杜仲叶总黄酮通过RhoA/ROCK信号通路参与脑出血大鼠神经功能修复  

Eucommia ulmoides leaves total flavonoids participate in the repair of neural function in rats with cerebral hemorrhage through RhoA/ROCK signaling pathway

作　　者：张秀峰[1] 李小菲 刘明[1] 王慧静 ZHANG Xiufeng;LI Xiaofei;LIU Ming;WANG Huijing(Department of Neurosurgery,the First Hospital Affiliated to Hebei North University,Zhangjiakou 075000,China)

机构地区：[1]河北北方学院附属第一医院神经外科,075000

出　　处：《天津医药》2023年第3期252-258,共7页Tianjin Medical Journal

基　　金：河北省医学科学研究课题计划项目(20190872)。

摘　　要：目的从Ras同源基因家族成员A(RhoA)/Rho相关卷曲螺旋蛋白激酶(ROCK)通路探讨杜仲叶总黄酮促进脑出血大鼠神经功能修复的机制。方法取SD雄性大鼠,用改良二次注血法建立脑出血模型,并按随机数字表法分为假手术组、模型组、杜仲叶总黄酮组、RhoA抑制剂组、RhoA激动剂组、杜仲叶总黄酮+RhoA激动剂组,每组20只。杜仲叶总黄酮组灌胃200 mg/kg杜仲叶总黄酮,RhoA抑制剂组腹腔注射1 mg/kg的RhoA抑制剂Y27632,RhoA激动剂组腹腔注射30μg/kg的RhoA激动剂U-46619,杜仲叶总黄酮+RhoA激动剂组灌胃200 mg/kg杜仲叶总黄酮的同时腹腔注射30μg/kg U-46619,模型组及假手术组灌胃10 mL/kg生理盐水,1次/d,连续7 d。给药结束后,观察大鼠行为变化,采用改良神经功能缺损评分(mNSS)对神经功能缺损症状进行评估;取脑组织后称质量,计算脑组织含水量,然后制备脑组织切片并计算脑血肿体积百分比;透射电镜观察血肿周围组织神经突触结构变化;TUNEL染色法观察血肿周围组织神经元凋亡;鬼笔环肽染色观察血肿周围组织神经元骨架变化;蛋白免疫印迹法检测血肿周围组织RhoA、ROCK、细胞骨架蛋白(F-actin)、丝切蛋白(cofilin)、磷酸化cofilin(p-cofilin)、促神经元及突触生长相关蛋白[神经生长因子(NGF)、神经营养因子3(NT3)、突触后致密蛋白-95(PSD-95)、突触素(SYP)]的表达。结果与假手术组相比,模型组大鼠神经功能缺损评分、脑组织含水量、脑血肿体积百分比升高,血肿周围组织神经元凋亡及骨架结构损伤,神经突触结构改变严重,RhoA/ROCK通路激活,促神经元及突触生长相关蛋白表达降低(P<0.05)。杜仲叶总黄酮或RhoA抑制剂干预治疗均可抑制RhoA/ROCK通路激活介导的神经元骨架结构改变,提高促神经元及突触生长相关蛋白表达,缓解脑出血后血肿周围组织神经元损伤及凋亡,促进神经功能修复(P<0.05)。RhoA激动剂可促进RhoA/ROObjective To explore the possible mechanism of eucommia ulmoides leaves total flavonoids promoting neurological repair in rats with cerebral hemorrhage from the Ras homolog gene family member A(RhoA)/Rho-associated coiled-coil protein kinase(ROCK)pathway.Methods SD male rats were used to establish cerebral hemorrhage model by the modified double injection method,and they were randomly divided into the sham operation group,the model group,the eucommia ulmoides leaves total flavonoids group,the RhoA inhibitor group,the RhoA agonist group,and the eucommia ulmoides leaves total flavonoids+RhoA agonist group,with 20 rats in each group.The eucommia ulmoides leaves total flavonoids group was intragaically treated with 200 mg/kg eucommia ulmoides leaves total flavonoids.The RhoA inhibitor group was intraperitoneally injected with 1 mg/kg RhoA inhibitor Y27632.The RhoA agonist group was intraperitoneally injected with 30μg/kg RhoA agonist U-46619,and the eucommia ulmoides leaves total flavonoids+RhoA agonist group was intraperitoneally injected with 200 mg/kg eucommia ulmoides leaves total flavonoids and 30μg/kg RhoA agonist U-46619.The model group and sham operation group were given 10 mL/kg normal saline once a day for 7 consecutive days.After the administration,the behavioral changes of rats were observed with the naked eye and the neurological deficit symptoms were assessed by the modified neurological severity score(mNSS).Brain tissue was taken and weighed,and the water content of the brain tissue was calculated according to the formula to evaluate the cerebral edema,then the brain tissue slices were prepared and the volume percentage of the cerebral hematoma was calculated according to the formula.Changes in the structure of nerve synapses in perihematoma tissue were observed by transmission electron microscope.Changes of neuronal apoptosis of perihematoma tissue were observed by TUNEL staining method.Changes in neuron skeleton of perihematoma tissue were detected by phalloidin staining.The expression levels of Rh

关 键 词：脑出血 ρA GTP结合蛋白质 RHO相关激酶类 杜仲叶总黄酮 神经功能修复 

分 类 号：R285.5[医药卫生—中药学]