Genipin 抗發炎機轉分析

第四章結果

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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82 Genipin inhibits lipopolysaccharide-induced acute systemic inﬂammation

in mice as evidenced by nuclear factor- j B bioluminescent imaging-guided

transcriptomic analysis

Q2 Chia-Cheng Li

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