CAJ | 학술논문

目的探讨依达拉奉减轻缺氧复氧引起的细胞毒性损伤及保护神经元细胞的分子作用机制. 方法建立缺氧缺糖复氧(OGD-R)神经元损伤模型,分空白对照组、OGD-R组、OGD-R加不同浓度依达拉奉组,检测不同时段乳酸脱氢酶(LDH)释放量、Caspase-3活性、Hoechst33258染色(计算核固缩比例),并进行比较选择适当浓度的依达拉奉及依达拉奉并特异性丝氨酸-苏氨酸蛋白激酶(AKT)阻断剂LY294002和MK2206对细胞分别进行预处理后再行OGD-R处理,用蛋白质免疫印迹法(Western blot)检测细胞外信号调节激酶(ERK)、AKT及Bcl-2蛋白表达,反转录聚合酶链反应(RT-PCR)检测脑源性神经生长因子(BDNF)、Bcl-2 mRNA表达,酶联免疫吸附试验(ELISA)检测BDNF蛋白表达.分析比较相应数据探讨依达拉奉在OGD-R模型中可能的保护机制. 结果依达拉奉预处理培养细胞减轻了缺氧复氧诱导的神经元损伤程度,LDH释放减少、Caspase-3活性和核固缩比例下降,且具有剂量依赖性;依达拉奉预处理后OGD-R,磷酸化ERK的表达量没有明显变化,而磷酸化AKT的表达量明显增加(P＜0.01).依达拉奉预处理可明显降低OGD-R处理细胞中LDH释放和核固缩比例(P＜0.01),但该过程可被MK2206抑制;与OGD-R组比较,依达拉奉预处理显著上调了BDNF和Bcl-2 mRNA的表达,且在OGD-R 8 h时尤其明显;ELISIA检测提示依达拉奉在同一时间点使BDNF蛋白表达增多;Western blot则显示依达拉奉上调了Bcl-2蛋白表达,这种作用可被MK2206抑制. 结论依达拉奉通过上调BDNF和Bcl-2的表达、抑制Caspase-3活性减轻缺氧复氧引起的神经元毒性损伤,磷脂酰肌醇3-激酶(PI3K)/AKT通路的激活可能是上述作用实现的重要途径之一.
목적 탐토의체랍봉감경결양복양인기적세포독성손상급보호신경원세포적분자작용궤제. 방법 건립결양결당복양(OGD-R)신경원손상모형,분공백대조조、OGD-R조、OGD-R가불동농도의체랍봉조,검측불동시단유산탈경매(LDH)석방량、Caspase-3활성、Hoechst33258염색(계산핵고축비례),병진행비교선택괄당농도적의체랍봉급의체랍봉병특이성사안산-소안산단백격매(AKT)조단제LY294002화MK2206대세포분별진행예처리후재행OGD-R처리,용단백질면역인적법(Western blot)검측세포외신호조절격매(ERK)、AKT급Bcl-2단백표체,반전록취합매련반응(RT-PCR)검측뇌원성신경생장인자(BDNF)、Bcl-2 mRNA표체,매련면역흡부시험(ELISA)검측BDNF단백표체.분석비교상응수거탐토의체랍봉재OGD-R모형중가능적보호궤제. 결과 의체랍봉예처리배양세포감경료결양복양유도적신경원손상정도,LDH석방감소、Caspase-3활성화핵고축비례하강,차구유제량의뢰성;의체랍봉예처리후OGD-R,린산화ERK적표체량몰유명현변화,이린산화AKT적표체량명현증가(P＜0.01).의체랍봉예처리가명현강저OGD-R처리세포중LDH석방화핵고축비례(P＜0.01),단해과정가피MK2206억제;여OGD-R조비교,의체랍봉예처리현저상조료BDNF화Bcl-2 mRNA적표체,차재OGD-R 8 h시우기명현;ELISIA검측제시의체랍봉재동일시간점사BDNF단백표체증다;Western blot칙현시의체랍봉상조료Bcl-2단백표체,저충작용가피MK2206억제. 결론 의체랍봉통과상조BDNF화Bcl-2적표체、억제Caspase-3활성감경결양복양인기적신경원독성손상,린지선기순3-격매(PI3K)/AKT통로적격활가능시상술작용실현적중요도경지일.
Objective To investigate the molecular mechanism how edaravone reduces cytotoxicity caused by hypoxia/reoxygenation-induced injury.Methods Ischemia model-hypoxia/ reoxygenation model which also called oxygen and glucose deprivation/reoxygenation model (OGD-R) was established.Cells were divided into control group,OGD-R group,OGD-R with edaravone of different concentration groups Cells of OGD-R model were pretreated with the appropriate concentration of edaravone or combine with the specific channel blockers LY294002 and MK2206 individually,the expression levels of ERK,AKT and Bcl-2 were detected with Western-blot,while the expression levels of BDNF and Bcl-2 mRNA detected with RT-PCR,the expression levels of BDNF protein detected with ELISIA,the levels of LDH releasing and Hoechst33258 staining used with homologue assay kit or regents.Results The pretreatment of cultured neurons with edaravone alleviated hypoxia/reoxygenation induced neuronal injury in a dose-dependent manner (LDH releasing reduced,Caspase-3 activity and ratio of nuclear pyknosis declined).The edaravone pretreatment did not increase significantly the p-ERK expression levels,but the amount of p AKT expression was significantly increased(P＜0.01).Pretreatment of edaravone(10 μmol/L) remarkably reduced the LDH release and declined the ratio of nuclear pyknosis after reoxygenation (P＜0.01),which was inhibited by MK2206.Compared to OGD-R,edaravone pretreatment markedly promoted BDNF and Bcl-2 mRNA expression at each time point,and the most pronounced effects occured at 8 h after OGDR.ELISIA analysis showed that BDNF protein level was significantly elevated after edaravone pretreatment at the same point.Western blot analysis indicted that edaravone pretreatment significantly enhanced Bcl-2 protein expression at 8 h after OGD-R,which was blocked by MK2206.Conclusions Edaravone may alleviate hypoxia/reoxygenation induced neuronal injury by enhancing BDNF and Bcl-2 expression and inhibiting Caspase-3 activity through activation of the PI3K/Akt pathway.