Extract
Note: Please read the complete
full text with Figures and Tables at
Introduction
Stroke, mostly caused by cerebral ischemia, is a multifactorial
disease involving the activation of myriads of death inducers, which
leads to injury of neurons by eliciting cascades of signal transduction
pathways[1]. The Qing Kai Ling injection (QKLI) is a
modified preparation of the "An Gong Niu Huang" pill,
a famous traditional Chinese medicament. In recent studies, QKLI
showed strong therapeutic effects on stroke in clinical usage. However,
this preparation is a compound comprising of many components. Its
therapeutic mechanism is very complex, with regard to both individual
and integrative effects. Baicalin and gardenin are two effective
compounds of QKLI. Baicalin, known as an antioxidant flavonoid in
vitro, functions as a biological response modifier[2,3],
and has been investigated for its neuroprotective effects against
glutamate/N-methyl-D-aspartate (Glu/NMDA) stimulation
and glucose deprivation in primary cultured rat brain neurons. Baicalin
was found to significantly reduce Glu/NMDA-increased lactate dehydrogenase
(LDH) release[4]. Baicalin is also a potent inhibitor
of endothelial cell prolifera-tion, migration, and differentiation[5].
Gardenin can significantly decrease the content of transcription
factor monocyte chemoattractant protein-1 (MCP-1) in rat brains
suffering focal ischemia[6]. cDNA microarray possesses
the ability to analyze the expression changes of hundreds, thousands,
or even tens of thousands of genes simultaneously. Several waves
of gene expression have been observed after focal cerebral ischemia[7];
however, a comparative study examining the pharmacological mechanisms
of different drugs has not been reported in the literature. Results
from the present study suggest that baicalin and gardenin play a
pharmacological role in focal cerebral ischemia by regulating gene
expression.
Materials and methods
Cerebral ischemia Forty-four Sprague-Dawley rats (250-280 g,
supplied by Beijing Weitong-Lihua Experimental Animal Center, Beijing,
China) were randomly divided into four groups: model, baicalin-treatment,
gardenin-treatment, and sham-operated group, respectively. Under
isofurane anesthesia, rats were subjected to occlusion of the left
middle cerebral artery (MCAO) using an intraluminal filament as
described by Haruo et al[8]. After 24 h of focal
cerebral ischemia, the rats were deeply re-anesthetized using halothane
and the left half of the brain was removed for total RNA isolation.
In the sham-operated rats, all procedures except for MCAO were carried
out. Baicalin or gardenin (National Institute for the Control of
Pharmaceutical and Biological Products, Beijing, China) was given
orally (40 mg/kg) 2 h after MCAO.
Measurement of cerebral infarction areas Eight rats from
each group were used to calculate the infarction ratio. In brief,
the cerebrum was removed and cut into five slices on the coronal
section at the location from which the distance to the prefrontal
is 1, 3, 5, and 7 mm, respectively. The slices were stained
in 4% 2,3,5-triphenyltetrazolium chloride (TTC) solution for 10
min[9]. Images of the slices were captured using a digital
camera (Color CCD camera TP-6001A, Topica Inc, Japan). We calculated
the areas of the infarction region using a Pathology Image Analysis
System (Topica Inc, Japan). The ratio of the infarction area to
the total slice area was calculated.
Total RNA isolation The left half of the brain from the
remaining three rats in each group was carefully dissected out from
the re-anesthetized rats under RNAase-free conditions. The total
RNA in each sample was extracted with Trizol reagent ( Invitrogen,
San Diego, CA, USA) using the procedure outlined in the manufacturer's
protocol.
Microarrays Hybridization was carried out on the rat "Biostar40S"
gene microarray (BioStar Genechip Incorporated, Shanghai, China),
which contained 4096 elements. The array provided a broad overview
of the rat genome, which was added to the high-quality array cDNA
libraries, and the sequence information, maps, and expression data
were placed into the public domain (NCBI, Nucleotide). All clones
used for production of the microarrays were sequence verified. Three
pieces of total RNA from the same group were pooled following transcription
into cDNA and were labeled with Cy5 and Cy3, respectively, and hybridized
on the array. Image files were processed using the Axon GenePix
4000B scanner(Axon, USA) and datasets were prepared according to
the routine procedures using Genepix 4.0 software (Axon, USA).
Data analysis Data sheets derived from the results of the
Genepix 4.0 analysis were further evaluated using Excel software
(Microsoft, USA). The microarrays were hybridized in triplicate
and each measurement containing the extracted total RNA from each
group was used to analyze each gene, which had three data points
of relative changes. Thus, a balance coefficient was calculated
to correct variations resulting from unequal amounts of fluorescent
dye fade; the average signal from all elements in the Cy3 channel
was divided by the average signal from all elements in the Cy5 channel,
which resulted in the balance coefficient. The Cy5 signal for each
element was then multiplied by the balance coefficient, prior to
calculating the expression ratio (Cy3/Cy5). A fold value of =2.0
or = -2.0 indicated that differences in the Cy3:Cy5 ratio were detected
at a 99% confidence level (information provided by Incyte, USA).
All probes fulfilling the criterion (=2.0 or = -2.0) were compiled[10-13].
Semiquantitative RT-PCR The procedures for isolating total
RNA using Trizol reagent (Invitrogen, Life Technology, Incorporated,
New York, NY, USA) and synthesizing first strand cDNA were exactly
as previously described[14]. The optimal polymerase chain
reaction (PCR) amplification conditions and cycle number were determined
experimentally to ensure specific signal generation. Glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) was considered to be stably expressed after
focal cerebral ischemia acted as an inter-reference gene. All amplifications
were carried out on a Gradient PCR System (Biometra, USA). cDNA
synthesis was carried out using 200 ng of mRNA with 5×106 U/L
reverse transcriptase (TaKaRa, Japan) in a total 100 µL reaction
mixture and incubated for 60 min at 48 °C. The primer pairs
were for ribosomal protein L19 (Rpl19): AAC AGA TCA GGA AGC TGA
TCA AGA; AGT CTT GAT GAT CTC CTC CTT CTT (NM 031103); casein kinase
II beta subunit (Csnk2b): CGG ACA TAA AGA TGA GTA GCT CTG A; GTG
GTG CCT AGA GGA CTT GGG TGT G (NM 031021); GAPDH: ACC ACA GTC CAT
GCC ATC AC; TCC ACC ACC CTG TTG CTG TA (NM 008084.1). After heating
to 95 °C for 5 min, each RT reaction mixture was used for PCR
amplification. The PCR mixture started with in an initial step of
5 min at 95 °C followed by 35 cycles of 30 s at 95
°C and an annealing temperature of 1 min at 64.5 °C for
Csnk2b mRNA, at 57.4 °C for Rpl19 mRNA and GAPDH, followed
by 72 °C for 10 min. Products of 5 µL from each PCR mixture
were dyed with 1 µL SYBR Green I Nucleic Acid Gel stain (Cambrex
Bio Science Rockland Incorporated, USA) and loaded on 2.5% agarose
gels, which were evaluated according to the intensity differences
of image electrophoresis using Gel Analyzing Imager (FuRi, Shanghai,
China).
Results
TTC staining Both baicalin and gardenin reduced the infarction
areas in cerebral ischemia rats by 6%-7% compared with the model
group. The ratio of differences was tested using the Student t-test
(Figure 1, Table 1).
Changes in gene expression after MCAO occlusion Genes were
classified into 12 categories by the International General Principle
(www.geneontology.org). One hundred and ninety-nine genes were significantly
upregulated which are involved in metabolism, signal transduction,
cell organization, response to stress, cell adhesion, transport,
apoptosis, and a variety of other processes. Another 12 genes showed
downregulated expression under the same conditions (Table 2).
Metabolism The largest increases in gene expression were shown
by genes involved in protein synthesis: the ribosomal proteins L10,
S5, S3a, S24, S6, S11, and L19, glycoprotein 38, eukaryotic translation
elongation factor 1 alpha and actin-related protein complex 1b.
The expression of genes related to cell metabolism changed with
increased expression of coenzyme A dehydrogenase, procollagen-lysine,
lactate dehydrogenase and ubiquinone oxidoreductase subunit B13.
Cell organization and adhesion Cell organization and adhesion genes
also showed prominent changes in expression. These included Fibronectin
1, Integrin, H2A histone family member and apolipoprotein M.
Signal transduction Increases in expression were shown by genes
involved in cellular signal transduction: CDK5, pyruvate kinase
3, phosphatidylinositol 4-kinase, and casein kinase II. In addition,
the G protein pathway suppressor showed decreased expression over
this interval.
Response to stress Several "stress" genes involved in
cellular responses to inflammation and injury were induced, including
Cd63 antigen, MRC OX-45 surface antigen and immunoglobulin superfamily
member. Another induced gene, glutathione peroxidase 1, was also
upregulated after focal cerebral ischemia.
Apoptotic genes The expression of several apoptotic effector genes
was altered. These included programmed-cell-death-8, Urmodulin,
and tumor protein translationally controlled genes.
Effect of baicalin on gene expression Increases in gene
expression were observed in 89 genes. These included genes involved
in metabolism, signal transduction, cell organization, responses
to stress, and transcription regulators. In addition to the induced
genes, 88 genes simultaneously showed decreased expression (Table
3).
Metabolism Metabolism-related genes showed prominent changes in
expression. These included ADP-ribosyla-tion factor, enolase, adenine
phosphoribosyltransferase, and palmitoyl-protein thioesterase. In
addition, histamine N-methyltransferase simultaneously showed
decreased expression.
Signal transduction The largest increases in expression were shown
by genes involved in protein tyrosine phosphatase receptor type
D and receptor type A. S100 calcium-binding protein A9 also showed
prominent changes. In addition, the expression of protein kinase
C-binding protein Zeta and surfactant-associated protein decreased.
Protein kinase is controlled by specific binding proteins, which
are believed to sequester each type of kinase to the region of a
neuron, such as the postsynaptic specialization or cell nucleus,
that requires its function[15]. The protein kinase C-binding
protein Zeta is one of these proteins. Arachidonic acid epoxygenase,
an adapter protein of the prostaglandin and leukotriene family of
intracellular messengers, also appears to play an important role
in the regulation of signal transduction in the brain and elsewhere[14].
Cell organization The expression of nucleolar phosphoprotein p130,
an adapter protein that participates in nucleolar disassembly and
cell cycle, decreased after MCAO. In addition, peroxisomal membrane
protein showed increased expression over this interval.
Transcription regulator Two genes related to the transcription
regulator were altered. These were spliceosome-associated protein
and DNA primase.
Effect of gardenin on gene expression Increases in gene
expression were observed for 68 genes. These included genes involved
in metabolism, signal transduction, cell organization, response
to stress, cell cycle, and cell mobility. In addition, two genes
showed decreased expression over this interval (Table 4).
Metabolism The expression of several genes involved in protein
synthesis and cell metabolism was altered. These included ADP-ribosylation
factor, HS-glycoprotein and deoxyribose-phosphate aldolase.
Signal transduction Changes in the expression of genes indirectly
related to signal transduction were observed, and consisted of increased
expression in nuclear receptor, adapter-related protein complex
and retinoid-inducible serine carbopeptidase precursor.
Response to stress Several "stress" genes involved in
cellular responses to oxidation were induced. The most striking
was the increased expression of glutathione-S-trans-ferase.
Cell mobility Myosin heavy chain and tropomyosin beta chain genes
that participate in cell mobility and cell cycle were also induced.
Semiquantitative RT-PCR The outcome of this study showed
that the expression of ribosomal protein RpL19 mRNA and Csnk2 mRNA
were consistently upregulated in the individual samples from MCAO
rats relative to sham-operated rats. Expression increased by 1.9-
and 2.1-fold, respectively, and showed the same trend as the results
of the microarray. The outcome of RT-PCR indicated that broad, array-based
gene expression measurements were reliable for determining gene
expression patterns in the brain (Figure 2).
Discussion
Messenger RNA is only an intermediate on the way to the production
of the eventual protein products. In the present study we explored
the potential role of cDNA microarrays for gene expression analysis
after focal brain ischemia and examined the differences in gene
expression after the action of different compounds of QKLI. An ischemia
period of 24 h was used because this is the maximum period
of cerebral ischemia that is compatible with neuroprotection[16-18].
To select results from microarray experiments with reliably altered
ratios, we filtered the results using two criteria[10-13]:
minimal fold-change values, and ratios reproducibly different from
unity. The hybridization ratio had to be at least two-fold higher
or lower than the control groups. Of these filters, the two-fold
ratio filter was by far the most restrictive and there was no uniform
standard across the different platforms of the arrays. We have listed
the information of differential gene expression according to the
functional classification of genes. In brief, baicalin and gardenin
appear to have a profound influence on the model by regulating the
expression of different genes or by acting on different metabolic
pathways. The genes related to cell metabolism presented striking
changes, and showed increased expression in both model and baicalin-treatment
groups in contrast to the gardenin-treatment group. Baicalin appears
to have a more prominent action on signal transduction in cells
than gardenin, including activation of ion channels, regulation
of kinase and phosphorylation of receptor proteins. In contrast,
the effects of gardenin in stress responses and anti-oxidation appear
to be more significant than those of baicalin.
Changes in gene expression after MCAO Prominent changes
were recorded in the ribosomal proteins S6, L6, S3a, S24, L5, L10,
L19, and S11 on the microarray[19]. In general, this
finding supports previous studies[20], perhaps reflecting
recovery from an early postischemic transcriptional defect. Integrin
is a transmembrane protein, and the adhesion effects between leucocytes
and cerebral micrangium cells mediated by integrin participated
in the injury and destruction of inflammation factors to tissues.
The increased expression of integrin after MCAO is, in general,
in line with a preceding report[21]. The increased expression
of protein kinase (PK) is mostly caused by the large amount of PK
released as a result of metabolic dysfunction, necrosis, ischemia
and hypoxia of cells, which occurs in acute cerebrovascular diseases,
and this finding was in accordance with the clinical diagnosis.
The increased expression of genes in the G protein pathway suppressor
1 may have contributed to the adaptive modulation of the body. In
general, G protein plays a role in signal amplification and in switching
the molecule on and off in the course of signal transduction. G
protein pathway suppressor was upregulated after focal cerebral
ischemia, which might occur in compensation for stress and signal
transduction regulation in the body[22,23]. Casein kinase
II is a necessary substance for cell survival, and increased expression
of casein kinease II is correlated with hyperplasia and the proliferation
of cells after cerebral ischemia. Cyclin-dependent kinase 5 (CDK5)
mRNA was found to be downregulated after focal brain ischemia, whereas
variable changes were noted at the protein level in focal ischemia[24].
Downregulation of phosphatases was noted, which would alter the
balance of protein phosphorylation in several cellular signaling
pathways; much more information is needed before any suggestions
regarding functional effects can be made. As the oxidation of glutathione
peroxidase might offer a new modulating mechanism of cellular signal
transduction[25], it was suggested that its increased
expression could provide a sign in response to stress in the body.
Effect of baicalin on gene expression Programmed-cell-death-8
was upregulated after MCAO with baicalin treatment. Furthermore,
novel mediators of cerebral ischemia inducing neuronal death have
also been found using cDNA microarrays. All these studies reinforce
the idea that common cell death pathways are activated in response
to neurondeath inducers, which promise to serve as potential therapeutic
targets for modulation to achieve neuroprotection[26].
The expression of the protein kinase C-binding protein Zeta was
downregulated in rats treated with baicalin, which resulted in a
decrease in the activity of protein kinase C (PKC). Aronowski et al[27]
observed that the activity of PKC in the brain cortex and hippocampus
decreased significantly after ischemia and this decrease could be
alleviated by the pre-treatment of the NMDA receptor antagonist.
Further studies need to examine whether baicalin acts as an antagonist
to the NMDA receptor. After all, the relationship between the changing
activity of PKC and neuron injury has not been elucidated completely
in many key pathophysiological procedures of ischemia, such as increment
of fermentation, acidosis, and deficiency of ATP production. The
prominent differential genes involved in protein tyrosine phosphatase
receptor type D and A are central to the course of signal transduction,
which has been implicated both in the regulation of cell growth
and the rearrangement of actin that is mediated by several receptor
tyrosine kinases. Differential gene expression showed that baicalin
played an important role in cell signal transduction and protein
phosphorylation after MCAO, and might act as a neuroprotectant.
Effect of gardenin on gene expression The differential
genes in the gardenin-treatment group showed extreme variation compared
with the baicalin-treatment group. Several genes encoding anti-oxidation
were upregulated. For example, an increase in Mu2 suggested that
Mu2 played an activation role in anti-oxidation responses, and was
regarded as one of the markers of internal anti-injury because of
its anti-oxidative and antidotal effects[25]. According
to the actions of Mu2, the anti-oxidative and antidotal effects
of gardenin might be one of the mechanisms of cerebral protection
after MCAO.
The most interesting phenomenon observed was that there was no
overlap in the genes showing differential expression in the three
groups despite the similar trends in expression. The results suggest
that there are considerable differences in the pharmacological effects
of baicalin and gardenin at the molecular level after MCAO. Compared
with the role of gardenin and bacalin, which are components of QKLI,
it appears that QKLI plays an integral role and has important therapeutic
effects requiring further investigation.
In conclusion, the cDNA microarray study not only confirmed that
changes in many genes contributed to cerebral ischemia, but also
suggested several potential targets for further investigation. Of
course, global expression of genes measured at the levels of mRNA
transcription obtained using microarray analysis should be viewed
from two different standpoints. Looking from mRNA towards protein,
one would ultimately like to test the increased production of the
proteins encoded by the upregulated mRNA or demonstrate a loss of
protein encoded by downregulated mRNA. Thus, it may be rewarding
to reconstruct the networks of regulation in response to ischemia
and, by inference, to hypoxia, using bioinformatic strategies[28].
Understanding such regulatory networks and the therapeutic mechanisms
of QKLI for ischemia-hypoxia responsive gene expression in neurons
may extend the relevance of these studies.
References
- 1 Hou ST, MacManus JP. Molecular mechanisms of cerebral ischemia-induced
neuronal death. Int Rev Cytol 2002; 221: 93-148.
- 2 Chen X, Nishida H, Konishi T. Baicalin promoted the repair
of DNA single strand breakage caused by H2O2
in cultured NIH3T3 fibroblasts. Biol Pharm Bull 2003; 26: 282-4.
- 3 Huang Y, Wong CM, Lau CW, Yao X, Tsang SY, Su YL, et
al. Inhibition of nitric oxide/cyclic GMP-mediated relaxation
by purified flavonoids, baicalin and baicalein in rat aortic rings.
Biochem Pharmacol 2004; 67: 787-94.
- 4 Lee HH, Yang LL, Wang CC, Hu SY, Chang SF, Lee YH. Differential
effects of natural polyphenols on neuronal survival in primary
cultured central neurons against glutamate- and glucose deprivation-induced
neuronal death. Brain Res 2003; 986: 103-15.
- 5 Liu JJ, Huang TS, Cheng WF, Lu FJ. Baicalein and baicalin
are potent inhibitors of angiogenesis: inhibition of endothelial
cell proliferation, migration and differentiation. Int J Cancer
2003; 106: 559-65.
- 6 Zhou JW. Gardenin and bacalin show the effect on MCP-1 of
rat brain suffered focal ischemia. Chin Arch Tradit Chin Med 2004;
22:1016-7.
- 7 Sharp FR, Lu A, Tang Y, Millhorn DE. Multiple molecular penumbras
after focal cerebral ischemia. J Cereb Blood Flow Metab 2000;
20: 1011-32.
- 8 Haruo N, Agasawa K, Yuya K. Correlation between cerebral blood
flow and histologic changes in a new rat model of middle cerebral
artery occlusion. Stroke 1989; 20: 1037-42.
- 9 Bederson JB. Evaluation of 2,3,5-triphenyltetrazolium chloride
as a stain for detection and quantification of experimental cerebral
infarction in rats. Stroke 1986; 17: 1304-8.
- 10 Jiang CH, Tsien JZ, Schultz PG, Hu Y. The effects of aging
on gene expression in the hypothalamus and cortex of mice. Proc
Natl Acad Sci USA 2001; 98: 1930-4.
- 11 Jin K, Mao XO, Eshoo MW, Nagayama T, Minami M, Simon RP,
et al. Microarray analysis of hippocampal gene expression
in global cerebral ischemia. Ann Neurol 2001; 50: 93-103.
- 12 Lee CK, Weindruch R, Prolla TA. Gene-expression profile of
the ageing brain in mice. Nat Genet 2000; 25: 294-7.
- 13 Sandberg R, Yasuda R, Pankratz DP, Barlow C. Regional and
strain-specific gene expression mapping in the adult mouse brain.
Proc Natl Acad Sci USA 2000; 97: 11038-43.
- 14 Hou ST, Callaghan D, Fournier MC, Hill I, Kang L, Massie
B, et al. The transcription factor E2F1 modulates apoptosis
of neurons. J Neurochem 2000; 75: 91-100.
- 15 Jaken S. An overlay assay for detecting protein kinase C-binding
proteins and substrates. Methods Mol Biol 2003; 233: 359-68.
- 16 Belayev L, Liu Y, Zhao W, Busto R, Ginsberg MD. Human albumin
therapy of acute ischemic stroke: marked neuroprotective efficacy
at moderate doses and with a broad therapeutic window. Stroke
2001; 32: 553-60.
- 17 Huh PW, Belayev L, Zhao W, Koch S, Busto R, Ginsberg MD.
Comparative neuroprotective efficacy of prolonged moderate intraischemic
and postischemic hypothermia in focal cerebral ischemia. J Neurosurg
2000; 92: 91-9.
- 18 Belayev L, Zhao W, Busto R, Ginsberg MD. Transient middle
cerebral artery occlusion by intraluminal suture: I. Three-dimensional
autoradiographic image-analysis of local cerebral glucose metabolism-blood
flow interrelationships during ischemia and early recirculation.
J Cereb Blood Flow Metab 1997; 17: 1266-80.
- 19 Guhaniyogi J, Brewer G. Regulation of mRNA stability in mammalian
cells. Gene 2001; 265: 11-23.
- 20 Schmidt-Kastner R, Zhang B, Belayev L, Khoutorova L, Amin
R, Busto R, et al. DNA microarray analysis of cortical
gene expression during early recirculation after focal brain ischemia
in rat. Brain Res Mol Brain Res 2002; 108: 81-93.
- 21 Zhang ZG, Chopp M, Tang WX, Jiang N, Zhang RL. Postischemic
treatment (2-4) with anti-CD11b and anti-CD18 monoclonal antibodies
is neuroprotective after transient (2 h) focal cerebral ischemia
in the rat. Brain Res 1995; 698: 79-85.
- 22 Bogousslavsky J, Paciaroni M, Gallai V. Glycoprotein (GP)
IIb/IIIa inhibitors for acute stroke treatment. Clin Exp Hypertens
2002; 24: 603-10.
- 23 Wannier-Morino P, Rager G, Sonderegger P, Grabs D. Expression
of neuroserpin in the visual cortex of the mouse during the developmental
critical period. Eur J Neurosci 2003; 17: 1853-60.
- 24 Hayashi T, Warita H, Abe K, Itoyama Y. Expression of cyclin
dependent kinase 5 and its activator p35 in rat brain after middle
cerebral artery occlusion. Neurosci Lett 1999; 265: 37-40.
- 25 Paolicchi A, Dominici S, Pieri L, Maellaro E, Pompella
A. Glutathione catabolism as a signaling mechanism. Biochem Pharma-col
2002; 64: 1027-35.
- 26 Read SJ, Parsons AA, Harrison DC, Philpott K, Kabnick
K, O'Brien S, et al. Stroke genomics: approaches to identify,
validate, and understand ischemic stroke gene expression. J Cereb
Blood Flow Metab 2001; 21: 755-78.
- 27 Aronowski J, Waxham MN, Grotta JC. Neuronal protection and
preservation of calcium/calmodulin-dependent PKII and PKC activity
by dextrorphan treatment in lobal ischemia. J Cereb Blood Flow
Metab 1993;13: 550-7.
- 28 Thieffry D. From global expression data to gene networks.
Bioessays 1999; 21: 895-9.
|
