Lysiak Jeffrey J, Bang Hyun J, Nguyen Quoc An T, Turner Terry T
Department of Urology Box 800422, University of Virginia Health System, Charlottesville, VA 22908, USA.
J Androl. 2005 Jan-Feb;26(1):129-35.
Ischemia-reperfusion (IR) of the testis results in testicular oxidative stress and germ cell-specific apoptosis. Nuclear factor kappa B (NF-kappaB) is a nuclear transcription factor involved in the control of a number of cellular processes, and its activation is part of the cellular stress response to a variety of factors including cytokine stimulation, irradiation, and IR. The present study investigates NF-kappaB activation after IR of the murine testis and potential downstream target genes of that activation. Mice were subjected to a period of testicular ischemia followed by 0-4 hours of reperfusion. Activation of NF-kappaB was assessed by 1) Western blot analysis of the NF-kappaB inhibitory protein, IkappaBalpha; 2) immunohistochemistry for IkappaBalpha; and 3) TranSignal NF-kappaB target gene array (107 genes) analysis. Results demonstrate that IkappaBalpha is phosphorylated on serine 32 reaching a peak by 2 hours after IR of the testis. A decrease in total IkappaBalpha was also noted at 2 hours after IR, consistent with the rapid degradation of the phosphorylated protein. Phosphorylation and degradation of IkappaBalpha is indicative of NF-kappaB activation. Immunolocalization revealed IkappaBalpha specifically in Sertoli cells of the murine testis. Results of the TranSignal target gene array revealed that the expression of 9 genes was consistently changed 2 hours after IR of the testis, 3 of which increased in expression and 6 of which were down-regulated. Most notably, high-mobility group nucleosomal binding domain 1 increased in expression while platelet-derived growth factor B and Wilms tumor homolog decreased. These results suggest that testicular IR releases the suppression of NF-kappaB by IkappaBalpha in Sertoli cells. Activation of the NF-kappaB pathway in the testis resulted in an alteration of expression of potential NF-kappaB target genes, some increased while others decreased. The specific roles of these genes in the testicular response to IR remains to be determined.
睾丸缺血再灌注（IR）会导致睾丸氧化应激和生殖细胞特异性凋亡。核因子κB（NF-κB）是一种参与多种细胞过程调控的核转录因子，其激活是细胞对包括细胞因子刺激、辐射和IR等多种因素产生应激反应的一部分。本研究调查了小鼠睾丸IR后NF-κB的激活情况以及该激活的潜在下游靶基因。对小鼠进行一段时间的睾丸缺血，随后再灌注0 - 4小时。通过以下方法评估NF-κB的激活：1）对NF-κB抑制蛋白IκBα进行蛋白质印迹分析；2）对IκBα进行免疫组织化学分析；3）进行TranSignal NF-κB靶基因阵列（107个基因）分析。结果表明，IκBα在丝氨酸32处磷酸化，在睾丸IR后2小时达到峰值。IR后2小时还观察到总IκBα减少，这与磷酸化蛋白的快速降解一致。IκBα的磷酸化和降解表明NF-κB被激活。免疫定位显示IκBα特异性存在于小鼠睾丸的支持细胞中。TranSignal靶基因阵列的结果显示，睾丸IR后2小时，9个基因的表达持续发生变化，其中3个基因表达增加，6个基因表达下调。最显著的是，高迁移率族核小体结合域1表达增加，而血小板衍生生长因子B和肾母细胞瘤同源物表达减少。这些结果表明，睾丸IR解除了支持细胞中IκBα对NF-κB的抑制。睾丸中NF-κB途径的激活导致潜在NF-κB靶基因表达发生改变，一些基因表达增加而另一些基因表达减少。这些基因在睾丸对IR反应中的具体作用仍有待确定。
