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目的:研究胎球蛋白(Fetuin)在大鼠局灶性脑缺血中的作用及与炎症的关系,对氧糖剥夺神经元损伤的保护作用。方法:大鼠大脑中动脉闭塞(middle cerebral artery occlusion,MCAO)60 min制备局灶性脑缺血再灌注(cerebral ischemia reperfusion)(MCAO/R)模型。经神经行为学特征、氯代三苯基四氮唑(2,3,5-triphenyltetrazolium chloride,TTC)染色、免疫组织化学、Western Blot及定量PCR方法分析:①MCAO/R不同时间段Fetuin在损伤梗塞区域的表达;②MCAO 30 min后静脉注射(intravenous injection,iv)Fetuin(500、50、25、5 mg·kg~(-1)),24 h后大脑梗塞体积及神经行为学特征;③MCAO后不同时间点(15、30、60、120 min)给予Fetuin(50 mg·kg~(-1),iv)24 h后脑梗塞体积的变化;④MCAO 30 min后给予Fetuin(50 mg·kg~(-1),iv),24 h后脑缺血区域巨噬细胞(macrophages,M)、小胶质细胞(microglia,MG)及肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)的变化;⑤Fetuin对体外氧糖剥夺/复氧(oxygen glucose deprivation/reoxygenation,OGD/R)神经元损伤的影响。结果:①大鼠脑缺血2 h损伤区域Fetuin mRNA及蛋白表达增强,48 h达高峰;②Fetuin改善神经行为学特征,缩小脑梗塞体积,作用呈量-效依赖关系;③MCAO后15、30、60 min给予Fetuin均缩小脑梗塞体积,作用强弱依次为15>30>60 min,120 min则无明显效果;④Fetuin减少M/MG在脑损伤区域的聚集,抑制TNF-α的表达;⑤Fetuin(1~100μmol·L~(-1))减少OGD/R神经元乳酸脱氢酶(lactate dehydrogenase,LDH)释放,与Fetuin浓度呈反向关系。结论:MCAO/R大鼠缺血性脑损伤组织自身表达Fetuin;外源性Fetuin改善脑缺血大鼠神经行为学特征,缩小缺血性脑梗塞体积,对OGD/R导致的神经元损伤有保护作用;提示Fetuin参与对神经元的保护而产生对脑缺血有益作用,其作用与抑制M/MG在病灶区域的聚集和TNF-α表达有关。
OBJECTIVE: To study the role of Fetuin in focal cerebral ischemia in rats and its relationship with inflammation and the protective effect of Fetuin on neuronal injury induced by oxygen-glucose deprivation. Methods: Cerebral ischemia reperfusion (MCAO / R) model was established by middle cerebral artery occlusion (MCAO) for 60 min in rats. The neurobehavioral characteristics, TTC staining, immunohistochemistry, Western Blot and quantitative PCR analysis: ①MCAO / R at different time points Fetuin in the injury of the infarct ; (2) Fetuin (500, 50, 25, 5 mg · kg -1) intravenous injection (iv) for 30 min after MCAO, cerebral infarct volume and neurobehavioral characteristics after 24 h; Fetuin (50 mg · kg -1) was given at the time points (15, 30, 60, 120 min), and the changes of cerebral infarct volume were observed after 24 h. ), iv), macrophages (M), microglia (MG) and tumor necrosis factor-α (TNF-α) in the ischemic area after 24 h. ⑤Fetuin on oxygen glucose deprivation / reoxygenation (oxygen glucose deprivation / reoxygenation, OGD / R) neuronal damage. Results: ① Fetuin mRNA and protein expression increased at 48 hours after focal cerebral ischemia in rats, and peaked at 48 hours. (2) Fetuin improved neurobehavioral characteristics and reduced infarct volume in a dose-dependent manner. ③ After 15, 30, Fetuin at 60 min reduced the volume of cerebral infarction, the effect was 15> 30> 60 min, followed by 120 min; ④Fetuin reduced the accumulation of M / MG in brain injury area and inhibited the expression of TNF-α; ⑤Fetuin (1 ~ 100μmol·L -1) decreased the release of lactate dehydrogenase (LDH) in OGD / R neurons, which was inversely related to the Fetuin concentration. CONCLUSION: Fetuin is expressed in MCAO / R rats with ischemic brain injury. Exogenous Fetuin can improve the neurobehavioral characteristics of ischemic brain and reduce the volume of ischemic cerebral infarction. The damage of neurons induced by OGD / R Suggesting that Fetuin may play a beneficial role in the protection of neurons against cerebral ischemia. Its role is related to the inhibition of the aggregation of M / MG and the expression of TNF-α in the lesion area.