CAJ | 학술논문

目的了解程序性坏死阻断剂necrostatin(Nee-1)对铝诱导的神经细胞凋亡的影响,并探讨Nec-1对Caspases凋亡通路的作用机制.方法用4 mmol/L铝染毒神经母细胞瘤细胞株(SH-SYSY)制备铝致神经细胞死亡模型,在不同浓度铝和(或)Nec-1作用下检测细胞活力的变化,用Heochst 33342/碘丙啶(PI)双染法观察细胞的凋亡和坏死现象,Annexin V/PI双染法定量细胞的凋亡率和坏死率.用Caspase-3、Caspase-8、Caspase-9酶活力的变化检测凋亡通路,用免疫细胞化学法检测核转录因子(NF-κB)和细胞色素c(Cyt-c)蛋白表达的变化.结果随着铝染毒剂量的加大,细胞活力明显下降;但加入60 μmol/L Nec-1后,细胞活力明显增强.差异有统计学意义(P<0.05). Nec-1浓度的提高使细胞活力明显上升,表现了明显的促进神经细胞生长、抑制细胞死亡的作用.凋亡和坏死的荧光显微镜观察及流式细胞术定量检测结果表明,随着铝染毒剂量的增加,神经细胞的凋亡率与坏死率明显上升,但在0～90μmol/L Nec-1作用下,Nec-1浓度越高,细胞的坏死率降低越明显,差异均有统计学意义(P<0.05,P<0.01).同时,虽然Nec-1是程序性坏死的阻断剂,但其对凋亡率也有明显的影响,可以使染铝细胞的凋亡率明显下降,差异有统计学意义(P<0.05,P<0.01).对Nec-1作用后细胞的凋亡相关酶Caspase-3、Caspase-8、Caspase-9活力变化检测结果表明,Nec-1对Caspase-9活力的影响不明显,且对线粒体通路的凋亡诱导分子Cry-c的作用也不明显;但对Caspase-8活力的影响明显,表现在可以明显降低各染铝细胞的Caspase-8活力,提高NF-κB蛋白表达量和在高剂量染铝细胞中降低Caspase-3的活力.结论 Nec-1可以降低铝诱导的神经细胞凋亡,并通过死亡受体通路Caspase-8上调NF-κB表达,降低细胞凋亡率.
목적 료해정서성배사조단제necrostatin(Nee-1)대려유도적신경세포조망적영향,병탐토Nec-1대Caspases조망통로적작용궤제.방법 용4 mmol/L려염독신경모세포류세포주(SH-SYSY)제비려치신경세포사망모형,재불동농도려화(혹)Nec-1작용하검측세포활력적변화,용Heochst 33342/전병정(PI)쌍염법관찰세포적조망화배사현상,Annexin V/PI쌍염법정량세포적조망솔화배사솔.용Caspase-3、Caspase-8、Caspase-9매활력적변화검측조망통로,용면역세포화학법검측핵전록인자(NF-κB)화세포색소c(Cyt-c)단백표체적변화.결과 수착려염독제량적가대,세포활력명현하강;단가입60 μmol/L Nec-1후,세포활력명현증강.차이유통계학의의(P<0.05). Nec-1농도적제고사세포활력명현상승,표현료명현적촉진신경세포생장、억제세포사망적작용.조망화배사적형광현미경관찰급류식세포술정량검측결과 표명,수착려염독제량적증가,신경세포적조망솔여배사솔명현상승,단재0～90μmol/L Nec-1작용하,Nec-1농도월고,세포적배사솔강저월명현,차이균유통계학의의(P<0.05,P<0.01).동시,수연Nec-1시정서성배사적조단제,단기대조망솔야유명현적영향,가이사염려세포적조망솔명현하강,차이유통계학의의(P<0.05,P<0.01).대Nec-1작용후세포적조망상관매Caspase-3、Caspase-8、Caspase-9활력변화검측결과 표명,Nec-1대Caspase-9활력적영향불명현,차대선립체통로적조망유도분자Cry-c적작용야불명현;단대Caspase-8활력적영향명현,표현재가이명현강저각염려세포적Caspase-8활력,제고NF-κB단백표체량화재고제량염려세포중강저Caspase-3적활력.결론 Nec-1가이강저려유도적신경세포조망,병통과사망수체통로Caspase-8상조NF-κB표체,강저세포조망솔.
Objective To study the effect of necroslatin (Nee-1) on apoptosis induced by aluminum (A1), and approach the mechanism. Methods Neural cell death model was made by 4 mmol/L Al treated neuroblastoma cells (SH-SY5Y). Cell viabilities were detected at different concentrations of Al and/or Nec-1. Heochst 33342 / PI double staining was used to observe apoptosis and (or) necrosis that were quantified by flow cytometry using Annexin V / PI double staining. Apoptotic pathway was tested by activities of Caspase-3, Caspase-8 and Caspase-9. In addition, the expression of NF-kB and Cyt-c was measured by immunocytochem-istry.Results Cell viabilities were significantly decreased with the increasing concentrations of Al (P<0.05), which could be significantly upregulated by 60 μmol/L Nec-1 (P<0.05) and were correlated with the concentrations of Nec-1 (P<0.05, P<0.01). Apoptosis and necrosis were observed under fluorescent microscope and quantified by flow cytometry, which suggested an increasing trend of apoptotic and necrotic rates (P<0.05, P< 0.01). Whereas, Nec-1 could not only decrease the necrotic rate but also apoptotic rate as well (P<0.05, P< 0.01). Data of Nec-1 on caspases activities showed that Nec-1 could not affect Caspase-9 activity (P>0.05) and Cty-c protein expression as well (P>0.05). However, Nec-1 could reduce Caspase-8 activity significantly (P< 0.05, P<0.01) and increase NF- k B protein expression(P<0.05, P<0.01) and finally decrease Caspase-3 activity (P<0.05 ). Conclusion Nec-1 could reduce cell apoptosis induced by Al, through Caspase-8 pathway, and up-regulate the expression of NF- k B protein.