Zhang Qin-li, Ji Xiu-liang, Guo Wei-li, Zhang Ce, Liu Cheng-yun, Niu Qiao
Department of Occupational Health, Shanxi Medical University, Taiyuan 030001, China.
Zhonghua Lao Dong Wei Sheng Zhi Ye Bing Za Zhi. 2010 Mar;28(3):175-80.
To study the effect of necrostatin (Nec-1) on apoptosis induced by aluminum (Al), and approach the mechanism.
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. Hoechst 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-kappa B and Cyt-c was measured by immunocytochemistry.
Cell viabilities were significantly decreased with the increasing concentrations of Al (P < 0.05), which could be significantly upregulated by 60 micromol/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-kappa B protein expression (P < 0.05, P < 0.01) and finally decrease Caspase-3 activity (P < 0.05).
Nec-1 could reduce cell apoptosis induced by Al, through Caspase-8 pathway, and up-regulate the expression of NF-kappa B protein.
研究坏死抑制因子（Nec-1）对铝（Al）诱导的细胞凋亡的影响，并探讨其机制。
用4 mmol/L Al处理神经母细胞瘤细胞（SH-SY5Y）建立神经细胞死亡模型。检测不同浓度的Al和/或Nec-1作用下的细胞活力。采用Hoechst 33342/PI双染法观察细胞凋亡和（或）坏死情况，并用Annexin V/PI双染法通过流式细胞术进行定量分析。通过检测Caspase-3、Caspase-8和Caspase-9的活性来检测凋亡途径。此外，采用免疫细胞化学法检测NF-κB和细胞色素C（Cyt-c）的表达。
随着Al浓度的增加，细胞活力显著降低（P<0.05），60 μmol/L Nec-1可使其显著上调（P<0.05），且与Nec-1浓度相关（P<0.05，P<0.01）。在荧光显微镜下观察到凋亡和坏死情况，并通过流式细胞术进行定量分析，结果显示凋亡率和坏死率呈上升趋势（P<0.05，P<0.01）。然而，Nec-1不仅可以降低坏死率，还可以降低凋亡率（P<0.05，P<0.01）。Nec-1对半胱天冬酶活性的数据显示，Nec-1不影响Caspase-9活性（P>0.05）和Cty-c蛋白表达（P>0.05）。然而，Nec-1可显著降低Caspase-8活性（P<0.05，P<0.01），增加NF-κB蛋白表达（P<0.05，P<0.01），最终降低Caspase-3活性（P<0.05）。
Nec-1可通过Caspase-8途径减少Al诱导的细胞凋亡，并上调NF-κB蛋白的表达。
