| BackgroundStroke is a common frequently-occurring disease, stroke is listed as the three kinds of deadly diseases with heart disease and malignant tumor, which has a extremely high death morbidity, and stroke is getting expanding and growing younger every year all over the world. Nowadays, in China, the death caused by stroke has exceeded cancer and cardiovascular disease, which became the first cause to death. Acute treatment is very vital for the prognosis of patient with stroke. Today we usually use intravenous thrombolysis as the main method for acute ischemic stroke and vascular occlusion stroke in clinical treatment. But, the patient who can go to the hospital within the efficient time and has thrombolysis are very limited. In addition, when the patient got vascular occlusion stroke, the repass rate of vessel is very low after intravenous thrombolysis, such as, the repass rate of on middle cerebral artery M1 is about 30%, and the rate on the end of the internal carotid artery is only 6%. Those disadvantages in the process of blood flow reconstruction、increasing the supply of blood and improving microcirculation not only limit the extensive use of thrombolysis drug in clinical practice, but also can cause the secondary serious damage after the recover of blood flow, called cerebral ischemia-reperfusion injury which seriously affects the quality of patient’s life.Codonopsis pilosula,Plants of the campanulaceae codonopsis pilosula genus,which is a tonic of chinese traditional medicine with a long history,sweet and natured, with the effect of tonifying middle-Jiao and Qi、nourishing fluid、strengthening spleen and tonifying lung, invigorating spleen and lung,Commonly used in qi-blood deficiency and blood deficiency chlorosis embolism.Modern pharmacological studies show that codonopsis pilosula has the functions of anti-oxidation, anti-aging, reducing blood fat and blood pressure, controlling cell division and differentiation, hematopoiesis. The main effective component of codonopsis pilosula is polysaccharide(CPPS), which as the key active substances with the effect of improving immune and anti-hypoxiaischemia. CPPS with nerve growth factor activity to improving microcirculation,anti-oxygen free radical, enhance immunity and play the role of nourishing and strong.It is just because of the pharmacological effects of CPPS, many experts and scholars have regarded it as a natural herb with great application value. CPPS has important research value in the protection of cerebral ischemia injury.In recent years, a growing number of studies have found that CPPS polysaccharide has an important research value in the field of cardiovascular and cerebrovascular diseases. This research aims at the study on the protection of CPPS polysaccharide to cerebral ischemia-reperfusion injury for rat, and discussing mechanism of the protective effects related to it.Objectives:The model of ischemia reperfusion injury in rats is established through line plug method in this subject,Studying on the protective of CPPS on the brain injury of rats and discussing its possible mechanism.Methods:1. Animal grouping and administration 120 male SD rats,SPF grade, weight(200g ±20g),They were randomly divided into four groups according to body weigh,The sham operation group, model group, 2 dose administration groups(1 g/kg,2 g/kg of CPPS),each group own 30 rats.According to the experimental design,administration groups needs to be intragastric administration one time everyday,4 consecutive days.Dosing administration volume is 1ml/100 g,the other two groups are given equal volume of distilled water.2. Animal model preparation MCAO( middle cerebral artery occlusion) rats were prepared by modified longa method, establishing cerebral ischemia reperfusion model in rats.3. neurological score Using longa neurological score, according to the 5-point scale principle to get neurobehavioral scores in rats after modeling in the time 12 h and24h.When the score is 2~4,showing the success of the model.4. Cerebral infarction area Rats brain tissue are separated through frozen section,new configuration 2,3,5 triphenyl tetrazolium chloride(2,3,5- triphenyltetrazolium chloride, TTC) dye staining in water bath with the dark surrundings, through digitalphotography, osiris software calculates each piece of brain infarct area and the whole slicebrain area,whic is used to calculate the percentage of brain infarcted area.5.Brain tissue cell ultrastructure pathological observation Rats brain tissue conventional embedding, slicing, staining, tobserving the different parts of the cell morphology in brain through ransmission electron microscope, taking pictures.6. Determination of oxidation index Preparation of brain tissue extracts, in strict accordance with the process of kit instructions, measuring the contents of malondialdehyde(MDA) and superoxide dismutase(SOD), glutathione oxidase(GSH- PX) activity.7. Determination emissions of lactate dehydrogenase Preparation of brain tissue extracts, in strict accordance with the process of lactate dehydrogenase(LDH) kit instructions, measuring LDH activity.8. Contents detection of inflammatory factors Separation of serum, diluting the multiple of concentrations, according to ELISA kit instructions, measuring content of inflammatory cytokines interleukin-1β( I L-1β) and tumor necrosis factor(TNF-α).9. Differential expression of brain tissue proteins Detection of apoptosis- related proteins Caspase-3, Bcl-2, Bax expression and autophagy-related protein Beclin-1differential expression through immunoblot(western blot), Chemiluminescence exposure, gel imaging analyzer analyzes photos, software of Image Lab 4.0 calculates the target protein gray value.10. Statistics processing and analysis methods Using SPSS3.0 statistical software to analyze datas.Comparison between the two groups of data is taken by t test, the experimental data is indicated by mean ± standard deviation((?)±s).Results:1. After 12h、24h, model groups and two dose CPPS groups all appear varying degrees of neurological dysfunction. Between the two groups, after 12 h, model group score is higher than the high-dose administration of CPPS group(2.27±0.70)(P<0.01), model group score is also higher than the low dose CPPS administration group(2.53±0.74)(P<0.05). After 24 h, model group score is higher than two doses CPPS administration groups, the difference was significant(2.40±0.83、2.20±0.68)(P<0.01).After TTC staining, the model group can be finded the obvious infarction.Between the two groups, infarct size of the model group is bigger than two doses CPPS administration group, the difference is significant(30.17±10.11、25.63±10.59)(P<0.05).Cell morphology of the sham group is intact,nucleolus is clear. Cell structures of model group are not clear, which was broken seriously. Compared with the model group, the neuronal morphology of two CPPS administration groups is normal, and the protective effect of the high dose group is better than the low dose group.2. Compared to the sham-operated group, MDA contens of model group was significantly increased(0.19±0.03)(P<0.01), GSH-PX, SOD activity reduced(2.70±0.18、38.64±5.60)(P<0.01). Compared to the model group,MDA contents of the two doses CPPS administration groups reduced(0.13±0.04、0.11±0.02), GSH-PX,SOD enzyme activity increased(4.33±0.18、5.21±0.11,48.36±2.52、56.29±3.41)(P<0.01). Compared to the sham-operated group, LDH activity in model group increased(3061.12±83.57)(P<0.01),Compared to the model group, LDH activity of the two doses CPPS administration groups reduced(2545.39±69.49、2503.76±117.82)(P<0.01).3. There are more contents of serum IL-1β, TNF-αin model group than the sham group(133.92±2.41、265.88±4.6)(P<0.01).compared with the model group, the serum IL-1β, TNF-αconcentrations in two doses CPPS administration group were significantly reduced(124.01±2.12、105.02±2.0,188.62±3.96、168.11±3.56)(P<0.01).4. Compared with model group, pro-apoptotic protein Caspase-3 and Bax expression of two doses administered CPPS group decreased, antiapoptotic protein Bcl-2 expression increased, the difference was significant, P <0.01.Compared with sham group, autophagy-related protein Beclin-1 expression in model group increased significantly. Compared with the model group, Beclin-1 expression of two doses CPPS administration group reduced. The difference was significant, P <0.05.Conclusion:CPPS plays the role of improving neurological dysfunction in rat cerebral ischemia reperfusion injury,which can reduce the infarct area, protecting the morphology integrity of neuronal. Improving antioxidant enzyme activity, inhibiting expression of inflammatory cytokines、reducing brain cell apoptosis and autophagy may be the protective mechanism of CPPS on rat cerebral ischemia-reperfusion injury. |
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