Cochlear Implant Research Program, University of Miami Ear Institute, Department of Otolaryngology, University of Miami, Miller School of Medicine, 1600 NW 10th Avenue, RMSB 3160, Miami, FL 33136-1015, USA.
Neuroscience. 2011 Aug 11;188:157-67. doi: 10.1016/j.neuroscience.2011.04.061. Epub 2011 May 7.
The objective was to determine the role of nuclear factor kappa B (NFκB) in dexamethasone base (DXMb) protection of auditory hair cells from tumor necrosis factor-alpha (TNFα)-induced loss on gene expression and cell signaling levels. Organ of Corti (OC) explants from 3-day-old rats were cultured under one of the following conditions: (1) media only--no treatment; (2) media+TNFα; (3) media+TNFα+DXMb; (4) media+TNFα+DXMb+NFκB-Inhibitor (NFκB-I); or (5) media+TNFα+DXMb+NFκBI-Scrambled control (NFκBI-C). A total of 60 organ of Corti explants (OC) were stained with FITC-Phalloidin after 96 h in culture (conditions 1-5) for hair cell counts and imaging of surface characteristics. A total of 108 OC were used for gene expression studies (i.e. B-actin, Bax, Bcl-2, Bcl-xl, and TNFR1) after 0, 24, or 48 h in vitro (conditions 1-4). A total of 86 OC were cultured (conditions 1-3) for 48 h, 36 of which were used for phosphorylated NFκB (p-NFκB) ELISA studies and 50 for whole mount anti-p-NFκB immunostain experiments. TNFα+DXMb exposed cultures demonstrated significant upregulation in anti-apoptotic Bcl-2 and Bcl-xl genes and downregulation in pro-apoptotic Bax gene expression; DXMb treatment of TNFα explants also lowered the Bax/Bcl-2 ratio and inhibited TNFR1 upregulation. After inhibiting NFκB activity with NFκB-I, the gene expression profile following TNFα+DXMb treatment now mimics that of TNFα-challenged OC explants. The levels of p-NFκB and the degree of nuclear translocation are significantly greater in TNFα+DXMb exposed OC explants than observed in the TNFα and control groups in the middle+basal turns of OC explants. These findings were supported by the results of the hair cell counts and the imaging results obtained from the whole mount OC specimens. DXMb protects against TNFα-induced apoptosis of auditory hair cells in vitro via activation of NFκB signaling in hair cell nuclei, and regulation of the expression levels of anti- and pro-apoptotic genes and a pro-inflammatory gene.
目的在于确定核因子 kappa B（NFκB）在地塞米松（DXM）保护听觉毛细胞免受肿瘤坏死因子-α（TNFα）诱导的基因表达和细胞信号水平损失中的作用。将来自 3 天大的大鼠的耳蜗器官（OC）外植体在以下条件之一下培养：（1）仅培养基 - 无处理；（2）培养基+TNFα；（3）培养基+TNFα+DXM；（4）培养基+TNFα+DXM+NFκB 抑制剂（NFκB-I）；或（5）培养基+TNFα+DXM+NFκB-I 随机对照（NFκBI-C）。在培养 96 小时后（条件 1-5），用 FITC-Phalloidin 对总共 60 个耳蜗器官外植体（OC）进行染色，以进行毛细胞计数和表面特征成像。总共 108 个 OC 用于基因表达研究（即 B-肌动蛋白、Bax、Bcl-2、Bcl-xl 和 TNFR1），在体外培养 0、24 或 48 小时后（条件 1-4）。总共培养了 86 个 OC（条件 1-3）48 小时，其中 36 个用于磷酸化 NFκB（p-NFκB）ELISA 研究，50 个用于全 mount 抗 p-NFκB 免疫染色实验。用 TNFα+DXM 处理的外植体显示抗凋亡 Bcl-2 和 Bcl-xl 基因的表达显著上调，促凋亡 Bax 基因的表达下调；DXM 处理 TNFα 外植体还降低了 Bax/Bcl-2 比值，并抑制了 TNFR1 的上调。用 NFκB-I 抑制 NFκB 活性后，TNFα+DXM 处理后的基因表达谱现在类似于 TNFα 挑战的 OC 外植体。与 TNFα 和对照组相比，OC 外植体中 TNFα+DXM 暴露的 OC 外植体中 p-NFκB 的水平和核易位程度明显更高。这些发现得到了从全 mount OC 标本获得的毛细胞计数和成像结果的支持。DXM 通过激活毛细胞核中的 NFκB 信号，调节抗凋亡和促凋亡基因以及促炎基因的表达水平，来保护体外 TNFα 诱导的听觉毛细胞凋亡。
