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

项目摘要 胃肠道综合征(GIS)仍然是一个严重的临床问题，没有有效的治疗方法。放射治疗 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，游离态的亚铁血红素能刺激活性氧种(ROS)和ACTs的产生 作为与Toll样受体4结合的致炎危险相关分子模式(DAMP) (TLR4)。 在这项研究中，我们将重点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，肠道中的髓样细胞或上皮细胞。我们的数据显示有大量的 术后患者结肠活检组织中HO-1阳性巨噬细胞的表达 放射治疗与非照射对照组或缺血肠组织与匹配的正常组织的比较。 此外，我们还发现髓系细胞特异性的HO-1缺失导致了异常的上皮细胞增殖 照射后肠道DNA损伤增加(磷酸化组蛋白H_2AX(H_2AXg))。我们 建议通过血红素清除剂血凝素(HX)和/或通过HO-1(或 生物制品)可能是治疗地理信息系统患者的潜在选择。我们新的初步数据显示 苯肼诱导的HX-/-小鼠结肠损伤和炎症水平升高 溶血。我们还证明了血红素通过与G-四链(G4)结合来影响基因表达。 基因组DNA中的二级结构，它是基因组稳定性、转录和复制的关键调节因素。 我们最先进的实验方法将使我们能够剖析髓系细胞中游离血红素的功能 结肠上皮细胞在治疗过程中引起炎症和组织损伤。此外，我们会评估 肠道微生物区系对HO-1/HX抗地理信息系统保护作用的影响具体地说，在这项提案中，我们 打算： 1.研究游离血红素在体外培养的结肠上皮细胞和髓系细胞中的作用。 2.确定髓系细胞表达的HO-1和血红素调节的肠道微生物区系多样性在 地理信息系统模型。 3.评价重组HX在大鼠局灶性脑缺血模型中的治疗作用。 4.活检组织中HO-1、HX、DNA损伤和增殖标记物及G4的染色特征 免疫组织化学染色与临床参数的相关性。 总而言之，这项研究将寻求定义一种新的角色和机制，通过这种新的作用和机制，游离的血红素作为一种 在组织损伤和肠道炎症等方面调节基因表达的关键因素 患有地理信息系统和/或结肠炎。这项研究不仅将对血红素生物学领域产生重大影响，而且还将 帮助制定治疗地理信息系统患者急需的治疗策略。

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.