川蛭通络胶囊对小鼠脑缺血再灌注损伤的保护作用及机制研究

Protective effects Chuanzhi Tongluo capsule on ischemia-reperfusion induced brain injury in mice

目的探讨川蛭通络胶囊对小鼠局灶性脑缺血再灌注损伤的保护作用。方法采用ICR小鼠中动脉阻断(MCAO)制备小鼠局灶性脑缺血再灌注模型,将ICR小鼠随机分为假手术组(sham)、MCAO/R组、川蛭通络胶囊治疗组(CZTLC),每组15只。其中CZTLC组于手术前5 d,口服灌胃给药2次/d。缺血60 min、再灌注24 h对小鼠进行神经行为学评分,深度麻醉取小鼠脑组织进行病理学TTC、HE及甲苯胺蓝染色;超氧化物歧化酶(SOD)试剂盒、丙二醛(MDA)测试盒分别测定脑组织SOD及MDA含量;ELISA检测肿瘤坏死因子-α(TNF-α)、白介素-1β(IL-1β)、IL-4及IL-10的表达变化;RT-PCR、Western Blot检测Bax、Bcl-2、Caspase-3基因及蛋白水平表达。结果HE染色、尼氏染色显示,与假手术组相比,MCAO/R可明显诱导左侧皮层和海马CA1区神经元损伤(P>0.05)。模型组大鼠脑组织损伤侧主要表现神经元细胞体积缩小,胞浆空泡变性,细胞间排列疏松,海马、皮质的神经元大部分萎缩;细胞内尼氏小体数量减少,染色变浅。与模型组比较,观察到川蛭通络胶囊治疗组小鼠脑组织区域神经元细胞损伤较轻微,表现为神经细胞的紧密排列和细胞内尼氏小体数量增加。同时,与假手术组相比,药物处理组海马CA1和皮层的正常神经元数量显著减少。假手术组小鼠脑组织SOD活性明显高于模型组,MDA水平明显低于模型组(P>0.05);与模型组相比,川蛭通络胶囊组小鼠脑组织中SOD活性显著升高,MDA水平显著降低(P>0.05)。模型组小鼠脑匀浆TNF-α、IL-1β及IL-6含量较假手术组明显升高,而IL-10含量明显降低(P<0.05)。CZTLC干预组小鼠脑匀浆TNF-α、IL-1β及IL-6含量较模型组明显降低,而IL-10含量明显升高(P<0.05)。CZTLC可显著下调凋亡蛋白caspase-3及Bax在基因水平及蛋白水平的表达,同时其亦明显上调Bcl-2的表达,与CAMO/R组进行对比差异有统计学意义(P<0.05)。结论CZTLC在小鼠脑缺血再灌注损伤中具有明确的保护作用,其机制与改善炎性反应及抑制细胞凋亡有关。

Objective To investigate the protective effects Chuanzhi Tongluo capsule(CZTLC)on ischemia-reperfusion induced brain injury in mice.Methods The ischemia-reperfusion injury(ICR)mice models were established by middle-cerebral-artery-occlusion(MCAO)surgery.The mice with ICR were randomly divided into three groups:sham operation group,MCAO/R group and CZTLC treatment group,with 15 mice in each group.The mice in CZTLC group were orally administrated twice a day for 5 days before MCAO.The neurological function scores were measured at 1h after ischemia and 24h after reperfusion.And the brain tissues of mice were taken out for pathological TTC,HE and toluidine blue staining.The levels of SOD,MDA,IL-1β,TNF-α,IL-6,IL-10 in cerebral tissues of mice were detected by ELISA.Western blot and quantitative reverse transcription polymerase chain reaction(qRT-PCR)were used to detect the expression levels of Bax,Bcl-2 and Caspase-3.Results MCAO/R could induce the neuron injury in left cortex and hippocampus CAI area(P>0.05).And CZTLC significantly decreased the neurobehavioral scores and infarct area,and improved the oxidation state of brain tissues.In additional,CZTLC significantly increased the levels of anti-inflammatory cytokines(IL-4 and IL-10),and decreased the expression levels of pro-inflammatory cytokines(TNF-αand IL-1β).Furthermore,CZTLC increased Bcl-2 levels and decreased the expression levels of Bax and caspase-3 mRNA and protein(P<0.05).Conclusion CZTLC has definite protective effects on cerebral ischemia-reperfusion injury in mice,and its action mechanism is related to improving inflammatory response and inhibiting cell apoptosis.