连翘苷对大鼠脑缺血耐受模型的保护作用及其机制研究

Protective Effect and Mechanism of Forsythin on Cerebral Ischemic Tolerance Model in Rats

目的:观察连翘苷对大鼠脑缺血耐受模型的神经保护作用,并探讨其可能的作用机制。方法:SD大鼠随机分为假手术对照组、脑缺血再灌注组、模型对照组、银杏叶提取物0.04 g/kg组、连翘苷0.1、0.2 g/kg组,各组灌胃给予相应药物或CMC-Na液,连续7 d。给药第1 d采取结扎双侧颈总动脉(CCA)10 min作为缺血预处理,给药第6 d进行第2次缺血损伤,采用线栓法制作大鼠大脑中动脉闭塞(MCAO)-再灌注模型。复灌22 h后,评价神经功能评分,以TTC染色观察脑梗死体积;分光光度法检测脑组织匀浆液中SOD、MDA、GSH-Px、CAT活力或含量;ELISA法检测脑组织匀浆液IL-1、IL-8、IL-10、TGF-β、TNF-α含量;免疫组化法检测缺血侧脑皮层TLR4和NF-κB p65的蛋白表达。结果:与假手术对照组比较,模型对照组大鼠神经功能障碍症状评分明显升高(P<0.05),脑梗死体积显著升高(P<0.01),神经功能缺失评分、脑梗死体积显著升高(P<0.01),脑组织中SOD、GSH-Px活力、CAT含量明显降低(P<0.05或P<0.01),MDA、IL-1、IL-8、IL-10、TGF-β、TNF-α含量明显升高(P<0.05或P<0.01),脑皮质区TLR4、NF-κBp65蛋白表达明显增强(P<0.01);说明造模成功;与模型对照组比较,连翘苷0.2 g/kg组能明显降低大鼠的神经功能评分,明显降低脑组织匀浆液中MDA含量(P<0.05),连翘苷0.1、0.2 g/kg组能明显降低脑梗死容积分数(P<0.05或P<0.01),能明显升高SOD、GSH-Px、CAT活力或含量(P<0.05或P<0.01),能明显降低IL-1、IL-8、TNF-α含量(P<0.05或P<0.01),明显升高IL-10、TGF-β含量(P<0.05),缺血侧脑皮层中TLR4、NF-κBp65蛋白表达明显降低(P<0.05或P<0.01)。结论:连翘苷能够提高大鼠脑缺血耐受模型的保护作用,减轻大鼠脑缺血的脑组织损伤,其机制可能与抗氧化作用及抑制炎症反应有关。

Objective:To investigate the neuroprotective effect of forsythin on the cerebral ischemic tolerance(CIT)model in rats and to explore the underlying mechanism.Methods:The SD rats were randomly divided into a sham control group,a cerebral ischemia-reperfusion injury(CIRI)group,a model group,a Ginkgo biloba extract(0.04 g/kg)group,and high-and low-dose forsythin(0.2 g/kg and 0.1 g/kg)groups.Rats were administered with corresponding drugs or CMC-Na for 7 days by gavage.On the 1 st day of administration,bilateral common carotid arteries(CCA)were ligated for 10 min as ischemic preconditioning.The second ischemic injury was performed on the 6 th day after administration,and the middle cerebral artery occlusion(MCAO)-reperfusion model was established in rats by the suture method.After 22 h of reperfusion,the neurological function scores were evaluated,and the volume of cerebral infarction was observed by TTC staining.The activities or levels of SOD,MDA,GSH-Px,and CAT were detected in brain tissue homogenate by spectrophotometry.The levels of IL-1,IL-8,IL-10,TGF-β,and TNF-αwere detected in the brain tissue homogenate by ELISA.The protein expression of TLR4 and NF-κB p65 in the ischemic cerebral cortex was determined by immunohistochemistry.Results:Compared with the sham control group,the model group showed higher score of neurological function(P<0.05),increased volume of cerebral infarction(P<0.01),blunted activities of SOD and GSH-Px,reduced content of CAT in brain tissues(P<0.05 or P<0.01),increased content of MDA,IL-1,IL-8,IL-10,TGF-β,and TNF-α(P<0.05 or P<0.01),and up regulated protein expression of TLR4 and NF-κB p65 in the cerebral cortex(P<0.01),which suggested successful modeling.Compared with the model control group,the high-dose forsythin(0.2 g/kg)group showed improved neurological function score and reduced level of MDA(P<0.05)in the brain tissue homogenate,while the forsythin(0.2 g/kg and 0.1 g/kg)groups displayed reduced volumes of cerebral infarction(P<0.05 or P<0.01),increased activities or levels of SOD,GSH-Px,and CAT(P<0.05 or P<0.01),reduced content of IL-1,IL-8,and TNF-α(P<0.05 or P<0.01),elevated content of IL-10 and TGF-β(P<0.05),and down-regulated protein expression of TLR4 and NF-κB p65 in the ischemic cerebral cortex(P<0.05 or P<0.01).Conclusion:Forsythin can improve the protective effect on the CIT model in rats and reduce the brain tissue damage caused by cerebral ischemia,and the mechanism may be associated with the antioxidant effect and the inhibition of inflammatory reactions.