CAJ | 학술논문

目的观察重组人红细胞生成素(rHuEP0)对戊四氮(PTZ)点燃的癫痫持续状态(status epilepticus,SE)的SD大鼠海马神经元磷酸化蛋白激酶B(p-PKB/p-Akt)和半胱氨酸天冬氨酸蛋白酶9(Caspase-9)表达的影响,应用磷脂酰肌醇3激酶(P13K)抑制剂LY294002进一步探讨rHuEPO作用的可能机制.方法采用PTZ点燃大鼠SE模型,将175只大鼠随机分为正常对照组(给予生理盐水腹腔注射)、PTZ组(腹腔注射PTZ点燃SE发作后30 min腹腔注射生理盐水)、rHuEPO组(SE发作后30 min腹腔注射rHuEPO 5000 U/kg)、LY294002组(SE发作后10 min脑室注射5 μlLY294002,SE发作后30 min腹腔注射rHuEPO 5000 U/kg)、二甲基亚砜(DMSO)组(SE发作后10 min脑室注射5μlDMSO,SE发作后30min腹腔注射rHuEPO 5000U/kg),并给予相应处理.检测大鼠行为学和脑电图的改变,免疫组织化学法观察p-Akt、caspase-9的表达,RT-PCR方法检测各组大鼠海马caspase-9 mRNA的表达;Western blotting方法检测各组大鼠海马Akt、p-Akt蛋白的表达.结果与PTZ组比较,rHuEPO组p-Akt免疫反应阳性细胞和P-Akt蛋白表达水平均增高,caspase-9免疫反应阳性细胞及caspase-9mRNA表达水平均降低(P<0.05);与rHuEPO组比较,LY294002组p-Akt免疫反应阳性细胞和p-Akt蛋白表达水平均降低,caspase-9免疫反应阳性细胞及caspase-9mRNA表达水平均增高(p<0.05).结论在SE大鼠模型中rHuEPO激活PI3K/Akt信号转导途径调整线粒体凋亡途径相关调控因子caspase-9,借以发挥抗凋亡的神经保护作用.
목적 관찰중조인홍세포생성소(rHuEP0)대무사담(PTZ)점연적전간지속상태(status epilepticus,SE)적SD대서해마신경원린산화단백격매B(p-PKB/p-Akt)화반광안산천동안산단백매9(Caspase-9)표체적영향,응용린지선기순3격매(P13K)억제제LY294002진일보탐토rHuEPO작용적가능궤제.방법 채용PTZ점연대서SE모형,장175지대서수궤분위정상대조조(급여생리염수복강주사)、PTZ조(복강주사PTZ점연SE발작후30 min복강주사생리염수)、rHuEPO조(SE발작후30 min복강주사rHuEPO 5000 U/kg)、LY294002조(SE발작후10 min뇌실주사5 μlLY294002,SE발작후30 min복강주사rHuEPO 5000 U/kg)、이갑기아풍(DMSO)조(SE발작후10 min뇌실주사5μlDMSO,SE발작후30min복강주사rHuEPO 5000U/kg),병급여상응처리.검측대서행위학화뇌전도적개변,면역조직화학법관찰p-Akt、caspase-9적표체,RT-PCR방법검측각조대서해마caspase-9 mRNA적표체;Western blotting방법검측각조대서해마Akt、p-Akt단백적표체.결과 여PTZ조비교,rHuEPO조p-Akt면역반응양성세포화P-Akt단백표체수평균증고,caspase-9면역반응양성세포급caspase-9mRNA표체수평균강저(P<0.05);여rHuEPO조비교,LY294002조p-Akt면역반응양성세포화p-Akt단백표체수평균강저,caspase-9면역반응양성세포급caspase-9mRNA표체수평균증고(p<0.05).결론 재SE대서모형중rHuEPO격활PI3K/Akt신호전도도경조정선립체조망도경상관조공인자caspase-9,차이발휘항조망적신경보호작용.
Objective To observe the effect of recombinant human erythropoietin (rhuEPO) on p-Akt and caspase-9 expressions in the hippocampus of rats with status epilepticus (SE) and explore the neuroprotective mechanism of rhuEPO. Methods Adult male SD rats were randomized into control, PTZ, rHuEPO, LY294002 group, and DMSO groups and treated with normal saline (NS), PTZ, PTZ+rHuEPO, PTZ+LY294002+rHuEPO, and PTZ+DMSO+rHuEPO, respectively. The behavioral and electroencephalogram (EEG) changes of the rats were recorded, and the expressions ofp-Akt and caspase-9 were detected using immunohistochemistry. The hippocampal expression of caspase-9 mRNA was detected using RT-PCR, and the expressions of Akt and p-Akt proteins were determined with Western blotting. Results The p-Akt-positive cell and p-Akt protein expression increased significantly while the caspase-9-positive cell and caspase-9 mRNA expression decreased in rHuEPO group as compared with those in PTZ group (P<0.05). LY294002 treatment prior to rHuEPO injection significantly abolished the effects of rHuEPO on caspase-9 and p-Akt immtmohistochemical positivity and caspase-9 mRNA and p-Akt protein expressions (P<0.05). Conclusion Administration of rHuEPO activates the PI3K/Akt signaling pathway in SE rats and increases the expression of p-Akt protein to regulate the expression of caspase-9, a regulatory factor of the mitochondrial-dependent apoptotic pathway, and therefore provides anti-apoptotic and neuroprotective effects.