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 marker, 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 Responsible for 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-κB/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-β, and Wnt/β-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-κB signaling/anti-carcinogenesis (quercetin), NF-κB signaling/anti-inflammation (ergosterol peroxide), NF-κB signaling/apoptosis (tanshinone IIA), NF-κB signaling/hypoxia (paeonol), Nrf2-antioxidant response element (ARE) signaling/chemoprevention (myricetin), PI3K-Akt signaling/chemoprevention (sulforaphane), PPAR-γ signaling/adipogenesis (aculeatin), reactive oxygen species (ROS) signaling/apoptosis (β-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 whichpreadipocytes 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-α). Among the components found in the extract of Toddalia asiatica, aculeatin was found to promote the differentiation of mouse 3T3-L1 preadipocytes into adipocytes [132]. DMA revealed the involvement of PPAR-γ target genes in the process of activation by aculeatin, which is not a ligand of PPAR-γ, 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).
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 3,3′-diindolylmethane from cruciferous vegetables [145], grifolin from Albatrellus confluens [153], paeoniflorin from Paeonia lactiflora [161], and quercetin from various dietary plants [161], and further analyzed by DMA. 3,3′-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, and downregulates 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, IκBα, IL1R (Interleukin-1 receptor), TNFR1, and TRAILR [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 Sarcodon aspratus suppresses inflammatory response in macrophages by inhibiting TNF-α secretion and down-regulating the expression of interleukin1α/β (IL-1α/β) through pathways such as C/EBPβ, ERK, JNK, MAPK, and NF-κB [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]. β-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-κB/CCL2-mediated apoptosis in leukemia cells [180]. 2,4,3′,5′-Tetramethoxystilbene extracted from 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-κB 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, skin care, 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 skn-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, anti-diabetic, 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), obovatol (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 about 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.