PUFA衍生物调节巨噬细胞极化状态促进糖尿病感染性溃疡创面修复的作用及机制研究

Macrophages are the main leukocytes in early stage of wounds. They are activated to phagocytose apoptotic polymorphonuclear cells, debris, and infectious microorganisms, and then coordinate later events of healing, including accelerating re-epithelialization, increasing the formation of granulation tissue, and enhancing angiogenesis. Heterogeneity of macrophages has recently been proposed. Macrophages can be functionally polarized into M1 (classically activated) and M2 (alternatively activated) phenotypes. M1 macrophages appeared in the early stage of wound healing, and the phenotype switches to M2 in the later stage, and the correct polarization program is very important for wound healing. However, under diabetic pathological conditions, M1 macrophages stay in the diabetic wounds for longer time than that in the normal wounds, which result in the long-term inflammation and impairs the wound healing. Bacteria are always found in the diabetic wounds and develop drug resistance by forming bacterial biofilm, which results in delayed wound healing. Bacterial biofilm can also change macrophage polarization state. Thus, when both diabetic pathological condition and bacteria exist in the diabetic infectious wounds, the macrophage polarization program and whether or not manipulating the macrophage polarization program can promote wound healing are still not clear. Docosahexaenoic acid (DHA), an essential polyunsaturated fatty acid(PUFA), can significantly promotes wound healing. RvD1, PD1, Maresin and 14S,21R-diHDHA are lipid mediators generated naturally from DHA during inflammation that have potent anti-inflammatory and protective functions. Our previous work showed that 14S,21R-diHDHA can inhibit macrophage inflammatory state and promote IL-10 generation, transplantation of 14S,21R-diHDHA treated macrophages into diabetic wounds can promote wound healing. Compared to DHA derivatives, Arachidonic acid(AA) derived lipid mediators 12S-HETE, 12S-HPETE and 15S-HPETE have reversed functions on macrophage inflammatory conditions and promote the secretion of inflammatory cytokines. Based on these studies, we will firstly establish diabetic infectious wound healing model on db/db diabetic mice, and then clarify the macrophage polarization state; determine the DHA and AA derived lipid mediator spectrums in different phenotypes of macrophages; evaluate and screen the lipid mediators which can adjust macrophage polarization state as well as clarify the mechanisms for lipid mediators adjusting macrophage polarization. At last, we will screen the best therapeutic regimen: adjusting macrophage polarization program by DHA and AA derived lipid mediators to promote diabetic infectious wound healing, which will provide a new lead for the development of better therapeutics used in treatment of diabetic infectious wound healing.

巨噬细胞极化状态紊乱是糖尿病创面修复缓慢的重要原因，而在严重感染的糖尿病皮肤溃疡中，由于糖尿病病理状态和病原微生物的共同影响，巨噬细胞极化状态及调节巨噬细胞极化状态能否促进溃疡创面修复尚不明确。多不饱和脂肪酸(PUFA)中二十二碳六烯酸(DHA)和花生四烯酸(AA)的衍生物对巨噬细胞炎性反应具有相反的调节作用。我们发现的一个全新DHA衍生物14S,21R-diHDHA，具有抑制巨噬细胞炎症因子产生的作用。移植由此活性脂质分子处理的巨噬细胞到伤口部位能够促进糖尿病创面修复。本项目拟利用严重感染的db/db小鼠糖尿病皮肤溃疡模型，研究皮肤溃疡伤口中巨噬细胞极化状态,明确DHA和AA的衍生物在巨噬细胞亚型中的代谢谱，并确定其对巨噬细胞极化的调节作用及机制；筛选并确定采用活性脂质分子修正巨噬细胞极化状态促进糖尿病感染性溃疡创面修复的最佳方案，以期为临床上提高糖尿病皮肤溃疡治愈率提供新的思路和方法。

本项目利用绿脓杆菌严重感染的db/db小鼠糖尿病皮肤溃疡模型，（1）阐明了绿脓杆菌感染伤口中巨噬细胞极性变化。绿脓杆菌感染伤口的愈合速度明显减慢。感染伤口中的巨噬细胞呈现混合激活的表型，细胞同时表达M1和M2巨噬细胞的相关标记分子，且M1巨噬细胞相关炎症因子的表达量增高的更显著。体外的研究发现绿脓杆菌可以激活巨噬细胞表达大量的炎症因子，同时增加IL-10的表达，但对M2巨噬细胞相关基因的表达没有显著影响。成纤维细胞在受到绿脓杆菌刺激后产生的条件培养液可以刺激巨噬细胞上调M2相关标记分子。因而，在细菌感染的糖尿病伤口中，巨噬细胞受到复杂的调控，呈现复杂的表型，而表达更多的炎症因子可能是伤口难以痊愈的重要原因。（2）明确细菌感染的伤口中DHA和AA的衍生物的的量的变化。同非糖尿病伤口组织相比，糖尿病伤口组织中DHA和AA的衍生物14S,21R-diHDHA、Maresin1、14,22-diHDHA、12(S)-HETE、15(S)-HETE、PGE2等脂类代谢分子表达量明显降低，但在细菌感染的伤口中这些脂质分子明显增高，证实受到炎症刺激后，脂类分子表达量的升高有可能是一种反馈调节。（3）阐述DHA和AA衍生物调节感染性伤口愈合的分子机制，包括对巨噬细胞表型的调节。DHA衍生物Maresin1、14,22-diHDHA可以促进细菌感染的糖尿病伤口修复，明显降低M1表型的巨噬细胞数量，略微增加M2细胞比例。AA衍生物12(S)-HETE、15(S)-HETE不具备促进伤口修复的作用。采用脂质体包裹氯膦酸盐静脉注射去除伤口中巨噬细胞，结果发现去除巨噬细胞后感染伤口修复明显减缓，且Maresin1、14,22-diHDHA的促进伤口修复作用消失。结果显示Maresin1、14,22-diHDHA可以抑制NF-Kb信号通路，从而减少炎症因子的产生，同是激活p38和STAT3信号通路，其调控巨噬细胞极性同IL-10的表达相关。此外，Maresin1、14,22-diHDHA调控巨噬细胞促进感染伤口修复的机制还包括Maresin1、14,22-diHDHA增强巨噬细胞的促血管生成作用及吞噬细菌的作用，且吞噬作用的增强同Maresin1、14,22-diHDHA上调Nrf2有关。本课题建立基于DHA和AA来源的活性脂质分子修正巨噬细胞极化状态、促进糖尿病感染性皮肤溃疡愈合的新方法