| Ischemic cerebrovascular illness is one of the common illnesses seriously threatening the health of people. During the process of arising and developing of this disease, it may cause focal cerebral ischemia-reperfusion injury(IRI), which remains the major barrier to the advancement of treatment and prognosis. The research of pathological physiological mechanism and preventing measures of focal cerebral ischemia-reperfusion injury has become an issue of growing concern in clinical practice. Radix astragali is widely used in therapy of Ischemic cerebrovascular disease, which has got more and more attention in cerebrovascular disease preventing. Cycloastragenol(CAG) is the aglycone of the Astragaloside IV, which is the effective position of radix astragali. CAG was confirmed to have a significant effect on telomerase activation in in vitro experiments. Telomerase is a kind of endogenous neural protection measures, overexpression of telomerase reverse transcriptase(TERT) can play a protective role in ischemia brain. The protective effect and mechanism of CAG against cerebral ischemia reperfusion injury are not yet clear. This study established the focal cerebral ischemia-reperfusion injury animal model and gave cycloastragenol intervention before model making, the effect of CAG on reducing cerebral ischemia reperfusion injury in rats was observed by detecting the neurological function scores and the percentage of cerebral infarction volume;The changes of TERT expression in brain tissues of rats were determined by immunohistochemistry and Western blot.and tested the effect of cycloastragenol on the apoptosis of focal cerebral ischemia-reperfusion injury neuron cells by TUNEL method. To investigate the molecular mechanism of CAG reducing cerebral ischemiareperfusion injury by inhibiting neuronal apoptosis and improving the expression of TERT. And to provide basis for prevention of focal cerebral ischemia-reperfusion injury with cycloastragenol.The Frist Part The research of Cycloastragenol reducing cerebral ischemia reperfusion injury in ratsObjective: To explore the effects of cycloastragenol on reducing cerebral ischemia reperfusion injury in rats.Method: 1 Preparation and group 60 male SD rats were randomly divided into sham operation group, model group, CAG low dose group(2.5 mg/kg/d), middle dose group(5.0 mg/kg/d), high-dose group(10 mg/kg/d) and Xuesaitong group(20 mg/kg /d),n=10. All drugs were adiministered by intragastric administration(i.g.), sham group and model group were given an equal volume of 0.5% CMC-Na suspension for consecutive week,in the last day the rats were treated of 1h later and then for modeling. 2 Preparation of focal cerebral ischemia-reperfusion model Focal cerebral ischemia reperfusion model of rats’ right middle cerebral artery occlusion(MCAO) was induced by improved suture method. After 24 h of cerebral ischemia-reperfusion injury, choosing rats with successful model for determining every index. 3 Detection of Garcia JH’s neurological scores After 24 h of cerebral ischemia-reperfusion injury, scoring nerve function according to Garcia JH’s neurological scores standards of rats with double-blind method. 4 Detection of the percentage of cerebral infarction volume After 24 h of cerebral ischemia-reperfusion injury, rats were decapitated, taken out brain, TTC stained and taken photographs to calculate the percentage of cerebral infarction volume.Results: 1.The effects of CAG on the Garcia JH’s neurological scores of rats Compared with sham-operated group, the Garcia JH nerve functional scoring for model group was significantly reduced(P<0.05); compared with model group, the Garcia JH nerve functional scoring for CAG high and medium dose group was upraised(P<0.05); CAG low dose group and model group were without significance difference(P>0.05). 2.The effects of CAG on the percentage of cerebral infarction volume of rats Compared with sham-operated group, the percentage of cerebral infarction volume for model group was significantly upraised(P<0.05); compared with model group, the percentage of cerebral infarction volume for CAG high and medium dose group and xuesaitong group was significantly reduced(P<0.05); CAG low dose group and model group were without significance difference(P>0.05).Summary: Cycloastragenol can improve nerve functional scoring of ischemia reperfusion injury and reduce the percentage of cerebral infarction volume of rats. Cycloastragenol can reduce cerebral ischemia reperfusion injury in rats.The Second Part Effects of Cycloastragenol on neuronal apoptosis and the expression of TERT in focal cerebral ischemia reperfusion ratsObjective: To observe effects of Cycloastragenol on neuronal apoptosis and the expression of TERT in rats with cerebral ischemia reperfusion injury. To explore the mechanism of Cycloastragenol reducing focal cerebral ischemia reperfusion injury.Detection of TERT protein expression: the male SD rats were randomly divided into 3 groups: sham operation group, model group, cycloastragenol group(10mg /kg/d). Modeling and administration were same as the first part. After 24 h of cerebral ischemia reperfusion, the ischemic cerebral cortex and ischemic penumbra were taken as the main materials and observation site, Immunohistochemistry and Western blot were used to detect the TERT expression.in the rats’ schemic cortex. Detection of neuronal apoptosis:Male SD rats were randomly divided into 5 groups : sham-operated group, model group, cycloastragenol group(10mg/kg/d), telomerase inhibitor ATZ group(20mg/kg/d),cycloastragenol((10mg/kg/d)and telomerase inhibitor ATZ group(20mg/kg/d). Modeling and administration were same as the first part. The apoptotic index of neurons in ischemic penumbra of all groups were detected by TUNEL staining after 24 h of cerebral ischemia reperfusion. Method:Result: 1. The expression of TERT protein determined by immunohistochemistry method: compared with sham-operated group, the expression of TERT for model group was upraised(P<0.05); compared with model group, the expression of TERT for cycloastragenol group was upraised(P<0.05). 2. Western blot indicated: compared with sham-operated group, the expression of TERT protein of model group was upraised(P <0.05); compared with model group, the expression of TERT protein of cycloastragenol group was upraised(P <0.05). 3. The outcome of TUNEL: compared with sham-operated group, the apoptotic index(AI) of model group, inhibitor group, inhibitor and cycloastragenol group were upraised(P<0.01);compared with model group,the apoptotic index(AI) of cycloastragenol group was significantly reduced(P<0.05); compared with cycloastragenol group, the apoptotic index(AI) of inhibitor and cycloastragenol group wasupraised(P<0.05); compared with inhibitor group, the apoptotic index(AI) of inhibitor and cycloastragenol group was without significance difference(P>0.05).Summary: 1.Cycloastragenol can improve the expression of TERT in rats’ brain tissues, presumably this is one of the mechanisms to reduce the cerebral ischemia-reperfusion injury. 2.Cycloastragenol can inhibit the neuron apoptosis in cerebral ischemia reperfusion rats, which is achieved by promoting the expression of TERT.Conclusion: 1.Cycloastragenol can improve nerve functional scoring of ischemia reperfusion injury and reduce the percentage of cerebral infarction volume of rats. Cycloastragenol can reduce ischemia reperfusion injury in rats. 2. Cycloastragenol can inhibit the neuron apoptosis in cerebral ischemia reperfusion rats, which is achieved by promoting the expression of TERT. |
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