黄芪多糖通过上调HIF-1α改善巨噬细胞代谢与功能发挥促进糖尿病创面愈合作用的研究

The typical pathological features of diabetic foot are susceptibility to infection and prolonged inflammatory reaction. We and others have found that the number of macrophages during the acute inflammation phase is reduced in diabetic wound and their immunocompetence is reduced. Immune activation of macrophages requires up-regulation of the glycolytic pathway through the transcription factor HIF-1α, whereas high glucose significantly inhibits HIF-1α expression and transcription, thereby affecting the activation of the glycolytic pathway. Our preliminary work found that the extract of astragalus membranaceus can reverse the down-regulation of HIF-1α by hyperglycemia and increase the infiltration of macrophages in diabetic wounds. Astragalus polysaccharides is the main active ingredient from astragalus membranaceus, and it was reported to enhance immune activity of macrophage. Therefore, we hypothesized that high glucose could inhibit the glycolysis of macrophage through suppressing HIF-1α, which could lead to abnormal immune activity of macrophage and delay the healing of diabetic wound. Astragalus polysaccharides could improve the expression of HIF-1α in macrophages, leading to normal activation of glycolysis and improved immune function of macrophages, thus promoting the repair of diabetic wounds. The aim of this study was to investigate the role of cellular metabolic pathways in the regulation of macrophage immune function using spontaneous diabetes mellitus mice model – Akita mice and mouse macrophages through the techniques of immunofluorescence, qPCR, immunoblotting, and siRNA, and to elucidate the effects and mechanisms of immune regulation and wound healing of astragalus polysaccharides.

糖尿病足典型特征为易感染和炎症反应迁延。我们和其他人的研究发现，糖尿病创面中重要的免疫细胞巨噬细胞数量减少且免疫活性降低。巨噬细胞免疫激活需要通过转录因子HIF-1α上调糖酵解通路，而高糖环境明显抑制 HIF-1α的表达和转录功能。我们前期工作发现黄芪提取物可逆转高糖对糖尿病创面中HIF-1α的抑制，调节糖尿病创面炎症反应；黄芪多糖是黄芪中具有免疫调节活性的主要成分。据此我们提出假说：高糖通过抑制HIF-1α/糖酵解通路，影响糖尿病创面中巨噬细胞免疫活性，导致创面难愈；黄芪多糖通过上调HIF-1α改善巨噬细胞糖酵解，改善巨噬细胞免疫功能，促进糖尿病创面修复。本研究以自发糖尿病Akita小鼠和小鼠巨噬细胞为研究对象，通过免疫荧光、qPCR、免疫印迹和siRNA等方法，探讨细胞代谢途径异常在调控糖尿病创面巨噬细胞免疫活性中的作用，阐明黄芪多糖免疫调控和促进糖尿病创面愈合的作用及机制。

糖尿病患者易于发生细菌感染，机制尚不明确。细菌菌壁成分脂多糖LPS通过激活巨噬细胞等免疫细胞上的先天性模式识别受体TLR4触发一系列信号传导级联通路，诱导炎症介质表达，这对于有效消除病原体至关重要。该系统的异常激活会导致免疫缺陷，败血性休克或诱发自身免疫。近年来的研究发现糖尿病代谢紊乱参与调控巨噬细胞的免疫反应。本研究应用棕榈酸（PA）预处理巨噬细胞，发现PA预处理组TNF-α在mRNA、蛋白和分泌水平均较对照组增加，而IL-1β在mRNA、蛋白和分泌水平均较对照组减少。PA预处理不影响巨噬细胞TLR4和Myd88的表达水平。LPS刺激后，与对照组相比，PA预处理组NF-κB磷酸化水平及核转位降低，ERK1/2磷酸化水平明显降低，p38、JNK磷酸化水平增加。PA处理抑制ERK及NF-κB通路上游的IKK磷酸化，增强p38、JNK上游的TAK1磷酸化。溴化棕榈酸预处理细胞24h及棕榈酸短时间处理或用etomoxir干扰棕榈酸代谢后，不会出现上述变化。ERK1/2通路抑制剂U0126-EtOH明显抑制IL-1β表达，而JNK通路抑制剂SP600125明显抑制TNF-α表达。巨噬细胞中IL-1β和TNF-α的表达受不同的信号通路调控，IL-1β表达主要受ERK1/2通路调节，而TNF-α的表达主要受P38通路和JNK通路调节。棕榈酸可能通过抑制LPS-TLR4通路活化后的ERK1/2通路而减少IL-1β表达,通过上调p38、JNK通路增加TNF-α表达，棕榈酸的这种作用可能与其在细胞内的代谢产物干扰LPS-TLR4信号通路有关。