Báo cáo khoa học: Transcriptome profiling analysis reveals multiple modulatory effects of Ginkgo biloba extract in the liver of rats on a high-fat diet pdf

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Báo cáo khoa học: Transcriptome profiling analysis reveals multiple modulatory effects of Ginkgo biloba extract in the liver of rats on a high-fat diet pdf

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Transcriptome profiling analysis reveals multiple modulatory effects of Ginkgo biloba extract in the liver of rats on a high-fat diet Xiaomei Gu 1, *, Zuoquan Xie 2, *, Qi Wang 1, *, Gang Liu 1 ,YiQu 1 , Lu Zhang 1,2 , Jiahu Pan 3 , Guoping Zhao 1,4 and Qinghua Zhang 1,2,4 1 National Engineering Center for Biochip at Shanghai, China 2 State Key Laboratory of Medical Genomics and Shanghai Institute of Hematology, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, China 3 School of Pharmacy, Fudan University, Shanghai, China 4 MOST-Shanghai Key Laboratory of Disease and Health Related Genomics, China Ginkgo biloba has been used for medical purposes for centuries in traditional Chinese medicine. The standard extracts of G. biloba leaves [G. biloba extract (GBE)] are now more widely used as dietary supplements or phytomedicines in Western countries. Both experimen- tal and clinical studies have demonstrated the cardio- vascular, cerebrovascular and neuroprotective effects of GBE [1–3]. Currently, the most common clinical uses of GBE include vascular dementia, Alzheimer’s disease, memory enhancement, intermittent claudica- tion, Raynaud’s syndrome and tinnitus of vascular ori- gin. The mixture of biologically active ingredients in GBE accounts for the pleiotropic effects, including antioxidant effects [4,5], inhibition of platelet aggrega- tion and thromboxane B 2 production [6,7], vasodila- tion [8,9] and modulation of cholesterol metabolism [10,11]. The active ingredients of the mixture, such as the flavonoids, have also been studied and have been Keywords Ginkgo biloba extract; high-fat diet; rat liver; regulation; transcriptome Correspondence Q. Zhang, State Key Laboratory of Medical Genomics and Shanghai Institute of Hematology, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200025, China Fax: +86 21 51320266 Tel: +86 21 51320288 E-mail: qinghua_zhang@shbiochip.com *These authors contributed equally to this work (Received 8 September 2008, revised 29 December 2008, accepted 5 January 2009) doi:10.1111/j.1742-4658.2009.06886.x Leaf extract of Ginkgo biloba (GBE) is increasingly used as a herbal medi- cine for the treatment of neurodegenerative, cardiovascular and cerebrovas- cular diseases. Several studies have demonstrated many protective effects of GBE in neurons, the endothelium and liver. In this study, we investigated the molecular mechanisms underlying the effects of GBE in disorders induced by long-term exposure to a high-fat diet (HFD). Rats were fed an HFD with or without the GBE product GBE50 for 19 weeks. We found that GBE50 reduced the development of fatty liver induced by an HFD and inhibited the commonly observed elevation of serum cholesterol and lactate dehydrogenase levels. Transcriptome profiling analysis showed that several genes were modulated by GBE50 in liver, including those involved in lipid metabolism, carbohydrate metabolism, vascular constriction, ion transportation, neuronal systems and drug metabolism. Notably, a number of genes coding for proteins involved in cholesterol metabolism were repressed, and some were upregulated. Fatty acid biosynthesis appeared to be repressed, whereas fatty acid metabolism appeared to be enhanced. In conclusion, using transcriptome profiling analysis, we demonstrated the molecular basis for the pleiotropic effects of GBE50, particularly those involved in lipid metabolism. This study provided new clues for further pharmacological study of GBEs. Abbreviations EST, expressed sequence tag; GBE, Ginkgo biloba leaf extract; HFD, high-fat diet; LDH, lactate dehydrogenase; PPAR, peroxisome proliferator-activated receptor; SREBP, sterol regulatory element-binding protein. 1450 FEBS Journal 276 (2009) 1450–1458 ª 2009 The Authors Journal compilation ª 2009 FEBS found to contribute to the antioxidant and ⁄ or free rad- ical-scavenging properties, prevent stroke and transient ischemic attack, and inhibit membrane lipid peroxida- tion [12]. In addition, ginkgolide B has been shown to be a potent platelet-activating factor antagonist [13]. A high-fat diet (HFD) is becoming an increasingly common dietary risk factor for health problems in modern society; such a diet contributes to the onset and ⁄ or development of various cardiovascular and cerebrovascular diseases, including hypertension, coro- nary artery disease and vascular dementia. In animal studies, an HFD has been found to elicit vascular dys- function and cardiac perivascular fibrosis in rats prior to the development of overt obesity, in the absence of hyperlipidemia [14]. Alzheimer’s disease may have developed as a result of an HFD, because amyloido- genesis and tau hyperphosphorylation have been found in cultured primary rat cortical neurons exposed to saturated fatty acids [15,16]. In addition, hepatic stea- tosis has been shown to develop in obese rats fed an HFD [17]. Because GBE has been used in the treatment of vari- ous cardiovascular and cerebrovascular diseases that can be induced by an HFD, we propose that a combi- nation of an HFD and GBE feeding may provide insights into the molecular mechanisms of GBE action in the treatment of these disorders. To test our pro- posal, we used transcriptomic strategies, which are now widely used in functional genomic research because gene expression analysis has become necessary to better understand the molecular mechanisms of her- bal medicine [18–20]. To investigate the molecular mechanisms underlying the pleiotropic protective effects of GBE, we fed rats an HFD or an HFD plus the GBE product GBE50 for 19 weeks. We then removed the livers and subjected them to transcriptomic profiling analysis using cDNA microarrays. Our results have revealed multiple effects of GBE on rat liver as well as the underlying molecular mechanisms mediat- ing these effects. Results and Discussion Body weight and biochemical parameters After 19 weeks of HFD (group H) or control diet (standard chow; group C) exposure, there was no sig- nificant difference in body weight between rats in the two groups. However, rats fed the HFD plus GBE50 (group HG) showed a significant increase in body weight. We also measured serum and hepatic total cholesterol content as well as serum high-density lipo- protein cholesterol content. We found that serum and hepatic total cholesterol content were significantly increased in group H over group C by 30% and 63%, respectively. Furthermore, serum lactate dehydroge- nase (LDH) increased three-fold in group H, reflecting the harm caused by exposure to a long-term HFD. The serum total cholesterol and LDH elevations found in group H were inhibited in group HG, suggesting that GBE50 prevented the harm caused by an HFD. In addition, we found no significant alterations in the levels of alanine aminotransferase, aspartate amino- transferase or creatinine kinase in either group H or group HG (Table 1). However, serum triglyceride levels were slightly decreased in group H (24%) in comparison with group C, although triglyceride levels were not significantly different in group HG (14%). No visible atherosclerotic plaques or vessel wall fatty streaks were detected in either group H or group HG (data not shown). Histological examination revealed that the liver lipid accumulation in group H was inhib- ited in group HG. Oil Red O staining of cryosectioned liver tissues showed scattered and weak droplets in Table 1. Body weight and serum biochemistry detection. HDL-c, high-density lipoprotein cholesterol; LDL-c, low-density lipoprotein choles- terol; ALT, alanine aminotransferase; AST, aspartate aminotransferase; CK, creatinine kinase. Group Group C (n = 4) Group H (n = 5) Group HG (n =4) Final body weight (g) 330 ± 11 352 ± 40 395 ± 21 a Serum total cholesterol (mmolÆL )1 ) 1.64 ± 0.08 2.04 ± 0.19 a 1.92 ± 0.04 a,c HDL-c (mmolÆL )1 ) 1.05 ± 0.01 1.45 ± 0.18 a 1.28 ± 0.06 a LDL-c (mmolÆL )1 ) 0.38 ± 0.07 0.42 ± 0.06 0.39 ± 0.04 Triglyceride (mmolÆL )1 ) 1.08 ± 0.17 0.82 ± 0.15 a 1.23 ± 0.24 ALT (UÆL )1 ) 120 ± 54 171 ± 132 134 ± 73 AST (UÆL )1 ) 187 ± 80 262 ± 96 181 ± 61 LDH (UÆL )1 ) 633 ± 513 2809 ± 1493 b 2106 ± 507 a,d CK (UÆL )1 ) 2279 ± 1622 2723 ± 867 1813 ± 1602 Hepatic total cholesterol (mgÆg )1 ) 2.16 ± 0.17 3.54 ± 0.84 a 2.84 ± 0.25 a a P < 0.01, b P < 0.05, versus group C; c P < 0.01, d P < 0.05, versus group H. X. Gu et al. GBE effects on rat liver gene expression FEBS Journal 276 (2009) 1450–1458 ª 2009 The Authors Journal compilation ª 2009 FEBS 1451 group C, whereas group H had positively stained lipid droplets that occupied a significantly greater area than in group C (60%). However, lipid droplets in group HG occupied a smaller area than in group H (< 30%) (Fig. 1). Global transcriptome modulation in rat liver with an HFD and GBE50 Using strict criteria to remove unreliable data, includ- ing weak signals, high expression levels or large vari- ability in group C, 5008 genes ⁄ expressed sequence tags (ESTs) were selected for later data analysis. The gene expression information is available on the website (http://www.shbiochip.com/research/GBE/rat) and in Table S1. The regulated genes were assigned to eight groups according to the gene expression patterns in group H and group HG (Fig. 2A). Among the total of 295 regulated genes ⁄ ESTs, 210 matched proper anno- tations based on the UniGene database, whereas 85 matched ESTs only (Table S2). On the basis of ontol- ogy annotations, the regulated genes were mainly those involved in lipid metabolism, carbohydrate metabo- lism, vascular constriction, ion transportation, neural systems and drug metabolism. Regulation of lipid and carbohydrate metabolism-related genes is discussed below, and detailed information on the other regulated genes is shown in Tables S1 and S2. To verify the alterations in levels of the genes identi- fied with microarray, eight lipid metabolism-associated genes were selected for real-time RT-PCR validation. As shown in Fig. 2B, the gene expression regulation patterns found through RT-PCR were consistent with data obtained through microarray, thereby demon- strating the reliability of the microarray results. Regulation of metabolism-related genes in group H In group H, 64 genes ⁄ ESTs were found to be upregulat- ed and 22 genes ⁄ ESTs were found to be downregulated in response to the HFD (Fig. S1 and Table S2). The HFD affected carbohydrate metabolism, which could result in insulin resistance and later development of dia- betes [21]. Upregulation of Foxa3, which codes for a transcription factor involved in glucose homeostasis by binding to the proglucagon gene G2 promoter element, and downregulation of an NADH-ubiquinone oxidore- ductase member gene (CI-B9) and the pyruvate kinase gene (Pklr), suggested impaired glucose metabolism in group H rats. In addition, excess intake of dietary fat caused deregulation of lipid metabolism. For example, the upregulated genes for an acyl-CoA synthetase mem- ber (Acsl1), pyruvate carboxylase (Pc), aspartoacylase 3 (Acy3) and solute carrier family 25 member 1 (Slc25a1) are involved in lipid biosynthesis, and the downregulat- ed genes for acetyl-CoA carboxylase beta (Acacb) and fatty acid-binding proteins (Fabp1 and Fabp5) contrib- ute to fatty acid metabolism. The repression of Hmgcr, which encodes the cholesterol biosynthesis rate-limiting enzyme HMG-CoA reductase, indicated that the endog- enous cholesterol biosynthesis was impaired as a result of the HFD. In addition, upregulation of the genes for the cytochrome P450 family member sterol 12-a-hydro- lase (Cyp8b1) and hydroxysteroid (17-b) dehydroge- nase 2 (Hsd17b2) suggested augmentation of steroid metabolism. Furthermore, the gene for sarcosine dehy- drogenase (Sardh), a flavoenzyme involved in the oxida- tive degradation of choline to glycine in rat [22], was upregulated. Transcriptome remodeling in group HG rat liver In group HG, 243 genes ⁄ ESTs were found to be regu- lated. Among the 22 genes ⁄ ESTs downregulated in group H, 11 were similarly downregulated in group HG, nine showed no significant difference from group C, and two (Slc4a1 and Tubb2b) were upregulat- ed as compared to group C. Of the 64 genes ⁄ ESTs upregulated in group H, 19 genes showed similar upregulation in group HG, 43 genes were not significantly different from group C, and two genes (Hsd17b2 and Trim28) were significantly downregulat- ed. The other 107 downregulated and 102 upregulated genes in group HG showed no significant upregulation or downregulation in group H, indicating that these genes were probably regulated by GBE50 in rat liver (Fig. S1 and Table S2). Fig. 1. Histology detection of rat liver. Oil Red O-stained frozen sample sections of livers from groups C, H and HG. Lipid deposits in liver were revealed by Oil Red O staining according to the method described by Bouma et al. [34]. GBE effects on rat liver gene expression X. Gu et al. 1452 FEBS Journal 276 (2009) 1450–1458 ª 2009 The Authors Journal compilation ª 2009 FEBS Lipid metabolism genes By comparison with expression in group H, the further repression of Hmgcr and the upregulation of Insig2, the upstream negative regulator of Hmgcr, probably contribute to impaired cholesterol biosynthesis. Furthermore, decreased expression of gene for the bile acid biosynthesis rate-limiting enzyme cholesterol 7-a- hydroxylase (Cyp7a1) and Cyp8b1 indicates that the conversion of cholesterol into bile acid may be repressed, which is consistent with our finding of upregulation of the gene for bile acid synthesis A B a b c d e f g h Fig. 2. (A) Box-plot presentation of gene expression regulation. Gene expression regulation was determined with the microarray data and compared with reference samples from group C. Detailed information is provided in Experimental procedures. A cut-off of 1.5-fold was used to identify regulated genes filtered with SAM. Eight groups of regulation patterns were obtained, and the number of genes is indicated above each graph. (B) Validation of eight genes with real-time RT-PCR. X. Gu et al. GBE effects on rat liver gene expression FEBS Journal 276 (2009) 1450–1458 ª 2009 The Authors Journal compilation ª 2009 FEBS 1453 negative regulator farnesoid X receptor (FXR or Nr1h4) and downregulation of the gene for bile acid biosynthesis circadian regulator Rev-ErbA alpha (Nr1d1) [23,24]. Our finding of significant downregula- tion of the gene for thyroid hormone-responsive pro- tein (Thrsp) in group HG indicates an inhibition of the lipogenic cascade in hepatocytes by GBE50 [25]. From our findings, we deduced that fatty acid bio- synthesis was repressed and metabolism was enhanced in animals exposed to an HFD with GBE50. Genes involved in fatty acid biosynthesis, such as Acacb, Acbd6 (acyl-CoA-binding domain containing 6), Scd2 (stearoyl-CoA desaturase 2) or associated genes, Sorbs3 (sorbin and SH3 domain containing 3) and Etnk1_predicted (ethanolamine kinase 1) were down- regulated or repressed, as was Acsl1, which was upreg- ulated in group H. The upregulation of the genes for peroxisomal 2,4-dienoyl-CoA reductase (Decr2) and glycerol kinase (Gyk) suggested enhanced glycerolipid and fatty acid metabolism. The upregulation of Cyp4a12 and downregulation of Fabp1 in both group H and group HG indicated that GB50E had no significant effect on these genes, whereas Fabp5 was further repressed in group HG. Carbohydrate metabolism and respiratory chain genes Downregulation of the genes encoding the glucose transporter Glut2 ( Slc2a2) and glycogen synthesis- associated enzyme UDP-glucose pyrophosphorylase 2 (Ugp2), glycogen synthase 2 (Gys2) and b4-galactosyl- transferase (B4galt3) supported the hypothesis that gly- cogenesis was impaired with GBE50 intake, although Foxa3 remained upregulated in group HG. In addi- tion, the decreased expression of Pc in group HG fur- ther suggested impaired glyconeogenesis. Upregulation of the insulin receptor substrate gene (Irs3) could con- tribute to glucose homeostasis and utilization enhance- ment in liver [26]. However, downregulation of mitochondrial protein genes (CI-B9, Atp6v0e1 and Nudfa8) indicates that the respiratory chain and oxida- tive phosphorylation pathway was repressed in group HG. Regulation of cholesterol metabolism-related gene expression by HFD and GBE50 in rat liver To better understand cholesterol metabolism regula- tion with the HFD and GBE50, real-time RT-PCR was employed to validate the microarray data, includ- ing genes that were not spotted or detected with the microarray (Table 2). The nuclear steroid receptor peroxisome proliferator-activated receptors (PPARs) are transcription factors that form heterodimers with retinoid X receptors to initiate the transcriptional regu- lation of target genes. Three subtypes of PPARs (a, b ⁄ d and c) and retinoid X receptors (a, b and c) have been identified, and the PPARs are mainly regulators of lipid metabolism [27]. In group H, genes for lipid metabolism regulators (Ppard, Pparg , and Rxra, Rxrb) were upregulated in rat liver, suggesting that lipid metabolism was activated. As the genes for low-density lipoprotein receptor (Ldlr) and scavenger receptor (Scarb1) are major members participating in choles- terol uptake, the upregulation of Ldlr and Scarb1 sug- gested enhanced uptake of cholesterol in liver of rats in group H. Furthermore, downregulation of Hmgcr suggested inhibition of endogenous cholesterol biosyn- thesis with HFD intake. In group HG, cholesterol metabolism genes were regulated in the liver in concert. Specifically, decreased expression of Ldlr and repression of Scarb1 suggested that the uptake of cholesterol was reduced. The insulin- induced gene Insig2 encodes an endoplasmic reticulum protein that blocks the processing of sterol regulatory element-binding proteins (SREBPs) by binding to SREBP cleavage-activating protein, thus preventing the latter from escorting SREBPs to the Golgi, and nega- tively regulating sterogenesis. The increased expression of Insig2 and the further repression of Hmgcr suggested that endogenous cholesterol synthesis was repressed. The upregulation of Nr1h4 and downregulation of Cyp7a1 could contribute to bile acid biosynthesis reduction. Meanwhile, the lipid metabolism regulators were also modulated. Ppard showed significantly higher expression in group HG than in group H, whereas Pparg, Rxra and Rxrb were restricted to express at normal level in group HG. Additionally, the upregu- lated expression of the ATP-binding cassette super- family members Abcg5 and Abcg8 in group H, which form heterodimers and promote biliary cholesterol excretion, was repressed in group HG, suggesting that the cholesterol excretion from liver might also be lessened by GBE50 (Table 2 and Fig. 3). Experimental procedures GBE GBE50 is a standardized GBE product that matches the standardized German product as EGb761. GBE50 is approved by the China State Food and Drug Administra- tion (SFDA) and is used clinically in China. GBE50 con- tains > 44.0% total flavonoids, > 24.0% flavonoglycoside and > 6.0% total terpen lactones. Ginkgolic acid is GBE effects on rat liver gene expression X. Gu et al. 1454 FEBS Journal 276 (2009) 1450–1458 ª 2009 The Authors Journal compilation ª 2009 FEBS controlled to be < 5 p.p.m. GBE50 products were kindly provided by Shanghai Xingling Pharmaceutical (Shanghai, China), manufactured under good manufacturing process conditions (lot no. 20030608). Animal diet and experiments Thirty male Wistar rats (3 weeks old, weighing 50–55 g) were obtained from Shanghai Laboratory Animal Center of the Shanghai Institute of Biological Sciences, Chinese Acad- emy of Sciences (Shanghai, China). Animals were housed in a temperature-controlled room (23–25 °C) for 19 weeks with a 12 h light ⁄ dark cycle, and allowed unrestricted access to pellet food and water. The rats were randomly divided into three groups of 10, including groups C, H and HG. Group C was fed standard chow and acted as the nor- mal control. Group H was fed an HFD, containing 8% lard, 7% egg yolk powder, 0.5% sodium cholate and 84.5% standard rat chow. No pure cholesterol was added. Group HG was fed the HFD supplemented with GBE50 (0.2%, w ⁄ w). The chows were manufactured and provided by the Shanghai Laboratory Animal Center. One to two animals in each group were killed for monitoring of cardio- vascular and liver alterations at 8, 12 and 15 weeks follow- ing initiation of the feeding paradigm. After 19 weeks, Table 2. Expression of cholesterol metabolism-related genes. The mean expression level of each gene in group C rats was converted to 1.00 as reference. RT-PCR was conducted on three samples with triplicate experiments in each. Gapdh was used as an internal control. Data for the microarray were derived from Table S3. Category and gene Group C Group H Group HG Microarray RT-PCR Microarray RT-PCR Microarray RT-PCR Uptake Scarb1 1.00 ± 0.25 2.03 ± 0.40 a 1.44 ± 0.52 Ldlr 1.00 ± 0.06 2.19 ± 0.35 b 0.49 ± 0.08 b Conversion Acat1 1.00 ± 0.24 1.75 ± 0.26 a 1.31 ± 0.48 Biosynthesis Insig2 1.00 ± 0.16 1.00 ± 0.08 1.25 ± 0.84 1.34 ± 0.14 1.81 ± 0.55 b 2.57 ± 0.45 b Hmgcr 1.00 ± 0.26 1.00 ± 0.39 0.80 ± 0.18 0.49 ± 0.13 a 0.55 ± 0.14 a 0.36 ± 0.12 a Secretion Abcg5 1.00 ± 0.07 2.34 ± 0.20 b 1.55 ± 0.25 a Abcg8 1.00 ± 0.02 1.76 ± 0.16 b 1.33 ± 0.03 b Bile acid metabolism Cyp7a1 1.00 ± 0.67 1.00 ± 0.51 0.65 ± 0.12 1.26 ± 0.04 0.16 ± 0.09 a 0.30 ± 0.00 a Cyp8b1 1.00 ± 0.41 1.00 ± 0.20 1.30 ± 0.30 0.76 ± 0.27 0.68 ± 0.08 0.76 ± 0.57 Regulators Nr1h4 1.00 ± 0.18 1.00 ± 0.47 0.96 ± 0.16 1.45 ± 0.74 1.41 ± 0.21 2.05 ± 0.52 a Ppard 1.00 ± 0.06 2.58 ± 0.46 b 6.41 ± 1.62 b Pparg 1.00 ± 0.01 2.08 ± 0.75 a 1.41 ± 0.33 Rxra 1.00 ± 0.19 3.07 ± 0.96 a 1.50 ± 0.49 Rxrb 1.00 ± 0.42 4.04 ± 1.35 a 1.67 ± 0.76 a P < 0.05, b P < 0.01, versus group C. Fig. 3. Ideogram illustration of regulated genes participating in cho- lesterol metabolism network modulated by an HFD and ⁄ or GBE50 in rat liver. Genes are framed, the key substances are circled, tran- scription factor genes are in ellipses, and the enzymes coded by each gene are in oblongs. Regulation of the genes (> 1.5-fold) is indicated by red (upregulation) and green (downregulation) in the background, and yellow indicates no significant difference in regula- tion from the control group. The left half represent regulation in group H and the right in group HG. The red line with arrow indi- cates positive regulation, the blocked green lines indicate negative regulation, and the dashed lines indicate indirect inhibition. The black lines indicate substance conversion or transportation flow. The dashed lines indicate multistep conversion. X. Gu et al. GBE effects on rat liver gene expression FEBS Journal 276 (2009) 1450–1458 ª 2009 The Authors Journal compilation ª 2009 FEBS 1455 which marked the end of the experiment, all animals were fasted overnight and decapitated in the morning. Livers were removed and resected, and stored in liquid nitrogen immediately for later mRNA analysis and cholesterol mea- surement or stored in 10% formalin buffer (pH 7.3) for his- topathological examination. Blood samples were clotted at room temperature to collect sera for biochemical measure- ments. All procedures were approved by Animal Experi- ment Committee of the School of Pharmacy Fudan University. Biochemical measurement and cholesterol detection The collected sera were assayed with a Roche-Hitachi Mod- ular P800 Chemistry analyzer and corresponding enzymatic reagents (Roche Diagnostics, Indianapolis, IN, USA) in the clinical laboratory of Zhongshan Hospital, affiliated to Fudan University. Hepatic cholesterol content was deter- mined as previously described [28], with a modification. Briefly, frozen stored liver tissues were weighed and homog- enized, lipids were extracted with isopropanol, and aliquots were used for colorimetric determination of total choles- terol using a cholesterol oxidase assay kit (Shanghai Jiemen Biotech Co., Shanghai, China). Liver tissue cholesterol con- tent was normalized to the liver tissue weight. Histopathological detection Liver tissues were fixed in 10% formalin, dehydrated and paraffin embedded. A series of 5-lm-thick slices were sub- jected to standard hematoxylin ⁄ eosin staining. For Oil Red O staining, frozen liver tissues were embedded in opti- cal coherence tomography and cryosectioned at 8 lm thick- ness, following standard procedures. The histology slides were cross-inspected by two pathologists. RNA preparation and cDNA microarray hybridization Rat liver total RNA was isolated using the Trizol reagent (Invitrogen) and purified with an RNAeasy column (Qia- gen). RNA quality was assessed with a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Homemade cDNA microarrays containing 10 200 rat genes or ESTs were fabricated as previously described [29–31]. The list of the genes on the microarray is available on the website http://www.shbiochip.com/research/GBE/rat, and the microarray platform was submitted to the GEO database with the accession number GPL4294. Microarray experi- ments were performed as previously described [29–31]. Equal amounts of RNA from four control rats were pooled for use as a reference and labeled with Cy3-dUTP in later experiments. RNA samples of 2 lg from group C (n = 4), group H (n = 5) and group HG (n = 4) were individually labeled with Cy5-dUTP. Hybridization was performed with each individual sample and the common labeled reference sample. After being washed, the microarray slides were scanned with an Agilent microarray scanner. Data analysis Raw intensities of spots were extracted from images by imagene v9.0 software, and the spots with a low signal-to- noise ratio (< 2) were automatically denoted ‘flag > 0’. Signal intensity normalization within each array was per- formed by Lowess regression, and the signal ratio was transformed to a log base 2 ratio. Further scale normaliza- tion between arrays was implemented using the median absolute deviation (MAD) approach. We successively filtered out genes denoted ‘flag > 0’, which represented more than 40% of the total number of arrays. We further eliminated gene sets with unreliable expression measures, on the basis of data from group C. Data were excluded if: (a) the signal of log ratios in the control group was signifi- cantly variable, as detected by a one-class comparison (setting false discovery rate = 0.01) using the software sam 2.20 [32]; (b) the standard deviation of the log ratios in the control group was > 1; or (c) the absolute value of the log ratio was > 0.585 (corresponding to a 1.5-fold change) in at least two samples in the control group. On the basis of the remaining dataset, we identified genes that were upregulated or downregulated in group H and group HG relative to control. This procedure was performed using a one-class comparison procedure (setting false discovery rate = 0.01) with sam software 2.20. In this analysis, we tested whether the log ratio values of each treatment group (H or HG) were significantly different from zero. The sig- nificant genes identified by the independent groups were used to perform hierarchical clustering of samples by using cluster 3.0, and the results were visualized using treeview (cluster and treeview were developed by Eisen et al. [33]). The regulated genes were classified into different groups on the basis of ontology, and the online database KEGG (http://www.genome.jp) was applied in gene func- tion pathway assignment. RT-PCR analysis Quantitative RT-PCR was conducted with an ABI Prism7000 sequence detection system (Applied Biosystems, Foster City, CA, USA) using SYBR Green I (TaKaRa Bio- tech, Dalian, China). Genes for RT-PCR were selected on the basis of the microarray data or because they were known to be involved in lipid metabolism but were not included in the gene list selected with microarray. The PCR primer sequences are listed in Table S3. Reactions were carried out in triplicate in a 25 lL volume for three animals GBE effects on rat liver gene expression X. Gu et al. 1456 FEBS Journal 276 (2009) 1450–1458 ª 2009 The Authors Journal compilation ª 2009 FEBS from each group. Following the PCR, the amplicon melting curve was checked for PCR specificity. Statistics The number of rats from each group used in each experi- ment is indicated in the figure legends. Values are presented as mean ± standard error of the mean. A two-tailed stu- dent’s t-test was used to calculate P-values, and a P-value < 0.05 was considered to be significant. Acknowledgements The authors thank B. F. Zhang, J. Fang, Y. H. Yang, S. J. Jiang, Z. D. Zhu, Y. Gao, L. B. Lin, Y. C. Chen, F. He and G. R. Zhu for their technical assistance, Q. Gao, G. A. Zhang, J. J. Xie and D. L. Xie for pro- viding the GBE50 products, and J. S. Han, H. S. Xiao and H. Y. Wang for their constructive comments and discussions. 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Proc Natl Acad Sci USA 95, 14863–14868. 34 Bouma ME, Amit N & Infante R (1979) Ultrastructural localization of apo-b and apo-c binding to very low density lipoproteins in rat liver. Virchows Arch B Cell Path 30, 161–180. Supporting information The following supplementary material is available: Doc. S1. Gene expression profile analysis. Fig. S1. Functional categories of genes modulated by an HFD and GBE50 in rat liver. Table S1. Gene expression dataset of rat livers after fil- tration. Table S2. Genes regulated in group H and group HG. Table S3. RT-PCR primer sequences. Table S4. Expression levels of selected genes. Table S5. Comparison of lipid metabolism gene expression in group CG and group HG. This supplementary material can be found in the online version of this article. Please note: Wiley-Blackwell is not responsible for the content or functionality of any supplementary materials supplied by the authors. Any queries (other than missing material) should be directed to the corre- sponding author for the article. GBE effects on rat liver gene expression X. Gu et al. 1458 FEBS Journal 276 (2009) 1450–1458 ª 2009 The Authors Journal compilation ª 2009 FEBS . Transcriptome profiling analysis reveals multiple modulatory effects of Ginkgo biloba extract in the liver of rats on a high-fat diet Xiaomei Gu 1, *,. suggesting that GBE50 prevented the harm caused by an HFD. In addition, we found no significant alterations in the levels of alanine aminotransferase, aspartate

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