糖尿病皮肤溃疡特异miRNA筛选及Wnt/β-catenin信号通路在积雪草苷与NO干预中的响应机制

Diabetic cutaneous ulcers (DCU) is one of the most serious complications of diabetes mellitus.Epidermal stem cells (ESCs) show great promise in treatment of burn patients, which is supported by results showing the ability of these cells of the hair follicle to regenerate epithelium of deep burn wounds. .MicroRNA(miRNA)are 19-24 nucleotide-long endogenously expressed non-coding single-strain RNAs that regulate the gene transcription. A large number of evidence suggested that miRNA play a significant role in cutaneous wound healing, and specific miRNA are expressed in specific phases, but there is no relational evidence of the expression in the DCU..The level of β-catenin may also play a role in determining the fate of ESCs. Whether ESCs can be applied to repair the DCU remains to be determined. In the mature skin, hair follicles undergo repeated cycles of growth (anagen), death/regression (catagen), and rest (telogen), and in each of these phases, Wnt/β-catenin was shown to play a role. Wnt/β-catenin signaling is up-regulated at the end of telogen and hereby promotes transition to anagen. Indeed, Wnt/β-catenin signaling provides one of the mitogenic stimuli that are necessary for hair growth. .Centella asiatica（L.）Urb. is a medicinal plant that has been used in folk medicine for hundreds of years in China. The active compounds include pentacyclic triterpenes, mainly centella total glucosides. Asiaticoside (AC) effective in improving treatment of small wounds, hypertrophic wounds as well as burns, psoriasis and scleroderma. .Nitric oxide (NO) is also a critical molecular signal and mediator for normal wound healing. Emerging wound-healing research with NO provided evidence of a critical dependency of effective cellular wound repair mediation on optimal wound NO bioactivity for the diabetic wound, but the production of endogenous NO in DCU is inadequate..Therefore, we proposed the ideas to find the miRNA differentially expressed in DCU and investigate the effect and mechanisms of the combination of AC and exogenous NO to find the target miRNA, target genes and drug targets in DCU treatment. The project will firstly collect the skin of DCU patients and analyse the differential expression of miRNA applying miRNA array technique and bioinformatics. Then, we evaluate comprehensively the effect and mechanisms of the drugs on DCU. From bedside to bench,where we intervene the DCU rats by combining AC with NO, and from inflammatory microenvironment, angiogenesis, protein expression,miRNA and ESCs mobilization to Wnt/β-catenin signaling pathway. Clarifying the target miRNA and the special efficacy of the traditional Chinese medicine for the healing of the wound in DCU. It will provide new targets and strategies for the diagnosis and treatment of DCU.

糖尿病皮肤溃疡（DCU）是糖尿病严重并发症之一，miRNA在创面修复中具有重要作用，但在DCU中的表达国内外尚无证据。积雪草苷（AC）是民族药积雪草的有效成分，有促进创面愈合、抑制瘢痕生长之作用。一氧化氮（NO）也证明可促进创面修复，但在DCU中内源性NO表达不足。本研究组前期实验发现AC可促进DCU大鼠溃疡皮肤愈合及上皮化，增加肉芽组织胶原及成纤维细胞和新生毛囊组织；且与外源性NO复方可抑制溃疡皮肤细菌生长。因此，本项目拟先筛选DCU患者特异性表达的miRNA并注释功能，再从临床到实验室，揭示AC加NO对DCU大鼠模型的疗效，进一步分析其对DCU特异性miRNA的影响，及对模型微环境中炎症水平、血管新生、胶原合成、表皮干细胞（ ESCs ）调动特点的影响，且基于Wnt/β-catenin信号通路揭示药物对DCU模型 ESCs 调动的下游事件及效应靶点的分子机制，为DCU的诊疗提供新策略。

糖尿病皮肤溃疡（DCU）是糖尿病严重并发症之一，是慢性难治性皮肤创伤。如何促进DCU早期愈合，降低其致残率和治疗费用，是临床治疗的重要课题之一。. 我们前期研究表明积雪草苷可以促进普通创面修复，但是否对于高糖环境引起的创面修复如DCU也具有有益的作用，国内外未见报道。本课题创新性地将既具有促进创面愈合、抑制瘢痕形成，又具有降糖作用，且有良好生物相容性和降解性的中药积雪草与NO进行结合，创制出复方积雪草生肌凝胶剂（CACMG），其既保证了叠加效应，又可使NO缓慢释放及改善创面高血糖状态。我们发现CACMG安全无毒，可促进DCU创面愈合，促进血管新生，使糖尿病大鼠创面再上皮化平均时间为13天，完全愈合平均时间缩短为15~16天；可抑制创面细菌增殖，抑制创面炎症因子IL-6、IL-8的表达，促进DCU创伤愈合的分子机制可能与Wnt/β-catenin信号通路有关。. 我们进一步对比了9例DCU患者溃疡处皮肤和30例健康人皮肤，通过miRNA基因芯片和qRT-PCR筛选验证出了26个差异表达的miRNAs；继而在糖尿病创伤动物模型中筛选出32个差异表达的miRNAs。令人兴奋的是，其中有且仅有miR-21-5p在DFU人类患者和糖尿病动物伤口皮肤中显著差异表达，这预示着可能仅miR-21-5p积极参与了哺乳动物糖尿病皮肤伤口愈合的过程，暗示了其独特的临床医学转化治疗价值。有望为民族药防治DCU提供一个新的靶点及策略。. 在该项目的资助下，进行了广泛的国内学术交流和合作（参加第十四届全国药用植物及植物药学术研讨会3人次，并应邀作学术大会分会场报告1次；中国东盟教育交流周5人次）；主持人继续申请到了国家自然科学基金1项，取得研究成果共计16项，其中IF>3的SCI论文5篇， CSCD北大中文核心5篇，其他核心论文2篇，会议论文3篇，在投SCI论文3篇，申请国家发明专利1项。以项目为依托，培养研究生4名，在读2名，组建了较成熟的研究糖尿病皮肤病的相关科研团队。