颅脑创伤后大鼠脑组织自噬表达变化及渥曼青霉素保护作用的研究

Expression of autophagy-related proteins in brain tissue and neuroprotective effect of wortmannin after traumatic cerebral injury in rats

目的探索颅脑创伤后大鼠脑组织自噬表达变化及渥曼青霉素的保护作用,为临床治疗脑外伤提供实验依据。方法将120只健康成年雄性SD大鼠随机分为3组[假手术组(A组)、颅脑创伤组(B组)、渥曼青霉素治疗组(C组)],每组40只。B、C组使用PinPoint^(TM)精密颅脑创伤撞击器、直径4mm的打击头制作中度创伤性脑损伤模型,其中C组于建模前10min侧脑室注入渥曼青霉素(0.5μg/kg),B组注入含有二甲基亚砜(DMSO)的PBS(25μl/kg);A组暴露硬脑膜但不撞击,建模前注入含有DMSO的PBS(25μl/kg)。采用Western blot检测自噬相关蛋白(LC3-Ⅱ,p62)表达水平,应用大鼠行为学评分观察大鼠颅脑创伤后神经功能的变化,采用干湿法检测脑组织含水量。结果与A组相比,B组和C组大鼠在颅脑创伤后24、72、168h脑组织LC3-Ⅱ表达水平增加,p62表达水平减少,LC3-Ⅱ/LC3-Ⅰ比值升高,差异均有统计学意义(均P<0.05)。与B组相比,C组大鼠在颅脑创伤后24、72、168h脑组织LC3-Ⅱ表达水平减少,p62表达水平增加,差异均有统计学意义(均P<0.05);伤后72、168h脑组织LC3-Ⅱ/LC3-Ⅰ比值降低(均P<0.05)。颅脑创伤后48、72h,C组大鼠行为学评分均高于B组(均P<0.05)。颅脑创伤后72h,B组和C组脑组织含水量均较A组明显增加(均P<0.05),但C组较B组明显减少(P<0.05)。结论渥曼青霉素能明显抑制颅脑创伤后过度的自噬反应,减轻脑水肿,改善大鼠神经功能。

Objective To investigate the expression of autophagy-related proteins in brain tissues and the neuroprotective effect of wortmannin after traumatic cerebral injury in rats. Methods One hundred and twenty healthy adult male SD rats were randomly divided into 3 groups: the sham operation group(A), the traumatic brain injury group(B) and the wortmannin group(C) with 40 animals in each group. Precision cortical impactor was used to establish medium traumatic brain injury model in groupB and C with a hit point of 4mm in diameter. The rats were pretreated with a single intracerebroventricular injection of wortmanninin group C, while with injection of DMSO in group B. The rats in group A received surgery to expose the dura without actualimpact, and were injected PBS before the modeling. The expression of microtubule-associated protein 1 light chain3B (LC3-Ⅱ)and p62 in brain tissues were detected by Western blot. The neurologic function of rats was assessed with behavioral score.Water content was evaluated by dry-wet analysis. Results The expression of LC3-Ⅱ was increased in groups B and Ccompared with group A at 24h, 72h and 168h after injury, the expression of p62 was decreased, and the ratio of LC3-Ⅱ/LC3-Ⅰwas increased (all P<0.05). The expression of LC3-Ⅱ was lower in group C than that in group B at 24h, 72h and 168h afterinjury, the expression of p62 was higher (all P<0.05) in group B and the ratio of LC3-Ⅱ/LC3-Ⅰ was decreased at 72h and 168h(all P<0.05) compared to group C. At 48h and 72h after injury, the behavioral scores in group C were significantly higher than thatin group B (all P<0.05). At 72h, the water contents of brain tissue in group B and C were higher than that in group A, while the water content in group C was significantly lower than that in group B(P<0.05). Conclusion Wortmannin can suppress the excessive autophagy, reduce the brain edema induced by traumatic cerebral injury and improve the neurological function in rats.