Gonzalez-Curiel Irma, Trujillo Valentin, Montoya-Rosales Alejandra, Rincon Kublai, Rivas-Calderon Bruno, deHaro-Acosta Jeny, Marin-Luevano Paulina, Lozano-Lopez Daniel, Enciso-Moreno Jose A, Rivas-Santiago Bruno
Medical Research Unit Zacatecas, Mexican Institute of Social Security-IMSS, Zacatecas, Mexico; Department of Immunology, Faculty of Medicine, Autonomous University of San Luis Potosi, San Luis Potosi, Mexico.
Medical Research Unit Zacatecas, Mexican Institute of Social Security-IMSS, Zacatecas, Mexico.
PLoS One. 2014 Oct 22;9(10):e111355. doi: 10.1371/journal.pone.0111355. eCollection 2014.
Diabetic foot ulcers (DFU) are one of the most common diabetes-related cause of hospitalization and often lead to severe infections and poor healing. It has been recently reported that patients with DFU have lower levels of antimicrobial peptides (AMPs) at the lesion area, which contributes with the impairment of wound healing. The aim of this study was to determine whether 1,25-dihydroxyvitamin D3 (1,25 (OH)2 D3) and L-isoleucine induced HBD-2 and LL-37 in primary cultures from DFU. We developed primary cell cultures from skin biopsies from 15 patients with DFU and 15 from healthy donors. Cultures were treated with 1,25 (OH)2D3 or L-isoleucine for 18 h. Keratinocytes phenotype was identified by western blot and flow cytometry. Real time qPCR for DEFB4, CAMP and VDR gene expression was performed as well as an ELISA to measure HBD-2 and LL-37 in supernatant. Antimicrobial activity, in vitro, wound healing and proliferation assays were performed with conditioned supernatant. The results show that primary culture from DFU treated with 1,25(OH)2D3, increased DEFB4 and CAMP gene expression and increased the production of HBD-2 and LL-37 in the culture supernatant. These supernatants had antimicrobial activity over E. coli and induced remarkable keratinocyte migration. In conclusion the 1,25(OH)2D3 restored the production of AMPs in primary cell from DFU which were capable to improve the in vitro wound healing assays, suggesting their potential therapeutic use on the treatment of DFU.
糖尿病足溃疡（DFU）是糖尿病相关住院最常见的原因之一，常导致严重感染和愈合不良。最近有报道称，DFU患者病变部位的抗菌肽（AMP）水平较低，这导致伤口愈合受损。本研究的目的是确定1,25 - 二羟基维生素D3（1,25(OH)2D3）和L - 异亮氨酸是否能在DFU原代培养物中诱导人β - 防御素2（HBD - 2）和人源抗菌肽LL - 37的产生。我们从15例DFU患者和15例健康供体的皮肤活检组织中建立了原代细胞培养物。培养物用1,25(OH)2D3或L - 异亮氨酸处理18小时。通过蛋白质免疫印迹法和流式细胞术鉴定角质形成细胞表型。进行了DEFB4、CAMP和维生素D受体（VDR）基因表达的实时定量聚合酶链反应（qPCR），并通过酶联免疫吸附测定（ELISA）测量上清液中的HBD - 2和LL - 37。用条件培养基上清液进行了体外抗菌活性、伤口愈合和增殖试验。结果表明，用1,25(OH)2D3处理的DFU原代培养物增加了DEFB4和CAMP基因表达，并增加了培养上清液中HBD - 2和LL - 37的产生。这些上清液对大肠杆菌具有抗菌活性，并诱导了显著的角质形成细胞迁移。总之，1,25(OH)2D3恢复了DFU原代细胞中AMP的产生，这些AMP能够改善体外伤口愈合试验，表明它们在DFU治疗中具有潜在的治疗用途。
