中受LPS所響影到的基因，全被genipin給反向調控。大多數的基因表現都被LPS給活化，而genipin則下調了LPS 所誘發的基因表現。只有5個基因，包括Endothelial-specific receptor tyrosine kinase 、 Notch-regulated ankyrin repeat protein (Nrarp) 、 SH3-binding kinase 1、Sucrose nonfermenting protein (SNF) -related kinase 以及 Lysosomal-associated protein transmembrane 5 (LAPTM5) 基因，分別是由LPS所下調，而geinpin活化了這些受LPS抑制基因表現。此外，在這些顯著差異的基因中，大約有三分之一的基因是屬於 chemokine ligand、chemokine receptor和IFN-induced protein基因。這些結果顯示， genipint可以抑制並且下調LPS所誘發的chemokine ligand、

chemokine receptor和IFN-induced protein。

在這些表現具有顯著差異的基因，我們也Genomatix Applications軟體及資料庫分析了基因與基因間的相互關聯度。基因間的關聯度是根據Genomatix Knowledge Base，以及DNA的啟動子 (promoter) 序列分析。有趣的是，大多數基因都直接連接NF-κB並且以NF-κB為一個核心的網路結構 (network) (圖4.16) 。這些結果顯示，NF-κB可能扮演著genipin調控基因network裡的主要角色。

4.3.5 利用 qPCR 驗證 genipin 新穎調控基因的表現

Microarray分析結果顯示，有三分之一有差異變化的基因屬於

chemokine ligand、 chemokine receptor及 IFN-induced protein族群的基

40 因，因此我們接著利用qPCR 的分析加以驗證microarray 的結果。我們利用qPCR將IFN-induced protein 這族群的基因進行定量分析，包含 Iigp1 和 Ifi202 基因。因為這些基因的表現在我們的Network分析裡顯示會直接的受到NF-κB調控。如表4.4所示，與mock組相比較下，LPS 組的 Iigp1 和 Ifi202 基因表現程度分別為6.62倍及55.4倍。然而，LPS 上調的Iigp1 和 Ifi202 基因在LPS/genipin組是被下調的。與mock組相比較下， LPS/genipin組的Iigp1 和 Ifi202 基因分別為5.17倍及28.33倍。

qPCR與microarray分析的數據一致，這結果顯示genipin 會下調LPS所

誘發的 Iigp1 和 Ifi202基因表現。

圖

圖4

圖 4 mg 我們

± 標比較

4.12 Gen g/kg) 和/或

們利用EL 標準差，與較***表示

ipin抑制L 或Genipin LISA偵測I 與mock組示p<0.001

LPS誘導以100 m IL-1β和TN 組比較，#

。

44 IL-1β和T mg/kg的劑 NF-α在血

##表示p<0

TNF-α產生劑量腹腔注血清裡的濃 0.01，###

生的效果注射方式給濃度。濃度

#表示p<0.

果。將 LPS 給予。之後度值以平均

.001；與L S (4

後，

均值

LPS

圖

圖臟和有表少有

4.14 以 G 和腎臟具有表現差異的有兩個基因

GO 分析在有差異性的基因進因的 GO

在 LPS 和性的基因。

進行組織及類別就會

46 和 LPS/gen 利用 Gen 及分到該類會被顯示出

nipin 兩個 ne Ontolo 類別，圖中出來.

個組別中腦 gy Tree M 中所示為

腦、心臟、

Machine 會

p<0.01 且

、肝

會將

且最

圖中，

後的在最

4.15 以 H

，腦、心臟的數據 log 最上方) 。

Hierarchic 臟、肝臟和腎

g2 值在圖

。基因的轉

al clusteri 腎臟具有圖中的以顏

轉錄表現上

47 ing 分析在有差異性的

顏色漸層的上升以紅色

7 在 LPS 和的基因。經的方式呈現

色表示，表

LPS/geni 經過標準化

現 (數值轉表現下降以

ipin 兩個組化 (Norma

轉換色差顯以綠色表示

組別

lize)

顯示

示。

圖

49

表4.4 qPCR 定量 Iigp1 和 Ifi202 表現程度。

Sample Average C

of target Average C

of GAPDH ΔC

ΔΔC

Relative to mock Iigp1

Mock 26.10±0.04 17.13±0.05 8.96±0.06 0.00±0.06 1.00

LPS 23.31±0.05 17.07±0.02 6.24±0.06 -2.72±0.06 6.62

LPS/Genipin 23.84±0.07 17.25±0.01 6.59±0.08 -2.37±0.08 5.17 Ifi202

Mock 32.11±0.11 17.13±0.05 14.97±0.12 0.00±0.12 1.00

LPS 26.25±0.04 17.07±0.02 9.18±0.05 -5.79±0.05 55.40

LPS/Genipin 27.40±0.02 17.25±0.01 10.15±0.02 -4.82±0.02 28.33

The ΔC

value is determined by subtracting the average C

value of GAPDH gene from the average C

value of target gene. The standard deviation of the difference is calculated from the standard deviations of the target gene and GAPDH gene.

The calculation of ΔΔC

involves subtraction by the ΔC

calibrator value. This is a subtraction of an arbitrary constant, so the standard deviation ofΔΔC

is the same as the standard deviation of ΔC

value.

49

50 第五章討論

5.1 NF-κB活體即時性肝炎報導平台之建立

本研究，我們利用 NF-κB 活體即時性肝炎報導平台，並以 silymarin 進行測試。利用這個平台我們發現 silymarin 可以依投予時間穩定的下降肝臟 CCl 4 所誘發的 NF-κB 活性。此外，NF-κB 活性與肝纖維化在基因轉殖鼠上所發出的冷光影像關係成正比，因此 NF-κB 活體即時性影像可以當作肝臟發炎及肝纖維化的即時偵測的平台。新興的生物冷光影像具有相當高靈敏度和即時性非侵入性的技術，可以即時的在活體的報導疾病的進程與評估治療的效果。這個技術目前已被應廣泛的應用在微生物感染的監控、腫瘤細胞的轉移、腫瘤標記及生物體-生物材料的交互作用 (62, 98-100) 。進一步，生物冷光影像照影也被用來預測小鼠肝腫瘤的大小 (101) 。我們先前的研究，已經構築帶有 NF-κB/luciferase 基因的基因轉殖鼠並且也直接的證實了 NF-κB 驅使的冷光可以用來評估生物體-生物材料間的交互作用、評估放射線對生物體的反應、評估香草醛 (vanillin) 在體內發炎性腸病的效果以及分析牛樟芝抗發炎的效果 (62, 64, 65, 102) 。本研究中，

我們成功的利用生物冷光影像照影評估 CCl ₄ 所誘發的肝損傷程度。

利用 CCl ₄ 進行誘發肝損傷是目前導致肝毒性的最佳的方法，並且也

是最常使用來篩選抗肝毒性或保肝藥物的模式 (48) 。CCl ₄ 的代謝機

轉主要是由 cytochrome p450 系統轉化成三氯甲基以及氯甲基過氧自

由基。這些來自於 CCl ₄ 的代謝物會跟生物體內的大分子形成共價鍵

結進而導致脂質過氧化，而此結果會造成肝臟細胞脂肪浸潤和肝臟漸

進式的從肝組織傷害到形成纖維化 (49) 。幾十年來，CCl ₄ 已經在許

多研究中被廣泛應用在誘發各種實驗動物肝損傷的平台上 (50, 51) 。

而且經由 CCl ₄ 所誘發的肝硬化的大鼠組織也顯示出與人類的肝硬化

51 相似 (52) 。傳統上，藉由致肝毒性物質所誘發的肝損傷以及肝纖維化可以利用肝組織的變化和血清生化指標中的丙胺酸轉胺酶、天門冬胺酸轉胺酶、鹼性磷酸酶及谷胺酸轉化酶濃度來進行評估 (53-55) 。由於由各種病因引起的持續性肝臟發炎進而導致肝纖維化又 NF-κB 在調節發炎反應發揮了關鍵作用 (1) 因此我們嚐試應用應用 NF-κB 基因轉殖鼠來報導 CCl ₄ 所誘發的肝纖維化。藉由天狼星紅染色和免疫組織化學染色的分析，可以顯示 CCl ₄ 所誘導的專一性肝臟 NF-κB 活化和冷光強度與肝纖維化成正相關。這個結果顯示，我們利用 NF-κB 生物冷光影像應用在 CCl ₄ 誘發肝纖維化的動物模式上是可行的。

Silymarin 是一個為人所熟知用來治療肝病的保肝劑 (56) 。它目前已有許多研究顯示可能具有抗氧化、抗脂質過氧化、抗纖維化、

細胞膜的穩定、免疫調節以及肝臟再生的活性 (57-61) 。 Silymarin 在許多實驗性的肝病研究平台上提供了很有效的保護作用。因此目前也被應用在臨床治療酒精性肝病、肝硬化、蕈類肝中毒以及藥物所引發的肝病等 (61) 。在本研究中，生物冷光影像照影顯示口服 silymain 可以專一的下降 CC 4 在肝臟所誘發的 NF-κB 的活性。而且藉著 silymarin 的投予治療，我們也觀察到降低 NF-κB 的活性跟降低肝纖維化的確有相當大的關聯，顯示 NF-κB 活體冷光影像照影可以用來評估在活體內 silymarin 的治療效果的可行性。更進一步，我們也發現，silymarin 在下降 CCl 4 所誘發的肝臟 NF-κB 活性具有很好的專一性。

接著，我們利用NF-κB活體冷光影像導引轉錄學平台分析進行探

勘新穎標的與silymarin的保肝機轉。先前的研究指出，silymarin抗纖

維化和抗發炎的效果主要是關係到TGF-β1 pathway (103) 。Silymarin

52 可透過下調大鼠在膽道纖維化的TGF-β1 mRNA進而抑制profibrotic

procollage-αa和TIMP-1基因的表現 (57) 。進一步，肝臟在受到鄰苯

三酚曝露下， silymarin 可以調節有關氧化壓力 (oxidative stress) 、細

胞週期 (cell cycle) 、細胞骨架 (cytoskeletal network) 、細胞-細胞附

著 (cell-cell adhesion) ，胞外基質、發炎及細胞凋亡的基因 (104) 。

本研究中， microarray的數據顯示在CCl ₄ 誘發肝纖維化之下， silymarin

調控TGF-β1-associated、TGF-β-induced apoptosis 及 TGF-β-mediated

pathway，此結果與先前的文獻一致。此外，我利用microarray分析

silymarin治療組的肝基因反應圖譜，發現silymarin除了可透過調控

TGF-β-mediated pathway 降低CCl 4 所誘發的NF-κB。更一步我們在這

microarray的數據中也發現了一些新穎的標的基因，在NF-κB活體即

時性影像導引轉錄體學的分析，某些Cox基因被silymarin 下調。我們

新發現silymarin可下調細胞骨架基因和粒線體電子傳遞鏈基因的表

現。目前已知CCl 4 可以誘發細胞骨架的再組織，並且進一步造成誘發

肝星狀細胞 (hepatic stellate cells) 分化成類肌纖維母細胞

(myofibroblast-like cells) (105) 。Silymarin 下調一群組成細胞骨架的

基因，我們推測silymarin 可能透過抑制細胞骨架的再組織而抑制肝

星狀細胞的轉型，進而達到改善肝纖維化的效果。 CCl 4 誘發肝纖維化

的過程牽涉到自由基產物，而自由基會顯著的導致整個粒腺體呼吸鏈

狀態的改變 (106-108) 。電子傳遞因子被歸納為4種複合體如：NADH

reductase、uccinate reductase、cytochrome c reductase及 Cox (109) 。

Cox扮演氧化機轉很重要的角色，並且是粒腺體電子傳遞鏈中電

子從cytochrome c傳送到氧分子

的

終點反應因子 (109) 。從先前的研

究與文獻中發現，在大鼠經CCl ₄ 誘發的肝纖維化中， NADH reductase

的活性會下降，且Cox 的活性會增加 (106, 108, 110) 。在我們的結

53 果也顯示，Cox的一些基因表現被CCl ₄ 給提高了。而NADH reductase 的下降與活性損失會導致電子連結到 O ₂ ^- 氧以及超氧化陰離子 (superoxide anion) 的產生，進而導致病態粒腺體的呼吸鏈氧吸收量上升。隨後， CCl ₄ 促進了電子轉移到氧分子上並且驅使粒線體生產ATP 進而提高Cox的活性 (108) 。另一方面，以前的研究顯示，從大鼠研究中發現silymarin會抑制粒線體氧氣的消耗量和增加NADH的還原酶還原鐵的活性大於基本值的量 (111, 112) 。此外，我們的結果顯示，

比較於Mock組，silymarin可以降低CCl4所誘導大於基礎表現量的Cox 基因的表現。這些結果也暗示，silymarin可能會抵消CCl ₄ 造成的粒線體電子傳遞鏈變化，因此可能關係到silymarin是如何改善CCl 4 所誘導肝纖維化的其中一個因素。

5.2 茵陳蒿湯及其組成在肝炎之療效分析

過往在中藥抗慢性肝炎在活體的研究，大多都是利用組織病理實驗以及血清生化指標加以評估，但這種方式需要長達數週至數個月的時間，而且需要犧牲相當數量的動物才能達到客觀的標準。除了活體模式外，以疾病分生為標的之細胞模式也被應用於中藥抗肝炎的搜尋。

雖然以疾病分生標的之細胞模式進行搜尋，確實可以加快藥物的評估，

但是體外試驗證實有效的藥物，進入活體試驗時的失敗率相當高。此外，有許多方法可以直接以活體試驗偵測肝炎分生標的的活性，例如：

免疫組織化學染色法 (immunohistochemistry stain; IHC) 、原位雜交

反應 (in situ hybridization) 等，但這些方法都無法即時性地反應動物

體內肝臟發炎的現象。以茵陳蒿湯為主要對象，以 NF-κB 基因轉殖

鼠配合四氯化碳誘發慢性肝炎，以即時性分子影像及 microarray 為分

析工具，配合組織染色切片、專一性纖維化蛋白染色，首先驗證茵陳

54 蒿湯在治療肝炎的效果，進一步從茵陳蒿湯拆解成單一中藥探討主要的療效藥物，並從該單味藥中找到肝炎的致效成份並從該單味藥中找到肝炎的致效成份。在前述的模式以 silymarin 測試的研究中，我們已經驗證該平台是非常可行的，而中藥的抗肝炎研究模式中，欠缺的部份如即時的評估療效，以及中藥投予週期、劑量等也可以利用此平台來加以分析。

以往有許多研究顯示，茵陳蒿湯在大鼠長期口服投予之下具有相當好的抑制肝纖維化 (35, 45-47) 。茵陳蒿湯透過抑制interferon (IFN) -γ和IL-12產生而改善小鼠被concanavalin A (con A)所誘發肝炎 (113) 。茵陳蒿湯在膽汁淤積性肝纖維化的大鼠模式中也有緩解肝纖維化的效果 (114) 。我們的結果也顯示，在利用活體冷光影像照影技術，CCl4 所誘發的肝炎經過4週的茵陳蒿湯治療的確有一定程度的緩解。茵陳蒿湯在許多不同的肝纖維化動物模式中，都有相當好的效果，進一步，

在這些研究中，也有被提出可能的致效成份是哪些。有文獻指出，茵陳蒿湯裡面的致效成份可能是大黃裡的Emodin (46) ，有的則指出可能是由Genipin所扮演致效成份 (47) 。在此研究中，我們將茵陳蒿湯拆方分為茵陳蒿、大黃及梔子三組各別進行測試。從結果當中發現，以大黃及梔子在降低CCl4所誘發的NF-κB效果非常的好，其中又以梔子的效果最為明顯。

梔子在歷史上廣泛的被應用在臨床治療急性及慢性的肝病。梔子

被研究最透徹的主要化物是genipin。因為現今研究大多以geniposide最

終代謝物genipin來探討治療的效果及其機轉。先前的研究指出，從梔

子萃取出來的geniposide可調節大鼠肝臟的cytochrome P-450-dependent

monooxygenases 、 glutathione 及 glutathione S-transferase (115) 。

Genipin可以抑制被IFN-gamma誘導過的巨噬細胞中IL-1beta、 IL-6以及

55 IL-12p70 的生合成 (113) 。Genipin抑制傷口誘發的細胞轉移和增殖，

同時也會下降collagen type I TGF-beta1 及 α-SMA 的mRNA 和蛋白質表現，另外也會抑制Smad2的訊息傳遞 (116) 。從我們的研究中也顯示，梔子無論在肝臟發炎指標NF-κB或是組織切片染色H&E的細胞完整度上來看都有很明顯的效果，另外利用天狼星紅染色觀察肝纖維化程度，在梔子組與CCl ₄ 組比較也有很明顯的勝出。因此，我們推測梔子中所含的genipin可能為茵陳蒿湯的致效成份之一。

5.3 Genipin抗發炎機轉分析

梔子的果實是一個在傳統中醫藥用草本植物，已用於治療發炎，

黃膽及肝病病上 (117) 。Genipin 是從geniposide代謝後的產物，而 geniposide一開始被發現於梔子 (118) 。Genipin已被用來作為在食品工業中的藍色著色劑 (119) 。它還被用來當作生物組織定固的交聯劑 (120) 。Genipin 目前被研究出來有許多的藥理活性，例如抗微生物、保肝活性及神經再生的效果 (121, 122) 。Genipin在抗局部發炎的潛力也有被研究發表 (42, 123) ; 然而，Genipin在體內對抗全身發炎的反應機制及療效還有待理清。有幾個研究顯示，在一些細胞模式中genipin呈現出很好的抗發炎效果，例如在LPS處理過的腦膠質細胞 (brain microglial cells) 、被LPS誘發的小鼠巨噬細胞、人類白血病細胞以及大鼠腎上腺嗜鉻細胞瘤細胞 (adrenal pheochromocytoma cells) (124, 125) 。此外，以往的研究顯示，genipin具有抑制角叉菜膠 (carrageenan) 誘發的大鼠足蹠腫脹、巴豆油誘發的小鼠耳腫脹、

concanavalin A誘導小鼠肝炎、鹽酸/乙醇誘導大鼠胃炎的重要外用消

炎作用，和LPS誘發的大鼠腦發炎 (42, 113, 123, 126) 。在這項研究

中，我們發現，genipin的腹腔給藥可以抑制NF-κB活性，並在許多器

56 官產生cytokine。這些結果顯示，genipin不僅可以外用，而且在全身體內抗發炎具有一定的潛力。

在這項研究中，我們利用即時性活體冷光影像監測genipin在體內抗急性全身性發炎模型的潛力。LPS是革蘭氏陰性菌外膜的主要組成結構成分。從革蘭氏陰性菌釋放LPS會活化一些細胞產生發炎性 cytokine，例如巨噬細胞和中性粒細胞。隨後生產的cytokine會導致的粘附分子表現及發炎性細胞聚集 (127) 。有研究指出，LPS結合 Toll-like receptor 4/CD14的複合體會活化NF-κB，然後上調發炎性 cytokine的基因表現，例如TNF-α、IL-1β和IL-6 (127) 。此外，以往的研究顯示，LPS在一些關鍵器官如肝，腎，脾，肺和心臟所誘發的發炎反應差異很大 (128) 。在本研究中，證明了LPS確實可以誘發在急性全身性發炎反應期的血清和器官的cytokine表現量增加。而LPS 誘發體內NF-κB活性與先前LPS引起NF-κB活化的全身性發炎反應結果相當吻合。此外， LPS會誘發NF-κB活性，所以結果顯示主要由LPS 處理後受到影響的主要器官大部份座落在心，肝，脾，腎。因此，這些研究結果顯示，抑制冷光強度可以用來反映在體內即時的發炎情況。

另外，除了先前已知的器官，LPS也對大腦產生了影響。

NF-κB生物冷光影像顯示LPS誘發大腦，心臟，肝臟，腎臟的 NF-κB活性會受到geinpin的抑制。我們進一步的分析這些器官的基因表現譜，以闡明genipin的抗發炎機制。GO分類結果顯示，有差異的表現基因大部份是屬於免疫相關的GO類別。此外，很有趣的發現，

genipin可以逆轉受到LPS影響的基因表現程度。值得注意的是，LPS

活化了大部份的基因，但卻下調了endothelial-specific receptor tyrosine

kinase，Nrarp，SH3-binding kinase，SNF-related kinase，和LAPTM5

基因的表現像。 LAPTM5是一種溶酶體蛋白，特別是表現在骨髓和

57 淋巴系統。LAPTM5會反向調控B細胞的細胞表面受體 (BCR) 的表現量以及B細胞的活化促使的小鼠細胞溶酶體的BCR降解 (129) 。 nrarp是一種小分子蛋白，轉錄由Notch signaling pathway調控。先前的研究已經證明，Nrarp會專一抑制T細胞的發育和造血系統 (130) 。 Endothelial-specific receptor tyrosine kinase是一種受體，最主要參與胚胎血管的形成。也有研究指出，Endothelial-specific receptor tyrosine kinase的補充和活化NF-κB2的A20結合抑制物，進而抑制NF-κB活化 (131) 。LPS下調endothelial-specific receptor tyrosine kinase、Nrarp和 LAPTM5基因的表現，暗示LPS可能促進B細胞活化、T細胞的發展和 NF-κB活化。然而，genipin可能會抑制掉透過對這些基因的上調，進而抑制B細胞活化，T細胞的發育和NF-κB的活化。

在79具有表現差異的基因中，74個基因被LPS上調和genipn下調，

這些基因有三分之一是屬於chemokine ligand、chemokine receptor、和 IFN-induced protein 基因。會吸引Helper T細胞T _H 1細胞趨化的基因，

chemokine (C-X-C motif) ligand 9 (CXCL9) 、CXCL10和chemokine (C-C motif) ligand (CCL5) ，分別在本研究中被LPS上調和genipin下調。

體外研究指出，CXCL9 (又稱為IFN-γ誘導的monokine) 和CXCL10 (又稱IFN-γ誘導蛋白10) 會刺激活化CXCR3陽性的T細胞和自然殺手細胞。而他們也是在傳染病和慢性發炎疾病時T細胞遷移的關鍵因子 (132) 。CCL5的 (也稱為RANTES；cytokine) 一般免疫反應後產生的，

並有助於增強和適應性往後的免疫反應 (133) 。除了這些chemokine，

有一些IFN-induced proteins也會受到LPS的影響。例如，IFN-inducible

GTPase和IFN-γ-inducible protein 47 都屬於免疫相關的GTP酶，在調

解先天免疫力去對抗細胞內的病原體扮演很關鍵角色 (134) 。

IFN-activated gene 202B (Ifi202) 是屬於IFN-inducible 200-protein家族，

58 在利用BWF1小鼠為實驗模式中發現，ifi202是一個重要的樹突狀細胞

的NF-κB活化因子，並參與IL-12的表現 (135) 。另外，helicase C

domain 1誘發的IFN可以作為一種病毒感染的細胞質傳感器和活化抗

病毒的免疫反應 (136) 。

59 第六章結論

我們是第一個利用NF-κB活體影像搭配microarray的分析，很成功的應用在對CCl 4 誘發的肝纖維化治療效果和silymarin新穎作用機制的評估和測定上。此疾病動物模式在對於肝臟發炎的研究上變得相當的便利，可在第一時間即時的觀察到發炎誘發情況，以及藥物治療狀況。

我們認為此方法極適合用來探堪目前有效的中醫藥並更深入的探堪複雜的中醫藥裡的致效成份。以此動物模式為基礎，利用現今中醫在臨床上常用在肝炎緩解的方劑-茵陳蒿湯來進行研究，進一步從茵陳蒿湯拆解成單一中藥探討主要的療效藥物，發現梔子對於抗肝纖維化有很好的效果，並從中進行文獻的分析找到genipin可能是肝炎的致效成份。

進一步，我們利用此平台對genipin進行抗發炎的機轉分析。我們發現，

急性全身性發炎模式中genipin呈現出系統性的抗發炎作用。進一步，

genipin 透過新穎的機制抑制發炎，包括下調 chemokine ligand ，

chemokine receptor，IFN-induced protein productions的表現量。這些結

果表明，genipin可能是一個治療肝炎及全身炎症反應的候選藥物。

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72 New functional genomics platform for analyzing the effects of Yin-Chen-Hao-Tang and its novel effective

components in liver disease

Graduate Institute of Chinese Medicine, China Medical University Advisor: Professor Tin-Yun Ho

Student: Chia-Cheng Li

Chronic liver disease is a global health problem that affects hundreds of millions of people. Liver damage followed by the persistent inflammation leads to hepatic fibrosis and cirrhosis. The inflammation is regulated by the transcription factor, nuclear factor-κB (NF-κB).

Therefore, we applied bioluminescent imaging-guided transcriptomic analysis to evaluate the feasibility of NF-κB-dependent bioluminescent image on the assessment of liver disease progression and therapeutic efficacies of herbs. Transgenic mice, carrying the luciferase genes driven by NF-κB, were given with carbon tetrachloride (CCl ₄ ) and/or silymarin.

In vivo NF-κB activity was evaluated by bioluminescent imaging, liver

fibrosis was judged by Sirius red staining and immunohistochemistry, and

gene expression profiles of silymarin-treated livers were analyzed by

DNA microarray. CCl ₄ enhanced the NF-κB-dependent hepatic

luminescence and induced hepatic fibrosis, while silymarin reduced the

CCl ₄ -induced hepatic luminescence and improved CCl ₄ -induced liver

fibrosis. Microarray analysis showed that silymarin altered the

transforming growth factor-β-mediated pathways, which play pivotal

roles in the progression of liver fibrosis. Moreover, we newly identified

that silymarin downregulated the expression levels of cytoskeleton

73 organization genes and mitochondrion electron-transfer chain genes, such

as cytochrome c oxidase Cox6a2, Cox7a1, and Cox8b genes. We further

applied this platform to evaluate the therapeutic potentials of

Yin-Chen-Hao-Tang and its novel effective components. We found that

Fructus Gardeniae, the component of Yin-Chen-Hao-Tang, was effective

for the treatment of liver fibrosis. Furthermore, genipin from Fructus

Gardeniae exhibited anti-fibrogenic and anti-inflammatory effects via

downregulation of chemokine ligand, chemokine receptor, and

interferone-induced protein productions. In conclusion, our data showed

the feasibility of NF-κB-dependent bioluminescent image on the

assessment of disease progression and therapeutic efficacies. Moreover,

the therapeutic potentials and mechainsms of Yin-Chen-Hao-Tang and its

constituents silynmarin and genipin were further identified by

bioluminescent imaging-guided transcriptomic analysis.

74 誌謝

感謝我的父母親的栽培，以及兄弟姊妹支持與鼓勵，讓我求學的過程一路順利。感謝我的指導教授侯庭鏞老師在研究上的協助與指點，

也感謝共同指導老師項千芸老師與吳世祿老師在研究技術與想法的

無私分享。感謝從大學、碩班及博班這 11 年來曾經一起在這實驗室

努力過的學長姊弟妹們互相幫忙與照顧，我永遠記得一個學長曾經對

我說「把實驗室當成你家廚房」，這句話我到現在才真正了解它的含

意。感謝那些曾經一起打拚與切磋的同學們。最後感謝所有曾經在我

身邊出現過的任何人，因為有你們的存在，我才能完成博士學程。

Identiﬁcation of novel mechanisms of silymarin on the carbon tetrachloride-induced liver ﬁbrosis in mice by nuclear factor- j B bioluminescent imaging-guided transcriptomic analysis

Chia-Cheng Li

, Chien-Yun Hsiang

, Shih-Lu Wu

, Tin-Yun Ho

^a,d,

^⇑

aGraduate Institute of Chinese Medicine, China Medical University, Taichung 40402, Taiwan

bDepartment of Microbiology, China Medical University, Taichung 40402, Taiwan

cDepartment of Biochemistry, China Medical University, Taichung 40402, Taiwan

dDepartment of Nuclear Medicine, China Medical University Hospital, Taichung 40447, Taiwan

a r t i c l e i n f o

Article history:

Received 13 September 2011 Accepted 14 February 2012 Available online 22 February 2012

Keywords:

Liver ﬁbrosis Silymarin Nuclear factor-

j

B Bioluminescent imaging DNA microarray Cytochrome c oxidase

a b s t r a c t

In this study, we applied bioluminescent imaging-guided transcriptomic analysis to evaluate and identify the therapeutic potentials and novel mechanisms of silymarin on carbon tetrachloride (CCl4)-induced liver ﬁbrosis. Transgenic mice, carrying the luciferase genes driven by nuclear factor-

j

B (NF-

j

B), were given with CCl4and/or silymarin. In vivo NF-

j

B activity was evaluated by bioluminescent imaging, liver ﬁbrosis was judged by Sirius red staining and immunohistochemistry, and gene expression proﬁles of silymarin-treated livers were analyzed by DNA microarray. CCl4enhanced the NF-

j

B-dependent hepatic luminescence and induced hepatic ﬁbrosis, while silymarin reduced the CCl4-induced hepatic lumines-cence and improved CCl4-induced liver ﬁbrosis. Microarray analysis showed that silymarin altered the transforming growth factor-b-mediated pathways, which play pivotal roles in the progression of liver ﬁbrosis. Moreover, we newly identiﬁed that silymarin downregulated the expression levels of cytoskel-eton organization genes and mitochondrion electron-transfer chain genes, such as cytochrome c oxidase Cox6a2, Cox7a1, and Cox8b genes. In conclusion, the correlation of NF-

j

B-dependent luminescence and liver ﬁbrosis suggested the feasibility of NF-

j

B bioluminescent imaging for the evaluation of liver ﬁbrosis progression and therapeutic potentials. Moreover, our ﬁndings suggested that silymarin might exhibit anti-ﬁbrotic effects in vivo via altering the expression of genes involved in cytoskeleton organization and mitochondrion electron-transfer chain.

1. Introduction

Liver ﬁbrosis is a pathological sequel of chronic inﬂammatory liver injury caused by various etiologies, such as hepatitis virus infection, autoimmune injury, alcohol, and toxins/drugs. Following hepatic inﬂammation and damage, hepatic stellate cells change to myoﬁbroblast-like cells and produce a large amount of extracellu-lar matrix like type I collagen. The accumulation of collagen in the hepatic parenchyma further leads to the ﬁbrosis of liver (Bataller and Brenner, 2005; Lotersztajn et al., 2005). Production of

proinﬂammatory cytokines, such as interleukin-1b, tumor necrosis factor- a and interferon- c , contribute to the progression of hepatic inﬂammation and sequential ﬁbrosis (Luedde and Schwabe, 2011).

The production of cytokines is further controlled by the transcrip-tion factor, nuclear factor- j B (NF- j B) (Baldwin, 1996). NF- j B is an inducible nuclear transcription factor that consists of heterodimers of RelA (p65), c-Rel, RelB, p50/NF- j B1, and p52/NF- j B2. NF- j B activity is activated by a large variety of stimuli, such as microbes, inﬂammatory cytokines, and physical and chemical stresses. When stimulated, NF- j B binds to the NF- j B-responsive element present in the promoters of inﬂammatory genes, resulting in the induction of gene expression and the inﬂammatory process. Accordingly, NF- j B is a critical molecule involved in the regulation of inﬂamma-tory cytokine production and inﬂammation (Bonizzi and Karin, 2004; Karin and Ben-Neriah, 2000; Siebenlist et al., 1994).

Moreover, controlling NF- j B activation has become a pharmaco-logical target, particularly in the chronic inﬂammatory disorders (Baeuerle and Baichwal, 1997).

doi:10.1016/j.fct.2012.02.025

Abbreviations: CCl4, carbon tetrachloride; Cox, cytochrome c oxidase; GAPDH, glyceraldahyde-3-phosphate dehydrogenase; H&E, hematoxylin and eosin; NF-

j

B, nuclear factor-

j

a

-SMA,

a

-smooth muscle actin; TGF-b, transforming growth factor-b.

⇑

Corresponding author. Address: Graduate Institute of Chinese Medicine, China Medical University, 91 Hsueh-Shih Road, Taichung 40402, Taiwan. Tel.: +886 4 22053366 3302; fax: +886 4 22053764.

E-mail address:[email protected](T.-Y. Ho).

Food and Chemical Toxicology 50 (2012) 1568–1575

Contents lists available at SciVerse ScienceDirect

Food and Chemical Toxicology

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / f o o d c h e m t o x

75 Silymarin, a ﬂavonoligan mixture of milk thistle (Silybum maria-num), is an important herbal hepatoprotective drug (Abenavoli et al., 2010). Silymarin possesses a variety of pharmacological activities, such as inﬂammatory, immunomodulatory, anti-oxidant, and anti-viral activities (Polyak et al., 2007; Saller et al., 2001; Shaker et al., 2010). Silymarin exhibits hepatoprotective effects by altering cytoplasmic membrane architecture and, in turn, preventing the penetration of hepatotoxic substances, such as carbon tetrachloride (CCl

), thioacetamide and

-galactosamine, into cells (Abenavoli et al., 2010; Basiglio et al., 2009). It also pos-sesses the anti-ﬁbrotic activity by retarding the activation of hepa-tic stellate cells (Chandan et al., 2008). Although the pharmacological mechanisms of silymarin have been reported, silymarin-altered hepatic gene expression proﬁles remained to be elucidated for the identiﬁcation of novel targets and mechanisms for silymarin-mediated protection in the liver.

Bioluminescence imaging is a sensitive and noninvasive tech-nique for real-time reporting and quantiﬁcation of therapy efﬁcacy in living animals (Hseu et al., 2011; Wu et al., 2009). This technique has been used for the assessment of host responses to biomaterials (Ho et al., 2007; Xiong et al., 2005). It has also been applied for imaging disease progression and diagnosis (Dothager et al., 2009;

Ottobrini et al., 2005). Microarray is a popular research and screen-ing tool for differentially expressed genes. Microarray-based gene expression patterns have been used to predict the candidate bio-markers, predict the therapeutic efﬁcacies of drugs, and recognize the toxic potential of drug candidate (Baur et al., 2006; Lamb et al., 2006; Suter et al., 2004). We have previously applied NF- j B biolu-minescent imaging-guided transcriptomic analysis to assess the host responses to biomaterials and ionizing radiation in vivo (Ho et al., 2007; Hsiang et al., 2009). In this study, we applied NF- j B bioluminescent image to evaluate both the progression of CCl

-in-duced liver injury and the therapeutic effects of silymarin. Micro-array analysis was further applied to globally elucidate the gene expression proﬁles of silymarin and to ﬁnd novel mechanisms of silymarin on CCl

₄

-induced liver injury. Our data showed the feasi-bility of NF- j B-dependent bioluminescent image on the assess-ment of disease progression and therapeutic efﬁcacies. Moreover, we newly identiﬁed that silymarin exhibited anti-ﬁbrotic effects in vivo via regulating transforming growth factor-b (TGF-b)-medi-ated pathways and altering the expression of genes involved in cytoskeleton organization and mitochondrion electron-transfer chain.

2. Materials and methods

2.1. Induction of liver ﬁbrosis and silymarin treatment

Mouse experiments were conducted under ethics approval from the China Medical University Animal Care and Use Committee. Transgenic mice, carrying the NF-

j

B-driven luciferase genes, were constructed previously (Ho et al., 2007).

CCl4-induced liver ﬁbrosis was performed as described previously (Sakaida et al., 2004). Silymarin was purchased from Sigma (St. Louis, MO) and suspended in dis-tilled water to a ﬁnal concentration 20 mg/ml. A total of 24 transgenic mice was randomly divided into three groups of eight mice: (1) mock, mice were intraperito-neally administered with 0.5 ml/kg olive oil twice a week for 12 weeks, (2) CCl4, mice were intraperitoneally administered with 0.5 ml/kg 10% CCl4in olive oil twice a week for 12 weeks, and (3) silymarin, mice were intraperitoneally administered with 0.5 ml/kg 10% CCl4in olive oil twice a week for 12 weeks, and silymarin was given orally at a dose of 200 mg/kg once a day from week 5 to 12 after CCl4

administration.

2.2. In vivo and ex vivo imaging of luciferase activity

For in vivo imaging, mice were anesthetized with isoﬂurane and injected intra-peritoneally with 150 mg luciferin/kg body weight. Five minutes later, mice were placed face up in the chamber and imaged for 1 min with the camera set at the highest sensitivity by IVIS Imaging System^Ò200 Series (Xenogen, Hopkinton, MA). For ex vivo imaging, mice were anesthetized and injected with luciferin intraperitoneally. Five minutes later, mice were sacriﬁced, and tissues were rapidly

removed, placed in the IVIS system, and imaged with the same setting used for in vivo studies. Photons emitted from tissues were quantiﬁed using Living Image^Ò software (Xenogen, Hopkinton, MA). Signal intensity was quantiﬁed as the sum of all detected photon counts from selected tissues and presented as photon/s.

2.3. Quantitative analysis of liver ﬁbrosis

For detecting hepatic ﬁbrosis, liver sections were stained with 0.1% Sirius red (Sigma, St. Louis, MO) in a saturated aqueous solution of picric acid (Panreac, Barcelona, Spain). One hour later, slides were rinsed in two changes of acidiﬁed water (0.5% glacial acetic acid in water), dehydrated in three changes of 100% eth-anol, cleared in xylene, mounted in a resinous medium, and then observed under a light microscope. Sirius red-positive areas were measured using Image-Pro Plus (Media Cybernetics, Bethesda, MD). The proportions of hepatic ﬁbrotic area (%) were calculated as areas occupied with red color/area of whole tissue.

2.4. Histological and immunohistochemical examination

Paraﬁlm-embedded liver tissues were cut into 5-

l

m sections and stained with hematoxylin and eosin (H&E). For immunohistochemistry, sections were deparaff-inized in xylene and rehydrated in graded alcohol. Endogenous peroxidase was quenched with 3% hydrogen peroxide in methanol for 15 min and the nonspeciﬁc binding was blocked with 1% bovine serum albumin at room temperature for 1 h.

Sections were incubated with antibodies against p65 (Chemicon, Temecula, CA), TGF-b1 (Santa Cruz, Santa Cruz, CA), or

a

-smooth muscle actin (

a

-SMA) (Santa Cruz, Santa Cruz, CA) at 1:50 dilution overnight at 4 °C and then incubated with biotinyl-ated secondary antibody (Zymed Laboratories, Carlsbad, CA) at room temperature for 20 min. Finally, slides were incubated with avidin–biotin complex reagent and stained with 3,3⁰-diaminobenzidine according to manufacturer’s protocol (Histostain^Ò-Plus kit, Zymed Laboratories, Carlsbad, CA). TGF-b1,

a

-SMA, and

NF-j

B-positive areas were measured using Image-Pro Plus (Media Cybernetics, Bethesda, MD) to quantify the expression levels of TGF-b1,

a

-SMA, and NF-

j

The proportions of TGF-b1,

a

-SMA, and NF-

j

B-positive areas were calculated as areas occupied with brown color/area of whole tissue.

2.5. Total RNA isolation

Total RNA was extracted from livers using the RNeasy Mini kit (Qiagen, Valen-cia, CA) and further treated with RNase-free DNase I (Qiagen, ValenValen-cia, CA) to re-move contaminating DNA. Total RNA was quantiﬁed using the spectrophotometer (Beckman Coulter, Fullerton, CA), and samples with A260/A280 ratios greater than 1.8 were further evaluated using Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA). The RNA sample with a RNA integrity number greater than 8.0 was accepted for microarray analysis.

2.6. Microarray analysis

Microarray analysis was performed as described previously (Cheng et al., 2010).

Brieﬂy, ﬂuorescent RNA targets were prepared from 5

l

g of total RNA using MessageAmp™ aRNA kit (Ambion, Austin, TX) and Cy5 dye (Amersham Pharmacia, Piscataway, NJ). Fluorescent targets were hybridized to the Mouse WG-6 Expression Bead Chip (Immunina, San Diego, CA) and scanned by an Axon 4000 scanner (Molecular Devices, Sunnyvale, CA). Number of replicates was three. The Cy5 ﬂuo-rescent intensity of each spot was analyzed by genepix 4.1 software (Molecular Devices, Sunnyvale, CA). The signal intensity of each spot was corrected by subtract-ing background signals in the surroundsubtract-ing. We ﬁltered out spots that signal-to-noise ratio was less than 0 or control probes. Spots that passed these criteria were normalized by the limma package of the R program using quantile normaliza-tion. Normalized data were tested for differential expression using Gene Expression Pattern Analysis Suite v3.1 (Montaner et al., 2006). Genes with fold changes P2.0 or 62.0 were further selected and tested enriched pathways on WebGestalt web site (http://bioinfo.vanderbilt.edu/webgestalt/login.php) by hypergeometric test.

2.7. Quantitative real-time polymerase chain reaction (qPCR)

The expression levels of cytochrome c oxidase genes (Cox6a2, Cox7a1, and Cox8b) were validated by qPCR. RNA samples were reverse-transcribed for 2 h at 37 °C with High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA). qPCR was performed by using 1

l

l of cDNA, 2 SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA), and 200 nM of forward and re-verse primers. The reaction condition was followed: 10 min at 95 °C, and 40 cycles of 15 s at 95 °C, 1 min at 60 °C. Each assay was run on an Applied Biosystems 7300 Real-Time PCR system in triplicates. The efﬁciency of PCR was measured by the serial dilution test. A 4-log dilution range was generated using 10-fold serial dilu-tions of the DNA with four concentration points at 10⁸, 10⁷, 10⁶, and 10⁵copies/

l

l. Fold changes were calculated using the comparative CT method. Primer sets used in this study were designed using Primer3 program (http://frodo.wi.mit.edu/

primer3/). The speciﬁcities of primer sets were analyzed by nucleotide BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Each primer set was able to amplify a

C.-C. Li et al. / Food and Chemical Toxicology 50 (2012) 1568–1575 1569

76

target DNA fragment from the respective gene with speciﬁcity. The primer set for each gene is followed: Cox6a2 forward, 5⁰-CAGAGAAGGACAGTGCCATTC-3⁰; Cox6a2 reverse, 5⁰-GAAGAGCCAGCACAAAGGTC-3⁰; Cox7a1 forward, 5⁰-CAATGACCTCCCA GTACACTTG-3⁰; Cox7a1 reverse, 5⁰-CCAAGCAGTATAAGCAGTAGGC-3⁰; Cox8b for-ward, 5⁰-TCCCAAAGCCCATGTCTCTG-3⁰; Cox8b reverse, 5⁰-CATCCTGCTGGAACCAT GAAG-3⁰; glyceraldahyde-3-phosphate dehydrogenase (GAPDH) forward, 5⁰-TCACC CACACTGTGCCCATCTATGA-3⁰; GAPDH reverse, 5⁰ -GAGGAAGAGGATGCGGCAGTGG-3⁰. Previous study has shown that the levels of GAPDH mRNA and protein in livers are consistent in mice given with CCl4(Hellerbrand et al., 1999). Therefore, we used GAPDH gene as the reference gene in this study.

2.8. Statistic analysis

Data were presented as mean ± standard error. Data were analyzed by one-way ANOVA and post hoc LSD test using PASW Statistics (SPSS) version 12. A p value less than 0.05 was considered as statistically signiﬁcant.

3. Results

3.1. Silymarin exhibited a steady decrease of CCl

-induced NF- j B activity in the liver

Transgenic mice were given with CCl

₄

and/or silymarin and im-aged for the NF- j B-driven luminescence on week 4, 6, 8, and 12. As shown in Fig. 1, administration of CCl

signiﬁcantly induced the NF- j B-dependent bioluminescent signal in the abdominal region as compared with mock group. Ex vivo imaging displayed that CCl

speciﬁcally induced the luminescence in the liver (Fig. 2). Oral administration of silymarin signiﬁcantly suppressed the CCl

-in-duced luminescent intensity in the abdominal region and the sup-pression displayed a time-dependent manner. Ex vivo imaging also

displayed that silymarin speciﬁcally reduced CCl

-induced NF- j B-driven bioluminescence in the liver. These ﬁndings suggested that CCl

induced NF- j B activation in the liver with speciﬁcity, while silymarin displayed a steady decrease of CCl

₄

-induced NF- j B activ-ity in the liver.

3.2. The decrease of NF- j B activity by silymarin in the liver was correlated with the improvement of liver ﬁbrosis

To evaluate the histological changes of liver and the degree of liver ﬁbrosis, we stained the hepatic sections with H&E and Sirius red. Hepatic ﬁbrosis is induced by the accumulation of collagen in the hepatic parenchyma (Bataller and Brenner, 2005). Sirius red is a strong anionic dye that has been used for the quantiﬁcation of col-lagen in tissue sections for many years (Jimenez et al., 1985;

Lopez-De Leon and Rojkind, 1985). Therefore, Sirius red-positive area can be a direct marker for the degree of liver ﬁbrosis. As shown in Fig. 3, no apparent pathological alternations were found in mock group. Sirius red-positive region in the mock group was appeared around the central vein but not in the hepatic paren-chyma. CCl

₄

damaged the lobular structure of liver, which was characterized by the inﬁltration of immune cells, hemorrhage, vac-uolar degeneration, and necrosis of hepatocytes. Sirius red-stained areas were clearly appeared in the boundaries of liver lobules and the proportion of the hepatic ﬁbrotic area was 3.86 ± 0.54%. In con-trast, silymarin improved the histological changes induced by CCl

. The CCl

-induced hemorrhage and necrosis in livers were amelio-rated by silymarin. Moreover, Sirius red-stained areas in the silymarin group were reduced as compared with CCl

group, and

Fig. 1. NF-

j

B-dependent bioluminescence in living mice. Transgenic mice were administered with CCl4and/or silymarin, and imaged at indicated periods. (A) In vivo imaging.

The color overlay on the image represents the photon/s emitted from the animal, as indicated by the color scales. Photos are representative images (n = 8). (B) Quantiﬁcation of photon emission from whole animal. Values are mean ± standard error (n = 8). ###p < 0.001, compared with mock.^⁄p < 0.05,^⁄⁄p < 0.01, compared with CCl4.

1570 C.-C. Li et al. / Food and Chemical Toxicology 50 (2012) 1568–1575

77 the proportion of ﬁbrotic areas (1.94 ± 0.29%) was signiﬁcantly de-creased by silymarin. These data suggested that silymarin im-proved the CCl

₄

-induced liver ﬁbrosis.

We further performed immunohistochemical staining to corre-late the liver ﬁbrosis with NF- j B activity. Liver sections were immunostained with a -SMA antibody to detect the presence of myoﬁbroblasts that produce collagen (Wells, 2005). Sections were also immunostained with antibody against TGF-b1, a cytokine playing a pivotal role in the liver ﬁbrosis (Lotersztajn et al., 2005). As shown in Fig. 4, there were many brown TGF-b1-positive cells and a -SMA-positive myoﬁbroblasts in the CCl

-treated liver.

However, oral administration of silymarin decreased the number of brown cells in the liver. The proportions of TGF-b1, a -SMA, and NF- j B-positive areas were increased in CCl

group and de-creased in silymarin group, suggesting that CCl

induced the expression of TGF-b1, a -SMA, and NF- j B, while silymarin inhibited the CCl

-induced TGF-b1, a -SMA, and NF- j B expression. Moreover, these ﬁndings suggested that silymarin ameliorated CCl

-induced liver ﬁbrosis, which was coincident with aforementioned histolog-ical data. Immunostaining with antibody against p65 revealed that there were many brown p65-positive cells in the CCl

-treated liver.

However, silymarin decreased the number of p65-positive cells in the liver. These data suggested that silymarin might improve

CCl

-induced liver ﬁbrosis via inhibition of NF- j B, TGF-b1, and

a -SMA. Moreover, the correlation between NF- j B activity, liver ﬁbrosis, and bioluminescent imaging suggested the feasibility of NF- j B-dependent bioluminescent imaging for the evaluation of therapeutic efﬁcacy of drugs for hepatic ﬁbrosis.

3.3. Analysis of gene expression proﬁle of silymarin in the CCl

-treated liver

We further analyzed the gene expression proﬁle of silymarin-treated liver by DNA microarray to identify the novel mechanisms of silymarin. In comparison with mock, 420 transcripts were upregulated and 439 transcripts were downregulated by 2-fold by CCl

. In comparison with CCl

, the expressions of 67 transcripts, including 2 upregulated and 65 downregulated transcripts, were altered with fold changes P2.0 or 62.0 by silymarin. These genes were further selected for pathway classiﬁcation. Table 1 shows that 34 pathways were signiﬁcantly altered by silymarin (p < 0.01). The half of pathways was associated with metabolism, while others were related to regulation of cellular process and nal transduction. TGF-b-associated pathways, including TGF-b sig-naling pathway, TGF-b-induced apoptosis and TGF-b-mediated pathway, were signiﬁcantly regulated by silymarin. Because

Fig. 2. NF-

j

B-dependent bioluminescence in individual organs. Transgenic mice were administered with CCl4and/or silymarin. Twelve weeks later, mice were sacriﬁced and organs were subjected to image. (A) Ex vivo imaging. The color overlay on the image represents the photon/s emitted from the organ, as indicated by the color scales. Photos are representative images (n = 8). (B) Quantiﬁcation of photon emission from organs. Values are mean ± standard error (n = 8). ###p < 0.001, compared with mock.

⁄⁄⁄p < 0.001, compared with CCl4.

C.-C. Li et al. / Food and Chemical Toxicology 50 (2012) 1568–1575 1571

78 TGF-b1 plays a pivotal role in the progression of liver ﬁbrosis, alteration of TGF-b-related pathways might contribute to the improvement of CCl

-induced liver ﬁbrosis by silymarin. Silymarin downregulated the expression levels of 65 genes in the CCl

-trea-ted liver. The genes with fold changes 64.0 are shown in Table 2. The half of silymarin-downregulated genes was associated with cytoskeleton organization and muscle contraction, while three genes, including Cox6a2, Cox7a2 and Cox8b genes, were related to mitochondrion electron-transport chain. These ﬁndings sug-gested that silymarin might improve the CCl

-induced liver ﬁbrosis via regulation the expression of genes involved in cytoskeleton organization and electron transport.

3.4. Veriﬁcation of expression levels of novel silymarin-regulated genes by qPCR

Microarray data showed that the expression of mitochondrial respiratory chain-related genes, including Cox6a2, Cox7a1 and Cox8b genes, were downregulated by silymarin. We further ap-plied qPCR to validate the transcriptional expression levels of these genes. As shown in Table 3, the expression levels of Cox6a2, Cox7a1, and Cox8b genes in CCl

group were 496.21-, 21.36-, and 240.38-fold higher, respectively, as compared with mock group.

However, CCl

-upregulated gene expression was downregulated by silymarin, and the expression levels of Cox6a2, Cox7a1, and Cox8b genes in silymarin group were 9.84-, 0.72-, and 0.7-fold, respectively, as compared with mock group. The consistent data from qPCR and microarray indicated that silymarin downregulated the CCl

-induced expression of Cox6a2, Cox7a1, and Cox8b genes.

4. Discussion

In this study, we found that silymarin exhibited a steady de-crease of CCl

-induced NF- j B activity in the liver, and the decrease of NF- j B activity by silymarin in the liver was correlated with the improvement of liver ﬁbrosis. During a steady decrease of CCl

-in-duced NF- j B-dependent luminescence by silymarin, microarray analysis of liver showed that silymarin altered the TGF-b-mediated pathways. Moreover, we newly identiﬁed that novel target genes like Cox genes were downregulated by silymarin, which was evi-denced by NF- j B bioluminescence imaging-guided transcriptomic analysis. Bioluminescence imaging is a sensitive and noninvasive technique for real-time reporting disease progression and quanti-fying therapy efﬁcacies in living animals. This technique has been used for monitoring tumor cell trafﬁcking, tumor targeting, and host-biomaterial interaction (Contag and Bachmann, 2002; Ho et al., 2007; Ottobrini et al., 2005; Xiong et al., 2005). It has also been used to predict hepatic tumor burden in mice (Sarraf-Yazdi et al., 2004). In previous studies, we have constructed the trans-genic mice carrying the NF- j B-driven luciferase gene and demon-strated the feasibility of NF- j B-dependent bioluminescent imaging for assessing the host-biomaterials interaction, elucidating the host response to ionizing radiation, evaluating the therapeutic ef-fects of vanillin in inﬂammatory bowel diseases, and analyzing the anti-inﬂammatory effects of Antrodia camphorata (Chang et al., 2011; Ho et al., 2007; Hseu et al., 2010; Wu et al., 2009).

In this study, we applied bioluminescent imaging to evaluate the progression of CCl

-induced liver damages. Liver injury induced by CCl

is the best-characterized mechanism of xenobiotic-induced

Fig. 3. Histological examination of liver by H&E and Sirius red staining. (A) Histological examination. Transgenic mice were administered with CCl4and/or silymarin. Twelve weeks later, mice were sacriﬁced, livers were excised, and sections were stained with H&E (100 magniﬁcation) or Sirius red (40 magniﬁcation). Photos are representative images (n = 8). (B) Quantiﬁcation of liver ﬁbrosis by Sirius red stain. Results are expressed as ﬁbrotic area (%), which was calculated as areas occupied with red color/area of whole tissue. Values are mean ± standard error (8 sections/group and 10 ﬁelds/section). ###p < 0.001, compared with mock.^⁄⁄⁄p < 0.001, compared with CCl4.

1572 C.-C. Li et al. / Food and Chemical Toxicology 50 (2012) 1568–1575

79 hepatotoxicity and a commonly used model for the screening of anti-hepatotoxic and/or hepatoprotective drugs (Weber et al., 2003). CCl

is metabolized by cytochrome p450 system and con-verted to trichloromethyl and trichloromethyl peroxy radicals.

The free radicals of CCl

bind covalently to macromolecules and cause lipid peroxidation, which results in the fatty inﬁltration of hepatocytes and the sequential liver damage and ﬁbrosis (Comporti et al., 2009). CCl

has been used extensively to induce liver injury in various animal models for decades. The experimen-tally induced cirrhotic response by CCl

in rats and mice are similar to liver cirrhosis in human (Weiler-Normann et al., 2007). Tradi-tionally, liver injury and liver ﬁbrosis induced by hepatotoxic sub-stances can be evaluated by histological changes and concentrations of alanine aminotransferase, aspartate aminotrans-ferase, alkaline phosphatase, and c -glutamyl transpeptidase in sera (Nanji et al., 2001; Sun et al., 2010; Tacke et al., 2005). Because the sustained hepatic inﬂammation induced by various etiologies leads to liver ﬁbrosis, and NF- j B plays a critical role in regulating inﬂam-matory responses (Luedde and Schwabe, 2011), we tried to apply NF- j B transgenic mice to report the liver ﬁbrosis induced by CCl

. CCl

induced the NF- j B-dependent luminescence in the liver with speciﬁcity and the NF- j B activation was correlated with liver ﬁbrosis, judged by Sirius red staining and immunohistochemical analysis. These ﬁndings indicated the feasibility of NF- j B biolumi-nescent imaging on the reporting of liver ﬁbrosis induced by CCl

. Silymarin is a well-known hepatoprotective agent for the treat-ment of liver diseases (Abenavoli et al., 2010). It possesses antiox-idative, antilipid peroxantiox-idative, antiﬁbrotic, membrane stabilizing, immunomodulatory, and liver regenerating activities (Polyak et al., 2007; Saller et al., 2001; Shaker et al., 2010). Silymarin offers a good protection in various models of experimental liver diseases.

It has also been applied clinically for alcoholic liver diseases, liver cirrhosis, Amanita mushroom poisoning, and drug-induced liver

diseases (Pradhan and Girish, 2006). In this study, bioluminescent imaging showed that oral administration of silymarin reduced the CCl

-induced NF- j B-dependent luminescent intensity in the liver with speciﬁcity. The correlation of the decreased NF- j B activity and the improved liver ﬁbrosis by silymarin, suggesting the feasi-bility of NF- j B-dependent bioluminescent imaging for the evalua-tion of therapeutic effect of silymarin in vivo.

NF- j B bioluminescent imaging-guided transcriptomic analysis was further applied for the evaluation of novel targets and mecha-nisms of silymarin-mediated protection in the liver. Previous stud-ies indicated that the anti-ﬁbrotic and anti-inﬂammatory effects of silymarin are associated with TGF-b1 pathway (Ai et al., 2010).

Silymarin suppresses the expression of proﬁbrotic procollagen- a

and TIMP-1 via downregulation of TGF-b1 mRNA in rats with bili-ary ﬁbrosis (Jia et al., 2001). Moreover, genes associated with oxi-dative stress, cell cycle, cytoskeletal network, cell-cell adhesion, extracellular matrix, inﬂammation, and apoptosis are altered by silymarin in pyrogallol-exposed liver (Upadhyay et al., 2010). In this study, microarray data showed that silymarin altered the TGF-b1-associated pathways, including TGF-b signaling pathway, TGF-b-induced apoptosis and TGF-b-mediated pathway, in CCl

-in-duced liver ﬁbrosis, which were in agreement with previous re-ports. Furthermore, we newly identiﬁed that silymarin downregulated the expression levels of cytoskeleton organization genes and mitochondrion electron-transfer chain genes. It has been known that CCl

treatment induces the reorganization of cytoskel-eton and, in turn, induces the differentiation of hepatic stellate cells into myoﬁbroblast-like cells (De Minicis et al., 2007).

Silymarin downregulated the expression of cytoskeleton compo-nent genes, suggesting that silymarin might suppress the transfor-mation of hepatic stellate cells via inhibiting cytoskeleton reorganization and thus ameliorate the ﬁbrosis of liver. Progression of CCl

-induced liver ﬁbrosis is associated with free radicals

pro-Fig. 4. Immunohistochemical examination of liver. Transgenic mice were administered with CCl4and/or silymarin. Twelve weeks later, mice were sacriﬁced, livers were excised, and sections were immunostained with antibodies against TGF-b1,

a

-SMA, and p65 (100 magniﬁcation). Quantiﬁcation of TGF-b1,

a

-SMA, and p65-positive areas (%) was shown at the bottom. Values are mean ± standard error (8 sections/group and 3 ﬁelds/section). Photos are representative images (n = 8).

C.-C. Li et al. / Food and Chemical Toxicology 50 (2012) 1568–1575 1573

80 duction that results in the signiﬁcant alternations in functional state of mitochondrial respiratory chain (Tanaka et al., 1987). The electron transporters are combined in four complex: NADH reduc-tase, succinate reducreduc-tase, cytochrome c reducreduc-tase, and Cox (Boyer, 1997). Cox plays a crucial role in oxidative metabolism, acting as

the terminal component of the mitochondrial electron-transport chain in which electrons are passed from cytochrome c to molecu-lar oxygen (Boyer, 1997). Previous studies showed that CCl

treat-ment decreases the activity of NADH reductase and increases the activity of Cox in rats with CCl

-induced liver ﬁbrosis (Krahenbuhl and Reichen, 1992; Shiryaeva et al., 2008; Tanaka et al., 1987). Our data also showed that the expression levels of Cox genes were ele-vated by CCl

. The decrease and damage of NADH reductase results in electron leakage to

O

oxygen and superoxide anion produc-tion, which lead to the increased oxygen consumption by the respi-ratory chain of pathologic mitochondria. Subsequently, the elevated activity of Cox by CCl

promotes the transfer of electrons to molecular oxygen and drive the ATP production of the mito-chondria (Shiryaeva et al., 2008). In contrast, previous study indi-cated that silymarin inhibits the oxygen consumption in mitochondria isolated from rats and increases the iron-reduced NADH reductase activity to the basal level (Chavez and Bravo, 1988; Pietrangelo et al., 2002). Moreover, our data showed that silymarin reduced the CCl

-induced expression levels of Cox genes to the basal levels as compared to mock. These ﬁndings suggested that silymarin might counteract the mitochondrion electron-trans-fer chain alteration by CCl

, which might be associated with the improvement of CCl

-induced liver ﬁbrosis by silymarin.

5. Conclusions

In conclusion, we applied for the ﬁrst time the in vivo NF- j B bioluminescent imaging and microarray analysis for the evaluation and identiﬁcation of the therapeutic potentials and novel mecha-nisms of silymarin in CCl

-induced liver ﬁbrosis. The correlation of NF- j B bioluminescence and liver ﬁbrosis suggested the feasibil-ity of NF- j B bioluminescent imaging on the evaluation of thera-peutic potentials of drugs for the treatment of liver ﬁbrosis.

Moreover, we newly identiﬁed that silymarin exhibited anti-ﬁbro-tic effects in vivo via regulating TGF-b-mediated pathways and altering the expression of genes involved in cytoskeleton organiza-tion and mitochondrion electron-transfer chain.

Conﬂict of Interest

The authors declare that there are no conﬂicts of interest.

Table 1

Pathway analysis of silymarin-altered genes with fold changes P2.0 or 62.0.

Pathway p Value^a

Regulation of cellular process/cell cycle and death

TGF-b signaling pathway 2.75 10⁷

p53-Mediated pathway 0.00171

Urea cycle and metabolism of amino groups 2.49 10⁵

Citrate cycle 2.45 10⁶

Arginine and proline metabolism 0.00016

Galactose metabolism 0.00034

Biosynthesis of steroids 0.00049

Glycine, serine and threonine metabolism 0.00070

Glycolysis/gluconeogenesis 0.00100

Butanoate metabolism 0.00098

Folate biosynthesis 0.00218

Pyruvate metabolism 0.00223

Fatty acid metabolism 0.00273

Bile acid biosynthesis 0.00273

Alanine and aspartate metabolism 0.00442

Glutathione metabolism 0.00544

Starch and sucrose metabolism 0.00601

Glycosaminoglycan degradation 0.00799

Glutamate metabolism 0.00921

Signal transduction

Adipocytokine signaling pathway 8.26 10⁵

IL6 signaling pathway 0.00016

PPAR signaling pathway 0.00039

Insulin signaling pathway 0.00047

Vitamin D3 signaling pathway 0.00067

RANKL signaling pathway 0.00548

TNF signaling pathway 0.00629

IGF signaling pathway 0.00655

Chemokine signaling pathway 0.00709

EGF signaling pathway 0.00770

PTH/PTHrP signaling pathway 0.00840

ap Value was calculated on WebGestalt web site by hypergeometric test.

Table 2

Expression levels of silymarin-downregulated genes in CCl4-treated liver.

Gene symbol

Description Fold changes^a

Acta1 Actin, alpha 1, skeletal muscle 90.21 ± 0.001

Myl1 Myosin, light polypeptide 1 77.05 ± 0.001

Tnni2 Troponin I, skeletal, fast 2 49.39 ± 0.001 Atp2a1 ATPase, Ca⁺²transporting, cardiac muscle, fast

twitch 1

48.46 ± 0.001

Mylpf Myosin light chain, phosphorylatable, fast skeletal muscle

41.90 ± 0.001

Mb Myoglobin 35.39 ± 0.002

Cox6a2 Cytochrome c oxidase, subunit VI a, polypeptide 2

28.43 ± 0.003

Cox8b Cytochrome c oxidase, subunit VIII b 18.60 ± 0.004

Eno3 Enolase 3, beta muscle 8.17 ± 0.009

Tnnt1 Troponin T1, skeletal, slow 7.60 ± 0.011

Tnnc1 Troponin C, cardiac/slow skeletal 7.56 ± 0.010 Cox7a1 Cytochrome c oxidase, subunit VIIa 1 6.67 ± 0.015 Eef1a2 Eukaryotic translation elongation factor 1

alpha 2

4.64 ± 0.022

EG433229 Predicted gene, EG433229, transcript variant 7 4.06 ± 0.016

aFold changes are mean ± standard error (n = 3).

Table 3

Expression levels of Cox6a2, Cox7a1, and Cox8b genes by qPCR.

Sample Average CT

of target

aThe DCTvalue is determined by subtracting the average GAPDH CTvalue from the average target gene CTvalue. The standard deviation of the difference is cal-culated from the standard deviations of the target gene and GAPDH.

bThe calculation of DDCTinvolves subtraction by the DCTcalibrator value. This is a subtraction of an arbitrary constant, so the standard deviation of DDCTis the same as the standard deviation of DCTvalue.

1574 C.-C. Li et al. / Food and Chemical Toxicology 50 (2012) 1568–1575

81 Acknowledgments

This work was supported by grants from National Science Coun-cil, Committee on Chinese Medicine and Pharmacy at Department of Health (CCMP100-RD-048), and China Medical University (CMU100-S-16, CMU100-S-34, and CMU100-TS-14).

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