Mechanism of EGF-Mediated Reduction of NEC

描述(申请人提供)：坏死性小肠结肠炎(NEC)是早产儿发病和死亡的主要原因，但原因不明，也没有有效的治疗方法。在大鼠NEC模型上，我们报道了表皮生长因子(EGF)能显著降低NEC的发生率和严重程度。这一建议的中心目标是阐明表皮生长因子介导的NEC还原的分子机制(S)。NEC患者出现加速的肠道细胞凋亡，而EGF对细胞的凋亡具有保护作用。我们的初步研究表明，EGF治疗NEC可以调节NEC损伤部位的促生存信号分子。具体目的1将通过评估EGF治疗(A)对患有NEC的新生大鼠回肠中促生存和促凋亡基因和蛋白的影响，(B)在Waved-2小鼠中(在非致病环境下EGF-R信号异常)，(C)在关键的促生存和促凋亡基因过度表达或被阻断的肠道细胞系中，阐明哪些促生存通路受EGF治疗的调控，以及(D)在给予EGF-R信号抑制剂的新生大鼠中。最近，我们发现肠-肝循环在NEC发病机制中起着至关重要的作用。我们的初步数据表明，给NEC新生大鼠应用EGF可下调胆汁酸(BA)水平，并调节BA转运体，提示EGF在BA体内平衡中起重要作用。在特定的目标2中，我们将通过评估EGF对NEC新生大鼠的BA转运的影响，(B)BA转运的基本成分过度表达或被阻断的IEC系，以及(C)Waved-2小鼠的变化，来探讨EGF在NEC过程中调节肠道BA转运的机制。肝素结合的表皮生长因子样生长因子(HB-EGF)是EGF相关多肽家族的一员。我们的初步结果表明，HB-EGF可降低实验性NEC的发生率，并增强EGF介导的NEC的减少。因此，HB-EGF联合EGF可能通过独特的非拮抗机制对NEC产生更好的保护作用。具体目的3将通过(A)确定降低新生大鼠NEC的最佳途径和剂量，(B)确定EGF和HB-EGF降低NEC的最佳组合，以及(C)利用体外肠道损伤模型来评价HB-EGF介导的NEC减少的机制，从而探讨HB-EGF介导的NEC减少的机制。这些研究将阐明EGF介导的NEC减少的机制，这是开始使用EGF作为人类NEC的预防或治疗方式所必需的信息。

DESCRIPTION (provided by applicant): Necrotizing enterocolitis (NEC) is a major cause of morbidity and mortality in premature infants but the cause is unknown and there are no effective treatments. Using the rat model of NEC, we reported that epidermal growth factor (EGF) markedly reduced the incidence and severity of NEC. The central goal of this proposal is to clarify the molecular mechanism(s) of EGF-mediated reduction of NEC. Accelerated intestinal apoptosis has been shown in patients with NEC, and EGF can protect cells from apoptosis. Our preliminary studies indicate that EGF treatment of NEC regulates pro-survival signaling molecules in the site of NEC injury. Specific Aim 1 will clarify which pro-survival pathways are regulated by EGF treatment of NEC by evaluating EGF treatment (a) on pro-survival and pro-apoptotic genes and proteins in the ileum of neonatal rats with NEC, (b) in waved-2 mice (aberrant EGF-R signaling under non-pathogenic circumstances), (c) in intestinal cell lines where crucial pro-survival and pro-apoptotic genes are over-expressed or blocked and (d) in neonatal rats given EGF-R signaling inhibitors. Recently, we have shown a crucial role of enterohepatic circulation in NEC pathogenesis. Our preliminary data indicate that administration of EGF to neonatal rats with NEC down-regulates luminal bile acid (BA) levels and regulates BA transporters, suggesting an important role of EGF in BA homeostasis. In Specific Aim 2, we will explore the mechanism by which EGF regulates BA transport in the intestine during NEC by evaluating EGF-mediated changes in (a) BA transport in neonatal rats with NEC, (b) IEC lines where essential components of BA transport are over-expressed or blocked, and (c) in waved-2 mice. Heparin-binding EGF-like growth factor (HB-EGF) is a member of the family of EGF-related peptides. Our preliminary results show that HB-EGF reduces the incidence of experimental NEC and enhances EGF-mediated reduction of NEC. Thus, HB-EGF with EGF may provide better protection against NEC through unique nonantagonistic mechanisms. Specific Aim 3 will examine the mechanisms of HB-EGF-mediated reduction of NEC by (a) identifying the optimal route and dose to reduce NEC in neonatal rats, (b) determining the optimal combination of EGF and HB-EGF to reduce NEC, and (c) utilizing an in vitro intestinal injury model to evaluate mechanisms of HB-EGF-mediated reduction of NEC. These studies will elucidate the mechanisms of EGF-mediated reduction of NEC, information that is essential to initiate use of EGF as a preventative or treatment modality for human NEC.