碱性成纤维细胞生长因子对创伤性脑损伤大鼠神经细胞自噬的影响

摘要

目的 探讨碱性成纤维细胞生长因子(bFGF)对创伤性脑损伤(TBI)大鼠神经细胞自噬的影响.方法 将120只健康成年雄性SD大鼠按随机数字表法分为假手术组、TBI+载体容量组和TBI+ bFGF治疗组,每组40只.PinPointTM精密皮质撞击器制作大鼠TBI模型,假手术组暴露硬脑膜而不撞击.TBI+ bFGF组于伤前1h鼻腔内给予250 μg/kg的人重组bFGF,而假手术组和TBI+载体容量组在伤前1h鼻腔内给予等量生理盐水.伤后6h用碘化丙啶(PI)染色观察坏死细胞情况.伤后24 h采用改良神经功能缺损评分(mNSS)评价bFGF对大鼠伤后神经功能的影响.伤后48 h采用于湿法测脑组织含水量;采用Westem blot法检测Beclin-1、P62蛋白和微管相关蛋白1轻链3(LC3)-Ⅱ/Ⅰ蛋白比值的水平;脑组织切片积分法计算脑组织损伤体积;免疫荧光染色测定神经元特异性核蛋白(NeuN)阳性细胞;TUNEL法观察细胞凋亡情况.结果 与假手术组[(4.0±1.2)％]比较,TBI+载体容量组[(54.3±10.1)％]和TBI+ bFGF组[(34.5±10.5)％]的坏死细胞数明显增加(P＜0.05),但TBI+ bFGF组坏死细胞数少于TBI+载体容量组(P＜0.05).与假手术组[(0.3±0.5)分]比较,TBI+载体容量组[(5.8±0.8)分]和TBI+ bFGF组[(4.7±1.1)分]的mNSS明显升高(P＜0.05),但TBI+ bFGF组mNSS低于TBI+载体容量组(P＜0.05).与假手术组[(76.7±0.7)％]比较,TBI+载体容量组[(79.2±0.5)％]和TBI+ bFGF组[(78.4±1.0)％]脑组织含水量有明显增加(P＜0.05),但TBI bFGF组脑组织含水量明显少于TBI+载体容量组(P＜0.05).与假手术组比较,TBI+载体容量组和TBI bFGF组Beclin-1蛋白、LC3-Ⅱ/Ⅰ蛋白比值水平均明显升高(P＜0.05),P62蛋白明显下降(P＜0.05),脑组织损伤体积明显增加(P＜0.05),NeuN阳性细胞数明显增加(P＜0.05),凋亡细胞数明显增加(P＜0.05);与TBI+载体容量组比较,TBI+ bFGF组Beclin-1蛋白和LC3-Ⅱ/Ⅰ蛋白比值水平的上调被明显抑制(P＜0.05),P62蛋白的下调被明显抑制(P＜0.05),脑组织损伤体积明显减少(P＜0.05),NeuN阳性细胞数和凋亡细胞数明显减少(P＜0.05).结论 bFGF能明显抑制TBI大鼠过度的自噬反应,减轻脑水肿,减少细胞凋亡和坏死,并改善神经功能.%Objective To investigate the effect of basic fibroblast growth factor (bFGF) on autophagy of nerve cells in rats after traumatic brain injury (TBI).Methods A total of 120 healthy adult male SD rats were randomly divided into sham group,TBI + vehicle group,and TBI + bFGF group by random number table method,with 40 rats in each group.PinPointTM Precision Cortical Impactor was used to simulate the pathological damage after TBI.In the sham group,the dura was exposed without impact.In the TBI + bFGF group,250 μg/kg of human recombinant bFGF was given in the nasal cavity 1 hour before the modeling,while in the sham group and TBI + Vehicle group,the same amount of saline was given in the nasal cavity 1 hour before the modeling.The necrotic cells were observed by propidium iodide(PI) staining 6 hours after injury.The effect of bFGF on the nerve function after TBI in rats was evaluated with modified neurological severity score (mNSS) 24 hours after injury.The water content of brain tissue was measured by dry and wet method 48 hours after injury.The ratio of Beclin-1,P62 protein and microtubule-associated protein 1 light 3 (LC3)-Ⅱ/Ⅰ protein was detected by western blot.The volume of brain injury was calculated by integral method of brain tissue section.The positive neuron specific nuclear protein (NeuN) cells were observed by immunofluorescence staining.The apoptotic cells were observed by TUNEL.Results Compared with the sham group [(4.0 ± 1.2) ％],the percentage of necrotic cells in TBI + vehicle group [(54.3 ± 10.1) ％] and TBI + bFGF group [(34.5 ± 10.5) ％] increased significantly (P ＜ 0.05),but the percentage of necrotic cells in TBI + bFGF group increased less than that in TBI + vehicle group (P ＜ 0.05).Compared with the sham group [(0.3 ± 0.5)points],the mNSS in the TBI + vehicle group [(5.8 ±0.8)points] and TBI + bFGF group [(4.7 ± 1.1) points] were significantly increased (P ＜ 0.05),but the mNSS of TBI + bFGF group was lower than that of TBI + vehicle group (P ＜ 0.05).Compared with the sham group [(76.7 ± 0.7) ％],the water content of brain tissue of TBI + vehicle group [(79.2 ± 0.5) ％] and TBI + bFGF group [(78.4 ± 1.0) ％] were significantly increased (P ＜0.05),but the water content of TBI + bFGF group was significantly lower than that of TBI + vehicle group (P ＜0.05).Compared with the sham group,protein expression of Beclin-1 and LC3-I/Ⅰ in TBI + vehicle group and TBI +bFGF group were significantly improved (P ＜ 0.05),P62 protein expression was significantly decreased (P ＜ 0.05),the volume of brain tissue injury was significantly increased (P ＜ 0.05),the number of positive NeuN cells increased significantly (P ＜ 0.05),and the number of apoptotic cells and apoptotic cells were significantly increased (P ＜0.05).Compared with the TBI + Vehicle group,the up-regulation of Beclin-1 protein and LC3-Ⅱ/Ⅰ protein ratio was obviously inhibited in the TBI + bFGF group (P ＜ 0.05),the down-regulation of P62 protien was significantly suppressed,the volume of brain tissue injury was significantly decreased,and the number of positive NeuN cells and apoptotic cells was significantly reduced (P ＜ 0.05).Conclusion The bFGF can significantly inhibit excessive autophagy in rats after TBI,reduce brain edema,reduce cell apoptosis and necrosis,and improve neural function.