PART 1 DNA Microarray-Based Screening And Characterization Of Traditional Chinese Medicine

Ryoichi Kiyama

Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba 305-8566, Ibaraki, Japan; kiyama.r@aist.go.jp; Tel.: +81-29-861-6189

Academic Editor: Christophe A. Marquette

Received: 16 December 2016; Accepted: 23 January 2017; Published: 30 January 2017

Abstract: The application of DNA microarray assay (DMA) has entered a new era owing to recent innovations in omics technologies. This review summarizes recent applications of DMA-based gene expression profiling by focusing on the screening and characterization of traditional Chinese medicine. First, herbs, mushrooms, and dietary plants analyzed by DMA along with their effective components and their biological/physiological effects are summarized and discussed by examining their comprehensive list and a list of representative effective chemicals. Second, the mechanisms of action of traditional Chinese medicine are summarized by examining the genes and pathways responsible for the action, the cell functions involved in the action, and the activities found by DMA (silent estrogens). Third, applications of DMA for traditional Chinese medicine are discussed by examining reported examples and new protocols for its use in quality control. Further innovations in the signaling pathway-based evaluation of beneficial effects and the assessment of potential risks of traditional Chinese medicine are expected, just as are observed in other closely related fields, such as the therapeutic, environmental, nutritional, and pharmacological fields.

Keywords: DNA microarray; traditional Chinese medicine; signaling pathway; estrogen; food chemicals

For more information please contact: Joanna.jia@wecistanche.com

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1. Introduction

Herbal medicine is an important part of the medical practices in traditional Chinese medicine (TCM) and consists of a variety of plant species [1,2]. While the effectiveness of herbal medicine has sometimes been considered doubtful [3,4], this could be due in part to the difficulty of controlling the quality of herbs and the amounts of their effective components. Thus, various methods have been used to confirm their efficacy and identify the effective components [5]. Modern technologies, such as high-performance liquid chromatography (HPLC) and quantitative reverse-transcription polymerase chain reaction (qRT-PCR), have been continually developed and utilized to replace conventional technologies for the comprehensive and cost-effective quality control of herbal medicine [6]. Although recent progress in the human and other genome projects and the development of a variety of omics technologies一such as genomics and transcriptomics一have contributed to the identification and utilization of effective components and quality control/authentication of medicinal plants [7-11], we are still searching for the best strategy for the maximum utilization of herbal medicine. This review focuses on DNA microarray-based gene expression profiling, a key technology in transcriptomics, and shows how researchers used this technology for the screening and characterization of TCM. In particular, cross-examination of the data on TCMs, and their constituent herbs, mushrooms, and dietary plants, has become quite important to evaluate the knowledge accumulated and to assess the advantages/disadvantages of TCM and its effective applications.

TCM includes practices such as acupuncture, moxibustion, Chinese herbal medicine, tui na, dietary therapy, tai chi, and qi gong, which is rooted in the ancient philosophy of Taoism and is more Microarrays 2017, 6,4; doi:10.3390/microarrays6010004 than 2500 years old [12]. The original TCM has been further developed and modified to adapt it to people of various nationalities and genetic backgrounds (giving rise to different types of health problems, dietary and nutritional customs/practices, and ways of thinking and beliefs about medicine), based on variations in the types of herbs and their ingredients in countries such as Japan and Korea, where Kampo and traditional Korean medicine (TKM), respectively, were developed. Therefore, herbal medicine includes a number of plant species and ways of processing them. Furthermore, herbal medicine is often used with other constituents such as mushrooms and dietary plants, and thus their extracts and effective chemicals are also discussed here.

DNA microarrays are a type of biotechnological device used to detect alterations in genomic DNA and mRNA and to monitor genes and their expressions associated with various functions; thus, they have been widely used in basic research and industrial research/development (reviewed by Kiyama & Zhu [13]). DNA microarray assay (DMA) has been used to screen and characterize useful materials among mixtures of chemicals and extracts of natural resources including plants. DMA has advantages and disadvantages compared with other technologies. It has been used as a diagnostic device, such as for the genotyping of drug-metabolizing genes and predicting the metastatic risk of breast cancer, which is attributable to its unique characteristic of providing sufficient complexity to differentiate the variations needed for diagnosis and the reliability needed to predict genotypes/gene expression profiles accurately.

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2. Herbal Medicine, Effective Chemicals, and Their Effects

2.1. Herbs, Mushrooms, and Dietary Plants Analyzed by DNA Microarray Assays

A number of herbs, mushrooms, and dietary plants, including those used as TCM in China, Korea (TKM), and Japan (Kampo), have been analyzed by DMA (Table 1).

Extracts from herbs have been analyzed by DMA, including the following: extracts of the whole, or parts such as flower and leaves, of alkanet root, American ginseng, barbed skullcap, beach vitex, beth root, black cohosh, cancer bush, Chinese figwort, boneset, dong Quai, field horsetail, greater celandine, kava, leigongteng, moutan, mum, orchid, pink lapacho, purple coneflower, salai, St. John's wort, turmeric, and wild yam. Meanwhile, the extracts of root, radix, or rhizome were made from Chinese goldthread, Chinese peony, cistanche, danshen, goldthread, Huang-qi, kudzu, and sheng-di-Huang; the extracts of seeds were from lotus, or the extracts enriched in essential oil were from lovage and turmeric.

The extracts from mushrooms were also analyzed after extraction of the whole or the fruiting body, such as that from buna-shimeji, caterpillar fungus, common mushroom, himematsutake, hiratake, lingzhi, lumpy bracket, maitake, and turkey tail; after extraction of the mycelium, such as that from caterpillar fungus, himematsutake, lingzhi, lumpy bracket, maitake, shiitake, and turkey tail; or after extraction of polysaccharides from lingzhi or triterpenes from hiratake, hoelen, and lingzhi.

Extracts were made from mixtures of TCM, such as the following: Danggui Buxue Tang, Guanxin No. 2 decoction, Huang-Lian-Jie-Du decoction, ISF-1, Kangxianling, PC-SPES, Pulsatillae decoction, Qingfei Xiaoyan Wan, Qinggan Huoxuefang, S/B remedy, Si-Wu-Tang, VI-28, Xiaoqinglong decoction, Xuefu Zhuyu decoction, and Zeng Sheng Ping (TCM); Chunggan, SH21B, and Youkongdan (TKM); or Boiogito, Bofutsushosan, Orengedokuto, Hochu-ekki-to, Inchin-ko-to, Juzen-taiho-to, Kososan, Saireito, Toki-shakuyaku-san, and Toki-to (Kampo).

Extracts were made from other dietary plants (including vegetables, fruit, and cereals), such as bilberry, bitter gourd, buckwheat, carob, Chinese mahogany, Chungkookjang (fermented soybean), ginger, gromwell, kothala himbutu, Marie Menard apple, and tarragon, or more common food materials such as apple, black raspberry, blueberry, broccoli, citrus, clove, garlic, ginkgo, grape, grapefruit, green tea, kiwi fruit, lychee, nectarine, oil palm, olive, peach, persimmon, pistachio, soybean, and sweetcorn.

2.2. Effective Chemicals Characterized by DNA Microarray Assays

After characterization of the extracts of herbs, mushrooms, or dietary plants, effective chemicals have been enriched or, in some cases, purified. They were then analyzed by DMA in order to identify the functions of interest or the signaling pathways involved (Table 2).

Abbreviations for assays are animal test (A), cell-proliferation assay (C), protein assay (such as Western blotting and immunoassay) (P), reporter-gene assay (R), and transcription assay (such as RT-PCR and DNA microarray assay) (T). ARE: antioxidant response element; ERK: extracellular signal-regulated kinase; HSP70: 70 kilodalton heat shock protein; IGF-1R: insulin-like growth factor 1 receptor; NF-kB: nuclear factor K-light-chain-enhancer of activated B cells; PGG: 1,2,3,4,6-Penta-O-galloyl-p-D-glucose; PI3K: phosphatidylinositol-3-kinase; PPAR: peroxisome proliferator-activated receptor; PUFA: polyunsaturated fatty acid; ROCK: Rho-associated protein kinase; ROS: reactive oxygen species; TCM: traditional Chinese medicine; TNFR1: tumor necrosis factor receptor 1

Pure chemicals analyzed by DMA are as follows: actein (a triterpene glycoside), aculeatin (a coumarin), baicalin (a flavone glycoside), berberine (an isoquinoline alkaloid), biochanin A, boswellic acid (a triterpene), brefeldin A (a lactone antibiotic), celastrol (a quinine methide triterpene), chelidonine (a tertiary alkaloid), curcumin (a diarylheptanoid), deoxycholic acid (a steroid acid), 3,3Z-diindolylmethane (an indole-3-carbinol derivative), emodin (an anthraquinone derivative), ergosterol peroxide (a steroid derivative), genistein (an isoflavone), ginsenosides Fl/Rbl/Re/Rgl/Rg3/Rhl (steroid glycosides/triterpene saponins), glycyrrhizin (a pentacyclic triterpenoid), grifolin (a farnesylphenol/sesquiterpenoid), (—)-hydroxycitric acid (a derivative of citric acid), p-hydroxyisovalerylshikonin (a naphthoquinone derivative), jasminoidin (a geniposide), ligustrazine (a tetrapyrazine), lycopene (a carotene), myricetin (a flavonol), obovatol (a biphenolic), paeoniflorin (a monoterpene glycoside), paeonol (an acetophenone derivative), 1,2,3,4,6-penta-O-galloyl-p-D-glucose (PGG), plumbagin (a naphthoquinone derivative), polysaccharide-K (Krestin) (a protein-bound polysaccharide), quercetin (a flavonol), resveratrol (a stilbenoid), saffron (a carotenoid), salvianolic acid B (a tanshinol/caffeic acid), sesamin/episesamin/sesamolin (lignans), siallyl trisulfide (an organosulfur compound), sparstolonin B (a xanthone/isocoumarin), sulforaphane (an isothiocyanate), tanshinone IIA (a phenanthrene-quinone derivative), 2,4,3’,5'-tetramethoxystilbene (a phenylpropanoid), and triptolide (a diterpenoid epoxide).

On the other hand, mixtures of chemicals analyzed by DMA are grape antioxidant dietary fiber (roughage/dietary fiber), oil palm phenolics, phytosterol mixtures (mixtures of steroid compounds), plant phospholipid/lipid conjugates, polysaccharides, and polyunsaturated fatty acids (PUFAs).

2.3. Biological/Physiological Effects Identified by DNA Microarray Assays

Biological/physiological effects and medicinal efficacy have been examined by DMA. To achieve this, a variety of assay systems have been used (Table 1), such as with different species (humans; animals, such as the chicken, dog, guinea pig, mouse, and rat; or microbes such as yeast and bacteria), tissues (brain, intestine, kidney, liver, lung, muscle, peripheral blood, or spleen) and cells (adenocarcinoma cells, alveolar epithelial cells, breast carcinoma cells, colon carcinoma cells, colorectal cancer cells, dendritic cells, dermal fibroblasts, endothelial cells, gingival fibroblasts, head and neck squamous cell carcinoma (HNSCC) cells, hepatoma cells, human umbilical vein endothelial cells (HUVECs), keratinocytes, lens tumor cells, leukemia cells, macrophages, neuroglial cells, oral squamous cell carcinoma cells, osteosarcoma cells, pancreatic cancer cells, peripheral blood mononuclear cells (PBMCs), preadipoctyes, prostate cancer cells, rat intestinal microvascular endothelial cells (RIMECs), retinal cells, or skin fibroblasts); the assays examining the statuses in vitro (using cultured normal or cancer cells, or yeast or bacterial cells, such as A549, BxPc-3, Caco-2, colo 205, DU145, ECV304, H9c2, HaCaT, HepG2, HCT-116, H4IIE, HL-60, Hs27, HT-29, J774.1,LT97, MCF-7, MDA-MB-231, MG-63, MonoMac6, NG108-15, PC-3, RAW 264.7, THP-1, 3T3-L1, UM1, UMSCC1, and YPK-1/4 cells) or in vivo (using tissues or cells from animals, or from healthy or diseased individuals); and DNA microarray platforms and assay protocols, such as those from ABioscience, Affymetrix, Agilent Technologies, Applied Biosystems, Clontech, GE Healthcare, Illumina, Mitsubishi Rayon, SuperArray, and Takara, or customized ones (see Section 3).

The biological/physiological effects analyzed are as follows: the functions/effects examined are angiogenesis modulation, anti-adipogenesis, anti-atherosclerosis/anti-arteriosclerosis, antibiotic effect, anti-carcinogenesis/anti-metastasis, antidepressant effect, anti-diabetic/anti-obesity effect, anti-endotoxin action, anti-fibrotic effect, anti-inflammation/anti-remodeling, anti-mitotic effect, apoptosis, cardioprotection, cell proliferation/differentiation, chemoprevention, cytotoxicity, DNA damage prevention, hepatotoxicity, immune response, inflammatory response, leukocyte function, neuromodulation/neuroprotection, skin aging prevention, stress response, and wound healing. The assays revealed the receptor-related signaling, such as by aryl hydrocarbon receptor (AhR), insulin receptor, peroxisome proliferator-activated receptor (PPAR), and Toll-like receptor (TLR), or hormone/growth-factor-related signaling, such as estrogen signaling, IFa/IFp signaling,

insulin-like growth factor 1 (IGF-1) signaling, and tumor necrosis factor-a (TNF-a)/tumor growth factor |31 (TGF-p1) signaling, or signal-mediator-related signalings, such as caspase-3, extracellular signal-regulated kinase (ERK), mitogen-activated protein kinase (MAPK), nuclear factor K-light-chain-enhancer of activated B cells (NF-kB), p53, and Wnt, or diseases/disorders, such as Alzheimer's disease, circulation disorders, gynecological diseases, lipid metabolism disorders, obstructive lung disease, and Parkinson's disease.

Meanwhile, the functions/effects identified by the analysis of pure chemicals (summarized in Table 2) are as follows: anti-carcinogenesis (actin, berberine, biochanin A, celastrol, chelidonine, genistein, ginsenoside Rg3, grape antioxidant dietary fiber, grifolin, lycopene, paeoniflorin, PGG, plant phospholipid/lipid conjugate, plumbagin, polysaccharide-K (Krestin), polysaccharides, PUFAs, quercetin, and salvianolic acid B); anti-atherosclerosis (brefeldin A and phytosterol mixture); anti-inflammation (ergosterol peroxide, glycyrrhizin, and phenol/paeoniflorin/albiflorin); immune response (celastrol, obovate, and triptolide);

anti-diabetic/anti-obesity response ((—)-hydroxycitric acid and ginsenoside Re); anti-infectious (berberine); apoptosis (curcumin, emodin, p-hydroxy isovaleryl shikonin, tanshinone IIA, and 2,4,3Z,5Z-tetra methoxy stilbene); anti-oxidative response (curcumin); adipogenesis/angiogenesis (aculeate and sparstolonin B); cardio-, neuro-, or vasoprotection (ligustrazine, oil palm phenolics, resveratrol, and saffron); cell proliferation (PUFAs); chemoprevention (boswellic acid, myricetin, and sulforaphane); estrogen signaling (3,3'-diindolylmethane, ginsenosides F1/Rb1/Rg1/Rh1, and glycyrrhizin); ischemic stroke (baicalin/deoxycholic acid/jasminoidin); hypoxia (phenol); life-span extension (curcumin and diallyl trisulfide); lipid metabolism (sesamin/episesamin/sesamolin); and Rho/ROCK (Rho-associated protein kinase) signaling (tanshinone IIA).

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3. Mechanisms of Action by Traditional Chinese Medicine

DNA microarrays for gene expression analysis can be categorized into two types, global and focused DNA microarrays, based on their application [13,182]. Global DNA microarrays contain thousands to hundreds of thousands of probes representing some or all of the cDNA, expressed sequence tags (ESTs), and various types of expression markers, such as those for the estimation of mRNA copy numbers within cells. Meanwhile, focused DNA microarrays contain a few dozen to thousands of probes designed for specific purposes, such as the study of tissue/cell-type specificity, functional specificity, and expression profiling. Focused DNA microarrays are sometimes more appropriate for the study of the mechanisms of action when the action is known, such as in the case of comparative risk assessment of chemicals and the prediction of cancer metastatic risks.

The genes used in customized or focused DNA microarrays for basic research and the development of applications of TCM are as follows: sets of human apoptosis genes [38], 96 cancer-related genes [24], 225 genes related to chemotaxis/antigen processing/cell signaling/ apoptosis/immune-related functions [28], mouse immunology-related genes [31], and 100 genes related to cardiac diseases, apoptosis, cell cycle/proliferation, cytokine/inflammatory, and antioxidation [43], for the study of herbs; genes related to growth factors/receptors, extracellular matrix components, proteases/inhibitors, and oncogenes/tumor suppressors [65], cell cycle-related genes [62,63], 172 human estrogen-responsive genes [53], and human pancreatic adenocarcinoma genes [64], for the study of mushrooms; sets of 3000 prostate-derived genes [78] and 1536 brain genes [72], for the study of TCM/TKM/Kampo; sets of 172 human estrogen-responsive genes [127], human drug metabolism-related genes [116], 209 inflammation/immune responsive genes [109], 2304 genes expressed in Caco-2 cells [115], 204 genes related to the immune response [121], and human apoptosis-related genes [130], for the study of dietary plants.

3.1. Genes and Pathways Res^onsiblefor to the Action

The signaling pathways analyzed by DMA are as follows (see Kiyama & Zhu [13]; Kiyama et al. [183]): MAPK (such as G protein-coupled receptor (GPCR)/MAPK, MAPK/c-Jun N-terminal kinase (JNK), and NF-kB/MAPK/ERK) and other (such as angiogenesis, ErbB/human epidermal growth factor receptor (HER), nuclear receptor, and ubiquitin/proteasome) signaling pathways, or apoptosis pathways (such as those for death receptor, infectious response, and p53-dependent apoptosis), autophagy pathways (such as those for phosphatidylinositol-3-kinase (PI3K)/Akt/mTOR signaling and starvation stress response), cell cycle/DNA damage pathways (such as G1/S checkpoint and G2/M DNA damage checkpoint signaling pathways), cellular metabolism pathways (such as AMP-activated protein kinase (AMPK) and insulin receptor signaling pathways), chromatin/epigenetic regulation pathways (such as those for DNA methylation, heterochromatin, and histone modification), cytoskeletal regulation and adhesion pathways (such as those related to actin, adherens junction, and microtubule dynamics), development and differentiation pathways (such as hedgehog, Notch, TGF-p, and Wnt/p-catenin signaling pathways), immunology and inflammation pathways (such as those for B-cell receptor signaling, cytokine receptor signaling, inflammatory response, rheumatoid arthritis, T-cell activation, and TLR-induced immune response), neuroscience pathways (such as Alzheimer's disease- and Parkinson's disease-related signaling pathways) and translational control pathways (such as eIF2, eIF4/P70S6K, and mTOR signaling pathways).

Since genes and pathways responsible for the action of TCM are related to various cell functions, it is almost impossible to understand the mechanisms of action just by studying the mixture of chemicals. There are cases in which effective chemicals (such as those shown in Table 2) were analyzed in order to understand specific mechanisms, such as Bax signaling/apoptosis (2,4,3‘,5'-tetramethoxystilbene), ERK signaling/anti-atherosclerosis (brefeldin A), ERK signaling/anti-carcinogenesis (grifolin), estrogen signaling (ginsenosides F1/Rb1/Rg1/Rh1 and glycyrrhizin), estrogen signaling/carcinogenesis (3,3'-diindolylmethane), HSP70 (a 70 kilodalton heat shock protein) signaling/anti-carcinogenesis (paeoniflorin), NF-kB signaling/anti-carcinogenesis (quercetin), NF-kB signaling/anti-inflammation (ergosterol peroxide), NF-kB signaling/apoptosis (tanshinone IIA), NF-kB signaling/hypoxia (paeonol), Nrf2-antioxidant response element (ARE) signaling/chemoprevention (myricetin), PI3K-Akt signaling/chemoprevention (sulforaphane), PPAR-y signaling/adipogenesis (aculeatin), reactive oxygen species (ROS) signaling/apoptosis (p-hydroxyisovalerylshikonin), Rho/ROCK signaling/cell migration (tanshinone IIA), skn-1 signaling/life-span extension (diallyl trisulfide), and tumor necrosis factor receptor 1 (TNFR1)-IGF-1R signaling/apoptosis (emodin). These signaling pathways are summarized in Figure 1.

3.2. Cell Functions Involved in the Action

The major cell functions analyzed by DMA for TCM include adipogenesis, anti-atherosclerosis, anti-carcinogenesis, anti-inflammation, apoptosis, carcinogenesis, chemoprevention, hypoxia, and life-span extension (Table 2; Figure 1).

Adipogenesis is a cellular differentiation process in which preadipocytes are transformed into differentiated adipocyte cells and involves features such as morphological change, growth arrest, lipogenic gene expression, and the production of hormones and growth factors (such as leptin and TNF-a). Among the components found in the extract of Toddalia Asiatica, aculeate was found to promote the differentiation of mouse 3T3-L1 preadipocytes into adipocytes [132]. DMA revealed the involvement of PPAR-y target genes in the process of activation by aculeatin, which is not a ligand of PPAR-y, suggesting the presence of additional signaling mechanisms.

Atherosclerosis is a chronic inflammatory response of white blood cells in arterial blood vessels, which is promoted by low-density lipoproteins (LDLs), carriers of cholesterol, and triglycerides, and results in the formation of atherosclerotic plaques that are rich in macrophages and foam cells. Estrogenic activity was detected by DMA-based gene expression profiling in the extract of Agaricus blazei, which was attributable to brefeldin A [138]. The extract has no estrogen receptor-dependent cell proliferation activity while showing activation of estrogen signaling (such as activation of ERK, Akt, and P70S6K) and beneficial effects for patients with high levels of oxidized LDLs (see Section 3.3).

Figure 1. Summary of actions and their mechanisms by the chemicals related to traditional Chinese medicine. The m can isms of action by the chemicals originally identified or isolated from medicinal herbs, mushrooms, and dietary plants (aculeatin, brefeldin A, ergosterol peroxide, grifolin, p-hydroxy isovaleryl shikonin, paeonol, quercetin, and tanshinone IIA) within the cytosol (blue area) or the nucleus (yellow area) are summarized. APP: Amyloid precursor protein; CCL2: chemokine (C-C motif) ligand 2; ERK: extracellular-signal-regulated kinase; p-HIV S: p-hydroxy isovaleryl shikonin; MAPK: mitogen-activated protein kinase; PPAR-丫： peroxisome proliferator-activated receptor y; PXR: pregnane X receptor; Rb: retinoblastoma protein; TNFR: tumor necrosis factor receptor; and TRAP1: tumor necrosis factor receptor-associated protein 1.

Carcinogenesis, alternatively referred to as oncogenesis or tumorigenesis, is a process by which normal cells are transformed into cancer cells characterized by uncontrolled cell division; it involves a progression of changes at the cellular, genetic, and epigenetic levels. Several chemicals exhibiting anti-carcinogenic effects were isolated or identified from natural products, such as 3,3Z-diindolylmethane from cruciferous vegetables [145], grifolin from Albatrellus confluence [153], paeoniflorin from Paeonia lactiflora [161], and quercetin from various dietary plants [161], and further analyzed by DMA. 3,3Z-Diindolylmethane is estrogenic and shows gene expression profiles favoring tumor promotion [145]. Grifolin acts negatively against the cell cycle and cell growth through inhibiting ERK and Rb pathways, downregulating the expression of cyclin D1, cyclin E, and CDK4 (a gene for a cyclin-dependent kinase), and upregulates the expression of CKI (a CDK inhibitor gene) [153]. Paeoniflorin enhances the expression of HSP70, which helps to protect cells from stress, and modulates the expression of CDC2, FOSL1, and EGR1, regulators of cell growth and proliferation [161]. Quercetin, on the other hand, induces p53-independent apoptosis by enhancing the expression of death-receptor or TNFR signaling genes, such as the genes for caspase-10, DFF45, FAS, IKBa, IL1R (Interleukin-1 receptor), TNFR1, and TRAILER [171].

Inflammation is a protective response to cell injury and involves the local vascular system, the immune system, and various cells within the injured tissue. Ergosterol peroxide produced by the Sarcodon apparatus suppresses inflammatory response in macrophages by inhibiting TNF-a secretion and down-regulating the expression of interleukin1 a/ p (IL-1 a/p) through pathways such as C/EBPp, ERK, JNK, MAPK, and NF-kB [147].

Apoptosis is the process of programmed cell death that may occur in multicellular organisms in response to various stresses, such as heat, hypoxia, increased intracellular calcium concentration, nutrient deprivation, receptor-ligand binding, radiation, and viral infection. Several chemicals are related to the promotion of apoptosis and thus have been used as effective components in herbal medicine. Emodin extracted from the rhizomes of Rheum palmatum showed testicular toxicity, including the induction of apoptosis, most likely through pathways such as IGF-1, TGF/Wnt, and TNFR1 signaling [146]. p-Hydroxyisovalerylshikonin extracted from Lithospermum erythrorhizon is an inhibitor of protein-tyrosine kinases and induces apoptosis by suppressing TRAP1, a TNF-associated protein and a member of the HSPs, as well as the production of ROS [155]. Tanshinone IIA found in the root of Salvia miltiorrhiza induces peroxisome proliferator-activated receptor (PXR)/NF-KB/CCL2-mediated apoptosis in leukemia cells [180]. 2,4,3’,5'-Tetramethoxystilbene extracted from the fruit, berries, and grapes is a derivative of resveratrol and a strong inducer of apoptosis by increasing the expression of tubulin, stress response, and pro-apoptotic genes [178].

Chemoprevention refers to the administration of a medication, such as drugs and vitamins, for the purpose of preventing disease or infection, and various chemicals have been developed especially for cancer chemoprevention. Myricetin [158] and sulforaphane [177] isolated from dietary plants show chemopreventive activity against cancer through activating Nrf2-mediated antioxidant response or PI3K/Akt signaling pathways, respectively.

Hypoxia is a condition in which a cell is deprived of adequate oxygen supply and has been shown to stimulate various biological and physiological responses. Paeonol isolated from Paeonia suffruticosa induces the expression of hypoxia-inducible genes, including hypoxia-inducible factor 1 (HIF-1)-target genes, through suppressing the NF-kB signaling pathway and inhibiting amyloid precursor protein (APP) activity [162].

Life extension has been studied in terms of slowing down or reversing the processes of aging in order to extend both the maximum and the average lifespan, and the effects of anti-aging products, nutrition, physical fitness, skincare, hormone replacements, vitamins, supplements, and herbs have been examined. Diallyl trisulfide isolated from garlic increases the longevity of nematodes through activation of the pro-longevity transcription factor gene skin-1 and the products of its target genes [144].

Conditions such as chronic (arthritis, asthma, cancer, diabetes, and viral diseases) and neurodegenerative (Parkinson's and Alzheimer's diseases) diseases have been treated with TCM [1],

among which some were investigated by DMA and explored by animal tests and/or clinical studies to eventually achieve clinical applications. Other than the cell functions discussed above, the diseases with extensive impacts were also investigated. For example, antidepressant, antidiabetic, anti-obesity, neuromodulation, and neuroprotection effects, and the treatments of neurological, Parkinson's, and Alzheimer's diseases associated with TMC and/or constituent herbs/mushrooms/dietary plants were studied by means of DMA (Table 1), or their effective components, such as ginsenosides (for diabetes), (—)-hydroxycitric acid (for obesity), obovate (for neuroinflammation), and salvianolic acid B (for neuroprotection), were studied by means of DMA (Table 2).

3.3. Activities Found by DNA Microarray Assays (Silent Estrogens)

Activities found by DMA are often detected as cell signals in specific pathways, such as angiogenesis, ErbB/HER, MAPK, nuclear receptor, and ubiquitin/proteasome signaling pathways, and/or in cell functions, such as apoptosis, autophagy, cell cycle/DNA damage/cytoskeletal formation, cellular metabolism, chromatin/epigenesis regulation, development/differentiation, immunology/inflammation response, neurological diseases, and translational control [183]. While most of these cell signaling pathways and cell functions can be detected by other technologies, there might be some activities that can be detected exclusively by DMA. One such activity is by a group of estrogens, silent estrogens, which show estrogenic gene expression profiles without showing positive effects on cell proliferation [13].

Estrogen is a female hormone that is responsible for various biological and physiological activities, including receptor-mediated stimulation of the proliferation of cells in tissues such as the breast and ovary. Several chemicals and mixtures of chemicals, such as brefeldin A [138], licorice extracts [150], and oil degradation products [184], were found to show gene expression profiles similar to that for estrogen, although they did not stimulate the proliferation of estrogen receptor-positive breast cancer MCF-7 cells. Although the signaling pathway for cell proliferation could theoretically be separated from those for other cell functions, this separation has not been possible because most of the cells examined for estrogenic activity contain estrogen receptors and the technologies used were not suited to such a purpose. Recent findings of more complicated signaling pathways/networks, such as autocrine/paracrine/homeostatic networks and crosstalk/bypassing of cell signals, include pathways not necessarily involving cell proliferation or the cells containing estrogen receptors [185,186]. Thus, estrogenic activity can be detected even for silent estrogens because DMA can separate various signaling pathways, and the similarity of chemicals can be analyzed at the levels of gene expression and cell signaling.

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