LL-37及其衍生肽促进糖尿病创面愈合的机制

Efficacy of antimicrobial peptide LL-37 and derivative peptides in diabetic wound healing

目的探讨LL-37及其衍生肽LL-37R对糖尿病创面愈合的影响。方法高糖条件下培养小鼠胚胎成纤维细胞NIH/3T3和人脐静脉内皮细胞(HUVECs),采用活/死染色及细胞计数试剂盒(CCK-8)检测LL-37和LL-37R对细胞活性的影响,划痕实验和成管实验评价对HUVEC细胞增殖和迁移能力的影响。选用36只雄性SD大鼠制作糖尿病创面模型,随机分为对照组、LL-37组和LL-37R组,每组12只。在背部制作直径2cm的圆形全层皮肤创面,伤后每2d分别于皮下注射生理盐水、LL-37、LL-37R,伤后第0、3、7、14、21天拍照,第21天取材进行苏木精-伊红(HE)染色、Masson染色和免疫组织化学染色评估创面愈合情况。采用ANOVA分析和非配对t检验进行数据分析。结果LL-37R能够缓解高糖环境下NIH/3T3细胞和HUVEC细胞的损伤,并且表现出促进HUVEC细胞迁移和成血管的能力。对于大鼠糖尿病创面,在造模后21d,LL-37组和LL-37R组的平均创面面积占初始创面的比例分别为(31.46±5.51)%、(18.01±4.29)%,均显著小于对照组[(41.10±4.54)%,t=3.01、2.83,P<0.05];HE染色结果显示,LL-37组和LL-37R组的创面表皮厚度分别为(73.06±17.67)、(70.35±4.60)μm,大于对照组[(26.94±11.61)μm,t=2.18、3.48,P<0.05],且LL-37R组基本完成了上皮化过程;Masson染色结果显示,LL-37R处理过后的真皮中胶原纤维比例更高(F=78.61,P<0.01),且排列更加有序和紧密;免疫组织化学染色结果表明,LL-37R组的CD31阳性血管密度为(221.62±18.69)个/mm^(2),与对照组[(100.38±11.94)个/mm^(2)]和LL-37组[(117.95±16.01)个/mm^(2)]比较,血管形成最为显著(F=17.02,P<0.01),且LL-37R组创面中被CD86标记的炎性巨噬细胞数量[(20.18±5.08)个/mm^(2)]明显少于LL-37组[(61.82±8.101)个/mm^(2)]和对照组[(236.00±17.89)个/mm^(2),F=95.59,P<0.01]。结论LL-37及其衍生肽能够通过促进血管再生、胶原沉积和调节炎症加速糖尿病创面的愈合。

Objective To investigate the effects of LL-37 and its derivative peptides LL-37R on diabetic wound healing.Methods The mouse embryonic fibroblast cell lines(NIH/3T3)and human um-bilical vein endothelial cells(HUVECs)were cultured under high glucose conditions.Cell compatibility of LL-37 and LL-37R was evaluated using live/dead staining and cell counting kit(CCK-8)assay,and their effects on the proliferation and migration of HUVECs were assessed through scratch and tube formation as-says.A total of 36 male SD rats were selected and randomly divided into control,LL-37,and LL-37R groups,with 12 rats in each group.Diabetic wound models were created by making a 2 cm diameter full-thickness skin lesion on the back.Physiological saline,LL-37,or LL-37R was injected subcutaneously ev-ery 2 days.Wound photographs were taken on days 0,3,7,14,and 21.On day 21,tissues were collect-ed for hematoxylin-eosin(HE)staining,Masson's trichrome staining,and immunohistochemistry staining to evaluate wound healing.Data were analyzed using ANOVA and unpaired t-tests.Results LL-37R alle-viated cell damage in NIH/3T3 and HUVEC cells under high glucose conditions and promoted HUVECs migration and angiogenesis.For diabetic wounds in rats,the mean wound area proportions of the LL-37 and LL-37R groups were(31.46±5.51)%and(18.01±4.29)%of the initial wound at 21st day post-model-ing,respectively,significantly smaller than the control group[(41.10±4.54)%,t=3.01,2.83,P<0.05].HE staining revealed a significant increase in epidermal thickness for LL-37 and LL-37R groups[(73.06±17.67),(70.35±4.60)μm,respectively]compared to the control group[(26.94±11.61)μm,t=2.18,3.48,P<0.05].Masson's trichrome staining showed more organized and dense collagen fiber arrangement in the LL-37R group(F=78.61,P<0.01).Immunohistochemical staining results showed that the CD31 positive vascular density in the LL-37R group was(221.62±18.69)vessels/mm^(2),significantly greater than in the control group[(100.38±11.94)vessels/mm^(2)]and LL-37 group[(117.95±16.01)vessels/mm^(2),F=17.02,P<0.01].In addition,the LL-37R group demonstrated a markedly lower count of CD86-positive inflammatory macrophages in the wound area,with(20.18±5.08)cells/mm^(2),than the LL-37 group[(61.82±8.101)cells/mm^(2)]and the control group[(236.00±17.89)cells/mm^(2),F=95.59,P<0.01].Conclusion LL-37 and its derivative peptide LL-37R can accelerate the healing of diabetic wounds by promoting vascular regeneration,collagen deposition,and inflammation regulation.