内皮高表达脂多糖相关因子1通过影响NF-κB信号通路促进糖尿病小鼠皮肤创面愈合的研究

Endothelial-overexpressed lipopolysaccharide-associated factor 1 promotes skin wound healing in diabetic mice by affecting the nuclear factor-kappa B signaling pathway

摘要:

目的 探讨内皮高表达脂多糖相关因子1（endothelial-overexpressed lipopolysaccharide-associated factor 1，EOLA1）对2型糖尿病小鼠皮肤创面促愈合效果及机制。方法 选取SPF级6周龄db/db糖尿病小鼠，构建全层皮肤创面模型，在创口周边注射EOLA1高表达慢病毒。小鼠分为正常对照组、空载体慢病毒组、EOLA1慢病毒组。除正常对照组外，拍照记录不同时期小鼠创口愈合情况，造模第15天取创口及周边皮肤，HE染色观察创面肉芽组织及炎性浸润，Masson染色观察创面胶原纤维沉积，免疫荧光检测ARG1、iNOS观察巨噬细胞分型，Western blot及Real-time PCR检测NF-κB信号通路IκB-α、p-IκB-α、p65蛋白表达和EOLA1、TLR4、TNF-α、IL-1β基因表达水平。结果 与空载慢病毒组相比，EOLA1慢病毒组小鼠皮肤伤口愈合速度加快，肉芽组织及胶原纤维形成增多。EOLA1慢病毒组M2型巨噬细胞标志物ARG1表达升高，M1型巨噬细胞标志物iNOS表达降低。EOLA1慢病毒组IκB-α蛋白表达升高，p-IκB-α蛋白表达降低，TNF-α、IL-1β基因表达水平下调。结论 EOLA1可抑制糖尿病小鼠皮肤创面局部炎症，加速创面愈合，其机制可能为通过上调巨噬细胞内IκB-α表达，封闭p65活性，从而抑制炎症因子TNF-α、IL-1β的释放，巨噬细胞的表型也表现为从促炎症的M1型向促愈合的M2型转变。提示EOLA1在糖尿病创面具有抗炎促愈的潜力。

Abstract:

Objective To investigate the effect of endothelial-overexpressed lipopolysaccharide-associated factor 1（EOLA1） on skin wound healing in mice with type 2 diabetes and the related mechanisms.Methods Specific pathogen-free db/db diabetic mice，aged 6 weeks，were used to establish a model of full-thickness skin wound，and the lentivirus with high EOLA1 expression was injected around the wound. The mice were divided into normal control group，empty vector lentivirus group，and EOLA1 lentivirus group. For all mice except those in the normal control group，photos were taken at different time points to observe wound healing； after the samples of the wound and the skin around the wound were collected on day 15 of modeling，HE staining was used to observe wound granulation tissue and inflammatory infiltration，Masson staining was used to observe collagen fiber deposition at the wound，immunofluorescence assay was used to measure ARG1 and iNOS for macrophage typing，and Western blot and real-time PCR were used to measure the protein expression levels of the nuclear factor-kappa B（NF-κB） signaling pathways proteins IκB-α，p-IκB-α，and p65 and the gene expression levels of EOLA1，Toll-like receptor 4（TLR4），tumor necrosis factor-α（TNF-α），and interleukin-1β（IL-1β）.Results Compared with the empty vector lentivirus group，the EOLA1 lentivirus group had a significantly higher speed of skin wound healing and significant increases in the formation of granulation tissue and collagen fibers. The EOLA1 lentivirus group had an increase in the expression of the M2 macrophage marker ARG1 and a reduction in the expression of the M1 macrophage marker iNOS，as well as an increase in the protein expression of IκB-α，a reduction in the protein expression of p-IκB-α，and downregulation of the gene expression levels of TNF-α and IL-1β.Conclusion EOLA1 can inhibit local skin wound inflammation and accelerate wound healing in diabetic mice，possibly by upregulating the expression of IκB-α in macrophages，blocking the activity of p65，and then inhibiting the release of the inflammatory cytokines such as TNF-α and IL-1β，and the phenotype of macrophages changes from M1 type for promoting inflammation to M2 type for promoting healing，suggesting that EOLA1 has the potential to protect diabetic wound from inflammation and promote wound healing.