Objectives: Diabetic foot ulcer (DFU), with a high rate of amputation and mortality, is a serious complication of diabetes. However, the therapeutic effect of diabetic foot is poor. This study aimed to investigate the effect of CD147 on epithelial-mesenchymal transition (EMT) process in DFU and molecular mechanisms.
Methods: Immunohistochemistry was used to reveal the expression of several proteins, such as CD147, E-cadherin, N-cadherin, Slug, and Phospho-RSK2 in DFU, non-diabetic refractory tissues, and wound margin tissues (normal blood glucose). Western blotting was used to analyze the expression of CD147 and Slug in HaCaT cells in the high-glucose environment. HaCaT cells with CD147 or RSK2 knockdown was constructed. Wound healing assay was used to test the migration capability of HaCaT cells with knockdown of CD147. Western blotting was used to detect the protein level of Slug in HaCaT cells with CD147 or RSK2 knockdown to investigate the effects of CD147 or RSK2 on EMT. Immunoprecipitation (IP) assay was used to detect the interaction between CD147 and RSK2.
Results: The expression levels of CD147 and Slug in the epithelial cells of marginal DFU tissues were significantly lower than those in non-diabetic refractory tissues and wound margin tissues (all P<0.05). CD147 and Slug expressions were down-regulated in HaCaT cells cultured with high glucose (all P<0.05). The migration ability of HaCaT cells with CD147 knockdown was decreased. Knockdown of CD147 or RSK2 significantly inhibited the expression of Slug. The direct interaction between RSK2 and CD147 was found via IP assay.
Conclusions: CD147 could cause DFU re-epithelialization obstacle via affecting RSK2-mediated Slug/EMT process, which might be an underlying mechanism for the slow healing of DFU.
目的: 糖尿病足溃疡(diabetic foot ulcer，DFU)是糖尿病的一种严重并发症，患者截肢及病死率很高，然而目前糖尿病足的治疗效果欠佳。本研究旨在探究CD147在DFU组织中对上皮间充质转化(epithelial-mesenchymal transition，EMT)进程的调控作用及其分子机制。方法: 采用免疫组织化学法检测CD147，E-cadherin，N-cadherin，Slug，磷酸化RSK2(p-RSK2)在DFU，非糖尿病相关的难愈合组织及血糖正常外伤组织中的表达。采用蛋白质印迹法检测高糖环境对HaCat细胞表达CD147和Slug的影响。构建CD147及RSK2敲减的HaCaT细胞系，采用划痕试验检测CD147敲减的角质形成细胞的迁移能力。进一步通过蛋白质印迹法检测细胞敲减CD147或RSK2对Slug相关EMT的影响。最后，采用免疫共沉淀实验验证RSK2与CD147的相互作用。结果: CD147和Slug在DFU边缘组织上皮细胞中的表达水平明显低于非糖尿病相关的难愈合组织及血糖正常外伤组织中的表达水平(均P<0.05)。高糖条件培养的HaCaT细胞CD147和Slug表达下调(均P<0.05)。敲减CD147的HaCaT细胞迁移能力下降，且CD147或RSK2敲减的HaCaT细胞Slug表达水平均降低。免疫共沉淀实验结果显示：CD147在293T细胞中可以与RSK2相互作用。结论: CD147通过影响RSK2介导的Slug/EMT进程，引起DFU再上皮化障碍。这可能是DFU组织愈合缓慢的机制之一。.
Keywords: CD147; RSK2; diabetic foot ulcer; healing.