| Objective1. The study is to test the hypothesis that Ligustrazine Hydrochloride can prevent endothelial dysfunction under oxidative stress.2. The study is to investigate the mechanism underlying tension oscillation of middle cerebral artery in the carotid artery ligature rat model and to test the hypothesis that Ligustrazine Hydrochloride has vascular protective effect in the model.3. The study is to observe the intracranial hemodynamic change in the patients of ischemic stroke accompanied by carotid stenosis and to find out the way Chinese Herbs is used clinically.Method1. The experiment carried out in animals(1)Endothelial dysfunction model was induced by incubation of H2O2 in vitro and then vascular tension measurement, western blotting determining expression level of eNOS, Nitrotyrosine and SOD-1 and endothelial cell super oxide anion observation in confocal image were carried out.The vasorelaxing effect was expressed as percentage reduction of evoked contraction. Nonlinear regression curve fitting was performed on individual cumulative concentration-response curves (GraphPad Prism software, Version 5.0) to estimate Emax as the maximal response and pD2 values as negative logarithm of drug concentration that induced 50% of the maximal response. Concentration-response curves were analyzed by two-way ANOVA followed by Bonferroni post-tests. Statistical significance was determined by one-way ANOVA followed by Bonferroni post-tests to analyze the data of western blotting and confocal image. A P-value of less than 0.05 was regarded as significant. All data are expressed as mean±SEM of n rins.(2)Vasomotion and neurological presence were observed in cerebral ischemia rat model induced by internal carotid ligature and Ligustrazine Hydrochloride treatment groups independently. The neurological presence was evaluated by Longma 4 grade 5 score neurological deficient scale. Significant difference was tested by two-tail student t test and A P-value of less than 0.05 was regarded as significant. Vasomotion was only expressed as tension oscillation observation omitting statistical analysis due to the irregular frequency and amplitude of vasomotion.2. The clinical investigationThe study was to investigate the cerebral ischemic stroke patients in the Cerebral Disease Center of Guangdong Provincial Hospital of TCM from Jan to December in 2007. Normal carotid artery and carotid stenosis (mild, moderate, severe, occlusion) groups were determined by the Color Doppler Ultrasound results and then the Transcranial Doppler results were analyzed. The average velocity of middle cerebral artery and anterior cerebral artery, VMCA, VACA, the velocity difference between cerebral artery of two sides,ABS(VLMCA-VRMCA) ABS (VLACA VRACA) and the velocity difference between middle and anterior cerebral artery, ABS (VMCA-VACA) were compared. SPSS 17.0 were applied to all the statics analysis. F test for variance equality and normal distribution evaluation were analyzed before all difference tests. Two-tailed student t test, Mann Whitney u test or Wilcoxion paired test was performed. Kruskal-Wallis test were performed when more than two groups were compared. A P-value of less than 0.05 was regarded as significant. The data are expressed as average±SD for parametric statics and mean±SEM for the rank sum test.Result1. The experiment results carried out in animals(1)Endothelium dependent relaxation were impaired by 1 hour incubation of 200μM H2O2 in SD rat aorta and catalase, NADPH oxidase inhibitor, Ligustrazine Hydrochloride, Losartan, Vitamin E could prevent the impairment, while NOS inhibitor worsened it. The mechanism underlying the preventive effect was not related to the eNOS, SOD-1 or Nitrotyrosine expression change.(2) H2O2 increased the super anion in the vascular endothelium cells significantly and pretreatment of NADPH oxidase inhibitor, SOD, Ligustrazine Hydrochloride could reduce it.(3)The neurological deficient score of internal carotid artery ligature model was significantly higher than the normal SD rat 6 hours after the operation, manifesting circling to the right.(4)Irregular tension oscillation of cerebral artery induced by serotonin or U46619 was found in the carotid artery ligature model while stable contraction to the plateau in normal SD rat. Stable contraction to the plateau induced by 60 mM high potassium was observed in either normal or ligature rat. Voltage-gated calcium channel, SR Ca2+-ATPase, Ca2+-sensitive Potassium channel, Na+-K+-ATPase blocker reduced the tension oscillation while voltage-gated channel opener increased it. Voltage-gated potassium channel blocker had no effect on the tension oscillation.(5)Incubation of 1 nM Ligustrazine Hydrochloride of 1 hour could totally inhibit the irregular tension oscillation of middle cerebral artery in the carotid ligature model. Treatment of Ligustrazine Hydrochloride by 4.11mg/kg vein injection could significantly suppress the irregular tension oscillation in the ligature model.2. Compared to the normal carotid artery group, VMCA,VACA,ABS (VMCA-VACA) of patients of cerebral ischemic stroke accompanied by carotid stenosis were significant higher (P<0.05) and ABS(VLMCA-VRMCA) or ABS(VLACA-VRACA) was no difference between tow groups(P>0.05).There was no difference of hemodynamic change among different levels of carotid stenosis(P>0.05). VMCA ipsilateral was less than that contralaterl while VMCA ipsilateral was more than that contralaterl (P<0.05) in the unilateral carotid stenosis patients.There was no difference between ABS(VMCA-VACA) of two sides (P>0.05) in the unilateral carotid stenosis patients.Conclusion1. Endothelium dependent relaxation impairment induced by H202 is successful and that superoxide anion produced by NADPH oxidase activated by H202 impairs the endothelium cell might be the underlying mechanism.2. Acute incubation of low dosage Ligustrazine Hydrochloride could partly prevent endothelium dependent relaxation impairment induced by H202 as an effectively antioxidant drug.3. Cerebral ischemia model induced by internal carotid artery ligature is successful and the increase of intracellular calcium originated from cell membrane and ER might play an important role in the irregular tension oscillation of cerebral artery in the model.4. Acute treatment of low dosage Ligustrazine Hydrochloride could inhibit the vasomotion in the carotid artery ligature model in both vitro and vivo experiments.5. Hemodynamic change is significant in the patients of cerebral ischemic stroke accompanied by carotid stenosis.
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