整合素连接激酶(ILK)通过调控自噬影响深度烧伤创面愈合

Integrin-linked kinase(ILK)affects deep burn wound healing by regulating autophagy

目的:探讨整合素连接激酶(ILK)在大鼠深度烧伤创面的表达变化及其对创面愈合的影响和机制。方法:10只Wistar大鼠随机分为正常组和实验组,每组5只,实验组大鼠建立背部皮肤深Ⅱ度烧伤模型,正常组大鼠不做任何处理,免疫组化染色和Western blot检测烧伤创面ILK表达;另取45只大鼠建立背部皮肤深Ⅱ度烧伤模型后随机分为对照组、阴性对照组和ILK组,每组15只,阴性对照组和ILK组大鼠创面分别注射pEGFP-C1慢病毒和pEGFP-C1-ILK慢病毒,对照组注射等量生理盐水;于1 d、7 d、10 d、14 d、21 d观察创面愈合情况,10 d时取创面组织,ELISA测定创面组织TNF-α、IL-1β、IL-6、IL-10表达;21 d时取创面组织,HE染色观察创面组织形态变化,免疫组化染色检测创面组织自噬标志蛋白轻链蛋白3(LC3)表达,Western blot检测自噬相关蛋白表达。结果:烧伤大鼠创面组织ILK表达较正常大鼠皮肤组织显著升高(P<0.05);ILK组大鼠创面愈合率在第7、14、21天时均显著高于对照组和阴性对照组(P<0.05);与对照组和阴性对照组比较,ILK组大鼠创面组织病变坏死程度明显降低,炎症细胞浸润减少,TNF-α、IL-1β及IL-6含量均显著降低(P<0.05),IL-10含量显著升高(P<0.05),LC3阳性表达平均光密度值显著降低(P<0.05),LC3-Ⅱ/Ⅰ显著降低(P<0.05),Beclin-1蛋白表达显著下调(P<0.05),p62蛋白表达显著上调(P<0.05)。结论:ILK在深度烧伤大鼠创面组织表达增加,过表达ILK可减轻创面炎症反应,抑制自噬,促进烧伤创面愈合。

Objective:To explore expression changes of integrin-linked kinase(ILK)on deep burn wounds in rats,and its influence and mechanism on wound healing.Methods:Ten Wistar rats were randomly divided into normal group and experimental group,with 5 rats in each group.Rats in experimental group were established a model of deep second-degree burns on back skin,and rats in normal group did not do any treatment.ILK expression in burn wounds was detected by immunohistochemical staining and Western blot.Another 45 rats were established model of deep second-degree burns on back skin and randomly divided into control group,negative control group and ILK group,with 15 rats in each group,wounds of negative control group and ILK group were injected with pEGFP-C1 lentivirus and pEGFP-C1-ILK lentivirus,respectively,control group was injected with same amount of saline;wound healing on 1 d,7 d,10 d,14 d,21 d was observed;wound tissue was taken on 10 d,ELISA was used to determine TNF-α,IL-1β,IL-6 and IL-10 expressions in wound tissue;wound tissue was taken at 21 d,HE staining was used to observe changes in wound tissue morphology,immunohistochemical staining was used to detect expression of autophagy marker protein light chain protein 3(LC3)in wound tissues,Western blot was used to detect expressions of autophagy-related proteins.Results:ILK expression in wound tissue of burn rats was significantly higher than that in skin tissue of normal rats(P<0.05);on 7 d,14 d and 21 d,wound healing rate of rats in ILK group was significantly higher than control group and negative control group(P<0.05);compared with control group and negative control group,degree of necrosis of wound tissue in ILK group was significantly reduced,infiltration of inflammatory cells was reduced,contents of TNF-α,IL-1βand IL-6 were significantly reduced(P<0.05),content of IL-10 was significantly increased(P<0.05),average optical density value of LC3 positive expression was significantly reduced(P<0.05),LC3-Ⅱ/Ⅰwas significantly decreased(P<0.05),expression of Beclin-1 protein was significantly down-regulated(P<0.05),and expression of p62 protein was significantly up-regulated(P<0.05).Conclusion:ILK expression is increasing in wound tissue of deep burn rats,whose over-expression can reduce inflammation of wound,inhibit autophagy and promote healing of burn wound.