胰岛素对烧伤血清诱导血管内皮细胞核因子κB核移位的抑制作用

Inhibitory effect of insulin on nuclear factor-kappa B nuclear translocation of vascular endothelial cells induced by burn serum

目的 了解胰岛素对烧伤血清诱导的血管内皮细胞NF-κB核移位的抑制作用及其相关机制.方法 体外培养人脐静脉内皮细胞（HUVEC）.按照随机数字表法将细胞分为5组：空白对照组,不加任何刺激因素常规培养;正常血清对照组和烧伤血清刺激组,分别用含体积分数20%健康人血清、体积分数20%烧伤患者血清的培养液培养;烧伤血清＋胰岛素处理组,在烧伤血清刺激组培养液成分的基础上添加胰岛素（终浓度1×10-7mol/L）进行培养;抑制剂预处理组,预先加入蛋白激酶B（PKB或Akt）特异性抑制剂LY294002（50μmol/L）孵育细胞,30 min后改用培养液（成分同烧伤血清＋胰岛素处理组）培养.6 h后,采用扫描电镜观察各组HUVEC损伤情况;用流式细胞仪检测细胞凋亡率;蛋白质印迹法检测细胞胞质磷酸化κB-α抑制蛋白（p-IκB-α）和磷酸化Akt（p-Akt）水平,以及胞核NF-κB-p65的蛋白表达变化.结果 （1）扫描电镜观察：与空白对照组HUVEC比较,烧伤血清刺激组、抑制剂预处理组细胞收缩明显,细胞间呈锯齿状连接或连接消失,胞核结构不规整.正常血清对照组及烧伤血清＋胰岛素处理组细胞结构有轻微改变,但细胞延展性及胞核结构明显好于烧伤血清刺激组.（2）细胞凋亡率：空白对照组为（15.7±2.2）%.烧伤血清刺激组为（28.5±2.3）%,烧伤血清＋胰岛素处理组为（22.3±1.8）%,抑制剂预处理组为（29.7±2.4）%,均明显高于空白对照组（F=14.288,P〈0.05或P〈0.01）;正常血清对照组细胞凋亡率为（17.0±2.5）%,与空白对照组比较差异无统计学意义（F=14.288,P〉0.05）.与烧伤血清刺激组相比,烧伤血清＋胰岛素处理组细胞凋亡率明显降低（F=14.288,P〈0.05）.（3）蛋白表达水平：与空白对照组相比,烧伤血清刺激组和抑制剂预处理组细胞胞质p-IκB-α与胞核NF-κB-p65的蛋白表达水平明显升高,p-Akt表达水平明显下降;正常血清对照组和烧伤血清＋胰岛素处理组3种蛋白水平均与空白对照组接近.结论 胰岛素通过调节磷脂酰肌醇3激酶/Akt信号通路抑制IκB-α磷酸化,继而限制NF-κB核移位,最终发挥改善内皮细胞功能的作用.

Objective To study the inhibitory effects of insulin on nuclear factor-kappa B （NF-κB） nuclear translocation of vascular endothelial cells induced by burn serum and its correlative mechanism. Methods Human umbilical vein endothelial cells （HUVECs） were cultured in vitro and divided into 5 groups： blank control group （BC, ordinary culture without any stimulation） , normal serum control group （NS, cultured with nutrient solution containing 20% healthy human serum） , burn serum stimulation group （BS, cultured with nutrient solution containing 20% burn human serum） , burn serum ＋ insulin treatment group （BI, cultured with nutrient solution containing 20% burn human serum and 1 × 10-7 mol/L insulin） ,inhibitor pretreatment group [IP, pretreated with 50 μmol/L protein kinase B （Akt） specific inhibitor LY-294002, then cultured with the same medium as used in BI group 30 minutes later] according to the random number table. Six hours later, the injury and apoptosis of HUVECs was respectively observed by the scanning electron microscope and determined by the flow cytometry. Meanwhile, the phosphorylation of inhibitor kappa B-α （p-IκB-α） and Akt （p-Akt） in cytoplasm, and the content of NF-κB-p65 in nucleus were determined with Western blot. Results （1） Compared with those in BC group, HUVECs in BS group shrank obviously with irregular nuclear structure, and intercellular links jagged or vanished. Slight change was observed in HUVECs structure in NS and BI groups, with the cell ductility and nuclear structure much better than those in BS group. （2） The apoptosis rates of HUVECs in BS group [（28. 5 ± 2. 3） %] , BI group [（22. 3 ±1.8）%], and IP group [（29. 7 ± 2. 4） %] were all obviously higher than that in BC group [（15.7 ±2.2）% , F =14.288, P 〈0.05otP 〈0.01]. There was no significant statistical difference between NS group [（17. 0 ± 2. 5） %] and BC group in apoptosis rate （F = 14. 288 , P 〉 0. 05）. The apoptosis rate of HUVECs in BI group was obviously lower than that in BS group （F = 14. 288 , P 〈0.05）. （3）Compared with those in BC group, the protein expressions of p-IκB-α in cytoplasm and NF-κB-p65 in nucleus were up-regulated, and the protein expression of p-Akt in cytoplasm was down-regulated in BS and IP groups. The expression levels of the three proteins in NS and BI groups were close to those in BC group. Conclusions Insulin could inhibit the IκB phosphorylation, and then restrict NF-κB nuclear translocation and improve the vascular endothelial cells function accordingly through regulating phosphatidylinositol 3 ki-nase/Akt pathway.