绿茶提取物介导Rho/ROCK通路缓解脑梗死大鼠脑组织损伤的实验研究

Experimental Study on Green Tea Extract Mediates Rho/ROCK Pathway to Relieve Brain Tissue Damage in Rats with Cerebral Infarction

目的:探讨绿茶提取物对Ras同源基因(Rho)/Rho相关螺旋卷曲蛋白激酶(ROCK)通路的调控作用及对脑梗死大鼠脑损伤的影响,初步探讨其作用机制。方法:用线栓法复制大鼠脑梗死模型,并随机分为假手术组、模型组、绿茶提取物组(200 mg/kg)、Rho抑制剂组(给予盐酸法舒地尔15 mg/kg)、Rho激动剂组(给予溶血性磷脂酸50 mmol/kg)、联合组(绿茶提取物+Rho激动剂),每组18只。给药结束后,观察大鼠行为变化;氯化三苯基四氮唑(TTC)染色观察脑梗死状况;尼氏及脱氧核糖核苷酸末端转移酶介导的黏性末端标记(TUNEL)染色法观察神经元损伤及凋亡情况;鬼笔环肽染色观察神经元细胞骨架变化;透射电镜下观察神经突触结构变化;免疫组化法观察Rho阳性表达水平;蛋白免疫印迹法(Western Blot)检测Rho/ROCK通路蛋白、促神经生长因子[神经生长因子(NGF)、脑源性神经营养因子(BDNF)、神经营养因子3(NT3)]、神经生长抑制因子[轴突生长抑制因子(NogoA)、髓鞘相关蛋白(MAG)、切丝蛋白(cofilin)]、突触再生重塑相关蛋白[突触后致密物质(PSD-95)、突触素(SYP)等]蛋白表达。结果:与假手术组比较,模型组大鼠神经功能缺损评分、脑梗死体积均增加,脑皮层神经元损伤和凋亡、突触小泡破裂融合等结构损伤及细胞骨架破坏严重,Rho/ROCK通路活化,及其介导的促神经生长因子表达降低,而神经抑制因子表达升高(P<0.05)。绿茶提取物及Rho抑制剂均可减轻脑皮层神经元损伤和凋亡、突触小泡破裂融合等结构损伤及细胞骨架破坏等病理损伤,降低神经功能缺损评分及脑梗死体积,抑制Rho/ROCK通路活化和神经抑制因子表达,促进促神经生长因子表达(P<0.05)。Rho激动剂可逆转绿茶提取物的上述作用(P<0.05)。结论:绿茶提取物可抑制Rho/ROCK活化,促进脑梗死后神经元及突触的再生及重塑,改善神经功能损伤,发挥脑保护作用。

Objective:To investigate the regulatory effect of green tea extract on Ras homologous gene(Rho)/Rho-related coiled protein kinase(ROCK)pathway,and its effect on brain injury in rats with cerebral infarction,and to preliminarily explore its mechanism.Methods:The rats were taken to establish the rat cerebral infarction model by suture method,and randomly divided into sham operation group,model group,green tea extract group(200 mg/kg),Rho inhibitor group(fasudil hydrochloride,15 mg/kg),Rho agonist group(lysophosphatidic acid,50 mmol/kg),combined group(green tea extract+Rho agonist),with 18 rats in each group.After the administration,the behavioral changes of the rats were observed.Triphenyl tetrazolium chloride(TTC)staining was used to observe the status of cerebral infarction.Nissl and terminal-deoxynucleoitidyl transferase mediated nick end labeling(TUNEL)staining methods were used to observe neuronal injury and apoptosis.Phalloidin staining was used to observe changes in neuron cytoskeleton.Transmission electron microscope was used to observe the changes of nerve synapse structure.Immunohistochemistry was used to observe the positive expression level of Rho.Western Blot method was used to detect the expression of Rho/ROCK pathway proteins,nerve growth promoting factor[nerve growth factor(NGF),brain-derived neurotrophic factor(BDNF),neurotrophic factor 3(NT3)],nerve growth inhibitor[neurite growth inhibitory factor(NogoA),myelin-associated protein(MAG),cofilin],synaptic regeneration remodeling-associated protein[post-synaptic dense substance(PSD-95)and synaptophysin(SYP)]and other proteins.Results:Compared with sham operation group,the neurological deficit score and cerebral infarction volume of model group increased,the structural damage such as neuronal damage,apoptosis,synaptic vesicle rupture,and fusion in the cerebral cortex,and the destruction of the cytoskeleton were serious,the Rho/ROCK pathway was activated,and the expression of NGF mediated by it decreased,while the expression of nerve inhibitory factor increased(P<0.05).Both green tea extract and Rho inhibitor could alleviate structural damage such as neuronal damage,apoptosis,synaptic vesicle rupture and fusion,and pathological damage such as cytoskeleton destruction,reduce neurological deficit score and cerebral infarction volume,inhibit the Rho/ROCK pathway activation and neuro suppressive factor expression,and promote the expression of NGF(P<0.05).Rho agonists could reverse the above effects of green tea extract(P<0.05).Conclusion:Green tea extract can inhibit the activation of Rho/ROCK to promote the regeneration and remodeling of neurons and synapses after cerebral infarction,improve nerve function damage,and play a protective effect on the brain.