CAJ | 학술논문

目的研究低氧诱导因子(HIF)-1α是否介导缺糖缺氧诱导的原代皮质神经元caspase非依赖性细胞死亡,Necrostain-1( Nec-1)能否抑制其表达起到保护作用.方法 (1)原代皮质神经元培养14d,予caspase抑制剂z-VAD.fmk(z-VAD)预保护30 min,同时分别予Nec-1 0.1,1,5,10,25,50 μmol/L,缺糖缺氧(OGD)2h、再灌注12h,通过LDH测细胞死亡情况；(2)予z-VAD作用后30 min后,缺糖缺氧(OGD)2h再灌注0,2,6,12,24,48 h,Westernblot检测HIF-1α蛋白表达情况,RT-PCR测HIF-1αRNA表达；(3)予z-VAD和Nec-125 μmol/L作用30min,OGD2h、再灌注12h后检测HIF-1α蛋白及RNA表达情况.结果 (1)予Nec-1 5μmol/L(6.97±0.06)后与未加Nec-1组(14.23 ±0.08)比较LDH水平明显下降(P＜0.05),Nec-1 25 μmol/L(2.21 ±0.05)时LDH降至正常水平与正常组(1.03±0.03)比较,P＞0.05)；(2)缺糖缺氧2h再灌注后HIF-1α蛋白表达增加,再灌注2h后(0.57±0.09)与正常组(0.24±0.01)相比差异有统计学意义(P＜0.05),再灌注12h(0.91 ±0.08)达高峰,HIF-1 αRNA表达无变化(P＞0.05)；(3)与未予Nec-1组(0.83±0.03)相比,Nec-1组(0.32±0.04) HIF-1 α蛋白表达明显降低(P＜0.05),HIF-1αRNA表达无变化(P＞0.05).结论 HIF-1α介导了缺氧缺氧诱导的原代皮质神经元caspase非依赖性死亡,Nec-1能够抑制HIF-1α的表达起到保护作用.
목적 연구저양유도인자(HIF)-1α시부개도결당결양유도적원대피질신경원caspase비의뢰성세포사망,Necrostain-1( Nec-1)능부억제기표체기도보호작용.방법 (1)원대피질신경원배양14d,여caspase억제제z-VAD.fmk(z-VAD)예보호30 min,동시분별여Nec-1 0.1,1,5,10,25,50 μmol/L,결당결양(OGD)2h、재관주12h,통과LDH측세포사망정황；(2)여z-VAD작용후30 min후,결당결양(OGD)2h재관주0,2,6,12,24,48 h,Westernblot검측HIF-1α단백표체정황,RT-PCR측HIF-1αRNA표체；(3)여z-VAD화Nec-125 μmol/L작용30min,OGD2h、재관주12h후검측HIF-1α단백급RNA표체정황.결과 (1)여Nec-1 5μmol/L(6.97±0.06)후여미가Nec-1조(14.23 ±0.08)비교LDH수평명현하강(P＜0.05),Nec-1 25 μmol/L(2.21 ±0.05)시LDH강지정상수평여정상조(1.03±0.03)비교,P＞0.05)；(2)결당결양2h재관주후HIF-1α단백표체증가,재관주2h후(0.57±0.09)여정상조(0.24±0.01)상비차이유통계학의의(P＜0.05),재관주12h(0.91 ±0.08)체고봉,HIF-1 αRNA표체무변화(P＞0.05)；(3)여미여Nec-1조(0.83±0.03)상비,Nec-1조(0.32±0.04) HIF-1 α단백표체명현강저(P＜0.05),HIF-1αRNA표체무변화(P＞0.05).결론 HIF-1α개도료결양결양유도적원대피질신경원caspase비의뢰성사망,Nec-1능구억제HIF-1α적표체기도보호작용.
Objective To explore whether hypoxia-inducible factor-1α( HIF-1α )is involved in oxygenglucose deprivation (OGD)induced caspase-independent cell death in primary cortical neurons and whether their expression is infected by necrostatin-1 ( Nec-1 ).Methods ( 1 ) Primary cerebrocortical neurons were cultured for 14 days.Pretred z-VAD.Fmk (z-VAD)and Nec-1 with 0.1,1,5,10,25 and 50 μ mol/L before the neurons were exposed to OGD for 2 hours and reoxygenated for 12 hours,then cell viability was determined by measure LDH level.(2)Pretred z-VAD before the neurons were exposed to OGD for 2 hours,then reoxygenated for 0,2,6,12,24and 48 hours.Then western blot analysis protein level of HIF-1 α ;rt-PCR check its RNA level.(3)Pretred z-VAD and Nec-1 with 25μ mol/L before the neurons were exposed to OGD for 2 hours and reoxygenated for 12 hours.Then western blot analysis protein level of HIF-1 α; rt-PCR check its RNA level.Result ( 1 ) When cells were pretread Nec-1 with 5 μ mol/L(6.97 ± 0.06),the level of LDH was lower than cells untreated( 14.23 ± 0.08 ) (P＜ 0.05);At 25 μmol/L( 2.21 ± 0.05),the level of LDH was essentially the same as that of the control( 1.03 ±0.03 ) (P＞0.05).(2)The protein level of HIF-1 αwas different from normal (0.24 ±0.01 ) when exposed to OGD for 2 hours and reoxygenated for 2 hours (0.57 ± 0.09) and was highest after cells were exposed to OGD for 2 hours and reoxygenated for 12 hours(0.91 ± 0.08 ) (P＜ 0.05 ).The RNA level of HIF-1 α when cells were exposed to OGD was not deferent from normal (P ＞ 0.05 ).( 3 ) When cells were pretread with Nec-1 (0.32 ± 0.04 ),the protein level of HIF-1α were lower than untreated(0.83 ±0.03) (P＜0.05),but the RNA level of HIF-1α had no deference(P ＞ 0.05).Conclusion HIF-1α was involved in cell' s caspase-independent cell death;Nec-1 can protect neurons through inhibiting the expression of HIF-1α.