AGEs/RAGE/NF-κB通路在氢气促进糖尿病创面愈合中的作用机制研究

Non-healing wound is a common complication of diabetes with high incidence rate. It remains difficult to treat and urgently waits to be solved in clinic. Advanced glycation end products(AGEs) accumulation is an important mechanism underlying diabetic refractory wound, closely associated with free radical damage. Further more binding its receptor RAGE can activate NF-κB pathway which also induces oxidative stress. It has been reported that hydrogen can specifically neutralize free radicals, such as •OH and ONOO-, and decrease the oxidative stress and inflammation response. Previously we found hydrogen can promote wound healing in DM mice. Although hydrogen had no significant effect on blood glucose level lowering. It can reduce the level of AGEs in fibroblasts treated by high glucose, and inhibit NF-κB activation. Therefore we hypothesize that hydrogen could reduce the production of AGEs or block AGEs/RAGE/NF-κB pathway activation, and thus promote wound healing in DM. We will make DM rat model induced by streptozotocin and investigate on endothelial cells and fibroblasts. We will determine the effects of hydrogen on diabetic wound healing and prove the inference. Several techniques will be employed such as Western Blot, immunohistochemical staining, and flow cytometry. This research may illustrate the mechanism accounting for hydrogen effects on diabetic wound healing, and provide the evidence for future hydrogen application in clinic.

难愈性创面是糖尿病常见的并发症，其发生率高，治疗困难，是临床亟待解决的问题。晚期糖基化终末产物（AGEs）蓄积可导致自由基损伤，而且与其受体（RAGE）结合可激活NF-κB进一步引起氧化应激，是导致糖尿病创面难愈的重要机制。有报道表明氢气可特异性中和自由基•OH与ONOO-，减轻氧化损伤。课题组前期研究发现：氢气能促进糖尿病小鼠创面愈合，对血糖无明显影响，而能降低高糖处理的成纤维细胞AGEs水平，抑制NF-κB激活。为此我们推测氢气是通过减少AGEs生成或者阻止AGEs/RAGE/NF-κB通路活化，而促进糖尿病创面愈合。本项目选择糖尿病大鼠创面及内皮细胞、成纤维细胞为研究对象，利用分子生物学、免疫组化染色、流式细胞检测等技术方法，进一步证实氢气在糖尿病创面愈合过程中的作用，同时对上述推论加以证明。研究结果将阐释氢气治疗糖尿病创面的重要机制，评价氢气的生物学作用，为氢气的临床应用提供依据。

晚期糖基化终末产物（AGEs）蓄积可导致自由基损伤，而且与其受体（RAGE）结合可激活NF-κB进一步引起氧化应激损伤，是导致糖尿病创面难愈的重要机制。课题组前期研究发现，氢气能促进STZ诱导的1型糖尿病小鼠创面愈合，对血糖无明显影响，而能降低高糖处理的成纤维细胞AGEs水平，抑制NF-κB激活。本项目选择糖尿病鼠创面及皮肤成纤维细胞、角质细胞为研究对象，进一步证实氢气在糖尿病鼠创面愈合过程中的作用，并检测氢气对AGEs/RAGE/NF-κB通路的影响。研究证实：氢气具有明显的促进糖尿病创面愈合的作用，可以减轻氧化应激损伤及炎症因子生成，细胞实验发现应用氢气处理可以明显降低AGEs、RAGE水平，抑制NF-κB过度激活，减少氧化产物生成及炎症因子ROS、8-OHdG、3-NT、MDA、IL-1β和TNF-α水平。研究结果在一定程度上阐释了氢气促进糖尿病创面愈合的作用机制，为氢气的临床应用提供重要依据。