Characterizing the role of the heme catabolism in tissue damage and inflammation.

项目摘要 胃肠道综合征（GIS）仍然是一个重要的临床问题，没有有效的治疗。放疗 和化疗导致出血和细胞死亡，导致血红蛋白和/或游离血红素的释放。 越来越多的数据表明，游离血红素刺激活性氧（ROS）的产生， 作为一种促炎性细胞因子相关分子模式（DAMP），通过与Toll样受体4结合 （TLR4）。 在本研究中，我们将着重了解游离血红素如何通过基因调控来调节炎症 在GIS模型中肠道的髓样或上皮细胞中。我们的数据显示， 结肠癌患者结肠活检组织中的浸润性血红素氧合酶-1（HO-1）阳性巨噬细胞（MCAH） 放射治疗与未照射对照或缺血肠与匹配的正常组织相比。 此外，我们发现髓系细胞特异性HO-1缺失导致上皮细胞异常增殖 和增加的DNA损伤（磷酸化组蛋白H2 AX（H2 AXg））。我们 提出通过血红素清除剂血红素结合蛋白（Hx）和/或通过HO-1（或 生物制品）可能是GIS患者的潜在治疗选择。我们新的初步数据显示 Hx-/-小鼠中结肠损伤和炎症水平升高，以响应苯肼诱导的 溶血我们还证明了血红素通过与G-四链体（G4）结合来影响基因表达。 基因组DNA中的二级结构是基因组稳定性、转录和复制的关键调节因子。 我们最先进的实验方法将使我们能够解剖游离血红素在骨髓中的功能， 结肠上皮细胞在治疗诱导的炎症和组织损伤。此外，我们会评估 肠道微生物群在HO-1/Hx对GIS的保护作用中的影响。具体而言，在本提案中，我们 打算： 1.探讨游离血红素在体外结肠上皮细胞和骨髓细胞中的作用。 2.确定骨髓细胞表达的HO-1和血红素调节的肠道微生物群多样性在 GIS模型。 3.在GIS模型中评估重组Hx的治疗潜力。 4.表征活检组织中HO-1、Hx、DNA损伤和增殖标记物以及G4的染色 并将免疫染色与临床参数相关联。 总之，这项研究将追求一个新的作用和机制，自由血红素作为一个新的定义， 在组织损伤和肠道炎症中调节基因表达的关键作用，例如在患者中观察到的 GIS和/或结肠炎本研究不仅对血红素生物学领域具有重要意义， 有助于为GIS患者制定急需的治疗策略。

Project Summary Gastrointestinal syndrome (GIS) remains a significant clinical problem with no effective treatment. Radiotherapy and chemotherapy lead to bleeding and cell death resulting in release of hemoglobin and/or free heme. Accumulating data suggest that free heme stimulates the production of reactive oxygen species (ROS) and acts as a pro-inflammatory danger-associated molecular pattern (DAMP) through its binding to Toll-Like Receptor 4 (TLR4). In this study, we will focus on understanding how free heme regulates inflammation through gene regulation in myeloid or epithelial cells in the gut in the GIS models. Our data indicate there are high numbers of infiltrating heme oxygenase-1 (HO-1)-positive macrophages (Mø) in colonic biopsies from patients after radiotherapy versus non-irradiated controls or in the ischemic intestine compared to matched normal tissues. Further, we showed that myeloid cell-specific deletion of HO-1 resulted in abnormal epithelial cell proliferation and increased DNA damage (phosphorylated histone H2AX (H2AXg)) in the intestine upon irradiation. We propose that removal of free heme by the heme scavenger, hemopexin (Hx), and/or by the activity of HO-1 (or biological products) may be potential therapeutic option for patients with GIS. Our new preliminary data showed heightened levels of colonic damage and inflammation in Hx-/- mice in response to phenylhydrazine-induced hemolysis. We also demonstrated that heme affects gene expression through binding to G-quadruplex (G4) secondary structures in genomic DNA, which are key regulators of genomic stability, transcription and replication. Our state-of-the-art experimental approaches will allow us to dissect functions of free heme in myeloid and colonic epithelial cells during treatment-induced inflammation and tissue damage. Moreover, we will assess the impact of the gut microbiota in the protective effects of HO-1/Hx against GIS. Specifically, in this proposal we intend to: 1. Characterize the role of free heme in colonic epithelial and myeloid cells in vitro. 2. Determine the role of myeloid cell-expressed HO-1 and heme-regulated gut microbiota diversity in the GIS models. 3. Assess the therapeutic potential of recombinant Hx in the GIS models. 4. Characterize the staining of HO-1, Hx, DNA damage and proliferation markers and G4 in biopsies from patients with GIS and correlate immunostaining with clinical parameters. In summary, this study will pursue the definition of a novel role and mechanisms by which free heme acts as a key player in regulating gene expression upon tissues injury and gut inflammation such as observed in patients with GIS and/or colitis. This study will not only have high impact on the field of heme biology but will also contribute to developing much needed treatment strategies for GIS patients.