硫化氢通过诱导内皮祖细胞Ang-1/Tie2信号途径改善2型糖尿病皮肤伤口愈合的机制

Diabetic foot ulceration is still a serious and intractable clinical issue, and the dysfunction of angiogenesis is a key reason of refractory wound healing in diabetic population.Our preliminary study revealed that there were delayed skin wound heaing course,decreased Ang-1 expression in skin tissue and deficient tube formation and adhesion function of EPC in db/db diabetic mice; and hydrogen sulfide(H2S)treatment accelerated their skin wound closure rate.This effect of H2S was related to the restoration of endothelial progenitor cell(EPCs) angiogenic function. However, it is not clear that what is the detailed mechanism of the improvement of EPC functions stimulating by H2S. In the present study,we will answer this scientific question by observing the wound healing course and the Ang-1/Tie2 signaling protein expression change in wound skin tissue through the in vivo study in db/db diabetic mice treated with H2S, H2S+Ang-1 inhibitor, or observing the angiogenic function alteration and the Tie2 post-receptor signaling protein change of EPCs by the in vitro study in diabetic EPCs treated with H2S+/- Ang-1 inhibitor or si-Ang-1,and the in vivo angiogenic study of Mitragel plug after injection of different EPCs into C57BL/6 mice, to elucidate the detailed mechanism of Ang-1/Tie 2 signaling in mediating the pro-angiogenic effect of H2S in diabetes. This optimal work will provide a potential therapeutic target for diabetic foot ulceration, and provide the scientific basis for the future clinical application of H2S in the occlusive artery disease and wound healing of lower extremities in diabetic populations.

血管新生障碍是糖尿病伤口愈合困难的关键病因。我们的前期研究表明，2型糖尿病小鼠皮肤伤口愈合减慢，表达Ang-1降低，内皮祖细胞（EPCs）功能降低，而硫化氢（H2S）可加速其皮肤伤口愈合，且作用与改善糖尿病EPCs功能有关。但尚不清楚H2S改善EPCs功能和伤口愈合的分子机制。本研究拟通过观察经H2S或H2S+Ang-1抑制剂治疗后糖尿病小鼠皮肤伤口愈合情况和皮肤Ang-1/Tie2通路信号蛋白表达的体内研究，和Ang-1抑制剂、siRNA 沉默Ang-1后，H2S对EPCs功能影响的改变和EPCs Tie2受体后信号蛋白表达变化的体外研究，以及小鼠EPC的Mitragel Plug血管新生体内研究，阐明EPCs 的Ang-1/Tie2信号通路在介导H2S促血管新生和伤口愈合中的作用机制，有望为糖尿病足溃疡的治疗寻找新靶点，为H2S在糖尿病下肢血管病变和足溃疡中的临床应用奠定科学基础。

血管新生障碍，炎症反应及并发神经病变是糖尿病伤口愈合困难的重要病因，从这三方面探讨糖尿病伤口愈合其中的分子机制与治疗方式已成为糖尿病研究领域的一个重要课题。本研究利用db/db小鼠伤口模型及患者伤口皮肤样本，首次揭示外源性硫化氢（H2S）供体或体外硫化氢处理的内皮祖细胞可促进糖尿病伤口愈合，且在体内和体外均可改善内皮祖细胞的粘附和小管形成功能，而用硫化氢合成酶抑制剂PAG抑制或沉默硫化氢后正常小鼠皮肤伤口愈合变慢，内皮功能受损。并阐明其分子机制主要是通过修复内皮祖细胞的功能和激活血管生成素-1从而刺激皮肤血管新生而实现；首次发现外源性硫化氢供体治疗可减轻因糖尿病小鼠受损的周围神经神经超微结构改变，显著改善髓鞘变性；同时发现海藻酸钙敷料能够通过抑制伤口炎症、MMP-9 mRNA及蛋白表达，上调伤口局部胶原蛋白I/II比例等途径促进糖尿病模型的溃疡愈合；首次证实负压吸引治疗（NPWT）主要通过减轻局部炎症反应，激活ATF-3，下调NF-κB等途径，提高Wagner3级以上重症糖尿病足患者的伤口愈合率。.本项目研究结果为寻求基于血管新生、神经损伤的糖尿病足溃疡新方法提供了理论依据，同时也为H2S，海藻酸钙敷料等新方法在糖尿病下肢血管，神经病变和足溃疡中的临床应用奠定了科学基础。