Mi Bobin, Liu Jing, Liu Yi, Hu Liangcong, Liu Yukun, Panayi Adriana C, Zhou Wu, Liu Guohui
Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
Division of Plastic Surgery, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA.
Cell Physiol Biochem. 2018;51(2):647-663. doi: 10.1159/000495320. Epub 2018 Nov 21.
BACKGROUND/AIMS: Antimicrobial peptides are effective promoters of wound healing but are susceptible to degradation. In this study, we replaced the GIGDP unit on the N-terminal of the endogenous human antimicrobial peptide hBD-2 with APKAM to produce A-hBD-2 and analyzed the effect on wound healing both in vitro and in vivo.
The effects of A-hBD-2 and hBD-2 on cytotoxicity and proliferation in keratinocytes were assessed by Cell Counting Kit-8 assay. The structural stability and antimicrobial activity of hBD-2 and A-hBD-2 were evaluated against Staphylococcus aureus. RNA and proteins levels were evaluated by real-time PCR and western blotting, respectively. Cell migration was evaluated using a transwell assay. Cell cycle analysis was performed by flow cytometry. Wound healing was assessed in Sprague-Dawley rats. Epidermal thickness was evaluated by hematoxylin and eosin staining.
We found that hBD-2 exhibited cytotoxicity at high concentrations and decreased the structural stability in the presence of high sodium chloride concentrations. A-hBD-2 exhibited increased structural stability and antimicrobial activity, and had lower cytotoxicity in keratinocytes. A-hBD-2 increased the migration and proliferation of keratinocytes via phosphorylation of EGFR and STAT3 and suppressed terminal differentiation of keratinocytes. We also found that A-hBD-2 elicited mobilization of intracellular Ca2+ and stimulated keratinocytes to produce pro- and anti-inflammatory cytokines and chemokines via phospholipase C activation. Furthermore, A-hBD-2 promoted wound healing in vivo.
Our data suggest that A-hBD-2 may be a promising candidate therapy for wound healing.
背景/目的：抗菌肽是伤口愈合的有效促进剂，但易降解。在本研究中，我们用APKAM替换内源性人抗菌肽hBD-2 N端的GIGDP单元以产生A-hBD-2，并在体外和体内分析其对伤口愈合的影响。
采用细胞计数试剂盒-8法评估A-hBD-2和hBD-2对角质形成细胞的细胞毒性和增殖的影响。评估hBD-2和A-hBD-2对金黄色葡萄球菌的结构稳定性和抗菌活性。分别通过实时PCR和蛋白质印迹法评估RNA和蛋白质水平。使用Transwell实验评估细胞迁移。通过流式细胞术进行细胞周期分析。在Sprague-Dawley大鼠中评估伤口愈合情况。通过苏木精和伊红染色评估表皮厚度。
我们发现hBD-2在高浓度时表现出细胞毒性，并且在高氯化钠浓度存在下结构稳定性降低。A-hBD-2表现出更高的结构稳定性和抗菌活性，并且在角质形成细胞中细胞毒性较低。A-hBD-2通过EGFR和STAT3的磷酸化增加角质形成细胞的迁移和增殖，并抑制角质形成细胞的终末分化。我们还发现A-hBD-2引起细胞内Ca2+的动员，并通过磷脂酶C激活刺激角质形成细胞产生促炎和抗炎细胞因子及趋化因子。此外，A-hBD-2在体内促进伤口愈合。
我们的数据表明A-hBD-2可能是一种有前景的伤口愈合候选治疗方法。
