Activation of nuclear factor kappa B during heat stress-induced neuronal apoptosis

doi:10.3969/j.issn.2095-4344.2014.11.001

Abstract

Abstract:

BACKGROUND: Hyperpyrexia can induce a wide range of cell apoptosis in organisms, but no study has introduced the mechanism of heat stress-induced neuronal apoptosis.

OBJECTIVE: To observe the effect of nuclear factor kappa B (NF-κB) signal pathway on heat stress-induced neuronal apoptosis through reactive oxygen species.

METHODS: Heat stress model was established in the cell incubator. Heat stress group of cells were incubated at 39, 41, 43 ℃ for 2 hours, while control group of cells were incubated at 37 ℃ in 5% CO₂ for 2 hours. Apoptosis was analyzed by flow cytometry using Annexin V-FITC/PI staining. The expression levels of caspase-3 and p-NF-κB65 were determined by western blot analysis. The amounts of intracellular reactive oxygen species were assayed by DCFH staining. In addition, the effect of MnTMPyP and PTDC on heat stress-induced apoptosis was also studied.

RESULTS AND CONCLUSION: 39 ℃ heat stress had no impact on the apoptosis, 41 ℃ heat stress induced a small amount of apoptosis (10.19%), and 43 ℃ heat stress triggered a large amount of apoptosis (43.02%). The expression of caspase-3 and p-NF-κB65 was increased, in a temperature-dependent manner. In addition, both MnTMPyP and PTDC significantly decreased the heat stress-induced apoptosis and expression of caspase-3 and p-NF-κB65. Experimental findings indicate that, the increase of intracellular reactive oxygen species may induce neuronal apoptosis, and NF-κB participates in the heat stress-induced neuronal apoptosis as the intermedial signal pathway.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

全文链接：

Key words: heat stroke, apoptosis, NF-kappa B

CLC Number:

R394.2

Liu Yun-song, Deng Xu-bin, Huo Shao-fen, Su Lei. Activation of nuclear factor kappa B during heat stress-induced neuronal apoptosis[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(11): 1641-1646.

Figures/Tables 2

2.1 热打击后神经元细胞凋亡、凋亡相关蛋白caspase-3及核转录因子抗-磷酸化核因子κB65蛋白表达不同温度热打击后使用流式细胞仪检测神经元细胞凋亡率，发现热打击组39 ℃对凋亡无影响，而随着热打击温度的增加神经元细胞凋亡逐渐增多，41 ℃凋亡率为10.19%，43 ℃凋亡率急剧上升到43.02%，与对照组37 ℃比较，热打击组41 ℃和43 ℃神经元细胞凋亡率差异有显著性意义(P < 0.01，图1A)。Westen blot检测凋亡关键蛋白caspase-3及核转录因子抗-磷酸化核因子κB65的表达发现，随着热打击温度的增加caspase-3及抗-磷酸化核因子κB65蛋白的表达逐渐增强，其表达趋势与神经元细胞凋亡一致(图1B)。 2.2 不同温度热打击后神经元细胞内活性氧水平检测结果使用DCFH-DA法检测不同温度热打击后细胞内活性氧水平，热打击神经元细胞2 h后继续在细胞培养箱孵育12 h。发现与对照组37 ℃比较，热打击39 ℃对细胞内活性氧水平无影响，而随着热打击强度的增加，细胞内活性氧水平明显增加，41 ℃和43 ℃热打击分别增加了近7倍和15倍(与对照组37 ℃比较，图2)。 2.3 检测活性氧特异性清除剂MnTMPyP对不同温度热打击后细胞凋亡、凋亡相关蛋白caspase-3及核转录因子抗-磷酸化核因子κB65蛋白表达的影响结果显示MnTMPyP可明显抑制热打击引起的细胞凋亡，与单纯43 ℃热打击组(47.42%)相比，MnTMPyP+43 ℃热打击细胞凋亡率(18.45%)明显减少，两组比较差异有显著性意义(P < 0.01，图3A)。Westen blot检测发现，43 ℃热打击组活化凋亡相关蛋白caspase-3及抗-磷酸化核因子κB65蛋白表达明显增强，MnTMPyP预处理后明显抑制了caspase-3及抗-磷酸化核因子κB65蛋白的表达(P < 0.01，图3B，C) 2.4 核因子κB活性抑制剂PDTC对热打击诱导细胞凋亡的影响结果显示：与单纯43 ℃热打击组(41.67%)相比，PDTC预处理后神经元细胞凋亡率(11.20%)明显减少，两组比较差异有显著性意义(P < 0.01，见图4A)。同样，PDTC也明显抑制了热打击诱导的活化caspase-3蛋白的表达(图4B)。"

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