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Metabolomics Coupled with Proteomics Advancing Drug Discovery toward More Agile Development of Targeted Combination Therapies

2013; Elsevier BV; Volume: 12; Issue: 5 Linguagem: Inglês

10.1074/mcp.m112.021683

1535-9484

Xijun Wang, Aihua Zhang, Ping Wang, Hui Sun, Gelin Wu, Wenjun Sun, Haitao Lv, Guozheng Jiao, Hongying Xu, Ye Yuan, Lian Liu, Dixin Zou, Zeming Wu, Ying Han, Guangli Yan, Wei Dong, Fangfang Wu, Tianwei Dong, Yang Yu, Shuxiang Zhang, Xiuhong Wu, Xin Tong, Xiangcai Meng,

Acute Myeloid Leukemia Research

To enhance the therapeutic efficacy and reduce the adverse effects of traditional Chinese medicine, practitioners often prescribe combinations of plant species and/or minerals, called formulae. Unfortunately, the working mechanisms of most of these compounds are difficult to determine and thus remain unknown. In an attempt to address the benefits of formulae based on current biomedical approaches, we analyzed the components of Yinchenhao Tang, a classical formula that has been shown to be clinically effective for treating hepatic injury syndrome. The three principal components of Yinchenhao Tang are Artemisia annua L., Gardenia jasminoids Ellis, and Rheum Palmatum L., whose major active ingredients are 6,7-dimethylesculetin (D), geniposide (G), and rhein (R), respectively. To determine the mechanisms underlying the efficacy of this formula, we conducted a systematic analysis of the therapeutic effects of the DGR compound using immunohistochemistry, biochemistry, metabolomics, and proteomics. Here, we report that the DGR combination exerts a more robust therapeutic effect than any one or two of the three individual compounds by hitting multiple targets in a rat model of hepatic injury. Thus, DGR synergistically causes intensified dynamic changes in metabolic biomarkers, regulates molecular networks through target proteins, has a synergistic/additive effect, and activates both intrinsic and extrinsic pathways. To enhance the therapeutic efficacy and reduce the adverse effects of traditional Chinese medicine, practitioners often prescribe combinations of plant species and/or minerals, called formulae. Unfortunately, the working mechanisms of most of these compounds are difficult to determine and thus remain unknown. In an attempt to address the benefits of formulae based on current biomedical approaches, we analyzed the components of Yinchenhao Tang, a classical formula that has been shown to be clinically effective for treating hepatic injury syndrome. The three principal components of Yinchenhao Tang are Artemisia annua L., Gardenia jasminoids Ellis, and Rheum Palmatum L., whose major active ingredients are 6,7-dimethylesculetin (D), geniposide (G), and rhein (R), respectively. To determine the mechanisms underlying the efficacy of this formula, we conducted a systematic analysis of the therapeutic effects of the DGR compound using immunohistochemistry, biochemistry, metabolomics, and proteomics. Here, we report that the DGR combination exerts a more robust therapeutic effect than any one or two of the three individual compounds by hitting multiple targets in a rat model of hepatic injury. Thus, DGR synergistically causes intensified dynamic changes in metabolic biomarkers, regulates molecular networks through target proteins, has a synergistic/additive effect, and activates both intrinsic and extrinsic pathways. Currently, a paradigm shift is occurring in that there is a new focus on agents that modulate multiple targets simultaneously, rather than working at the level of single protein molecules (1Drews J. Drug discovery: a historical perspective.Science. 2000; 287: 1960-1964Crossref PubMed Scopus (2240) Google Scholar). Multiple-target approaches have recently been employed to design medications that are used to treat atherosclerosis, cancer, depression, psychosis, and neurodegenerative diseases (2van der Greef J. Martin S. Juhasz P. Adourian A. Plasterer T. Verheij E.R. McBurney R.N. The art and practice of systems biology in medicine: mapping patterns of relationships.J. Proteome Res. 2007; 6: 1540-1559Crossref PubMed Scopus (159) Google Scholar). 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Interestingly, traditional Chinese medicine (TCM), 1The abbreviations used are:D6,7-dimethylesculetinDAdiscriminant analysisGgeniposideHIhepatic injuryOPLSorthogonal projection to latent structuresPCAprincipal components analysisRrheinTCMtraditional Chinese medicineUPLC-HDMSultra-performance liquid chromatography–high definition mass spectrometryYCHTYinchenhao Tang. 1The abbreviations used are:D6,7-dimethylesculetinDAdiscriminant analysisGgeniposideHIhepatic injuryOPLSorthogonal projection to latent structuresPCAprincipal components analysisRrheinTCMtraditional Chinese medicineUPLC-HDMSultra-performance liquid chromatography–high definition mass spectrometryYCHTYinchenhao Tang. which is a unique medical system that assisted the ancient Chinese in dealing with disease, has advocated combinatorial therapeutic strategies for 2,500 years using prescriptions called formulae (14Wang L. Zhou G.B. Liu P. Song J.H. Liang Y. Yan X.J. Xu F. Wang B.S. Mao J.H. Shen Z.X. Chen S.J. Chen Z. Dissection of mechanisms of Chinese medicinal formula Realgar-Indigo naturalis as an effective treatment for promyelocytic leukemia.Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 4826-4831Crossref PubMed Scopus (636) Google Scholar). Typically, formulae consist of several kinds of crude drugs that originate from medicinal plants, animals, or minerals; one represents the principal component and is called the monarch drug in TCM, and the others serve as adjuvant components that facilitate the delivery of the principal component to the disease site within the body. More specifically, according to the rules of TCM theory, the famous formulae include four elements: the monarch (which plays the most important role in the formula), the minister (which increases the effectiveness of the monarch herb), the assistant (which helps the monarch and minister herbs reach their target positions), and the servant (which can reduce the adverse effects and/or increase the potency of the whole formula). In formulae, the herbs work together harmoniously to achieve an ideal therapeutic outcome. Therapeutic regimens that include more than one active ingredient are commonly used clinically in Chinese medicine (15Keith C.T. Borisy A.A. Stockwell B.R. Multicomponent therapeutics for networked systems.Nat. Rev. Drug Discov. 2005; 4: 71-78Crossref PubMed Scopus (631) Google Scholar). The therapeutic efficacy of TCM is usually attributed to its synergistic properties, its capacity for minimizing adverse reactions, or its improved therapeutic efficacy. Synergism is a core principle of traditional medicine, or ethnopharmacology, and plays an essential role in improving the clinical efficacy of TCM. It is believed, at least in regard to some formulae, that multiple components can hit multiple targets and exert synergistic therapeutic effects (14Wang L. Zhou G.B. Liu P. Song J.H. Liang Y. Yan X.J. Xu F. Wang B.S. Mao J.H. Shen Z.X. Chen S.J. Chen Z. Dissection of mechanisms of Chinese medicinal formula Realgar-Indigo naturalis as an effective treatment for promyelocytic leukemia.Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 4826-4831Crossref PubMed Scopus (636) Google Scholar). A scientific explanation for this type of synergy would certainly promote the reasonable and effective application of TCM and help to encourage rational approaches to the safe combination of healthcare systems from various cultures. Multidrug combinations are increasingly important in modern medicine (16Zhang X.W. Yan X.J. Zhou Z.R. Yang F.F. Wu Z.Y. Sun H.B. Liang W.X. Song A.X. Lallemand-Breitenbach V. Jeanne M. Zhang Q.Y. Yang H.Y. Huang Q.H. Zhou G.B. Tong J.H. Zhang Y. Wu J.H. Hu H.Y. de Thé H. Chen S.J. Chen Z. Arsenic trioxide controls the fate of the PML-RARalpha oncoprotein by directly binding PML.Science. 2010; 328: 240-243Crossref PubMed Scopus (609) Google Scholar, 17Wang X. Zhang A. Sun H. Future perspectives of Chinese medical formulae: chinmedomics as an effector.OMICS. 2012; 16: 414-421Crossref PubMed Scopus (145) Google Scholar, 18Chait R. Craney A. Kishony R. Antibiotic interactions that select against resistance.Nature. 2007; 446: 668-671Crossref PubMed Scopus (336) Google Scholar). However, the precise mechanisms through which formulae function are poorly understood and must be addressed using a molecular approach. 6,7-dimethylesculetin discriminant analysis geniposide hepatic injury orthogonal projection to latent structures principal components analysis rhein traditional Chinese medicine ultra-performance liquid chromatography–high definition mass spectrometry Yinchenhao Tang. 6,7-dimethylesculetin discriminant analysis geniposide hepatic injury orthogonal projection to latent structures principal components analysis rhein traditional Chinese medicine ultra-performance liquid chromatography–high definition mass spectrometry Yinchenhao Tang. Yinchenhao Tang (YCHT), which was recorded in Shanghanlun, a classic resource on TCM written by Zhongjing Zhang (150–215 A.D.), is one of the most famous Chinese herbal formulae. YCHT consists of Artemisia annua L. (the monarch herb), Gardenia jasminoides Ellis (the minister herb), and Rheum Palmatum L. (the assistant and servant herb) and has been used for more than a thousand years to treat jaundice and liver disorders (19Zhang Z.J. Shanghanlun. Peo Hygiene, Beijing2005Google Scholar). Pharmacological studies and clinical practice have shown that it can be used clinically to treat cholestasis, hepatitis C, primary biliary cirrhosis, liver fibrosis, and cholestatic liver diseases (20Zhang A. Sun H. Wang X. Jiao G. Yuan Y. Sun W. Simultaneous in vivo RP-HPLC-DAD quantification of multiple-component and drug-drug interaction by pharmacokinetics, using 6,7-dimethylesculetin, geniposide and rhein as examples.Biomed. 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Sun W. Yuan Y. Pharmacokinetics screening for multi-components absorbed in the rat plasma after oral administration traditional Chinese medicine formula Yin-Chen-Hao-Tang by ultra performance liquid chromatography-electrospray ionization/quadrupole-time-of-flight mass spectrometry combined with pattern recognition methods.Analyst. 2011; 136: 5068-5076Crossref PubMed Scopus (130) Google Scholar). Interestingly, dimethylesculetin (D) has been shown to be effective in treating hepatic injury (HI) by exerting hepato-protectivity, and it contributes directly to the therapeutic effect of YCHT (24Murat Bilgin H. Atmaca M. Deniz Obay B. Ozekinci S. Taşdemir E. Ketani A. Protective effects of coumarin and coumarin derivatives against carbon tetrachloride-induced acute hepatotoxicity in rats.Exp. Toxicol. Pathol. 2011; 63: 325-330Crossref PubMed Scopus (69) Google Scholar). Geniposide (G), a primary component of the fruits of Gardenia jasminoids Ellis, exhibits various pharmacological properties, including antioxidant, anti-inflammatory, and hepato-protective effects (25Yin F. Liu J. Zheng X. Guo L. Xiao H. Geniposide induces the expression of heme oxygenase-1 via PI3K/Nrf2-signaling to enhance the antioxidant capacity in primary hippocampal neurons.Biol. Pharm. Bull. 2010; 33: 1841-1846Crossref PubMed Scopus (59) Google Scholar, 26Zheng X. Yang D. Liu X. Wang N. Li B. Cao H. Lu Y. Wei G. Zhou H. Zheng J. Identification of a new anti-LPS agent, geniposide, from Gardenia jasminoides Ellis, and its ability of direct binding and neutralization of lipopolysaccharide in vitro and in vivo.Int. Immunopharmacol. 2010; 10: 1209-1219Crossref PubMed Scopus (50) Google Scholar, 27Ma T. Huang C. Zong G. Zha D. Meng X. Li J. Tang W. Hepatoprotective effects of geniposide in a rat model of nonalcoholic steatohepatitis.J. Pharm. Pharmacol. 2011; 63: 587-593Crossref PubMed Scopus (88) Google Scholar). Intriguingly, a recent study showed that rhein (R), a metabolite of anthranoids and a major component of Rheum Palmatum L., helps to ameliorate liver fibrosis (28Guo M.Z. Li X.S. Xu H.R. Mei Z.C. Shen W. Ye X.F. Rhein inhibits liver fibrosis induced by carbon tetrachloride in rats.Acta Pharmacol. Sin. 2002; 23: 739-744PubMed Google Scholar, 29Lin Y.L. Wu C.F. Huang Y.T. Phenols from the roots of Rheum palmatum attenuate chemotaxis in rat hepatic stellate cells.Planta Med. 2008; 74: 1246-1252Crossref PubMed Scopus (26) Google Scholar, 30Sheng X. Wang M. Lu M. Xi B. Sheng H. Zang Y.Q. Rhein ameliorates fatty liver disease through negative energy balance, hepatic lipogenic regulation, and immunomodulation in diet-induced obese mice.Am. J. Physiol. Endocrinol. Metab. 2011; 300: E886-E893Crossref PubMed Scopus (79) Google Scholar). Studies indicate that D, G, and R have all been used as marker compounds in quality control for YCHT (31Wang X. Lv H. Sun H. Jiang X. Wu Z. Sun W. Wang P. Liu L. Bi K. Quality evaluation of Yin Chen Hao Tang extract based on fingerprint chromatogram and simultaneous determination of five bioactive constituents.J. Sep. Sci. 2008; 31: 9-15Crossref PubMed Scopus (23) Google Scholar). It is noteworthy that a previous study reported the synergistic effects of DGR based on the pharmacokinetics of the main effective constituents of YCHT (32Zhang A. Sun H. Yuan Y. Sun W. Jiao G. Wang X. An in vivo analysis of the therapeutic and synergistic properties of Chinese medicinal formula Yin-Chen-Hao-Tang based on its active constituents.Fitoterapia. 2011; 82: 1160-1168Crossref PubMed Scopus (98) Google Scholar, 33Wang X. Zhang A. Han Y. Wang P. Sun H. Song G. Dong T. Yuan Y. Yuan X. Zhang M. Xie N. Zhang H. Dong H. Dong W. Urine metabolomics analysis for biomarker discovery and detection of jaundice syndrome in patients with liver disease.Mol. Cell. Proteomics. 2012; 1: 370-380Abstract Full Text Full Text PDF Scopus (209) Google Scholar). These results demonstrate the clinical efficacy of DGR and indicate the need for further research regarding the mechanics of this formula. It has been proposed that DGR-based combination treatment for HI produces a synergistic effect. However, although much is known regarding the interactions among D, G, and R at pharmacokinetic sites, there is little knowledge regarding the compound's synergistic properties. Understanding the synergistic effects of DGR represents an even greater challenge because multilayered regulation might be involved, with the three compounds having overlapping but distinct target properties. In order to gain insight into the complex biochemical mechanisms that underlie this effective HI therapy, we conducted an investigation incorporating advanced technologies using metabolomic, proteomic, and biochemical analyses throughout the treatment process for HI (which has been shown to respond specifically to these agents). In analyzing the formula design in TCM, here we use the treatment of HI with DGR as a working model. D, G, and R—which are derived from Artemisia annua L., Gardenia jasminoids Ellis, and Rheum Palmatum L., respectively—were used as the active compounds in YCHT, and the efficacy and mechanisms of the DGR combination as used to treat HI were tested both in vivo and in vitro. This is the first study that investigates the unique synergistic effect of combination dosing and provides support for the popular view that traditional Chinese formulae require multiple components to exert their combined effects. Acetonitrile (HPLC grade) was purchased from Merck (Darmstadt, Germany). Methanol (HPLC grade) was purchased from Fisher (USA). Distilled water was purchased from Watson's Food & Beverage Co., Ltd. (Guangzhou, China), and formic acid (HPLC grade) was purchased from the Beijing Reagent Company (Beijing, China). Leucine enkephalin was purchased from Sigma-Aldrich, and carbon tetrachloride (CCl4) was purchased from the Chemicals Factory (Tianjin, China). Glycerol was supplied by the Chemicals Factory. Other chemicals, except as noted, were analytical grade. D, G, and R were isolated within our laboratory and identified via spectral analyses, primarily NMR and MS. After identification, the substances were further purified via HPLC to yield authorized compound with a purity of at least 99%. Freeze-dried YCHT powder was produced by our laboratory. The assay kits were purchased from the Nanjing Jiancheng Biotech Company (Nanjing, China). The other reagents that were used in the two-dimensional electrophoresis were purchased from Bio-Rad. Male Wistar rats were bred and maintained in a specific pathogen-free environment. The animals were allowed to acclimatize in metabolic cages for 1 week prior to treatment. The animals were randomly assigned to various groups and treated with D, G, and/or R at the doses indicated in the supplementary material. The experiments were performed with the approval of the Animal Ethics Committee of Heilongjiang University of Chinese Medicine, China. Blood was collected from the hepatic portal vein, and plasma was separated via centrifugation at 4,500 rpm for 5 min at 4 °C, flash frozen in liquid nitrogen, and stored at −80 °C until the liver function tests and proteomics analyses were performed. Urine was collected daily (at 6:00 a.m.) from the metabolic cages at ambient temperature over the course of the entire procedure and centrifuged at 10,000 rpm at 4 °C for 5 min; the supernatants were then stored frozen at −20 °C for subsequent metabolomic analysis. We collected plasma samples in heparinized tubes, kept them on ice for 1 h, and centrifuged them at 4,500 rpm for 15 min at 4 °C. We quantified the levels of plasma alanine transaminase, aspartate transaminase, alkaline phosphatase, glutathione peroxidase, and superoxide dismutase activity and the malondialdehyde, triglyceride, glutamyl transferase, cholesterol, total protein, direct bilirubin, and total bilirubin content using assay kits according to the manufacturer's instructions. The rat livers were removed immediately after plasma collection and stored at −70 °C until analysis. The livers were fixed in 4% neutral buffered formaldehyde at 4 °C and embedded in paraffin. The liver tissue was stained with H&E for histopathological analysis. Immunohistochemistry was performed using antibodies against Fas and BCL-2. TUNEL staining was performed to detect and quantify apoptotic cells using the in situ cell death detection kit. The sections were viewed and photographed using standard fluorescent microscopic techniques. Urine and serum were collected for UPLC-HDMS analysis. For the reversed-phase UPLC analysis, an ACQUITY UPLC BEH C18 column (50 mm × 2.1 mm inner diameter, 1.7 μm, Waters Corp., Milford, MA) was used. The column temperature was maintained at 35 °C, the flow rate during the mobile phase was 0.50 ml/min, and the injection volume was fixed at 2.0 μl. Mobile phase A involved the use of 0.1% formic acid in acetonitrile, whereas mobile phase B involved the use of 0.1% formic acid in water. For the urine UPLC-HDMS (Waters Corp., Milford, MA) analysis, the optimal conditions for positive ion mode were as follows: capillary voltage of 2,500 V, desolvation temperature of 350 °C, sample cone voltage of 35 V, extraction cone voltage of 3 V, microchannel plate voltage of 1,600 V, collision energy of 4 eV, source temperature of 110 °C, cone gas flow of 50 l/h, and desolvation gas flow of 600 l/h. A lock mass of leucine enkephalin at a concentration of 200 pg/ml in acetonitrile (0.1% formic acid):H2O (0.1% formic acid) (50:50, v/v) for positive ion mode ([M+H]+ = 556.2771) was employed via a lock-spray interface. The data were collected in centroid mode, the lock-spray frequency was set at 5 s, and the lock-mass data were averaged over 10 scans. A "purge-wash-purge" cycle was employed when the auto-sampler was used, with 90% aqueous formic acid used for the wash solvent and 0.1% aqueous formic acid used as the purge solvent, which ensured minimal carry-over between injections. The mass spectrometry full-scan data were acquired in the positive ion mode from 100 to 1,000 Da with a 0.1-s scan time. For the plasma UPLC-HDMS analysis, the desolvation gas flow was 600 l/h, and the other parameters were the same as for the urine. The MS data were generated and recorded using MassLynx V4.1 (Waters Corp., Milford, MA), MarkerLynx Application Manager (Waters Corp., Milford, MA) was used for peak detection, and EZinfo 2.0 software (which is included in MarkerLynx Application Manager and can be applied directly) was used for the principal component analysis (PCA), partial least squares–discriminant analysis (PLS-DA), and orthogonal projection to latent structures (OPLS) analysis. "Unsupervised" data were analyzed using PCA, and "supervised" analysis was conducted using PLS-DA and OPLS. Putative markers were extracted from S-plots that were constructed following the analysis using OPLS, and markers were chosen based on their contribution to the variation and correlation within the data set. The processed data were then analyzed using EZinfo 2.0 software. The potential biomarkers were matched with the structure message of metabolites acquired from available biochemical databases, the Human Metabolome Database, and the Kyoto Encyclopedia of Genes and Genomes. Two-dimensional polyacrylamide gel electrophoresis tests were performed. Protein spots with more than a 3-fold change in density (paired Student's t test yielding p ≤ 0.05) with consistent increases or decreases were considered as differentially expressed and were selected for further identification via a MALDI-TOF-MS/MS analysis. Details regarding the immobilized pH gradient (IPG)-2-DE and image analysis, MALDI-TOF-MS/MS analysis, and Gene Ontology (GO) functional analysis can be found in the supplementary material. All experiments were performed at least in triplicate to ensure reproducibility. All statistical analyses were performed using Student's t test. Differences with a p value of 0.05 or less were considered significant. Assays were performed in triplicate, and the results are expressed as mean ± S.D. The efficacy of combination therapy with D, G, and R was compared with the efficacy of monotherapy with each of the three components individually in a rat model of HI. There was significant variation between the biochemical indicators for the control and model groups after CCl4 treatment (supplementary Table S1). This indicates that the HI model successfully replicated the disease. The model group had higher alanine transaminase, aspartate transaminase, alkaline phosphatase, r-glutamyl transferase, triglyceride, total cholesterol (TC), malondialdehyde, total bilirubin, direct bilirubin, and total protein values but had lower levels of superoxide dismutase and glutathione peroxidase than the control animals. Each treatment group was treated back to baseline levels (i.e. those of the control group), which demonstrates that these drugs had a therapeutic effect in the rat HI model. Our data show that the DGR combination statistically intensified the therapeutic efficacy relative to the control condition or monotherapy using D, G, or R (supplementary Table S1). Interestingly, the DGR protocol decreased the levels of alanine transaminase, aspartate transaminase, alkaline phosphatase, total bilirubin, direct bilirubin, glutamyl transferase, malondialdehyde, and total protein but increased the levels of glutathione peroxidase, triglyceride, and total cholesterol. These results indicate that DGR combination therapy exerted a synergistic effect and yielded better therapeutic effects than did the approaches that were based on the use of D, G, or R as a single agent. These data provide evidence of the synergy that is created with co-administration. Among the various monotherapies, D showed the most potent therapeutic efficacy. Our data also indicate that D is the principal component of the formula, whereas G and R serve as adjuvant ingredients. To confirm the protective effects of DGR in treating liver tissue damage, histological, TUNEL, and immunohistochemical analyses were performed on liver tissue that was obtained from HI rats and compared with tissue from control rats. Microscopic analyses of H&E- and TUNEL-stained liver sections showed that DGR significantly decreased hepatocyte necrosis, fibrotic area, and hepatocyte apoptosis levels, making them comparable to those in normal liver (Fig. 1A). The histopathological examination of liver sections that were stained with H&E revealed numerous apoptotic hepatocytes and the accumulation of massive necrosis with intralobular hemorrhage in the livers of HI rats (Fig. 1A). Further analysis revealed multiple and extensive areas of portal inflammation and hepatocellular necrosis in the HI group and a moderate increase in inflammatory cell infiltration. In the portal areas, Kupffer cells were detected within the sinusoids. The degree of necrosis was clearly lower in the CCL4-treated rats that received YCHT, the DGR combination, or bitherapies using various combinations of D, G, and R (Fig. 1A). Minimal hepatocellular necrosis and inflammatory cell infiltration and mild portal inflammation were observed in rats that were treated with either the DGR combination or YCHT as compared with animals that were treated with the control or with monotherapies or biotherapies