Chronic activation of neutral ceramidase protects β-cells against cytokine-induced apoptosis
Chronic activation of neutral ceramidase protects β-cells against cytokine-induced apoptosis
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DOI：
10.1111/j.1745-7254.2008.00781.x
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发表时间：
2008-05-01
影响因子：
8.2
通讯作者：
Liu, Chao
中科院分区：
文献类型：
--
作者：
Zhu, Qun;Jin, Jun-fei;Liu, Chao
目的：研究中性神经酰胺酶（N-CD酶）在胰岛素分泌细胞系INS-1中的活性和表达，及其在细胞对细胞因子应答中的作用。研究方法：进行HPLC、蛋白质印迹和定量实时PCR以检测用细胞因子混合物（5 ng/mL白细胞介素-1 β、10 ng/mL TNF-α和50 ng/mL干扰素-γ）处理的INS-1细胞中N-CD酶的活性和表达。N-CDase-siRNA转染可特异性抑制INS-1细胞中N-CDase的表达和活性。Annexin V-异硫氰酸荧光素/碘化丙啶流式细胞术用于评估INS-1细胞中的凋亡。结果：INS-1细胞具有一定的基础N-CD酶活性，细胞因子诱导N-CD酶激活呈时间依赖性延迟。结果，N-CD酶的活化在刺激后8 h首次检测到。在16 h达到峰值，并在24 h保持升高。细胞因子还上调INS-1细胞中N-CD酶的mRNA和蛋白表达。此外，当N-CD酶活性被RNA干扰抑制，苦参碱诱导的INS-1细胞凋亡显着增加。结论：N-CD酶途径在INS-1细胞中是活跃的，并且N-CD酶的慢性活化参与β细胞对细胞因子的病理反应，潜在地提供针对细胞因子毒性的保护。
Aim: To investigate the activity and expression of neutral ceramidase (N-CDase) in the insulin-secreting cell line INS-1 and its role in the cellular response to cytokines. Methods: HPLC, Western blotting, and quantitative real-time PCR were performed to detect the activity and expression of N-CDase in INS-1 cells treated with a cytokine mixture (5 ng/mL interleukin-1 beta, 10 ng/mL TNF-alpha, and 50 ng/mL interferon-gamma). The expression and activity of N-CDase in the INS-1 cells were specifically inhibited using N-CDase-siRNA transfection. Annexin V-fluorescein-isothiocyanate/propidium iodide flow cytometry was used to assess apoptosis in the INS-1 cells. Results: The INS-1 cells exhibited some basal N-CDase activity, and cytokines induced a time-dependent delay in the activation of N-CDase. As a result, the activation of N-CDase was first detectable at 8 h after stimulation. It peaked at 16 h and remained elevated at 24 h. Cytokines also upregulated the mRNA and protein expression of N-CDase in the INS-1 cells. Furthermore, when N-CDase activity was inhibited by RNA interference, cytokine-induced apoptosis in the INS-1 cells was markedly increased. Conclusion: The N-CDase pathway is active in INS-1 cells, and the chronic activation of N-CDase is involved in the pathological response of beta-cells to cytokines, potentially providing protection against cytokine toxicity.