Protective Effects Of Total Flavones Extracted From Chrysanthemum Morifolium On Rat Brain Against Cerebral Ischemia/reperfusion Injury

BackgroundStroke has been identified medically as one of the major killers to threaten the human beings, and has turned into a healthy problem worldwide. Stroke has the high mortality and disability rate, and it has been proved that over 80% of the people above 35 years old are prone to stroke to different extent. Although thrombolytic drugs, such as tissue plasminogen activator, prourokinase, low-molecular-weight heparin, or ancrod, have significantly improved the clinical outcome in patients with acute ischemic stroke, the targets of these drugs are thrombolysis and/or prevention of further intravascular clot formation to rescue neurons from ischemic stress, but not to protect neurons under ischemia.Flavonoids has been used in traditional Chinese medicine fore thousands of years, and are consumed regularly with the human diet. On average, the daily USA diet was estimated to contain approximately 1 g of mixed flavonoids. The flavonoids are found in fruits, vegetables, nuts, seeds, herbs, spices, stems, flowers, as well as tea and red wine. They are prominent components of citrus fruits and other food sources. They are low molecular weight compound composed of three-ring structure with different substituents. A resurgence of interest in traditional Chinese medicine during the past two decades, together with an expanded effort in pharmacognosy, has rekindled interest in the flavonoids and the need to understand their interaction with mammalian cells and tissues. The flavonoids have long been recognized to possess anti-inflammatory, antioxidant, antiallergic, hepatoprotective, antithrombotic, antiviral, and anticarcinogenic activities.Chrysanthemum morifolium contains flavonoids such as luteolin, apigenin and acacetin. Recently it has been reported that total flavones extracted from plants, such as total flavones of ginkgo biloba, total flavones of hawthorn leaves protect brain against hypoxia or ischemia injury, and has inhibitory effect on ischemia-induced apoptosis of neurons. Choi et al. found that flavones in extracts of scutellaria baicalensis can protect neuronal cells from apoptosis and inhibit protein oxidation. And our previous study also demonstrated that water extract of chrysanthemum morifolium not only has significant inhibiting effect on liver microsomer, brain mitochondria, and heart mitochondria against lipid peroxidation induced endogenously or by hydroxy radicals, but also can significantly attenuate the decrease in superoxide dismutase (SOD) activity and the elevation of malondialdehyde (MDA) content in the myocardium. However, the effect of total flavones extracted from chrysanthemum morifolium on ischemia/reperfusion-injured brain is not known.AimThe aim of the present study was to investigate the protective effect of total flavones extracted from chrysanthemum morifolium on rat brain against transient focal cerebral ischemia/reperfusion injury by occluding the middle cerebral artery and the possible mechanism.Methods1. Establishment of MCAO modelMCAO model was established by an intraluminal middle cerebral artery (MCA) blockade. After 90 min of ischemia and reperfusion for 22 h, rats were decapitated.2. Neurological function scoring, the measurement of cerebral infarction and the brain edema formationAfter reperfusion, neurological deficit was evaluated by using a 10-point neurological function scoring system, then the rats were decapitated immediately. Each brain was sliced coronally at 2-mm intervals, soaked in 2% solution of 2,3,5-triphenyl tetrazolium chloride (TTC) in 0.01 mol/L PBS (pH 7.4), and fixed in 10% buffered formalin. The ratio of infarct area over the whole area was quantified by an image analysis system (Image J, NIH). Hemispheric swelling (edema) was expressed as the percent increase in area of the ipsilateral brain hemisphere over the contralateral hemisphere.3. Measurement of the SOD activity and the MDA content in brain tissueThe sample of brain homogenate was prepared, and the SOD activity and the MDA content were measured.4. Measurement of reactive oxygen speciesThe sample of brain homogenate was prepared, reactive oxygen species (ROS) generation was measured spectrophotofiuorometrically. H2DCFDA was used as the indicator. Upon oxidation by ROS, H2DCF yields dichlorofluorescein (DCF) which fluorescence intensity represents the content of ROS.5. The isolation of brain mitochondriaThe brain mitochondria was isolated according to the procedure described by Kosenko et al.6. Measurement of the mitochondrial swellingMitochondrial swelling was induced by Ca2+, absorbance of isolated brain mitochondria at 540 nm was measured by spectrophotometer continuously.7. Measurement of TFCM components in cerebrospinal fluid and plasmaSamples of cerebrospinal fluid and plasma were prepared respectively, high performance liquid chromatography (HPLC) assay for analysis of TFCM components in these samples was used (using a mobile phase methanol: 0.2% phosphoric acid aqueous solution 55:45, V/V).Results1. Effect of TFCM on the neurological deficit scores in rats subjected to 1.5 h of middle cerebral artery occlusion and 22 h of reperfusionTreatment with TFCM (50, 100, 200 mg/kg, ip) significantly and dose-dependently decreased neurological deficit scores compared with the model group.2. Effect of TFCM on cerebral infarctionAdministration of TFCM at 50, 100 or 200 mg/kg produced 30.18%, 45.78% or 53.24% reduction compared with the model group in the ratio of infarct area over the whole area, respectively.3. Effect of TFCM on cerebral edema formationAdministration of TFCM at 50, 100 or 200 mg/kg produced 27.85%, 34.27% or 42.58% reduction compared with the model group in the percent increase in area of the ipsilateral brain hemisphere over the contralateral hemisphere, respectively.4. Effects of TFCM on the activity of superoxide dismutase (SOD) and the content of malondialdehyde (MDA) after cerebral ischemia/reperfusion in ratAdministration of TFCM at 50, 100 or 200 mg/kg significantly attenuated the decrease in SOD activity and the elevation of MDA content compared with the model group.5. Effect of TFCM on the generation of ROS in rat brainAdministration of TFCM at 100 mg/kg produced significantly reduction in the fluorescent intensity of rat brain compared with the hypoxia group.6. Effect of TFCM on Ca2+-induced mitochondrial swellingAdministration of TFCM at 50, 100 or 200 mg/kg significantly attenuated the Ca2+-induced mitochondrial swelling compared with the Ca2+ only group, and this effect was antagonized by atractyloside.7. TFCM components in cerebrospinal fluid and plasmaLuteolin and apigenin were detected in cerebrospinal fluid, and the content of apigenin was higher. However the levels of luteolin and apigenin were significantly lower than that of plasma.Conclusions1. TFCM has significantly protective effect on rat brain against cerebral ischemia/reperfusion injury.2. The underlying mechanism may be with relation to its antioxidant action and consequently inhibiting the mitochondrial swelling.3. Luteolin and apigenin, as two major components of TFCM, can directly penetrate the blood-brain barrier.