Pharmacodynamic Study Of Breviscapine Proliposome On Cerebral Ischemia Reperfusion Injury In Rats

IntroductionIschemia reperfusion injury is referred to the ischemic injury of tissue cells resulted from the ischemia and hypoxia of local tissue and organs,and the exacerbation of cellular dysmetabolism and disorganization after the recovery of blood reperfusion under certain conditions.lschemia reperfusion not only can retrieve the dying cells,but also can exacerbate cell injury,and even lead to death. Ischemic cerebrovascular disease has been considered one of the principal diseases resulted in disability and death of patients.Breviscapine is the flavonoids extracted from Erigeron breviscapus(Vant.). Breviscapine is the mixture of 4'-hydroxy-β-D-pyangluconate methyl ester and scutellarin,and the latter content is about more than 95 per cent.Breviscapine has extensive physiological activity,including protective effect for neurons and nerve cells in cerebral ischemic regions,antagonism of super oxide free radicals, extenuation of cerebral edema and inflammatory reaction,and abatement of ischemia and reperfusion injury.But it has low biological availability.Clinically,it is mainly used for the treatment of cerebral infarction,coronary heart disease,and angina pectoris.Liposome,as a targeted drug carrier,belongs to a new dosage form of targeted drug delivery system.It has some advantages for increasing drug solubility, enhancing drug target,and decreasing drug toxicity.Pro-liposome is a liposome disperse system existed in solid form after spraying dry and freezing dry.Dispersion medium is added into pro-liposome to re-disperse to form liposome.By the preparation technique of liposome,PS can improve the bioavailability and therapeutic effect.This study is to investigate the protective effect PS on cerebral ischemia reperfusion injury in rats,and to estimate the safety of PS.Methods1.The establishment of MCAO/R rat model and the evaluation of nerve pathological injuryThe middle cerebral artery occlusion-reperfusion rat model was established by the Longa technique.The assessment standard of Longa'4 scores was applied to assess the neural function of the rats after cerebral ischemia-reperfusion.The rats with 1-3 score were used in the follow experiments.2.TTC staining of brain tissueThe rats were decapitated and taken out of brain at the 4th hour after reperfusion. The brain was cut into 5 pieces along coronal plane.TTC staining was taken at 37℃in water bath.Taking photographs after paraformaldehyde fixation.3.Calculation of cerebral infarct area of by professional image analysis software ipp6.04.Determination of water content in brainWater content in brain(%)=(brain wet weight-brain dry weight)/brain wet weight×100%5.The model of inferior vena cava thrombosis in rats established by line occlusionRats were anesthetized by chloral hydrate.The inferior vena cava was separated and was ligated below the left renal vein6.The model of carotid artery thrombosis in rats established by using of Fecl₃Rats were anesthetized by chloral hydrate.The right common carotid artery was separate 2cm.A small piece of filter paper containing 20ul FeCl₃ solution was pasted in carotid artery for 30 min7.Clotting time were detected by capillary method8.Acute toxicity experimentLD₅₀ experiment was taken.The maximum dosage experiment was taken because it was not detected by LD₅₀.9.Statistical methodsData were expressed as mean±s and compared by ANOVA by using SPSS statistical program.The level of the statistical significance was P＜0.05.Results1.The results of brain infarct size quantitative analysisCompared with the model group,PS dose-dependently decreased cerebral ischemia-reperfusion infarct size.Infarct sizes were decreased from 0.239±0.051 in model groups to 0.141±0.036 and 0.113±0.029 in PS groups(P＜0.05,vs model). Compared with Bre groups,the infarct size was decrease from 0.185±0.008 to 0.141±0.036 in middle-dose group(P＜0.05)。2.The results of brain water content determinationCompared with the model group,PS dose-dependently decreased the degree of cerebral edema.Water content was decreased from79.72±0.46 in model groups to 78.68±0.46 and 78.39±0.19 in PS groups(P＜0.05,vs model).3.The results of inferior vena cava thrombosisCompared with the model group,PS dose-dependently reduced the weight of thrombus.Wet-weight of thrombus were decreased from27.34±18.06 to 23.92±20.09 and 7.46±6.20 in PS groups(P＜0.01,vs model).Dry-weight were decreased from8.42±5.85 to 7.49±6.30 and 7.49±6.30 in PS groups(P＜0.01,vs model).There were no significant differences between in Bre group and in PS middle-dose group. 4.The results of carotid artery thrombolysis experimentCompared with the model group,PS dose-dependently reduced the weight of thrombus,Thrombus weight were decreased from3.00±1.43 to 2.38±1.05 and 1.89±0.86 in PS groups(P＜0.01,vs model).5.The results of mice coagulation time test experimentCompared with the model group,PS can prolong the coagulation time significantly(P＜0.01).Coagulation times were prolonged from 59.5±11.3 to 115.8±18.6 and 118.0±17.8 in PS groups(P＜0.01,vs model).Compared with the Bre group,PS model group can prolong the coagulation time significantly(P＜0.01).6.The results of acute toxicity test experimentThere were no mice died in the three groups of PS(18000mg/kg,9000mg/kg, 4500mg/kg) in LD₅₀ detection.Maximum dosage experimentation was taken by 18000mg/kg oral dose twice a day.There was no abnormal behavior or death in mice within 14 days.It was found that there was no organ abnormality in gross anatomy.ConclusionCompared with model group,PS could remarkably reduce the brain infarct size of rats,and dose-dependently reduced water content of brain tissue,inhibited thrombosis, promoted thrombolysis,and prolonged the coagulation time of mice.PS could significantly inhibit brain ischemia-reperfusion injury.PS was more effective than Breviscapine in the same dosage.The acute toxicity experimental results show that:abnormal behavior,abnormal organs or death did not appear in mice maximum dosage experiment(18000mg/kg×2), which indicates that the PS would be a safe drug.