Materials
Puerarin, 2,3,5-triphenyltetrazolium (TTC), aprotinin, cremophor EL, leupeptin, lucigenin, N-formyl-Met-Leu-Phe (fMLP), and bovine serum albumin (BSA) were purchased from Sigma (St. Louis, MO). Ficoll-Paque plus was purchased from Amersham (Buckinghamshire, HP, UK). Puerarin was dissolved in solvent (cremophor: ethanol: normal saline at 1: 1: 4) for the in vivo studies, and dissolved in 0.5% DMSO for the in vitro studies.
MCAO-induced transient focal cerebral ischemia in rats
Male Wistar rats (250~300 g) were used in this study. All animal experiments and care were performed according to the Guide for the Care and Use of Laboratory Animals (National Academy Press, Washington, DC, 1996). Before undergoing the experimental procedures, all animals were clinically normal, were free of apparent infection or inflammation, and showed no neurological deficits.
Animals were anesthetized with a mixture of 75% air and 25% O2 gases containing 3% isoflurane. The rectal temperature was maintained at 37 ± 0.5°C. The right middle cerebral artery (MCA) was occluded as described in our previous report [14]. Briefly, the right common carotid artery was exposed, and a 4-0 monofilament nylon thread (25 mm) coated with silicon was then inserted from the external into the internal carotid artery until the tip occluded the origin of the MCA. After closure of the operative sites, the animals were allowed to awake from the anesthesia. During another brief period of anesthesia, the filament was gently removed after 1 h of MCAO. An observer blinded to the identity of the groups assessed the neurological deficits at 1 and 24 h after reperfusion (before euthanization) by the forelimb akinesia (also called the postural tail-hang) test, whereas the spontaneous rotational test was used as a criterion for evaluating the ischemic insult [15]. Animals not showing behavioral deficits at the above time points after reperfusion were excluded from the study. On the other hand, reperfusion was also ensured by an improvement in ipsilateral local blood flow to at least 60% of the baseline following an initial sharp decrease to about 50–60% of the baseline caused by MCAO as determined using a continuous laser Doppler flowmeter (LDF; Oxford Array™, Oxford Optronix, Oxford, UK) with a standard needle probe (pp-051). For the continuous measurement of local regional cerebral blood flow (rCBF), the rat was fixed in a stereotaxic frame. The skull was exposed, and a burr hole in a diameter of 2 mm was drilled on the right side of the skull at 2 mm posterior and 5 mm lateral to the bregma to accommodate the probeholder. LDF was used to estimate blood flow continuously during ischemia and reperfusion.
Rats were euthanized by decapitation after 24 h of reperfusion. The brains were cut into 2-mm coronal slices starting 1 mm from the frontal pole. Each stained brain (2% TTC) slice was drawn using a computerized image analyzer (Image-Pro plus). The calculated infarct areas were then compiled to obtain the infarct volume (mm3) per brain. Infarct volumes were expressed as a percentage of the contralateral hemisphere volume using the formula: (the area of the intact contralateral [left] hemisphere – the area of the intact region of the ipsilateral [right] hemisphere) to compensate for edema formation in the ipsilateral hemisphere [14].
All animals were divided into four groups: (1) a sham-operated group; (2) a solvent-treated (solvent) group (cremophor: ethanol: normal saline at 1: 1: 4); and groups treated with a single dose of (3) 25 or (4) 50 mg/kg, i.p. of puerarin. In the group treated with the solvent or puerarin, rats were given the isovolumetric solvent or puerarin (25 or 50 mg/kg) 10 min before MCAO.
Neurobehavioral test
The sensorimotor integrity was conducted to assess the neurobehavior at 1 and 24 h after MCAO in rats [15]. Five categories of motor neurological findings were scored: 0, no observable deficit; 1, forelimb flexion; 2, forelimb flexion and decreased resistance to lateral push; 3, forelimb flexion, decreased resistance to lateral push and unilateral circling; 4, forelimb flexion, unable or difficult to ambulate.
Determination of the expressions of HIF-1α, iNOS, and active caspase-3 in MCAO-insulted brain
The expressions of HIF-1α, iNOS, and active caspase-3 in the brain at 24 h after MCAO-reperfusion injury were analyzed by Western blotting as described by Rodrigo et al. [16] with minor modifications. The MCAO-insulted and sham-operated rats were anesthetized with chloral hydrate (400 mg/kg, i.p.), then the apex of the heart was penetrated with a perfusion cannula inserted through the left ventricle into the ascending aorta. Perfusion with ice-cold phosphate-buffered saline (PBS) was performed, and an incision was made in the right atrium for venous drainge. Brains were removed freshly and sectioned coronally into four sequential parts from the frontal lobe to the occipital lobe. The third parts of the right hemisphere was separately collected, snap-frozen in liquid nitrogen, and stored at -70°C. The frozen tissues were placed in homogenate buffer and homogenized, then sonicated for 10 s three times at 4°C. The sonicates were subjected to centrifugation (10,000 g).
The supernatant (50 μg protein) was subjected to SDS-PAGE and electrophoretically transferred to PVDF membranes (0.45 μm; Hybond-P; Amersham). After incubation in blocking buffer and being washed three times with TBST buffer (10 mM Tris-base, 100 mM NaCl, and 0.1% Tween 20; pH 7.5), the blots were treated with an anti-HIF-1α polyclonal antibody (pAb, 1: 1000; R&D, Minneapolis, MN), an anti-iNOS monoclonal antibody (mAb; 1: 3000, BD Biosciences, San Jose, CA), and an anti-active caspase-3 pAb (1: 250; Biovision, Mountain View, CA), or an anti-α-tubulin mAb (1: 2000; Santa Cruz Biotech, Santa Cruz, CA) in TBST buffer overnight. Blots were subsequently washed with TBST and incubated with secondary horseradish peroxidase-conjugated goat anti-mouse mAb or donkey anti-rabbit IgG (Amersham) for 1 h. The blots were then washed, and the immunoreactive protein was detected using film exposed to enhanced chemiluminescence detection reagents (ECL+ system; Amersham). The bar graph depicts the ratios of quantitative results obtained by scanning reactive bands and quantifying the optical density using Videodensitometry (Bio-1D version 99 image software).
Isolation of total RNA and reverse-transcription polymerase chain reaction (RT-PCR)
Fresh brains were removed, separated into ipsilateral and contralateral hemispheres, snap frozen in liquid nitrogen and stored at -70°C until used as stated above. Total RNA was isolated from the ipsilateral cortex by a commercially available kit according to the manufacturer's instructions (TRIzol, Gibco). For each RT-PCR, 0.5 mg of the RNA sample and 0.2 μM of primers were reverse-transcribed and amplified in a 50-μl reaction mixture of commercially available reagents (SUPERSCRIPT One-Step RT-PCR with PLATINUM Taq Kit, Invitrogen) containing a 1× reaction mixture and 0.2 μM of an RT/Taq mixture in one cycle of 30 min at 50°C for reverse transcription and one cycle at 95°C for 3 min, followed by 40 cycles at 95, 62, and 72°C for 30, 40, and 40 s, respectively; with a single extension step at 72°C for 5 min followed by 4°C for amplification in a thermal cycler (GeneAmp PCR system 2400, Perkin-Elmer). For visualization and quantification by densitometry of each RT-PCR, a 10-μl aliquot was subjected to electrophoresis on a 1.5% agarose gel using a mini horizontal submarine unit (HE 33) containing 0.5 mg/ml ethidium bromide to allow UV-induced fluorescence (TCP-20.M, Vilber Lourmat). Preliminary experiments were performed to determine the range of amplification cycles and the beginning RNA substrate within the linear phase of the exponential increase of the PCR products for each particular primer pair.
Determination of respiratory bursts in human neutrophils
Superoxide anion production of neutrophils was measured by the method of lucigenin-enhanced chemiluminescence (LCL) as described by Hsiao et al. [17] with some modifications. Washed neutrophil suspensions (2 × 106 cells/ml) in modified Hank's balanced salt solution (HBSS) containing 1 mM CaCl2 and 0.5 mM MgCl2 were dispensed into wells of a standard scintillation microplate. Before the assay, cells were preincubated with the solvent control (0.5% DMSO) or various concentrations of puerarin (10, 20, and 50 μM). Then, 20-μl aliquots of lucigenin were added at a final concentration of 100 μM. The basal LCL was recorded for 1 min by a microplate luminometer (Orion®, Berthold, Germany) at 37°C, and cells were immediately stimulated with fMLP (800 nM). The luminescent light was continuously recorded for 5 min. The chemiluminescent signal was represented as relative light units per second (RLU/s). The results of LCL intensity (as increments in the signal intensity) were determined by measuring basal and stimulator-induced peak values and calculating the difference between them.
Antioxidative activity of puerarin in rat brain homogenate preparations
Rat brain homogenates were prepared from brains of freshly killed Wistar rats, and the peroxidation in the presence of iron ions was measured by the thiobarbituric acid (TBA) method, as described by Braughler et al. [18] with some modifications. In brief, whole brain tissue, excluding the cerebellum, was washed and homogenized in 10 volumes of ice-cold Krebs buffer. The homogenate was centrifuged, and the supernatant was used immediately for the lipid peroxidation assays. The reaction mixture with puerarin or vehicle solution (0.5% DMSO) was incubated for 10 min, then stimulated by the addition of ferrous ions (200 μM). The reactions were terminated by adding a trichloroacetic acid solution and the TBA-reactive substance (TBARS) reagent. After boiling for 15 min, samples were cooled and extracted with n-1-butanol. The extent of lipid peroxidation was estimated as TBARS and was read at 532 nm in a spectrophotometer (Hitachi, Model U3200). Tetramethoxypropane was used as a standard, and the results were expressed as nanomoles of malondialdehyde equivalents per milligram protein of the supernatant of rat brain homogenates. The protein contents of the brain homogenates and other preparations were determined with the Bio-Rad method.
Electron spin resonance (ESR) spectrometry
ESR spectra were recorded on a Bruker EMX ESR spectrometer in the H2O2/NaOH/DMSO system as described previously [13]. Briefly, 100 μl of DMSO and the same volumes of 25 mM NaOH and puerarin (200 and 500 μM) were mixed in a test tube, followed by the addition of 10 μl DMPO and 100 μl of 30% H2O2. The reaction mixture was aspirated into a quartz flat cell and set in the ESR apparatus; scanning was begun 1 min after all reagents were mixed. The rate of free radical-scavenging activity was defined by the following equation: inhibition rate = 1-[signal height (puerarin)/signal height (solvent control)] [13].
Statistical analysis
The experimental results are expressed as the means ± S.E.M. and are accompanied by the number of observations. Student's unpaired t-test was used to determine significant differences in the study of MCAO-induced cerebral ischemia. The other experiments were assessed by the method of analysis of variance (ANOVA). If this analysis indicated significant differences among the group means, then each group was compared using the Newman-Keuls method. A P value of < 0.05 was considered statistically significant.
