内皮高表达脂多糖相关因子1（EOLA1）在糖尿病足慢性溃疡中的作用及其机制研究

Diabetic foot chronic ulcer (DFU) is a major complication of diabetes mellitus and often precedes lower extremity amputation. Immune dys-regulation represents the hallmark of diabetes mellitus that could contribute to prolonged inflammation causing transformation of wounds into chronic ulcers. However, the exact mechanism of inflammatory immune regulation in the pathogenesis of DFU is largely unknown. We have identified and successful cloned endothelial-overexpressed lipopolysaccharide-associated factor 1 (EOLA1) in 2004. Our study recently showed that EOLA1 acts as a transcriptional coactivator of MT2A protein and a negative regulator on LPS induced inflammatory cytokine production. Interestingly, EOLA1 was found to be significantly down-regulated in the tissues from DFU compared with non-diabetic acute trauma, indicating EOLA1 may regulate chronic inflammation in diabetes mellitus leading to DFU. Therefore, our goal in this application is to establish the functional role of EOLA1 and its mechanistic effect on the control of inflammatory immune response in DFU. We plan to collect biopsy samples from DFU in compared with non-diabetic trauma patients, and characterize the expression pattern of EOLA1 with the correlation of inflammatory gene expression in those tissues. We will further investigate the mechanistic effects of EOLA1 on the regulation of inflammatory genes both in primary human and mouse macrophage cultures using multiple molecular and genetic approaches. We will specifically focus on the inhibition action of EOLA1 on NF-κB active pathway, which is a major regulatory component for inflammatory immune response. The intervention effect will be employed in over expression EOLA1 on the local tissues of ulcer healing in db/db type 2 diabetes mice with trauma model. The work thus represents the novel regulatory pathway of inflammatory immune response and provides valuable information for the functional role of EOLA1 in the control of inflammation, wound healing of diabetes mellitus leading to DFU.

糖尿病足慢性溃疡（DFU）是糖尿病最难治疗的并发症之一，常导致截肢。其主要临床表现是创面慢性炎症迁延，但发病机制不清楚。最近我们在DFU创面组织发现内皮高表达脂多糖相关因子1（EOLA1）蛋白表达水平显著降低，EOLA1是我们早年发现的炎症负调节转录辅活化子，其表达降低可能在DFU创面慢性炎症发生发展中起重要作用。因此，我们拟收集DFU早期和晚期创面组织标本，检测明确EOLA1基因表达的时空特点，分析EOLA1与创面炎症反应和巨噬细胞M1/M2表型转变的关系；同时以人和小鼠原代巨噬细胞为模型进一步探索EOLA1调控免疫炎症反应的分子机制，明确EOLA1通过金属硫蛋白2A（MT2A）发挥其抑制NF-κB炎症信号通路的作用靶点；最后在db/db 2型糖尿病小鼠创面模型中观察高表达EOLA1基因对局部炎症反应和创面愈合的干预效果，阐明EOLA1在DFU中潜在的抗炎促愈作用及其免疫学调控机制。

糖尿病足慢性溃疡（DFU）是糖尿病最难治疗的并发症之一，常导致截肢。其主要临床表现是创面慢性炎症迁延，但发病机制不清楚。本课题研究中，我们收集了临床糖尿病足急性期（<4周）和慢性期（>4周）创面标本进行检测，结果发现糖尿病急性创面表达内皮高表达脂多糖相关因子1（EOLA1）基因水平高，而慢性创面表达EOLA1基因水平低，局部炎症持续，创面经久不愈，表明我们早年发现的EOLA1这一炎症负调节转录辅活化子，其表达降低在DFU创面慢性炎症发生发展中起重要作用。为了进一步了解EOLA1基因发挥炎症负调控作用的分子机制，我们在培养的巨噬细胞中转染EOLA1高表达慢病毒，通过分子生物学方法验证了EOLA1在巨噬细胞内通过与金属硫蛋白2A（MT2A）相互作用促进IκB-α转录，并且在细胞浆中与IκB-α直接结合抑制其磷酸化，从而增强了IκB-α在细胞浆中封闭核因子NF-κB活化的能力，减少了NF-κB转位进行细胞核的水平，从而抑制了后续细胞炎症因子的表达释放和细胞炎症反应的程度。最后，为验证我们观察到的临床现象和细胞实验结果，我们构建了EOLA1基因敲除鼠，联合db/db糖尿病鼠制备创面模型，在创面局部注射EOLA1高表达慢病毒，观察创面局部炎症反应、炎症因子表达、巨噬细胞表型变化和创面愈合速度，结果证实高表达EOLA1可以抑制局部巨噬细胞炎症活性，加速糖尿病创面的愈合。我们的研究结果阐明EOLA1在DFU中潜在的抗炎促愈作用及其免疫学调控机制，为未来糖尿病足慢性创面的治疗提供新的理论依据。