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NEUROPHARMACOLOGY
Department of Pharmacology, Shanghai Institute of Materia Medica (D.-M.C., X.C., X.-Z.Z.) and The Research Centre for Proteome Analysis, Key Lab of Proteomics, Institute of Biochemistry and Cell Biology (R.Z.), Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China; and Department of Research and Development (L.X.), Jiangsu Hengrui Medicine Company, Shanghai, China
Received for publication
March 10, 2006
Accepted
July 12, 2006.
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Abstract |
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B pathway are associated with the mechanisms of PF preconditioning.
). The most effective drug, tissue plasminogen activator, the only Food and Drug Administration-approved thrombolytic drug used in ischemia, is widely employed in clinic. However, it has a very narrow therapeutic time window of 3 h, and only 1 to 3% of ischemic stroke patients have been benefited (Pulsinelli et al., 1997). Therefore, the current focus of stroke research requires investigation of novel neuroprotective agents and strategies, which could prolong the therapeutic window.
Paeoniflorn (PF), the principal component of Paeoniae radix ("Shaoyao" in Chinese), has been widely investigated as antioxidant, cognitive enhancer, and endothelium-dependent vasodilator (Goto et al., 1996; Ryu et al., 2001; Tabata et al., 2001). Recently, our laboratory found that PF could produce a dose-dependent decrease in both neurological impairment and infarct volume in acute transient cerebral ischemia through activating adenosine A1 receptor in a manner different from its classic agonists (Liang et al., 2005; Liu et al., 2005b).
Ischemic preconditioning (IPC) is an endogenous mechanism by which brief episodes of a sublethal insult induce a robust protection against the deleterious effects of subsequent, prolonged lethal ischemia. There are two kinds of protection: "classic IPC" and "delayed IPC." Various drugs can mimic IPC, which is termed pharmacological preconditioning (PPC) (Wakahara et al., 2004). The mechanisms of IPC and PPC have been under intensive investigation; however, it remains uncertain. Moreover, ischemic neurodegeneration seems to be multifactorial in that a complex series of toxic reactions including inflammation, energy depletion, and mitochondrial injury lead to the damage of neurons. Thus, the fundamental intention is to determine which of these factors constitute the primary event and whether they act in concurrence in the pathogenic process (Dhodda et al., 2004). This has led to the current notion that many drugs bind to more than one target, drugs directed against a single target will be ineffective, and rather a single drug or combination of drugs with pluripharmacological properties may be more attractive.
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B
, anti-NF-B p65, anti-phospho-ERK, anti-phospho-JNK, anti-phospho-p38 MAPK, anti-inducible nitric-oxide synthase (iNOS), anti-tyrosine hydroxylase (TH), anti-brain-derived neurotrophic factor (BDNF), anti-S-100 
, anti-heat shock protein 70 (HSP70), anti-Bcl-2, anti-Bax, anti-regulator of G protein signaling (RGS)2, anti-RGS4, anti-RGS9, anti-tubulin 5, anti-
-enolase, anti-cytochrome c, and anti--synuclein. Chemicals employed for gel electrophoresis were purchased from Bio-Rad (Hercules, CA).
Animals
Male SD rats weighing 220 to 250 g (Shanghai Experimental Animal Center of Chinese Academy of Sciences) were used in the present studies. Animals were allowed to acclimatize for at least 7 days before experimentation. The animals were housed in individual cages under light-controlled conditions and at room temperature. Food and water were available ad libitum. All animals received postoperative care in compliance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH publication 80-23, revised 1996).
Experimental Design
The rats were randomly divided into the following 11 groups (n = 10 for each group): 1, MCAO (90 min) and reperfusion (24 h) at 48 h after PF (10 mg/kg) administration; 2, MCAO (90 min) and reperfusion (24 h) at 48 h after PF (20 mg/kg) administration; 3, MCAO (90 min) and reperfusion (24 h) at 48 h after PF (40 mg/kg) administration; 4, MCAO (90 min) and reperfusion (24 h) at 24 h after PF (20 mg/kg) administration; 5, MCAO (90 min) and reperfusion (24 h) at 5 days after PF (20 mg/kg) administration; 6, MCAO (90 min) and reperfusion (24 h) at 7 days after PF (20 mg/kg) administration; DPCPX (1 mg/kg i.p., selective A1 receptor antagonist) 1 h before group 2; 8, naloxone hydrochloride (10 mg/kg s.c., nonselective opioid receptors antagonist) 1 h before group 2; 9, DPCPX (1 mg/kg i.p.) 48 h before group 1; 10, naloxone hydrochloride (10 mg/kg s.c.) 48 h before group 1; and 11, sham group.
Physiological parameters were monitored before, at the time of MCAO, 30 min after reperfusion, and 24 h after reperfusion, and the rectal temperature was monitored with a rectal probe and maintained at 37 ± 1 °C throughout the intraoperative period using a heating pad and a heating lamp.
Establishment of MCAO
The experimental MCAO rat model was conducted as described previously (Takano et al., 1997), with minor modification. In brief, the rats were anesthetized with choral hydrate (300 mg/kg i.p.). The left common carotid artery, external carotid artery, and internal carotid artery were exposed through a ventral midline incision. The external carotid was ligated distally, and the pterygopalatine artery was ligated at its origin. An arteriotomy was made in the external carotid allowing the introduction of a 4-0 surgical nylon monofilament with its tip rounded by heat. The filament was gently advanced into the internal carotid artery 20 mm past the carotid artery bifurcation, thereby occluding the origin of the middle cerebral artery. After occlusion for 90 min, the filament was gently withdrawn to permit reperfusion.
Determination of Neurological Symptoms
The severity of neurological symptoms was determined after transient MCAO and reperfusion according to the method of described previously (Sydserff et al., 2002) with a slight modification as follows: score 0, no apparent neurological deficits; score 1, contralateral forelimb flexion; score 2, decreased resistance to lateral push; score 3, spontaneous movement in all directions and contralateral circling when pulled by tail; and score 4, spontaneous circling. The above behavioral observations were carried out in a blinded manner.
Measurement of Infarct Size
Coronal sections of the brain were cut into 2-mm slices and immersed in 2% TTC solution at 37°C for 15 min, followed by 10% formaldehyde solution. The image of each slice was captured by digital camera. The infarct area and hemisphere area were traced and quantified by an image analysis system (Adobe Image Ready 7.0; Adobe Systems, Mountain View, CA). The infarct volume was corrected by standard methods (contralateral hemisphere volume - volume of nonischemic ipsilateral hemisphere) with infarcted volume expressed as a percentage of the contralateral hemisphere.
RNA Extraction and Reverse Transcription-Polymerase Chain Reaction (PCR)
Animals were killed 24 h after reperfusion, and the ipsilateral cortex was dissected from the MCA territory in the frontoparietal cortex (underlying MCA immediately distal to the region of occlusion) and used immediately for total RNA extraction with the TRI-REAGENT-LS extraction kit. The expression of RNAs was determined by real-time PCR (COX-2) or semiquantified reverse transcription-PCR (Kir6.1 and Kir6.2). The COX-2 primers were: forward, 5'-CCATGTCAAAACCGTGGTGAATG-3'; and reverse, 5'-ATGGGAGTTGGGCAGTCATCAG-3' (374 bp). The amplification reaction consisted of 35 cycles of denaturation (94°C, 30 s), annealing (68°C, 30 s), and elongation (72°C, 45 s). The -actin primers were: forward, 5'-AAGATGACCCAGATCATGTT-3'; and reverse, 5-TTAATGTCACGCACGATT T-3' (286 bp). The Kir6.1 primers were: forward, 5'-GAAGGAGAGGTGGTGCTATTCA-3'; and reverse, 5'-GTTGCTCCTCCTCATGGAGTTGT-3' (480 bp). The Kir6.2 primers were: forward, 5'-GCCATCATCCTTCCACCTCAGTT-3'; and reverse, 5'-CAGGCACTTCCGAAGCAAGTAT-3' (303 bp).
Western Blot Analysis
The isolated cortex identical to the sampling sites of RNA extraction was homogenized in lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, and protease inhibitor cocktail), centrifuged at 3000g for 10 min. Protein concentration was determined by Bio-Rad protein assay. Samples were electrophoresed in SDS-polyacrylamide gel electrophoresis gels and transferred onto a polyvinylidene difluoride membrane. The membrane was blocked with 5% nonfat milk in 1x Tris-buffered saline and 0.1% Tween 20 at 25°C for 1 h and subsequently incubated overnight at 4°C with appropriate primary antibody diluted in Tris-buffered saline/Tween 20 [Tris-buffered saline, 0.1% (v/v) Tween 20]. After incubation with horseradish peroxidase-conjugated secondary antibodies for 1 h, the blots were developed with chemiluminescence reagent and exposed to X-ray film.
Proteomics Analysis
Preconditioning Procedure. PF (40 mg/kg i.p., 24 h) was given to induce pharmacological preconditioning in normal rats, and saline was vehicle treatment in sham normal rats (n = 8 for each group).
Protein Sample Preparation. After 24-h preconditioning, the ipsilateral hippocampus, striatum, and cortex were homogenized on ice with a tissue tearer in lysis buffer (40 mM Tris, 8 M urea, 4% CHAPS, and a mixture of protease inhibitors). The suspension was centrifuged at 10,000g for 10 min, and the supernatant was centrifuged further at 25,000g for 45 min. Protein concentration was determined using the Bio-Rad protein assay.
Two-Dimensional Gel Electrophoresis. Samples of approximately 1.0 mg were applied on immobilized pH 4 to 7 linear gradient strips (Bio-Rad) in rehydration buffer (8 M urea, 2% CHAPS, 65 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 0.2% pI 3-10 ampholytes) and focused at 100,000 V/h. An equilibration buffer was prepared that contained 50 mM Tris-HCl, pH 8.8, 6 M urea, 30% glycerol, and 2% SDS. Immobilized pH gradient strips were equilibrated in 2% dithiothreitol and 2.5% iodoacetamide for 15 min, respectively. The two-dimensional (2D) separation was performed on 12.5% homogeneous polyacrylamide gels in a Bio-Rad mini-PROTEAN II gel apparatus (Hoefer Scientific Instruments, San Francisco CA). The gels were stained with Coomassie Blue.
Image Analysis. The gels were quantified using the PDQuest 7.4.1 software (Bio-Rad). Spot detection and matching between six gels (three from PF-treated gels and three from sham gels) were performed automatically, followed by manual matching, and statistical analysis was performed by Dunnett's test.
MALDI-TOF MS, 2D Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometry (LC-ESI-MS/MS), and Protein Identification. The protein spots of interest were punched out of gels and destained. The tryptic peptides were extracted with 60% acetonitrile and 5% trifluoroacetic acid onto a MALDI target plate. MALDI-TOF-MS analysis was performed on a Voyager DE-RP MALDI-TOF mass spectrometer (Applied Biosystems, Foster City, CA). Orthogonal 2D-LC-ESI-MS/MS was performed using a Proteome X Workstation (Thermo Finnigan, San Jose, CA). Trypsin autocleavage peaks were used as internal standards for the mass calibration. The above data were all subjected to database searches for protein identification using a MASCOT search engine. Multiple databases such as NCBInr and Swiss-Prot (http://www.expasy.org/sprot/) were searched to yield more comprehensible results.
Statistics
For statistical analysis, a standard software package (SPSS for Windows 10.1) was used. All data were given as means ± SD. Differences between groups were compared by using Dunnett's test. P < 0.05 was considered significant.
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Results |
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Mortality Rate after PF Preconditioning or DPCPX and Naloxone Pretreatment. Apart from PF (10 mg/kg, 48 h) and PF (20 mg/kg, 7 days) groups, the mortality of other PF pretreatment groups was significantly decreased compared with the MCAO group. The mortality of DPCPX (1 mg/kg) and naloxone (10 mg/kg) was similar to that of the MCAO group. However, the mortality of the DPCPX + PF group was significantly increased compared with PF (20 mg/kg, 48 h) group. With pretreatment of naloxone before PF preconditioning, the mortality was not changed compared with PF preconditioning (Table 2).
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PF Preconditioning in Rat MCAO Model. Histological analysis revealed a mean area of 41.7 ± 2.2% total infarct volume of the right hemisphere after 90 min MCAO followed by 24-h reperfusion (n = 10). PF pretreatment (48 h before MCAO, 20 or 40 mg/kg) significantly reduced the magnitude of ischemic lesion, with 16.2 ± 2.0% and 15.4 ± 4.9% respectively, decreases of 61 and 63% (P < 0.01, P < 0.01, n = 10). The neurological deficits caused by MCAO could also be inhibited by PF preconditioning (Fig. 2, A-C). PF (20 mg/kg i.p.) administered 24 h, 48 h, and 5 days before MCAO effectively ameliorated the ischemic infarct and neurological deficits in a time course-dependent manner (Fig. 2, D-F).
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Involvement of Adenosine A1 Receptors and Regulator of G Protein Signaling Proteins. When rats were injected with DPCPX (1 mg/kg i.p.) or naloxone (10 mg/kg s.c.) alone, 48 h before MCAO, no significant neuroprotective effect was found (P > 0.05 versus control, n = 10). However, when DPCPX (1 mg/kg i.p.) was administered 1 h before group 2 treatment, the neuroprotection induced by PF preconditioning was abolished. In contrast, naloxone (10 mg/kg s.c.) administered 1 h before group 2 treatment had no effects on the ameliorated infarct size and neurological symptoms (Fig. 3). Therefore, adenosine A1 receptor, one of the G protein-coupled receptors, was involved in PF preconditioning. In view of the fact that the regulator of G protein signaling (RGS) protein family has important roles in G protein-coupled receptor signal transduction, three different types of RGS proteins were investigated by Western blot (Fig. 4). Ischemia evoked the decline of RGS2, whereas with enhancement of RGS4 and RGS9 expression, these changes were significantly antagonized by PF preconditioning.
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Involvement of Cyclooxygenase-2,5-Lipoxygenase and ATP-Sensitive Potassium Channel. Because arachidonic acid cascade of inflammatory reaction was involved in cerebral ischemia, the expression of two rate-limiting enzymes, COX-2 and 5-LOX, was investigated in PF preconditioning. After MCAO and reperfusion, the expression level of COX-2 gene in brain cortex was increased rapidly (the cycle number decreased to 19.9 ± 0.8, P < 0.01 versus that of sham rats, n = 3, Fig. 5A). PF (20 mg/kg i.p., 48 h before MCAO) pretreatment significantly inhibited the up-regulation of COX-2 mRNA expression (the cycle number decreased to 22.4 ± 1.1, P < 0.01 versus that of MCAO rats, n = 3). It is known that opening of ATP-sensitive potassium (KATP) channels during ischemia is necessary for delayed preconditioning (Yoshida et al., 2004). Pore-forming KATP channel proteins, Kir6.1 and Kir6.2, were up- and down-regulated, respectively, thereby resulting in a significant down-regulation of the Kir6.2/Kir6.1 ratio in ischemia. However, the changes of Kir6.1 and Kir6.2 transcriptional expression and their ratio in ischemia were substantially reversed by PF preconditioning (Fig. 5B). Likewise, PF preconditioning also inhibited the overexpressed COX-2 and 5-LOX protein levels in ischemia (Fig. 5, C and D).
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, and Heat Shock Protein 70. A substantial increase in the expression of iNOS (Shen et al., 2006), HSP70 (Liu et al., 2005a), and S-100 activity (Buttner et al., 1997) after cerebral ischemia has been reported. Our results revealed that the overexpression of iNOS, HSP70, and S-100 were induced by ischemia and decreased significantly by PF preconditioning (Fig. 6A, D, and E).
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). In our experiments, the expression of TH and BDNF was up- and down-regulated, respectively, during ischemia and normalized nearly to sham levels by PF preconditioning (Fig. 6, B and C). Involvement of Apoptosis-Related Proteins. Although changes of apoptosis-related proteins have been considered crucially important in ischemia, the roles these proteins play in pharmacological preconditioning have not been elucidated. The down-regulated Bax proteins and overexpressed cytochrome c after MCAO and reperfusion as compensatory mechanisms were normalized to sham levels by PF preconditioning. However, the overexpression of Bcl-2 in ischemia was significantly inhibited by PF preconditioning (Fig. 7).
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B Signaling. Since many above proteins induced by ischemia including COX-2, 5-LOX, iNOS, HSP70, and apoptosis-related proteins are reported to be regulated, at least in part, by a series of upstream kinases collectively known as MAPK or NF-B signaling, the effects of PF preconditioning on ischemia-induced activation of MAPK and NF-B signaling effectors were examined. The JNK and p38 MAPK were significantly activated via phosphorylation in ischemia, and these changes were partially suppressed by PF preconditioning. However, the phosphorylation of ERK decreased significantly in ischemia and was partially reversed by PF preconditioning. The expression of NF-B and IB was significantly increased or decreased by ischemic insult; however, it was completely reversed by PF preconditioning (Fig. 8).
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Effects of PF Preconditioning on Brain Proteins Expression. To test the effects of PF preconditioning on brain proteins expression, 2-DE technique was carried out. Approximately 700 to 
850 spots were observed in 2D gels from PF and sham brain samples stained with Coomassie Blue (Fig. 9). The relative levels of 42 proteins (mean of three gels for each sample) are given in Table 3. Among them, 20 proteins were elevated, and 22 were reduced (Fig. 10; Tables 4 and 5). The identities of these proteins were revealed by MALDI-TOF and LC-ESI-MS/MS analysis.
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Immunoblot Validation of Differentially Expressed Proteins. Among the identified proteins, tubulin 5, -enolase, and -synuclein were selected for Western blot analysis (Fig. 11). The expression patterns of the selected proteins were in agreement with 2-DE results (Table 4 and Table 5). The expression of tubulin 5 and -enolase was depressed significantly after MCAO; however, it was enhanced by PF preconditioning. In contrast, the expression of -synuclein was increased in ischemia, and PF preconditioning decreased it. These results of Western blot analysis attested to the reliability of proteomics analysis.
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). The multifunctional targets involved in PF preconditioning are elucidated as follows.
Adenosine Receptors and Signaling Pathway. Because the selective A1 receptor antagonist DPCPX could cancel the beneficial effects of PF preconditioning, whereas nonselective opioid receptor antagonist naloxone had no effect on it, the adenosine A1 receptor, rather than opioid receptors, was involved in PF preconditioning. It has been reported that PF could interact with A1 receptors (Liu et al., 2005b) and enter the central nervous system.
As GTPase-activating proteins, RGS2 and RGS4 were specifically considered as inhibitors of Gq- or Gi-mediated signaling, respectively (Heximer et al., 1999). PF preconditioning promotes RGS2 expression but inhibits RGS4 expression, indicating that PF pretreatment can attenuate Gq signaling but promote Gi-mediated signaling involved in ischemia. Furthermore, the expression of the brain-specific regulator of RGS9 was increased in ischemia, and this change was inhibited by PF preconditioning, consistent with a report (Bouhamdan et al., 2004) suggesting that the abundance of striatal RGS9 was increased in Parkinson's disease.
It has been shown that ischemic preconditioning causes the release of adenosine and the resultant activation of adenosine A1 receptor, then may activate K+ channels, thereby conferring ischemic tolerance (Reshef et al., 2000). It is known that opening of KATP channels, consisting of the inwardly rectifying K+ channel family (Kir6.1 and Kir6.2) and the sulfonylurea receptor isoforms (SUR1, SUR2A and SUR2B) during ischemia, is necessary for delayed preconditioning (Yoshida et al., 2004). Consistent with this conclusion, PF preconditioning substantially reversed the changes of Kir6.1 and Kir6.2 transcriptional expression and their ratio in ischemia. Therefore, suggestions can be made that A1 receptor-RGS-KATP signaling may be involved in PF preconditioning.
Arachidonic Acid Cascade of Inflammatory Reaction and Nitric Oxide System. COX-2 and 5-LOX are rate-limiting enzymes in the metabolism that convert arachidonic acid to prostaglandins and leukotrienes, which lead to enhanced vascular permeability, inducing the formation of vasogenic edema. The up-regulated COX-2 mRNA, COX-2, and 5-LOX protein expression after MCAO were substantially inhibited by PF preconditioning, consistent with reports showing that COX-2-deficient mice had a significant reduction in the brain injury (Sasaki et al., 2004), and translocation of 5-LOX to membrane was associated with reperfusion (Ohtsuki et al., 1995). PF preconditioning could also inhibit toxic NO production through decreasing iNOS expression, increasing glyoxalase 1 and dimethylarginine dimethylaminohydrolase expression. Evidence has been shown that iNOS null mice had smaller infarcts after focal ischemia (Park et al., 2006), overproduced NO could inactivate glyoxalase 1 (Mitsumoto et al., 1999), and dimethylarginine dimethylaminohydrolase can regulate the generation of nitric oxide (Tran et al., 2003).
Markers of Neuronal Damage. Increased neuron-specific enolase expression (Fontella et al., 2005) and decreased -enolase expression (Sensenbrenner et al., 1997) were reported in oxygen and glucose deprivation-induced in vitro ischemia and in vivo ischemia, respectively. Consistent with these data, PF pretreatment could decrease the expression of neuron-specific enolase and increase the expression of -enolase, which may contribute to its preconditioning.
In addition, PF preconditioning also inhibited the overexpression of synaptosomal-associated protein of 25 kDa (SNAP25); TH, the rate-limiting enzyme of dopamine biosynthesis (Soriano et al., 1997); and S-100 , an astroglial protein (Ahlemeyer et al., 2000), suggesting that inhibition of dopaminergic neurons lost, and microglial activation is one of the outcomes of PF preconditioning.
Mitochondrial Damage-Related Molecules. Apoptosis-related proteins, microtubule-related proteins, chaperones, ubiquitin-proteasome system, and ATP synthase are mitochondrial damage-related molecules involved in cerebral ischemia. Mitochondrial injury is reported to cause cytochrome c release into the cytosol that initiates the apoptotic cell death cascade (Carboni et al., 2005). Our results demonstrated the increased expression of Bcl-2 and cytochrome c and decreased expression of Bax in ischemia as a compensatory mechanism, and these changes were normalized by PF preconditioning. As the major components of neuronal microtubules, the expression of tubulin 5 was increased, whereas tubulin chain 15 was decreased significantly after PF preconditioning in 2D gel analysis, and the subsequent Western blot analysis confirmed these changes. Diminished expression of tubulin 5 is a putative marker for ischemic injury, correlating to the vulnerability in ischemia-induced neuronal death (Dewar and Dawson, 1997).
Microtubule breakdown induced by ischemia may impair trafficking, leading to cytoplasmic accumulation of HSP70 and peptidyl-prolyl cis/trans isomerases (PPIases) into large abnormal protein aggregates (Ramakrishnan et al., 2003; Liu et al., 2005a). Consistent with these data, PF preconditioning down-regulated the protein expression of HSP70 and PPIase. However, down-regulation of HSP70 after PF preconditioning is in contrast to the report showing that transgenic mice overexpressing HSP70 reduced mitochondrial cytochrome c release and diminished apoptotic cell death after MCAO (Kokubo et al., 2002).
The expression of proteasome and ubiquitin-conjugating enzyme E2N, two main components of ubiquitin-proteasome system (Prasad et al., 2002), were significantly increased by PF preconditioning (Table 4). However, -synuclein, a presynaptic protein involved in synaptic vesicle trafficking, was down-regulated by PF preconditioning (Table 5).
The activity of cytochrome c oxidase was reported to increase significantly as a compensatory mechanism to maintain cellular energy levels after MCAO before evident ischemic damage (Inoue et al., 1996). PF preconditioning normalized the overexpression of cytochrome c oxidase, indicating that integrity of mitochondrial oxidative phosphorylation protection might be involved in the neuroprotection of PF preconditioning.
As a major consumer of ATP under ischemic conditions, the mitochondrial ATP synthase switches its catalytic activity from ATP synthesis to ATP hydrolysis when the electrochemical gradient across the inner membrane collapses (Iijima et al., 2003). In the present studies, PF preconditioning alone significantly down-regulated the expression of ATP synthase, indicating that the ability of PF to maintain ATP levels was beneficial.
MAPK and NF-B Pathways. Studies have shown that activation of the ERK signaling pathway was critical for IPC-induced neuroprotection, and the p38 pathway could mediate neuronal damage by counteracting ERK signaling in models of apoptosis and free radical damage in vitro (Jiang et al., 2005). Consistent with these data, PF preconditioning potently restrained the activation of p38 MAPK and JNK, however, significantly promoted the activation of ERK.
Our results also revealed that ischemia induced the overexpression of NF-B and decreased expression of its endogenous inhibitor IB- (I-B), whereas these changes were antagonized by PF preconditioning, consistent with the report showing that NF-B and subsequent degradation of I-B are the key events in ischemia and reperfusion (Song et al., 2005).
In conclusion, we showed for the first time that a delayed neuroprotection could be induced by PF preconditioning and that several molecular and protein changes are observed in the mechanisms of PF preconditioning. These results may have important significance in the treatment of ischemic stroke.
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Footnotes |
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D.-M.C. and L.X. contributed equally to this work.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: MCA, middle cerebral artery; PF, paeoniflorn; IPC, ischemic preconditioning; PPC, pharmacological preconditioning; DPCPX, 8-cyclopentyl-1,3-dipropylxanthine; TTC, 2,3,7-triphenyltetrazolium chloride; COX, cyclooxygenase; 5-LOX, 5-lipoxygenase; NF, nuclear factor; ERK, extracellular signal-regulated kinase; JNK, c-Jun NH2-terminal kinase; MAPK, mitogen-activated protein kinase; iNOS, inducible nitric-oxide synthase; TH, tyrosine hydroxylase; BDNF, brain-derived neurotrophic factor; HSP70, heat shock protein 70; RGS, regulator of G protein signaling; MCAO, middle cerebral artery occlusion; PCR, polymerase chain reaction; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; 2D, two-dimensional; MALDI, matrix-assisted laser desorption ionization; TOF, time-of-flight; MS, mass spectrometry; LC-ESI-MS/MS, liquid chromatography electrospray ionization tandem mass spectrometry; SNAP25, synaptosomal-associated protein of 25 kDa; PPIase, peptidyl-prolyl cis/trans isomerase; I/R, ischemia and reperfusion injury; 2-DE, two-dimensional electrophoresis.
Address correspondence to: Dr. Xing-Zu Zhu, Department of Pharmacology, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 555 Zu Chong Zhi Road, Zhangjiang Hi-Tech Park, Pudong Shanghai 201203, China. E-mail: xzzhu{at}mail.shcnc.ac.cn
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