Protective Effects of Scutellarin and Breviscapine on Brain ... : Journal of Cardiovascular Pharmacology (original) (raw)
INTRODUCTION
Erigeron breviscapus (vant.) Hand-Mazz. (Fig. 1A) is an important traditional drug in China. It belongs to the family of compositae and is mainly distributed in southwest China. Breviscapine is the extract from this plant. Many recent studies have confirmed the neuroprotection and anticoagulation effects of breviscapine.1-3 Its preparations are widely used in clinics to treat cerebral insufficiency and peripheral circulatory problems. A high-performance capillary electrophoresis with electrochemical detection method was performed for the determination of the pharmacologically active ingredients in breviscapine and its extract phytopharmaceuticals. Under the optimal conditions, 9 ingredients: baicalein, naringenin, scopoletin, kaempferol, apigenin, scutellarin, luteolin, caffeic acid, and protocatechuic acid were separated from breviscapine.4 Scutellarin (shown in Fig. 1B), a known flavone 7-O-glucuronide with a molecular weight of 462.21, is considered the primary active ingredient of breviscapine. The present study was designed to study the protective effects of scutellarin on cerebral and myocardial ischemia and to compare these effects of scutellarin with its mixture, breviscapine.
(A) Erigeron breviscapus (vant.) Hand and (B) the structure of scutellarin.
MATERIALS AND METHODS
Animals
Male Sprague-Dawley (SD) rats were provided by the animal center of our university. The rats were housed with controlled temperature (23-25°C) and lighting (8:00-20:00 light, 20:00-8:00 dark) with free access to food and drinking water. All the animals used in the experiment received humane care. All surgical and experimental procedures were in accordance with institutional animal care guidelines.
Determination of Myocardial Infarction (MI) in Rats
The MI was performed on SD rats of 200 to 250 g according to a well-accepted technique.5,6 Under ether anesthesia, the heart was exteriorized via a left thoracotomy, and left anterior descending (LAD) arteries was ligatured with 6-0 polypropylene suture between the pulmonary outflow tract and left atrium. Then the beating heart was quickly returned to its normal position, the thorax was closed, and the air was removed. Rats were returned to cages with the previously mentioned condition. Four hours after the coronary artery ligature, the MI size was measured according to the previous methods.7,8 Briefly, the rats were killed by an overdose of pentobarbital. The left ventricle was isolated and cut into 4 to 5 slices perpendicular to the cardiac long axis. The slices were stained for 30 min at 37°C in 0.1% solution of nitro blue tetrazolium (NBT) phosphate buffer. The normal tissue was stained in blue, while necrotic tissue remained unstained. Stained and unstained tissues were isolated and weighed separately. The MI size was expressed as a fraction of the total left ventricular weight.
Assessment of Apoptosis
Rats were killed by an overdose of pentobarbital 24 hours after LAD coronary artery ligature. The hearts were isolated and cut into 4-5 slice perpendicular to the cardiac long axis, then fixed in 10% buffered formalin and embedded in paraffin preparing for apoptosis detection of myocardial cells. Apoptosis was assessed from TdT-mediated dUTP-biotin nick end labeling (TUNEL)-stained sections (TUNEL; Boster, China). The process was performed according to the manufacturer's instructions. Briefly, the section was treated with 250 μL of proteinase K solution, followed by a treatment with 50 μL of terminal deoxynucleotidyl transferase buffer. After removing the buffer, the section was reacted with diaminobenzidine enzymatic substrate kit. Controlling reaction time by microscope and observed by high power microscope (×400).
Cerebral Ischemia in Rats
Rats were anesthetized with chloral hydrate (300 mg/kg, IP). Focal cerebral ischemia was induced by occlusion of the middle cerebral artery (MCA) using an intraluminal filament technique according to the previously described method.9,10 Briefly, after a midline neck incision had been made, the left common and external carotid arteries were isolated and ligatured. A nylon monofilament (Ethilon) coated with silicon resin was introduced through a small incision into the common carotid artery and advanced to a position 18 mm distal from a carotid bifurcation for occlusion of the MCA. Scutellarin and breviscapine were administered intravenously via the ranine vein.
Brain samples were obtained 24 hours after MCA occlusion, and coronal sections of 2-mm thickness were immediately stained with 2% 2,3,5-triphenyltetrazolium chloride (TTC) as previously described.11 The infarct region was in white, and the normal region was in red. The infarct area and hemisphere areas of each section were traced and quantitated by an image analysis system (Microsystems Type DM LB2, Leica, Germany). The possible interference of brain edema to infarct volume was corrected by standard methods (contralateral hemisphere volume - volume of nonischemic ipsilateral hemisphere), with infarct volume expressed as a percentage of the contralateral hemisphere.
Protocol
Experiment 1: Effect of Scutellarin and Breviscapine on MI in Rats
Experiment 1 was performed in 6-week-old male SD rats. Forty-two animals were randomly divided into 7 groups: control group, scutellarin groups (5, 15, and 50 mg/kg) and breviscapine groups (50, 150, and 500 mg/kg), n = 6 in each group. Scutellarin and breviscapine (Lokis Pharmaceuticals, Beijing, China) dissolved in the 5% solution of glucose (pH 7.4) by sodium hydroxide were administered via abdominal cavity immediately after LAD ligature. Four hours later, the heart was dissected to measure the infarct size.
Experiment 2: Effect of Scutellarin on the Apoptosis of Myocardial Cells in MI Rats
Experiment 2 was performed in 12 male SD rats aged 6 weeks. They were randomly divided into 2 groups: control and scutellarin 50 mg/kg groups (n = 6 in each group). Scutellarin and the vehicle were given via abdominal cavity immediately after LAD ligatures. Twenty-four hours after ligature, hearts were dissected for apoptosis detection with above-mentioned methods.
Experiment 3: Effects of Scutellarin and Breviscapine on Cerebral Infarction in Rats.
Experiment 3 was performed in 42 male SD rats aged 8 weeks. They were randomly divided into 7 groups: control group, scutellarin groups (5, 15, and 50 mg/kg) and breviscapine groups (5, 15, and 50 mg/kg) (n = 6 in each group). Drugs and vehicle were administered via ranine vein immediately after MCA occlusion with above-mentioned methods. Twenty-four hours later, the brain samples were obtained to analyze infarct size.
Experiment 4: Acute Toxicity Test
Experiment 3 was performed in KM mice with body weight of 18 to 25 g, either sex. Mice were fasted for 24 h before drug administration. Drugs and vehicle were administered via abdominal cavity at the dosage of 1500 to 3500 mg/kg in scutellarin groups and 1500 to 4000 mg/kg in breviscapine groups. Toxic signs and death were recorded up to the fourteenth day, when the test was terminated. LD50 value was calculated by the Litchfield-Wilcoxon method.12
Statistics
Investigators were blind to the procedures during the determination of MI and cerebral infarction, weighing, and counting of apoptosis cells. Statistical analysis data are expressed as the mean ± SD. The comparisons among pre- and post-operation or drugs were made by paired t tests; comparisons between two groups were made by unpaired _t_-tests. The comparisons among three or more groups were made by one-way analysis of variance (ANOVA). Statistical significance was accepted at the 5%.
RESULTS
Effects of Scutellarin and Breviscapine on MI in Rats
The average infarct size of the experimental rats, expressed as a ratio of infarct region weight to left ventricle weight, is presented in Table 1. Meanwhile, the inhibition ratio of infarct size [(MI size of control − MI size of treatment)/MI size of control] is shown in Figure 2. Compared with control group, the average MI size was significantly decrease by scutellarin at both 15 and 50 mg/kg; and the inhibition ratio was significantly increased. This effect of scutellarin was dose-dependent from 5 to 50 mg/kg. However, the infarct size was not significantly changed in breviscapine-treated groups (50 to 500 mg/kg).
The effect of scutellarin and breviscapine on acute myocardial infarction in rats
The difference of inhibition ratio [(MI size of control − MI size of treatment) / MI size of control] between scutellarin and breviscapine groups in rats subjected to 4 h of left anterior descending (LAD) occlusion. MI, myocardial infarction.
Effect of Scutellarin on Apoptosis of Myocardial Cells in MI Rats
Apoptotic index (/HP) was defined as average number of TUNEL-positive nuclei in 5 fields at ×400 magnification in the infarct region. As shown in Figures 3A and 3B, fewer apoptotic cells were seen in scutellarin (50 mg/kg) group than in control group (34.7 ± 4.25 versus 41.8 ± 2.77%/HP, P < 0.05, n = 6 in each group).
Photomicrograph (light microscopic amplification × 400) of myocardium with TUNEL staining in the infarct region of myocardium 24 h after coronary ligature in rats without (A) or with (B) scutellarin (50 mg/kg) treatment. (C) In the infarct region, the number of TUNEL-positive nuclei was significantly reduced by scutellarin. P < 0.05 versus control group.
Effects of Scutellarin and Breviscapine on the Cerebral Infarction in Rats
Focal brain ischemia was induced by MCA occlusion with the intraluminal filament technique. Sections are shown in Figure 4A. For the summary shown in Figure 4B, the data are the sum of 5 brain slides from each animal and 6 animals in each group. Compared with control, there were significant decreases in infarct volume in both scutellarin and breviscapine-treated groups from 5 to 50 mg/kg (scutellarin: 44.0 ± 9.00%, 40.7 ± 6.00%, and 33.3 ± 5.00% versus 58.4 ± 7.00%; breviscapine: 43.9 ± 3.00%, 43.1 ± 3.00%, and 42.6 ± 10.0% versus 58.4 ± 7.00%). According to the inhibition ratio [(infarction volume of control − infarction volume of treatment)/infarction volume of control], the effect of scutellarin was dose-dependent (5 mg/kg: 25.4%; 15 mg/kg: 32.3%; 50 mg/kg: 44.1%; P < 0.01), and breviscapine was not (Fig. 4C).
(A) Typical sections of ischemic brain in rats subjected to 24 h of middle cerebral artery (MCA) occlusion with or without scutellarin or breviscapine treatment. MCA occlusion produced an ipsilateral focal infarct (white area) as shown by 2,3,5-triphenyltetrazolium chloride (TTC) staining. Upper: control group. Middle: breviscapine treatment (from left to right: 5, 15, 50 mg/kg). Lower: scutellarin group (from left to right: 5, 15, 50 mg/kg). (B) Scutellarin and breviscapine significantly reduced infarct volume compared with control rats. There was significant difference between 3 dosages groups in scutellarin, while not in breviscapine. (C) The difference of inhibition ratio [(infarction volume of control − infarction volume of treatment) / infarction volume of control] between scutellarin and breviscapine. **P < 0.01, ***P < 0.001 versus control group. †P < 0.05, ††P < 0.01 versus scutellarin (5 mg/kg). #P < 0.05 versus scutellarin (15 mg/kg).
Acute Toxicity
The intraperitoneal LD50 values for KM mice were 2402 mg/kg in scutellarin and 2682 mg/kg in breviscapine. All deaths occurred in rats of either sex during 6 to 72 hours after drug administration. Hypersensitivity, shivering, hemorrhage were observed before death.
DISCUSSION
In China, breviscapine has been approved to be used in the treatment of cerebral ischemic diseases by the State Food and Drug Administration. Many recent studies show that breviscapine is effective on cerebral ischemia, but no direct evidence has shown its effects on cardiac ischemia. The present work found that breviscapine significantly decreased the brain infarct volume in rats with MCA occlusion. However, this drug had no protective effects against cardiac ischemia in rats with LAD artery ligature.
Breviscapine is not a chemically pure substance, but a mixture containing at least 9 molecules. It was reported that these 9 molecules were baicalein, naringenin, scopoletin, kaempferol, apigenin, scutellarin, luteolin, caffeic acid, and protocatechuic acid.4 Among these molecules, scutellarin is the main component (about 80%). Scutellarin is a polyphenolic flavonoid compound. Flavonoids exist naturally and show high free radical-scavenging activity.13 The previous studies demonstrated that scutellarin was a good ROS scavenger in vitro.14 It is well established that glutamate is extensively released during and after ischemia, and activation of various subtypes of its receptor causes direct and indirect Ca2+ influx into the cell, resulting in neuronal damage.15 Scutellarin possesses potent antioxidative properties and protects cells from oxidative glutamate toxicity.16 Numerous studies have been conducted regarding the differential roles of NOS isoforms and their temporal NO production in the pathogenesis of ischemic brain injury.17-20 eNOS-derived NO is thought to be beneficial for promoting collateral circulation and microvascular flow, whereas nNOS- and iNOS-derived NO is detrimental in the ischemic brain. Scutellarin (50 mg/kg) downregulated iNOS expression and upregulated eNOS expression, which partly accounts for its protective effect on brain damage induced by cerebral ischemia/reperfusion.3 Therefore, it would be interesting to show upregulation of eNOS in cardiac and cerebral tissue in response to scutellarin and to test whether eNOS inhibition blocks the reduction in infarct size by scutellarin.
In the isolation of a substance from a plant, it is very common for the effectiveness to decrease with the increase in the molecular purity. In this case, it is believed that other molecules may be synergistic with the target substance and are eliminated with the purification. In the present study, it is not the case. It was found that, the effects of scutellarin (pure molecule) on cardiac and cerebral ischemia are much great than those of breviscapine. If scutellarin was the sole molecule with cardiovascular activity, the effects of breviscapine would reach the levels of scutellarin with increasing dose by about 20%. However, even a large dose (by 10 times) of breviscapine could not produce a similar effect as scutellarin. Therefore, the only explanation is that the effects of other molecules such as scopoletin, caffeic acid, or protocatechuic acid, may produce a reverse effect against scutellarin. However, we have not found any evidence to show a reverse action of these components. It is interesting to study the effects on cardiac and/or cerebral ischemia of the 8 other components in breviscapine.
It is well known that some differences exist between brain microvascular endothelial cells and coronary microvascular endothelial cells. Recently, it was reported that the differences may be in several aspects, including cytokines and growth-related molecules, stress-related proteins, signal transduction proteins, metabolic enzymes, and others.21 In the brain, the endothelial layer of the blood-brain barrier forms a tight interface between blood and neuronal tissue, with active transport systems that mediate the directed transport of nutrients into the central nervous system or toxic metabolites out of the central nervous system and create a milieu essential for the function of the underlying neuronal cells. On the other hand, in the heart, coronary microvessels play a pivotal role in determining the supply of oxygen and nutrients to the myocardium by regulating the coronary flow conductance and substance transport. Many neurohumoral mediators significantly affect coronary microvascular control in an endothelium-dependent manner. According to obtained results, we speculate that myocardium may be more sensitive than cerebral tissue to some components producing reverse effects in breviscapine.
CONCLUSION
The results demonstrated that the protective effects of scutellarin on cardiovascular and cerebrovascular ischemia were better than its mixture, breviscapine, in rats.
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Keywords:
scutellarin; breviscapine; myocardial infarction; cerebral ischemia; apoptosis
© 2007 Lippincott Williams & Wilkins, Inc.