促红细胞生成素对大鼠创伤性脑损伤的神经保护作用

Neuroprotective effect of erythropoiet in treatment of traumatic brain injury in rats

目的研究促红细胞生成素（EPO）对创伤性脑损伤（TBI）大鼠的神经保护作用，并探讨内质网应激反应及Caspase-12介导的凋亡在EPO神经保护中的作用。方法140只成年雄性SD大鼠，按随机数字表法分为假手术组、假手术＋EPO治疗组（EPO组）、TBI组和TBl＋EPO治疗组，各组35只。采用液压脑损伤方法制作大鼠TBI模型，伤后6h腹腔注射EPO（5000U／kg）。采用TUNEL染色法观察各组大鼠伤后24h损伤皮层阳性染色细胞率，使用改良神经功能评分（mNSS）评估各组干预后1，4，7，21，28，35d神经功能的改变。采用Western blot和免疫荧光染色检测各组伤后24h损伤皮层Caspase-12和内质网应激反应标志性蛋白C／EBP同源蛋白（CHOP）表达水平的改变。结果TUNEL染色结果显示，TBI组大鼠伤后24h损伤皮层阳性细胞率显著增多[（30．3±2．3）％]（P〈0．01），与TBI组相比，TBI＋EPO组能显著降低阳性细胞率[（14．6±1．5）％]（P〈0．01）。与TBI组相比，TBI＋EPO组在伤后7～35d均能显著降低mNSS评分（P〈0．05）。Westernblot结果显示，TBI组伤后24h损伤皮层Caspase-12和CHOP蛋白的表达水平较假手术组显著提高（P〈0．01）；与TBI组相比，TBI＋EPO组能够显著下调皮层Caspase-12和CHOP表达水平（P〈0．01）。免疫荧光染色结果显示，TBI组伤后24h皮层的Caspase-12和CHOP染色阳性细胞率较假手术组显著提高（P〈0．01）；TBI＋EPO组伤后24hCaspase-12和CHOP染色阳性细胞率较TBI组显著降低（P〈0．01）。结论外源性EPO对TBI大鼠有明确的神经保护作用。EPO可能通过抑制TBI后内质网应激反应和Caspase-12介导的凋亡发挥其神经保护作用。

Objective To evaluate the neuroprotective effects of erythropoiet （EPO） for traumatic brain injury （TBI） in rats and investigate the possible role of endoplasmic reticullum stress response and Caspase-12-induced apoptosis. Methods According to the random number table, 140 male Sprague- Dawley rats were divided into sham injury group, sham injury plus EPO group, TBI group and TBI plus EPO group, with 35 rats per group. TBI was induced by a fluid percussion device. EPO （5 000 U/kg in saline） was administered intraperitoneally at 6 hours after injury. The rate of TUNEL positive cells in injured cortex were measured to evaluate cell apoptosis status. Neurological function was assessed at days 1, 4, 7, 21, 28 and 35 after intervention using a modified neurological severity score （mNSS）. At 24 hours after injury, the expressions of Caspase-12 in injured cortex and C/EBP-homologous protein （CHOP） which was the symbol of ERS response were measured by Western blot and immunofluorescent staining to assess the changes of ERS response after TBI and EPO treatment. Results TUNEL-positive staining cell density was significantly increased by （ 30. 3 ±2.3 ） % in the injured cortex 24 hours after injury （P 〈 0.01 ）. Compared with TBI group, TBI plus EPO group had a significant decrease of the positive rate of TUNEL cells [ （ 14.6 ± 1.5 ） % ] （ P 〈 0.01 ）. Compared with TBI group, mNSS score significantly was decreased in TBI plus EPO group at 7-35 days after injury （ P 〈 0.05 ）. At 24 hours It after injury, the results of Western blot showed that the expression levels of Caspase-12 and CHOP in the injured cortex in TBI group were higher than those in sham group, but that in TBI plus EPO group was lower than those in TBI group （ P 〈 0.01 ）. At 24 hours after injury, the results of immunofiuorescent staining showed the rates of Caspase-12 and CHOP positive cells in the injured cortex in TBI group were higher than sham group （P 〈 0.01 ）. But the rates of Caspase-12 and CHOP positive cells in TBI plus EPO group was lower than that in TBI Group （ P 〈 0.01 ）. Conclusion Exogenous EPO has significant neuroprotective effects on TBI rats. EPO may exert its neuroproective effects through suppression of ERS response and inhibition of Caspase-12-induced apoptosis.