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JKM > Volume 35(4); 2014 > Article
Hwang, Shin, and Moon: Classification of the Efficacy of Herbal Medicine Alterations in Neuronal Hypoxia Models through Analysis of Gene Expression

Abstract

Objectives:

cDNA microarray is an effective method to snapshot gene expression. Functional clustering of gene expressions can identify herbal medicine mechanisms. Much microarray data is available for various herbal medicines. This study compares regulated genes with herbal medicines to evaluate the nature of the drugs.

Methods:

Published microarray data were collected. Total RNAs were prepared from dissociated hippocampal dissociate cultures which were given hypoxic shock in the presence of each herbal medicine. Up- or downregulated genes higher than Global M value 0.5 were selected, clustered in functional groups, and compared with various herbal treatments.

Results:

1. Akt2 was upregulated by Acorus gramineus SOLAND, Arisaema amurense var. serratum NAKAI and Coptis chinensis FRANCH, and they belong to Araceae herb. 2. Nf-κb1, Cd5, Gnγ7 and Sgne1 were upregulated by Arisaema amurense var. serratum NAKAI, Coptis chinensis FRANCH and Rheum coreanum NAKAI. 3. Woohwangcheongsim-won, Sohaphyang-won and Scutellaria baicalensis GEORGI downregulated Scp2 and upregulated Tsc2. Woohwangcheongsim-won and Sohaphyang-won upregulated Hba1 and downregulated Myf6. 4. Sohaphyang-won and Scutellaria baicalensis GEORGI downregulated Slc12a1. 5. Woohwangcheongsim-won and Arisaema amurense var. serratum NAKAI upregulated Rarα, Woohwangcheongsim-won and Coptis chinensis FRANCH downregulated Rab5a and Pdgfrα, and Woohwangcheongsim-won and Rheum coreanum NAKAI upregulated Plcγ1 and downregulated Pla2g1b and Slc10a1.

Conclusions:

By clustering microarray, genes are commonly identified to be either up- or downregulated. These results will provide new information to understand the efficacy of herbal medicines and to classify them at the molecular level.

Introduction

Apoptosis, due to reactive oxygen species (ROS), serves an important role in degenerative disorders of the nervous system. Because apoptosis results in numerous semi-permanent side effects even after treatment, there has been a lot of research on a treatment method that can effectively decrease apoptosis1,2).
Research on discovering biological, pathological mechanisms through genetic regulation has been increasing since the completion of the genetic map of several species, including that of human beings3, 4). Microarray, a technique to find the function and structure of genes, makes it possible to determine the general expression of an entire gene within a cell. It is a major tool in function genomics research5,6).
Previous experimental research reports that some herbal medicines or extracts delay apoptosis due to hypoxia, and the medicine’s gene expression has been studied through the microarray. By analyzing the function of genes where alteration in expression occurred and the neuron’s genetic expression alteration by each medicine and treatment, it is possible to infer the medicine’s mechanism714). However, although each herbal medicine may have functioned by different mechanisms, there has been no report about the instance of mutual comparison and interpretation by merging Korean medicine theory.
The author analyzed experimental studies that observed expressed genes from herbal medicines or extracts in neurons where apoptosis has occurred under hypoxia and interpreted the influence on gene function related to the medicine’s neuron protection and anti-oxidation from the point of view of Korean medicine.

Research Target and Method

1. Research Target

Clustering is an analyzing method that classifies the genes that have been identified with expression alteration through microarray by similar groups. Hierarchical clustering is a method that groups genes with similar characteristics and analyzes them. It is useful in understanding a herbal medicine and treatment’s properties based on the expressed genes16).
This study limited its target to experimental research that used clustering to investigate altered expression of genes through microarray after inducing apoptosis in neurons under hypoxia and treating with herbal medicines or extracts.
As a result, with similar analyzing methods, we detected results from observing gene alteration. We compared and analyzed based on the research findings that use Acorus gramineus SOLAND7), Arisaema amurense var. serratum NAKAI8), Pinellia ternata BREIT.9), Scutellaria baicalensis GEORGI10), Coptis chinensis FRANCH11), Rheum coreanum NAKAI12), Woohwangcheongsim-won13), and Sohaphyang-won14), that are frequently used in cerebrovascular disease.

2. Research Method

Among the genes expressed through microarray, we compared expressed genes with global M figure higher than +0.5 and lower than −0.5. We compared Acorus gramineus SOLAND, Arisaema amurense var. serratum NAKAI, and Pinellia ternata BREIT., which belong to the araceae herb and has function of dispel Dam-eum (phlegm-retained fluid); and compared Pinellia ternata BREIT., Coptis chinensis FRANCH, and Rheum coreanum NAKAI that are expressed in similar class; and observed whether there is an overlap of expressed genes between different medicines. Then, we compared the gene expression of Woohwangcheongsim-won and Sohaphyang-won that are used for acute stroke and observed an overlap of expressed genes with other medicines.

Results

1. Comparison of microarray methods

Table 1 shows the microarray method and the mRNA separation process from neurons induced of apoptosis due to ischemic damage from each study.

2. Clustering of herbal medicine’s genes

Table 2 shows the classification of each herbal medicine based on reported research findings. We excluded findings of global M figures within ±0.5because there is a difference in the global M figure which is the standard for each study.

3. Analysis of gene alternation between medicines

We investigated genes that demonstrate similar alteration between medicines.
  1. Comparison of global M figure of Acorus gramineus SOLAND and Arisaema amurense var. serratum NAKAI

    Thymoma viral proto-oncogene 2 (Akt2) altered in the same direction in the research on Acorus gramineus SOLAND and Arisaema amurense var. serratum NAKAI, which belong to the araceae herb. Table 3 shows the result of the analysis.
  2. Comparison of global M figure of Acorus gramineus SOLAND and Coptis chinensis FRANCH

    Akt2 altered in the same direction in the research on Acorus gramineus SOLAND and Coptis chinensis FRANCH. Table 4 shows the result of the analysis.
  3. Comparison of global M figure of Coptis chinensis FRANCH and Pinellia ternata BREIT.

    CD3 molecule, delta (Cd3δ) altered in the same direction in the research on Coptis chinensis FRANCH and Pinellia ternata BREIT. Table 5 shows the result of the analysis.
  4. Comparison of global M figure of Coptis chinensis FRANCH and Arisaema amurense var. serratum NAKAI

    A total of 25 types of genes were altered in the identical direction. Table 6 shows the result of the analysis. 2 types of genes related to apoptosis, 13 types of genes related to growth & maintenance, 2 types of genes related to cell cycle, 4 types of genes related to response to stress, 11 types of genes related to signal transduction, 3 types of genes related to transcription, 18 types of genes related to physiological process, and 3 types of genes related to immune response.
  5. Comparison of global M figure of Coptis chinensis FRANCH and Rheum coreanum NAKAI

    A total of 29 types of genes were altered in the identical direction. Table 7 shows the result of the analysis. There were 2 types of genes related to apoptosis, 3 types of genes related to cell cycle, 10 types of genes related to response to stress, 11 types of genes related to signal transduction, 5 types of genes related to transcription, 17 types of genes related to physiological process, and 7 types of genes related to immune response.
  6. Comparison of global M figure of Arisaema amurense var. serratum NAKAI and Rheum coreanum NAKAI

    A total of 5 types of genes were altered in the identical direction. Table 8 shows the result of the analysis. There were 1 type of gene related to apoptosis, 1 type of gene related to growth & maintenance, 2 types of genes related to response to stress, 3 types of genes related to signal transduction, 2 types of genes related to transcription, 2 types of genes related to physiological process, and 2 types of genes related to immune response.
  7. Comparison of global M figure of Arisaema amurense var. serratum NAKAI, Coptis chinensis FRANCH and Rheum coreanum NAKAI

    A total of 4 types of genes were altered in the identical direction in Arisaema amurense var. serratum NAKAI, Coptis chinensis FRANCH, and Rheum coreanum NAKAI. Table 9 shows the result of the analysis.

4. Comparison between Woohwangcheongsim-won and Sohaphyang-won and each medicine

We compared the genes that are identical from the research findings on Woohwangcheongsim-won and Sohaphyang-won which are used for acute stroke and compared the genes that were up- and down-regulated in the same direction from the research findings with other medicines.
  1. A total of 4 types of genes were identical. Table 10 shows the result of the analysis. There were 3 types of genes related to growth & maintenance, 1 type of gene related to signal transduction, and 1 type of gene related to transcription.

  2. Comparison of global M figure of Woohwangcheongsim-won and Scutellaria baicalensis GEORGI

    Tuberous sclerosis 2 (Tsc2) and sterol carrier protein 2 (Scp2) were altered in the identical direction in the research on Woohwangcheongsim-won and Scutellaria baicalensis GEORGI. Table 11 shows the analysis result.
  3. Comparison of global M figure of Sohaphyang-won and Scutellaria baicalensis GEORGI

    A total of 3 types of genes were identical. Table 12 shows the result of the analysis. There were 3 types of genes related to growth & maintenance and 1 type of gene related to signal transduction. Fig. 4 shows the genes that are upregulated in the three studies on Woohwangcheongsim-won, Sohaphyang-won and Scutellaria baicalensis GEORGI.
  4. Comparison of global M figure of Woohwangcheongsim-won and Arisaema amurense var. serratum NAKAI

    Retinoic acid receptor, alpha (Rarα) was altered in the identical direction in the research on Woohwangcheongsim-won and Arisaema amurense var. serratum NAKAI. Table 13 shows the analysis result.
  5. Comparison of global M figure of Woohwangcheongsim-won and Coptis chinensis FRANCH

    Ras-related protein 4a (Rab4a) and platelet derived growth factor receptor, alpha polypeptide (Pdgfrα) were altered in the identical direction in the research on Woohwangcheongsim-won and Coptis chinensis FRANCH. Table 14 shows the analysis result.
  6. Comparison of global M figure of Woohwangcheongsim-won and Rheum coreanum NAKAI

    A total of 3 types of genes were identical. Table 15 shows the result of the analysis. There were 2 types of genes related to growth & maintenance, 1 type of gene related to response to stress and 1 type of gene related to signal transduction.

Discussion

There has been a lot of research on preventing apoptosis due to damage because regeneration is difficult once the neuron has been damaged. When a neuron is damaged due to ischemia/hypoxia, along with necrosis in the center, apoptosis in the penumbra occurs17,18). Although apoptosis in the center is unpreventable because it occurs quickly, the penumbra goes through a delayed neuronal death, allowing time to minimize apoptosis19). Delayed neuronal death occurs through diverse mechanisms, but the main process is the production of ROS due to excitotoxicity. In other words, under hypoxia, there is an over-release of glutamate which over-activates the α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor and N-methyl-D-aspartate (NMDA) receptor that pass Ca2+, thereby activating the apoptotic protein20,21). When the calcium ion is overly entered into the mitochondria in the neuron, ROS is produced. Because of the overly produced ROS, the permeability of the mitochondria is altered, and the mitochondrial membrane potential (MMP) is lost. As a result, energy (ATP) production is hindered, and apoptosis occurs2,21,22). Meanwhile, the neuron, under hypoxia, operates a self-defense system17)- it boosts angiogenesis, activates apoptosis defense protein and cell survival protein, etc. - in order to protect the neuron23).
Recently, there has been a lot of research on the protective effects on the neuron of herbal medicines or extracts. Most look for pathological mechanisms and gene expression processes under the assumption that a particular protein expression alteration induces a pathological mechanism. However, this method does not consider the mutual interaction between proteins but focuses on the fragmentary role, which makes it difficult to know the overall connection because limited mechanisms can be understood even with the result.
cDNA microarray is a method that looks at large scale gene expression alteration simultaneously by using a DNA chip to integrate the gene in high density. It is a high-tech gene analysis method that allows understanding of diverse alterations in a neuron during pathological process at the same time and observation of the overall alteration expression6. Also, because cDNA microarray estimates the amount of expression of the gene by the quantitative alteration strength between the experimental group and the control group, it finds the expression direction and the strength of gene expression in a short period of time. Therefore, recently it has been used in diverse bio-science research24). Meanwhile, there are many methods to analyze the expression types of genes found from the cDNA microarray results, but to discover the mutual relationship of genes, the clustering method which groups by class according to their functions is often used. In other words, the clustering method has an advantage of knowing the medicine’s point of action from a wider point of view because it observes the overall directionality rather than the individual gene expression25).
There recently has been a lot of research that tries to find the characteristics of herbal medicines or extracts through cDNA microarray. This study compared and analyzed research reports limited to those that observed gene expression alteration through the clustering method in the neuronal hypoxia model among research on the gene expression of herbal medicines using the cDNA microarray method. Target herbal medicines were Acorus gramineus SOLAND7), Arisaema amurense var. serratum NAKAI8), Pinellia ternata BREIT.9),, Scutellaria baicalensis GEORGI10), Coptis chinensis FRANCH11), Rheum coreanum NAKAI12), Woohwangcheongsim-won13), and Sohaphyang-won14). We interpreted their significance by discovering the genes that were upregulated in common by these medicines through the clustering analysis method of cDNA microarray.
From previous research, it was difficult to find a common ROS-producing cell for Acorus gramineus SOLAND, Arisaema amurense var. serratum NAKAI, Pinellia ternata BREIT., Scutellaria baicalensis GEORGI, Coptis chinensis FRANCH, Woohwangcheongsim-won, and Sohaphyang-won because of excellent ROS removal ability, and they were helpful in maintaining MMP. In the case of Acorus gramineus SOLAND, Arisaema amurense var. serratum NAKAI, Scutellaria baicalensis GEORGI, Woohwangcheongsim-won, and Sohaphyang-won, genes that remove ROS were expressed a lot. For example, in Acorus gramineus SOLAND, Arisaema amurense var. serratum NAKAI,, Rar α was upregulated, which is a gene that inhibits apoptosis7,8). In research on Woohwangcheongsim-won, Sohaphyang-won, Scutellaria baicalensis GEORGI, and Arisaema amurense var. serratum NAKAI, catalase, which is an antioxidant enzyme, was upregulated8,10,13,14). On the other hand, in Pinellia ternata BREIT., Coptis chinensis FRANCH, and Rheum coreanum NAKAI, genes that remove ROS were regulated relatively less than with Acorus gramineus SOLAND, Arisaema amurense var. serratum NAKAI, Scutellaria baicalensis GEORGI, Woohwangcheongsim-won, and Sohaphyang-won; protein tyrosin phosphatase, non-receptor type 11 (Ptpn11) and Cd3 δ which are cell-supporting genes in their roots, and C-reactive protein (Crp) and platelet-activating factor receptor (Ptafr) which are genes related to the immune system, were upregulated a lot9,11,12). In research on Pinellia ternata BREIT., tubulin, beta 5 (Tubβ5), transforming growth factor alpha (Tgfα), Ptpn11, neuroblastoma ras oncogene (Nras), and platelet-derived growth factor alpha polypeptide (Pdgfα) which are related to cell growth and differentiation were upregulated9. In research on Coptis chinensis FRANCH, Rheum coreanum NAKAI, Defb3 which is a response to stress gene was upregulated and nuclear factor-kappaB (Nf-κb) which is related to neuron survival was upregulated11,12).
In the study on Acorus gramineus SOLAND, Arisaema amurense var. serratum NAKAI, and Pinellia ternata BREIT., which are all medicines that belong to the araceae herb, through the cDNA microarray analysis research of Acorus gramineus SOLAND and Arisaema amurense var. serratum NAKAI, we observed that Akt2, which is known to be the most important gene in cell survival, was commonly upregulated. There were no observed genes that were up- or downregulated commonly when Acorus gramineus SOLAND, Pinellia ternata BREIT., Pinellia ternata BREIT., and Arisaema amurense var. serratum NAKAI were combined. Also, in Acorus gramineus SOLAND and Coptis chinensis FRANCH, only Akt2 was commonly upregulated. In sum, Acorus gramineus SOLAND, Arisaema amurense var. serratum NAKAI, and Coptis chinensis FRANCH shared an upregulation of Akt2 [Global M = 0.9 (Acorus gramineus SOLAND), 0.6 (Coptis chinensis FRANCH), 0.7 (Arisaema amurense var. serratum NAKAI)].
Akt2 is an important gene in cell survival; it inhibits apoptosis in case of myogenic differentiation of serum removal state, and is known to inhibit apoptosis in divided cells26). Therefore, from the fact that Akt2 was commonly upregulated in Acorus gramineus SOLAND, Arisaema amurense var. serratum NAKAI, and Coptis chinensis FRANCH, we can infer that these medicines are related to effects of inhibiting apoptosis. Acorus gramineus SOLAND, Arisaema amurense var. serratum NAKAI, and Coptis chinensis FRANCH all have controlled humidity function27), and although it is difficult to conclude the function mechanism by Akt2 alone, it is a gene that should be considered for future research.
In Arisaema amurense var. serratum NAKAI, Coptis chinensis FRANCH, and Rheum coreanum NAKAI, four types of genes including Nfκb1 [Global M = 0.6 (Arisaema amurense var. serratum NAKAI), 2.0 (Coptis chinensis FRANCH), 1.2 (Rheum coreanum NAKAI)], Cd5 [Global M – 1.0 (Arisaema amurense var. serratum NAKAI), 1.7 (Coptis chinensis FRANCH), 0.5 (Rheum coreanum NAKAI)], Gng7 [Global M = 0.8 (Arisaema amurense var. serratum NAKAI), 1.6 (Coptis chinensis FRANCH), 0.6 (Rheum coreanum NAKAI)], and Sgne1 [Global M = 0.7 (Arisaema amurense var. serratum NAKAI), 0.7 (Coptis chinensis FRANCH), 0.7 (Rheum coreanum NAKAI)] were regulated in common. For Nf-κb, p50 and p65 that are subunits are activated due to stimulus of glutamic acid in the neuron which accelerates apoptosis or p50, p65, c-Rel subunits are activated due to stimulus of interleukin (IL)-1 β which protects from apoptosis28). If the density of inhibitor-κBα (IκBα) is decreased, IL-1βstimulates Nf-κ b and protects the neuron29). Caspase 3, caspase 2l, Bcl2-associated X protein (Bax), which are apoptosis boosting genes, or IL-1β, which is an apoptosis inhibiting gene, were not expressed in research of Arisaema amurense var. serratum NAKAI, Coptis chinensis FRANCH, and Rheum coreanum NAKAI. Caspase 2 (Casp2, Global M = −0.6) which is an apoptosis related gene was expressed in research of Rheum coreanum NAKAI. Unlike the caspase family, which is the general apoptosis boosting gene, because casp2 exhibits diverse functions such as acceleration or inhibition of apoptosis30), even with the result of the upregulation of Nf-κb, it is difficult to infer if the upregulation of Nf-κb boosts or inhibits apoptosis. Guanine nucleotide binding protein, gamma 7 (Gnγ7) which is a gene that produces guanine nucleotide binding protein (G protein) is known to exhibit downregulation in esophageal cancer or pancreatic cancer31). In this study, Gnγ7 was commonly upregulated in Arisaema amurense var. serratum NAKAI, Coptis chinensis FRANCH, and Rheum coreanum NAKAI. These medicines do not share an identical function from the point of view of Korean Medicine. It is difficult to find a consistent function because we can only infer similar functions such as controlled humidity function (Arisaema amurense var. serratum NAKAI, Coptis chinensis FRANCH), dispelled Dam-eum (phlegm-retained fluid) (Arisaema amurense var. serratum NAKAI, Rheum coreanum NAKAI), and dispelled Dam-eum (phlegm-retained fluid) (Rheum coreanum NAKAI). However, it is worth considering for future research as multiple genes were commonly expressed.27)
For Coptis chinensis FRANCH, 25 types and 29 types of commonly expressed genes were each observed by the mutual comparison between Coptis chinensis FRANCH, Arisaema amurense var. serratum NAKAI, Coptis chinensis FRANCH, and Rheum coreanum NAKAI. From the result of Coptis chinensis FRANCH and Pinellia ternata BREIT., only Cd3δ was expressed commonly and only Woohwangcheongsim-won and Rab-4a, Pdgfrα genes were expressed commonly. In reference to Coptis chinensis FRANCH, there are combined studies with Scutellaria baicalensis GEORGI, because it is expected to function similarly due to its clear heat effect. There was no gene that was expressed commonly between Scutellaria baicalensis GEORGI and Coptis chinensis FRANCH in this study. This result lends the possibility that Coptis chinensis FRANCH and Scutellaria baicalensis GEORGI may function in a different mechanism rather than the clear heat function in the same mechanism. Future research on this aspect is necessary.
From the observation results of common gene expression of Sohaphyang-won and Scutellaria baicalensis GEORGI, solute carrier family 12, member 1(Slc12a1) [Global M = −0.5 (Sohaphyang-won), −1.1 (Scutellaria baicalensis GEORGI)], Scp2 were expressed commonly. Also, from the observation result of Woohwangcheongsim-won and Scutellaria baicalensis GEORGI’s gene expression, Tsc2 and Scp2 were found to be expressed commonly. In all Scutellaria baicalensis GEORGI, Woohwangcheongsim-won and Sohaphyang-won, Tsc2 [Global M = 0.7 (Woohwangcheongsim-won), 0.5 (Sohaphyang-won), 0.6 (Scutellaria baicalensis GEORGI)] and Scp2 [Global M = −1.3 (Woohwangcheongsim-won), −0.8 (Sohaphyang-won), −0.7 (Scutellaria baicalensis GEORGI)]’s expression alteration was observed.
Tsc2 is a tumor suppressor gene, and it stimulates cell growth when phosphorylated by Akt32). When there is low energy, AMP-activated protein kinase (AMPK) phosphorylates Tsc2 and protects the cell from apoptosis33). In this study, in all Woohwangcheongsim-won, Sohaphyang-won and Scutellaria baicalensis GEORGI, Tsc2 was upregulated, and this is identical to the apoptosis inhibiting function. Meanwhile, Scp2 is a DNA combined protein that supports the chromosome structure in the former part of meiosis. It is involved in the transport and metabolism within the cholesterol cell and plays an important role in the production of macrophage foam cell which causes atherosderosis34). Therefore, the downregulation of Scp2 in Woohwangcheongsim-won, Sohaphyang-won, and Scutellaria baicalensis GEORGI research could be interpreted as a reduction of the lipid moving into the neuron35). Through this study, genes that are commonly expressed were identical in Scutellaria baicalensis GEORGI which is an ingredient medicine of Woohwangcheongsim-won and Woohwangcheongsim-won and Sohaphyang-won. Future research should study the gene that is expressed in the identical direction as the function research of the ingredient medicine of Woohwangcheongsim-won or Sohaphyang-won.
4 types of genes – hemoglobin, alpha 1 (Hbα1) [Global M = 0.9 (Woohwangcheongsim-won), 1.7 (Sohaphyang-won)], Tsc2, Scp2, and myogenic factor 6 (Myf6) [Global M = −0.6 (Woohwangcheongsim-won), −0.5 (Sohaphyang-won)] – were found to be commonly expressed in the research on Woohwangcheongsim-won and Sohaphyang-won. Hb α1, which is a type of hemoglobin that has oxygen transport ability, was upregulated in Woohwangcheongsim-won and Sohaphyang-won. This shows an upregulation of oxygen transporter under hypoxia, which means that these medicines have a significant effect in inhibiting apoptosis.
Myf6 is a myocyte differentiation gene and when muscles are created36), it is upregulated in the muscle while its expression is limited at the mRNA level37. In this study, Myf6 was downregulated in Woohwangcheongsim-won and Sohaphyang-won, but we could not explain the reason the myocyte related gene was expressed in the neuron. Also, it is difficult to find the reason for the downregulation of Slc12a1, which is a cotransporter of Na(+)-K(+)-2Cl(−)38).
In this study, when expressed genes of Woohwangcheongsim-won and Arisaema amurense var. serratum NAKAI were compared, Rarα was simultaneously upregulated [Global M = 1.0 (Woohwangcheongsim-won), 0.6 (Arisaema amurense var. serratum NAKAI)]. Rarα is a receptor in the retinoic acid that is derived from the activation of vitamin A. Retinoic acid is a necessary component in the creation of the nervous system and internal organs, and it is indispensable during the initial creation of the nervous system and the hindbrain39). Based on the finding that Rarα was upregulated in Woohwangcheongsim-won and Arisaema amurense var. serratum NAKAI, it appears that it protects the neurons under hypoxia.
When expressed genes of Woohwangcheongsim-won and Coptis chinensis FRANCH were compared, Rab4a [Global M = −0.6 (Woohwangcheongsim-won), −0.6 (Coptis chinensis FRANCH)] and Pdgfra [Global M = −0.6 (Woohwangcheongsim-won), −1.3 (Coptis chinensis FRANCH)] were commonly downregulated. Early endosome is limited by the gradual activation of PKC and Rab4a, and Rab4a recycles gene endocytic40). It is difficult to find a reasonable explanation for the downregulation of Rab4a in Woohwangcheongsim-won and Coptis chinensis FRANCH. Pdgfrα is upregulated in medulloblastoma, and by boosting cell division, it creates undivided neuroblasts or neuroglia, thereby being involved in the generation of nerve tumors. The reason this gene is downregulated in Woohwangcheongsim-won and Coptis chinensis FRANCH may be related to neuron protection, but it is difficult to explain in more detail41).
When expressed genes of Woohwangcheongsim-won and Rheum coreanum NAKAI were compared, phospholipase C and gamma 1 (Plcgγ1) [Global M = 0.6 (Woohwangcheongsim-won), 0.8 (Rheum coreanum NAKAI)] were commonly upregulated, and phospholipase A2, group IB, pancreas (Pla2g1b) [Global M = −0.6 (Woohwangcheongsim-won), −0.7 (Rheum coreanum NAKAI)], and solute carrier family 10, member 1(Slc10a1) [Global M = −0.8 (Woohwangcheongsim-won), −1.3 (Rheum coreanum NAKAI)] were commonly downregulated.
Plcγ1 induces the creation of blood vessels, and the upregulation under hypoxia can be understood as a defense mechanism against cell damage42). Pla2g1b is synthesized in the acinar cell of the pancreas and then released in the intestine during food intake. It is known to accelerate obesity and diabetes by weight loss induced diet43). Meanwhile, Slc10a1 is known to be the transporter of Na+ and bile acid44), and it is difficult to explain its downregulation by Woohwangcheongsim-won and Rheum coreanum NAKAI.
Thus is the comparison and analysis result of the medicines’ characteristics using the clustering analysis method based on the experimental research that compared the gene expression of single herbal treatment and herbal medicines through the cDNA microarray. Despite limitations, through the analysis result of cDNA microarray, we could collect information related to the characteristics of genes commonly expressed between individual herbal treatments or medicines. If we continue the relations analysis research systematically with the herbal medicine mechanism, we hope to better understand the details of the mechanism and the role of herbal treatments and medicines in the future.

Conclusion

We found the following conclusions through the comparison and analysis results using the cDNA microarray analysis method and functional clustering method of gene expression alteration due to herbal treatments or single herbal medicines within the apoptosis induced neuron under hypoxia model.
  1. Akt2 was upregulated by Acorus gramineus SOLAND, Arisaema amurense var. serratum NAKAI and Coptis chinensis FRANCH, and they belong to Araceae herb.

  2. Nf-κb1, Cd5, Gnγ7 and Sgne1 were upregulated by Arisaema amurense var. serratum NAKAI, Coptis chinensis FRANCH and Rheum coreanum NAKAI.

  3. Woohwangcheongsim-won, Sohaphyang-won and Scutellaria baicalensis GEORGI downregulated Scp2, and upregulated Tsc2. Woohwangcheongsim-won and Sohaphyang-won upregulated Hba1, and downregulated Myf6.

  4. Sohaphyang-won and Scutellaria baicalensis GEORGI downregulated Slc12a1.

  5. Woohwangcheongsim-won and Arisaema amurense var. serratum NAKAI upregulated Rarα, Woohwangcheongsim-won and Coptis chinensis FRANCH downregulated Rab5a and Pdgfrα, and Woohwangcheongsim-won and Rheum coreanum NAKAI upregulated Plcγ1 and downregulated Pla2g1b and Slc10a1.

Fig. 1.
Gene distribution expressed by Scutellaria baicalensis GEORGI, Woohwangcheongsim-won and Sohaphyang-won. A. Upregulated genes. B. downregulated genes.
skom-35-4-36f1.tif
Table 1.
Comparison of Experimental Procedures for Microarrays of Each Herb Extract or Composites.
Concentration Added DIV Inducing hypoxia Microarray platform Company Reference
Acorus gramineus SOLAND 10μg/mℓ DIV 12 DIV 14 TwinChipTM Rat-5K Digital Genomics Park et al. (2007)7
Arisaema amurense var. serratum NAKAI 10μg/mℓ DIV 12 DIV 14 TwinChipTM Rat-5K Digital Genomics Koh et al. (2003)8
Pinellia ternata BREIT. 2.5μg/mℓ DIV 12 DIV 14 TwinChipTM Rat-5K Digital Genomics Kwon et al. (2005)9
Scutellaria baicalensis GEORGI 20μg/mℓ DIV 12 DIV 14 TwinChipTM Rat-5K Digital Genomics Kim et al. (2004)10
Coptis chinensis FRANCH 2.5μg/mℓ DIV 12 DIV 14 TwinChipTM Rat-5K Digital Genomics Hwang et al. (2005)11
Rheum coreanum NAKAI 2.5μg/mℓ DIV 10 DIV 13 Rat 44K 4-Plex Gene Expression platform(Agilent) Digital Genomics Lee et al. (2009)12
Woohwangcheongsim-won 20μg/mℓ DIV 12 DIV 14 TwinChipTM Rat-5K Digital Genomics Park et al. (2004)13
Sohaphyang-won 20μg/mℓ DIV 12 DIV 14 TwinChipTM Rat-5K Digital Genomics Paik et al. (2004)14

All experiments were performed using E18 rat embryonic hippocampal neurons in culture grown in the neurobasal medium supplemented with B27.

All components were diluted or extracted with water and added directly to the medium.

* In all cases hypoxic shock was given for 3 hrs in 2% O2/5% CO2 and total mRNAs were extracted 24 hrs after shock.

Table 2.
The Number of Genes Up- or Downregulated by Each Treatment
Acorus gramineus SOLAND Arisaema amurense var. serratum NAKAI Pinellia ternata BREIT. Scutellaria baicalensis GEORGI Coptis chinensis FRANCH Rheum coreanum NAKAI Woohwangcheongsim-won Sohaphyang-won
Apoptosis upregulated 1 12 6
downregulated 1 2 5 6

Growth & maintenance upregulated 6 17 2 6 3
downregulated 8 4 3 18 3

Cell cycle upregulated 1 2 1 15 5
downregulated 1 1 1 15 8 1

Response to stress upregulated 1 5 1 27 17 5
downregulated 1 16 11 2

Signal transduction upregulated 12 3 1 56 22 5 1
downregulated 2 2 2 1 41 16 6

Transcription upregulated 1 4 1 1 20 11 2
downregulated 2 3 15 17 5 1

Physiological process upregulated 8 185 24
downregulated 16 129 12

Immune response upregulated 3 1 19 11 1
downregulated 1 10 6 3
Table 3.
An Upregulated Gene by Both Acorus gramineus SOLAND and Arisaema amurense var. serratum NAKAI
Acorus gramineus SOLAND Arisaema amurense var. serratum NAKAI
Growth & maintenance upregulated 1
downregulated -

Cell cycle upregulated 1
downregulated -
Table 4.
An Upregulated Gene by Both Acorus gramineus SOLAND and Coptis chinensis FRANCH
Acorus gramineus SOLAND Coptis chinensis FRANCH
Growth & maintenance upregulated 1
downregulated -

Cell cycle upregulated 1
downregulated -
Table 5.
An Upregulated Gene by Both Coptis chinensis FRANCH and Pinellia ternata BREIT
Coptis chinensis FRANCH Pinellia ternata BREIT.
Signal transduction upregulated 1
downregulated -

Physiological process upregulated 1
downregulated -

Immune response upregulated 1
downregulated -
Table 6.
Genes Up- or Downregulated by Both Coptis chinensis FRANCH and Arisaema amurense var. serratum NAKAI
Coptis chinensis FRANCH Arisaema amurense var. serratum NAKAI
Apoptosis upregulated 2
downregulated -

Growth & maintenance upregulated 13
downregulated -

Cell cycle upregulated 2
downregulated -

Response to stress upregulated 4
downregulated -

Signal transduction upregulated 10
downregulated 1

Transcription upregulated 3
downregulated -

Physiological process upregulated 18
downregulated -

Immune response upregulated 3
downregulated -
Table 7.
Genes Up- or Downregulated by Both Coptis chinensis FRANCH and Rheum coreanum NAKAI
Coptis chinensis FRANCH Rheum coreanum NAKAI
Apoptosis upregulated 2
downregulated -

Cell cycle upregulated 3
downregulated -

Response to stress upregulated 10
downregulated -

Signal transduction upregulated 10
downregulated 1

Transcription upregulated 4
downregulated 1

Physiological process upregulated 17
downregulated -

Immune response upregulated 6
downregulated 1
Table 8.
Genes Upregulated by Arisaema amurense var. serratum NAKAI, and Rheum coreanum NAKAI
Coptis chinensis FRANCH Rheum coreanum NAKAI
Apoptosis upregulated 1
downregulated -

Growth & maintenance upregulated 1
downregulated -

Cell cycle upregulated
downregulated

Response to stress upregulated 1
downregulated -

Signal transduction upregulated 3
downregulated -

Transcription upregulated 2
downregulated -

Physiological process upregulated 2
downregulated -

Immune response upregulated 2
downregulated -
Table 9.
Genes Upregulated by Arisaema amurense var. serratum NAKAI, Coptis chinensis FRANCH and Rheum coreanum NAKAI
Arisaema amurense var. serratum NAKAI Coptis chinensis FRANCH Rheum coreanum NAKAI
Apoptosis upregulated 1
downregulated -

Growth & maintenance upregulated 1
downregulated -

Response to stress upregulated 2
downregulated -

Signal transduction upregulated 3
downregulated -

Transcription upregulated 1
downregulated -

Physiological process upregulated 1
downregulated -

Immune response upregulated 2
downregulated -
Table 10.
Genes Up- or Downregulated by Both Woohwangcheongsim-won and Sohaphyang-won
Woohwangcheongsim-won Sohaphyang-woI
Growth & maintenance upregulated 2
downregulated 1

Signal transduction upregulated 1
downregulated -

Transcription upregulated -
downregulated 1
Table 11.
Genes Up- or Downregulated by Both Woohwangcheongsim-won and Scutellaria baicalensis GEORGI
Woohwangcheongsim-won Scutellaria baicalensis GEORGI
Growth & maintenance upregulated 1
downregulated 1

Signal transduction upregulated 1
downregulated -
Table 12.
Genes Up- or Downregulated by Both Sohaphyang-won and Scutellaria baicalensis GEORGI
Sohaphyang-won Scutellaria baicalensis GEORGI
Growth & maintenance upregulated 1
downregulated 2

Signal transduction upregulated 1
downregulated -
Table 13.
An upregulated Gene by Both Woohwangcheongsim-won and Arisaema amurense var. serratum NAKAI
Woohwangcheongsim-won Arisaema amurense var. serratum NAKAI
Apoptosis upregulated 1
downregulated -

Transcription upregulated 1
downregulated -
Table 14.
Genes downregulated by Both Woohwangcheongsim-won and Coptis chinensis FRANCH
Woohwangcheongsim-won Coptis chinensis FRANCH
Growth & maintenance upregulated -
downregulated 1

Signal transduction upregulated
downregulated 2
Table 15.
Genes Up- or Downregulated by Both Woohwangcheongsim-won and Rheum coreanum NAKAI
Woohwangcheongsim-won Rheum coreanum NAKAI
Growth & maintenance upregulated -
downregulated 2

Response to stress upregulated
downregulated 1

Signal transduction upregulated 1
downregulated -

References

1.. Honig LS, Rosenberg RN. Apoptosis and neurologic disease. Am J Med. 2000; 108:4. 317–30.
crossref

2.. Rego AC, Oliveira CR. Mitochondrial Dysfunction and Reactive Oxygen Species in Excitotoxicity and Apoptosis: Implications for the Pathogenesis of Neurodegenerative Diseases. Neurochem Res. 2003; 28:10. 1563–74.
crossref

3.. International Human Genome Sequencing Consortium. Initial sequencing and analysis of the human genome. Nature. 2001; 209:860–921.


4.. Arabidopsis Genome Initiative. Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature. 2000; 408:796–815.
crossref

5.. Tanaka TS, Jaradat SA, Lim MK, Kargul GJ, Wang X, Grahovac MJ, et al. Genome-wide expression profiling of mid-gestation placenta and embryo using a 15,000 mouse developmental cDNA microarray. Proc Natl Acad Sci USA. 2000; 97:9127–32.
crossref

6.. Harrington CA, Rosenow C, Retief J. Monitoring gene expression using DNA microarrays. Curr Opin Microbiol. 2000; 3:285–91.
crossref

7.. Park DJ, Jung SH, Moon IS, Lee WC, Shin GC. Microarray Analysis of Alteration in Gene Expression by Acori graminei rhizoma (AGR) Water-Extract in a Hypoxic Model of Cultured Rat Cortial Cells. J Life Science. 2007; 17:1. 150–61.
crossref

8.. Koh KD. Effects of Arisaema amurense var. serratum NAKAI(南星) on the modulation of ROS, MMP and Gene Expression in a Hypoxic Model of Cultured Rat Cortical Cells. The graduate school of Dongguk Uni. 2003.


9.. Kwon GR. Rizoma(半夏) on Prevention of Cortical Neuronal Cell Death and Gene Expression. The graduate school of Dongguk Uni. 2005.


10.. Kim SB, Chung SH, Shin GC, Lee WC. Effects of Scutellaria baicalensis GEORGI on Gene Expression in a Hypoxic Model of Cultured Rat Cortical Cells. Korean J Orient Int Med. 2004; 25:4–2. 324–36.


11.. Hwang JW, Kim KH, Shin GC, Moon IS. Efeects of Gene Expression by Coptidis chinesis FRANCH. in a Hypoxic Model of Cultured Rat Cortical Cells. Korean J Orient Int Med. 2011; 32:2. 301–21.


12.. Lee HS, Lee JY, Moon IS. Microarray Analysis of Gene Expression by Rhei Rhizoma Water Extracts in a Hypoxia Model of Cultured Neurons. J Life Sci. 2009; 19:1. 21–33.
crossref

13.. Park DW, Kim WS, Bae CH, Jeong SH, Shin GC, Lee WC. Effects of Woohwangcheongsim-won Gene Expression in a Hypoxic Model of Cultured Rat Cortical Cells. J Korean Oriental Med. 2004; 25:3. 123–36.


14.. Paik JW, Lee YH, Kim WS, Jeong SH, Shin GC, Lee WC. Effects of Sohaphyang-won Gene Expression in a Hypoxic Model of Cultured Rat Cortical Cells. J Korean Oriental Med. 2004; 25:2. 127–37.


15.. Sturn A, Quackenbush J, Trajanoski Z. Genesis: cluster analysis of microarray data. Bioinformatics. 2002; 18:1. 207–8.
crossref

16.. Eisen MB, Spellman PT, Brown PO, Botstein D. Cluster analysis and display of genome-wide expression patterns. Proc Natl Acad Sci USA. 1998; 95:25. 14863–8.
crossref

17.. Zimmermann KC, Green DR. How cells die: Apoptosis pathways. J Allergy Clin Immunol. 2001; 108:4. 99–103.
crossref

18.. Hochachka PW, Lutz PL. Mechanism, origin, and evolution of anoxia tolerance in animals. Comp Biochem Physiol B: Biochem Mol Biol. 2001; 130:4. 435–59.
crossref

19.. Won MH. Noeheohyeole Uihan Jiyeonseong Singyeongseposaui Gijeon. Biochemistry News. 2002; 22:2. 158–68.


20.. Banasiaka KJ, Xiab Y, Haddad GG. Mechanisms underlying hypoxia-induced neuronal apoptosis. Prog Neurobiol. 2000; 62:3. 215–49.
crossref

21.. Wang H, Yu SW, Koh DW, Lew J, Coombs C, Bowers W, et al. Apoptosis-Inducing Factor Substitutes for Caspase Executioners in NMDA-Triggered Excitotoxic Neuronal Death. J. Neurosci. 2004; 24:48. 10963–73.
crossref

22.. Nieminen AL. Apoptosis and necrosis in health and disease: Role of mitochondria. Int Rev Cytol. 2003; 224:29–55.
crossref

23.. Oosthuyse B, Moons L, Storkebaum E, Beck H, Nuyens D, Brusselmans K, et al. Deletion of the hypoxia-response element in the vascular endothelial growth factor promoter causes motor neuron degeneration. Nature Genet. 2001; 28:131–8.
crossref

24.. Oh MK. Research Trends of Bioinformatics Using DNA Microarray Data. Pros Ind Chem. 2006; 9:5. 43–8.


25.. Shih JH, Michalowska AM, Dobbin K, Ye Y, Qiu TH, Green JE. Effects of pooling mRNA in microarray class comparisons. Bioinformatics. 2004; 20:18. 3318–25.
crossref

26.. Fujio Y, Mitsuuchi Y, Testa JR, Walsh K. Activation of Akt2 Inhibits anoikis and apoptosis induced by myogenic differentiation. Cell Death Differ. 2001; 8:12. 1207–12.
crossref

27.. Sinmunpungchulpangongsa. Sinpyeonjungyakdaesajeon. Chopan: 1981. p. 113–9. p. 333–7. p. 2107–16.


28.. Pizzi M, Goffi F, Boroni F, Benarese M, Perkins SE, Liou HC, et al. Opposing Roles for NF-κ B/Rel Factors p65 and c-Rel in the Modulation of Neuron Survival Elicited by Glutamate and Interleukin-1β. J Biol Chem. 2002; 277:20717–23.
crossref

29.. Pizzi M, Sarnico I, Boroni F, Benetti A, Benarese M, Spano PF. Inhibition of IκBα phosphorylation prevents glutamate-induced NF-κB activation and neuronal cell death. Acta Neurochir. 2005; 93:59–63.
crossref

30.. Krumschnabel G, Sohm B, Bock F, Manzl C, Villunger A. The enigma of caspase-2: the laymen’s view. Cell Death Differ. 2009; 16:195–207.
crossref

31.. Ohta1 M, Mimori K, Fukuyoshi Y, Kita Y, Motoyama K, Yamashita K, et al. Clinical significance of the reduced expression of G protein gamma 7 (GNG7) in oesophageal cancer. Brit J Cancer. 2008; 98:410–7.
crossref

32.. Inoki K, Li Y, Zhu T, Wu J, Guan KL. TSC2 is phosphorylated and inhibited by Akt and suppresses mTOR signalling. Nat Cell Biol. 2002; 4:648–57.
crossref

33.. Inoki K, Zhu T, Guan KL. TSC2 Mediates Cellular Energy Response to Control Cell Growth and Survival. Cell. 2003; 115:5. 577–90.
crossref

34.. Offenberg HH, Schalk JAC, Meuwissen RLJ, Aalderen M, Kester HA, Dietrich AJJ, Heyting C. SCP2: A major protein component of the axial elements of synaptonemal complexes of the rat. Nucl Acids Res. 1998; 26:11. 2572–9.
crossref

35.. Liang WS, Dunckley T, Beach TG, Grover A, Mastroeni D, Walker DG, et al. Gene expression profiles in anatomically and functionally distinct regions of the normal aged human brain. Physiol Genomics. 2007; 28:311–22.
crossref

36.. Kassar-Duchossoy L, Gayraud-Morel B, Gomès D, Rocancourt D, Buckingham M, Shinin V, et al. Mrf4 determines skeletal muscle identity in Myf5: Myod double-mutant mice. Nature. 2004; 431:466–71.
crossref

37.. Hinitsa Y, Osborna DPS, Carvajalb JJ, Rigbyb PWJ, Hughes SM. Mrf4 (myf6) is dynamically expressed in differentiated zebrafish skeletal muscle. Gene Expression Patterns. 2007; 7:7. 738–45.
crossref

38.. Hannemann A, Christie JK, Flatman PW. Functional Expression of the Na-K-2Cl Cotransporter NKCC2 in Mammalian Cells Fails to Confirm the Dominant-negative Effect of the AF Splice Variant. J Biol Chem. 2004; 284:35348–58.
crossref

39.. Gavalas A, Krumlauf R. Retinoid signalling and hindbrain patterning. Curr Opin Genet Dev. 2000; 10:4. 380–6.
crossref

40.. Hellberg C, Schmees C, Karlsson S, Åhgren A, Heldin CH. Activation of Protein Kinase C Is Necessary for Sorting the PDGF β-Receptor to Rab4a-dependent Recycling. Mol Biol Cell. 2009; 20:2856–63.
crossref

41.. MacDonald TJ, Brown KM, LaFleur B, Peterson K, Lawlor C, Chen Y, et al. Expression profiling of medulloblastoma: PDGFRA and the RAS/MAPK pathway as therapeutic targets for metastatic disease. Nat Genet. 2001; 29:143–52.
crossref

42.. Lawson ND, Mugford JW, Diamond BA, Weinstein BM. phospholipase C gamma-1 is required downstream of vascular endothelial growth factor during arterial development. Genes Dev. 2003; 17:1346–51.
crossref

43.. Hui DY, Cope MJ, Labonté ED, Chang H-T, Shao J, Goka E, et al. The phospholipase A2 inhibitor methyl indoxam suppresses diet-induced obesity and glucose intolerance in mice. Brit J Pharmacol. 2009; 157:7. 1263–9.
crossref

44.. Mita S, Suzuki H, Akita H, Hayashi H, Onuki R, Hofmann AF, et al. Inhibition of Bile Acid Transport across Na+/Taurocholate Cotransporting Polypeptide (SLC10A1) and Bile Salt Export Pump (ABCB 11)-Coexpressing LLC-PK1 Cells by Cholestasis-Inducing Drugs. Drug Metab Dispo. 2009; 34:9. 1575–81.
crossref

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