Pannexin1蛋白-MAPK信号通路在体外冲击波疗法调控表皮干细胞促进糖尿病慢性创面愈合的机制研究

Chronic diabetic wounds show the delayed or disturbed healing processes and remain the great challenges for clinicians. The noninvasive and effective methods are urgently needed to promote healing process of chronic wounds. Recent studies showed that extracorporeal shock waves therapy (ESWT), as a new non-invasive method of treatment, is effective in stimulating stem cells proliferation and promoting the healing of non-healing fractures. And although its use in the treatment of chronic wounds is rare, the clinical pilot trials demonstrate that ESWT can also significantly improve the healing process of chronic wounds with different etiologies, including diabetic chronic wounds. The underlying mechanisms of ESWT for wound healing remain unclear. According to the studied of ESWT on non-healing fractures, the physiological explanation for the beneficial effect of ESWT involves increases in proliferation and differentiation of stem cells, growth factors release, and angiogenesis, the release of cellular adenosine triphosphate (ATP) and subsequently activating purinergic receptors and mitogen-activated protein kinases (MAPK) signaling. Although present in different organs, the proliferation and differentiation of stem cells seem to be driven by a limited set of signaling pathways, including those involving transforming growth factor beta (TGF-β), platelet-derived growth factor (PDGF) and MAPK signal pathways that can be triggered by mechanical stress. These intracellular signaling pathways are typically affected by cellular communication networks, including those mediated by pannexin-based channels which can be activated specificly by mechanical stimulation. Based on these, we hypothesize that ESWT with mechanical energy may promote Pannexin1 (Panx1) at epidermal stem cells (ESCs) plasma membrane and ATP release by Panx1-based channels, binding of ATP to P2X7 receptor, and then activate MAPK pathways, which mediate the proliferation, migration and differentiation of ESCs and finally promoting healing process and tissue repair of chronic wounds. However, further experimental data are necessary to confirm the hypothesis. So, this study aims to investigate if ATP release is an underlying mechanism through which ESWT promote ESCs function and tissue repair of chronic wounds with cellular, and animal experiments. In cell experiments, ESCs were used to determine the effect of ESWT on activated the proliferation, migration and differentiation of ESCs. Extra-cellular ATP release into the culture supernatant from ESCs was determined with a specific ATP Bioluminescence Assay Kit. Expression and phosphorylation of MAPK in ESCs were measured by Western Immunoblotting. In animal experiments, to investigate the influence of ESW on the histological features of chronic diabetic wounds and wound healing, a dorsal full-thickness skin wound in a streptozotocin-induced diabetic SD rat model was used. ESWT (High-energy group:0.10mJ/mm2；Low-energy group: 0.03 mJ/mm2, 500pules/cm2, 8 Hz/s) was applied to treat the wound twice every week for 2 weeks. The wound healing process, histological changes, proliferation, migration and differentiation of ESCs, and cytokine were observed by HE and immunohistochemical staining at pre-, 3, 7, 14, and 28 days after treatment. Protein expression and mRNA of MAPK singling pathways are validated using immunohistochemical analysis of topical peri-wounding tissues. The results of the study may provide an overview of ESWT start-up and mechanisms for stimulating wound healing and tissue repair from the histology, cellular level, and protein and gene levels. And understanding these mechanisms may contribute to the development of new and effective non-surgical therapeutic strategies and provide experimental basis for clinical management of chronic wounds.

糖尿病（DM）慢性创面（CW）是目前创伤研究领域的难点问题。本课题组临床研究表明：体外冲击波疗法（ESWT）治疗CW有效，但其机制尚不清楚。研究证实：ESWT有效治疗难愈性骨折的主要机制是其促进多种干细胞的增殖和分化，效应靶点可能是细胞膜特异性应力感应受体Panx1蛋白。据此，我们提出研究假说：ESWT可刺激创面表皮干细胞（ESCs）Panx1蛋白，激活MAPK信号通路，加速ESCs增殖，进而促进CW愈合。为验证之，本研究拟通过细胞和动物实验，观察ESWT对皮肤特异性ESCs增殖、迁移和分化的影响，测定Panx1蛋白、ATP释放、P2X7受体和MAPK信号通路基因及蛋白表达水平，并行CM动物模型在体验证，以期从组织-细胞-蛋白-基因水平逐步深入地揭示ESWT调控ESCs功能进而促进CW愈合的启动机制和效应通路，为CW治疗机制研究提供新靶点，最终为ESWT在再生医学领域应用提供理论依据。

糖尿病（DM）慢性创面是目前创伤研究领域的难点问题。前期临床研究表明：体外冲击波疗法（ESWT）治疗DM慢性创面有效，但其机制尚不清楚。本项目通过细胞和动物实验，证实ESWT可促进DM慢性创面微环境中功能受到抑制的人表皮HaCaT细胞的增殖活性和迁移能力，且其生物学效应呈现剂量相关性中、低剂量的冲击波能量刺激可促进HaCaT细胞的增殖活性和迁移能力，而高剂量的冲击波能量则产生抑制效应。；而刺激HaCaT细胞膜上特异性机械感应受体Pannexin1，导致细胞内ATP外向释放，激活MAPK信号通路（p38和ERK）是ESWT促进糖尿病慢性创面修复的重要机制；并通过DM小鼠慢性创面动物模型，进一步在体证实了ESWT可促进DM慢性创面愈合的有效性，验证了上述生物学效应机制。本项目研究结果从在组织-细胞-蛋白-基因水平逐步深入地揭示ESWT调控人表皮HaCaT细胞功能进而促进DM慢性创面愈合的启动机制和效应通路，为慢性创面的治疗机制研究提供新靶点，最终为ESWT在再生医学领域应用提供理论依据。