miR-21/PDCD4/NF-κB通路在血小板抗菌促糖尿病溃疡愈合中的作用及分子机制

Infection is one of the contributing factors for non-healing diabetic ulcer (DU) . Thus exploring new antimicrobial strategy poised an important practical significance. Studies suggested that platelet-rich plasma (PRP) can promote DU healing with anti-inflammatory function, but its mechanism is unclear. Recent studies revealed that miR-21 plays a key effect on antimicrobial, cell proliferation and migration via regulation of nuclear transcription factor (NF-κB) expression. Our previous study found that miR-21 expression was down-regulated in Staphylococcus aureus (S. aureus) infested mice during wound healing process. After PRP intervention, only miR-21 and IL-10 expression were up-regulated, while PDCD4 or NF-κB still down-regulated. Extrapolating from these preliminary experiment results, we propose a hypothesis: PRP promotes DU anti-microbial and wound healing through regulating PDCD4 expression and NF-κB pathway activation. The study intends to observe miR-21 regulates PDCD, NF-κB expression and IL-6, IL-10, TGF-β1 secretion through different glucose levels of HaCaT cells and infected mice DU induced by S. aureus. Further, miR-21 interacts with PDCD4 and its effect on downstream molecules expression and NF-κB signaling pathway activation through Northern blot and RT-PCR in vitro study. Meanwhile, the influence of miR-21 on DU antimicrobial and wound healing will be investigated in vivo study. Through above research, it is expected to reveal molecular mechanisms and healing effects of miR-21 released by platelet for DU treatment, which may provide a theoretical basis and new targets for prevention and treatment of recalcitrant wound.

感染是阻碍糖尿病溃疡（DU）愈合的关键因素。研究提示富血小板血浆 (PRP) 具有抗感染促DU愈合作用，但其机制尚不清楚。新近研究提示miR-21参与调控NF-κB发挥抗感染促修复作用。我们前期研究发现小鼠金黄色葡萄球菌（S. aureus）DU愈合中miR-21表达下降；PRP干预后miR-21、IL-10上调，而PDCD4和NF-κB下调。结合文献调研，我们提出PRP通过miR-21调控PDCD4和NF-κB的表达及通路活化发挥拮抗感染性炎症并促进DU愈合的假设。本项目拟以HaCaT细胞和小鼠S. aureus DU为模型，选用Northern blot等方法离体研究miR-21与PDCD4的相互作用及对下游分子表达和NF-κB信号通路活化的影响；在体验证miR-21抗感染促愈合作用。期望揭示血小板分泌miR-21在PRP治疗DU中的作用及分子机制，为防治难愈性创面提供依据和新靶点。

糖尿病足溃疡（DFUs）是糖尿病最严重和治疗费用最高的慢性并发症之一，病重者可发生截肢和死亡。感染是加重创面损伤和阻碍愈合的重要危险因素。富血小板血浆（PRP）是从全血中分离出的血小板浓缩物，通过凝血酶和钙剂激活PRP后得到的富血小板凝胶（PRG）被证实在治疗DFUs中展现出良好的抗感染和促进愈合作用，但具体机制尚不明确。近来研究发现血小板可产生和释放多种microRNA，其中microRNA-21（miR-21）在血小板中为高表达且与创面愈合密切相关。结合有报道microRNA-21可能通过NF-kb信号通路调控炎症反应，以及研究证实PDCD4是受miR-21调控的靶分子，我们推测PRG可能通过miR-21调控PDCD4介导NF-κB通路活性改变在PRG治疗DFUs中发挥作用。. 鉴于DFUs容易合并金黄色葡萄球菌（S. aureus）感染，我们将人角质形成细胞（HaCaT细胞）与S. aureus在高糖环境下共培养建立体外感染创面模型。采用PRP、PRG或富血小板凝胶萃取液（EPG）干预本模型，研究干预物对细胞和细菌生长的影响，以及干预前后细胞内miR-21、PDCD4的表达变化，NF-κB信号通路的活性改变以及相关炎症因子水平的变化。. 结果显示S. aureus浓度依赖性地损伤HaCaT细胞增殖，刺激细胞内PDCD4表达上调，激活NF-kb信号通路，引起促炎因子IL-6和TNF-α分泌增加，抗炎因子IL-10分泌减少。PRG在本模型中均有抑菌作用，能够保护HaCaT细胞对抗S. aureus损伤并促进细胞增殖。采用EPG干预本模型后可上调HaCaT细胞内miR-21水平，下调PDCD4水平，抑制NF-κB信号通路活性及减少IL-6和TNF-α分泌。在没有S. aureus感染的情况下，EPG直接作用HaCaT细胞仍能引起上述改变，提示EPG具有直接抗炎作用。. 本研究建立的体外高糖感染创面模型为研究DFUs的修复机制提供了一种有价值的工具，同时为PRP的基础和临床应用提供了参考方法及证据支持。研究结果表明在PRG治疗DFUs的过程中，miR-21可能通过下调PDCD4抑制NF-kb信号通路活性从而抑制炎症反应，最终加速创面愈合。microRNA-21作为一个有前景的干预靶点，可能为糖尿病足溃疡等难治性创面的防治防治提供新的策略。