Role of SLc11a1 Gen in Warm Ischemia-Reperfusion Injury

描述（由申请人提供）： 虽然手术技术和药物干预的改进改善了肝切除术、出血性休克和器官移植的结果，但由于缺血再灌注（I/R）损伤导致的损伤、排斥和最终器官衰竭仍然是临床环境中常见的问题。因此，开发新的治疗方式来预防或最小化I/R损伤将具有巨大的益处。大量证据支持枯否细胞（KC）负责I/R损伤的早期阶段，并为更严重的多形核白细胞（PMN）介导的晚期损伤奠定基础。然而，KC和PMNs介导各自I/R损伤阶段的机制尚未完全清楚。这反映在传统的治疗干预模式在实验和临床层面都缺乏成功。我们已经确定了一个基因，Slc 11 a1，这是诱导在I/R损伤，并介导生产的促炎和抗炎细胞因子。与此一致的是，在肝I/R损伤的早期阶段，在Slc 11 a1-/-小鼠中观察到血浆TNF-α水平降低。Slc 11 a1可能具有调节KC和PMN铁稳态的功能，其表达水平可能最终决定肝脏是否通过调节I/R损伤早期和晚期的损伤程度而免受I/R损伤。为了验证这一假设，我们提出了三个目标：（1）我们将确定Slc 11 a1基因的破坏是否保护小鼠肝脏免受I/R损伤。为了研究这一点，我们将在小鼠肝脏模型中观察I/R损伤的早期和晚期。将采用标准生化和组织学技术评估血液转氨酶和H&E肝脏切片，结合分子技术监测Slc 11 a1的蛋白质和mRNA水平以及促炎和抗炎细胞因子。(2)我们将确定Slc 11 a1基因的破坏是否减弱了I/R损伤期间KC和浸润性PMN的激活/引发。Slc 11 a1介导KC和PMN的活化。因此，我们将从I/R的肝脏中分离这些细胞，并使用标准的生物化学和分子技术通过观察自发的超氧阴离子和NO产生以及NF-κ B活化来评估其活化/引发程度。(3)我们将确定Slc 11 a1基因激活KC和PMN的机制。如果破坏Slc 11 a1通过调节铁稳态的介导来保护肝脏免受I/R损伤，那么在I/R损伤之前对Slc 11 a1 +/+小鼠进行去铁胺预处理应该保护肝脏免受I/R介导的损伤，如对Slc 11 a1-/-小鼠所观察到的那样。此外，Fe 2SO 4预处理应逆转在Slc 11 a11-/-小鼠中观察到的保护作用。与目标1相同，我们将使用标准生化和组织学技术评估肝损伤。这些研究将确定Slc 11 a1调节肝脏I/R损伤的分子机制，并将为I/R介导的器官损伤的治疗干预提供新的靶点。

DESCRIPTION (provided by applicant): While improvements in surgical techniques and pharmacological interventions have improved the outcome of liver resections, hemorrhagic shock, and organ transplantation, injury, rejection and eventual organ failure due to Ischemia-reperfusion (I/R) injury continue to be common place in the clinical setting. Accordingly, the development of novel treatment modalities to prevent or minimize I/R injury would be of tremendous benefit. Substantial evidence exist to support the notion that Kupffer cells (KC) are responsible for the early-phase of I/R injury, and sets the stage for the more severe polymorphonuclear leukocyte (PMN)-mediated late-phase injury. However, mechanisms by which KC and PMNs mediate their respective phases of I/R injury are not fully understood. This is reflected in the lack of success with conventional modes of therapeutic intervention both at the experimental and clinical levels. We have identified a gene, Slc11a1, which is induced during I/R injury, and mediates the production of pro-and anti-inflammatory cytokines. Consistent with this is the reduced levels of plasma TNF-alpha observed in Slc11a1-/- mice during the early phase of liver I/R injury. Slc11a1 proposed function of the regulating iron homeostasis in KC and PMNs, and its level of expression may ultimately determine if the liver is protected from I/R injury by modulating the degree of injury during both the early- and late-phases of I/R injury. To test this hypothesis we propose three aims: (1) We will determine if disruption of the Slc11a1 gene protects the mouse liver from I/R injury. To investigate this, we will look at both the early- and late-phases of I/R injury in the mouse liver model, in vivo. Standard biochemical and histological techniques will be employed to evaluate blood transaminases and H&E liver sections, in conjunction with molecular techniques to monitor the protein and mRNA levels of Slc11a1 and pro-and anti-inflammatory cytokines. (2) We will determine if disruption of the Slc11a1 gene blunts the activation/priming of KC and infiltrating PMNs during I/R injury. Slc11a1 mediates the activation of KC and PMNs. Therefore, we will isolate these cells from livers subjected to I/R, and use standard biochemical and molecular techniques to evaluate their degree of activation/priming by looking at spontaneous superoxide anion and NO production, and NF-kappaB activation. (3) We will determine the mechanism(s) by which the Slc11a1 gene elicits activation of KC and PMNs. If disruption Slc11a1 protects the liver from I/R injury by modulating mediation of iron homeostasis, then deferoxamine pretreatment of Slc11a1+/+ mice before I/R injury should protect the liver from I/R mediated injury, as observed for Slc11a1-/- mice. Furthermore, pretreatment with Fe2SO4 should reverse the protection observed in Slc11a11-/- mice. As in Aim #1, we will assess liver injury with use of standard biochemical and histological techniques. These studies will identify molecular mechanisms by which Slc11a1 modulates liver I/R injury, and will provide a novel target for therapeutic intervention in I/R-mediated organ injury.