整合素连接激酶信号通路在糖尿病大鼠皮肤病变及创面愈合中的作用

目的探讨整合素连接激酶（ILK）信号通路在糖尿病大鼠皮肤病变及创面愈合中的作用。方法将36只SD大鼠按随机数字表法分为糖尿病组和非糖尿病组，每组18只。糖尿病组大鼠腹腔注射10g／L链脲佐菌素（60mg／kg），非糖尿病组大鼠腹腔注射等量的柠檬酸钠缓冲液。糖尿病组大鼠建模成功2周后，切除2组大鼠背部两侧2处2cm×2cm的全层皮肤致伤。伤后第1、3、7、10、14、21天，2组各取3只大鼠，对创面进行拍照观察并计算创面愈合率。2组剩余15只大鼠于伤后第3、7、10、14、21天各取3只，切取背部的未致伤皮肤组织和创面中心组织。HE染色观察未致伤皮肤组织形态，测量全层皮肤和表皮的厚度；实时荧光定量RT—PCR法检测未致伤皮肤组织ILK、蛋白激酶B（Akt）和糖原合成酶激酶3p（GSK-3β）的mRNA表达；蛋白质印迹法检测未致伤皮肤组织ILK、Akt、磷酸化Akt、GSK-3β、磷酸化GSK-3β和创面组织ILK、磷酸化Akt的蛋白表达。对数据行两独立样本t检验、单因素方差分析、SNK检验和析因设计方差分析。结果（1）伤后，非糖尿病组大鼠创面痂皮干燥，脱落后见鲜红色肉芽组织，无分泌物，创面愈合速度快；糖尿病组大鼠创面痂皮下有分泌物，易脱落，脱落后见粉红色肉芽组织，有较多分泌物，创面愈合缓慢。除伤后第3天外，伤后各时相点糖尿病组大鼠的创面愈合率明显低于非糖尿病组（t值为3．858～13．738，P〈0．05或P〈0．01）。（2）伤后第3天，非糖尿病组大鼠的未致伤皮肤组织皮下毛囊和血管组织丰富，表皮由复层细胞如基底细胞和KC构成；糖尿病组大鼠的未致伤皮肤组织皮下毛囊和血管组织稀少，表皮几乎只有单层细胞。伤后3～21d，非糖尿病组大鼠未致伤皮肤组织全层皮肤和表皮的厚度无明显变化，糖尿病组大鼠未致伤皮肤组织全层皮肤和表皮的厚度分别由伤后第3天的（1074±66）、（15．1±3．8）汕m逐渐变薄至伤后第21天的（785±122）、（9．7±2．1）μm。伤后各时相点，非糖尿病组大鼠的未致伤皮肤组织全层皮肤和表皮厚度均明显厚于糖尿病组（t值为4．620～23．549，P值均小于0．001）。（3）伤后3—21d，糖尿病组大鼠未致伤皮肤组织中ILK和Akt的mRNA表达量明显低于非糖尿病组（t值分别为d．779、3．440，P值均小于0．05）；2组大鼠未致伤皮肤组织中GSK-3βmRNA表达量相近（t：0．363，P〉0．05）。（4）伤后3～21d，糖尿病组大鼠未致伤皮肤组织中ILK、Akt和磷酸化Akt蛋白的表达量均明显低于非糖尿病组（t值为2．630～6．209，P〈0．05或P〈0．01）；2组大鼠未致伤皮肤组织中GSK-3β蛋白的表达量相近（t=0．652，P〉0．05）；糖尿病组大鼠未致伤皮肤组织中磷酸化GSK-3β蛋白的表达量明显高于非糖尿病组（t=4．131，P〈0．001）。2组大鼠伤后各时相点创面组织中ILK蛋白的表达量相近（t值为0．381～2．440，P值均大于0．05）。除伤后第3天外，非糖尿病组大鼠伤后各时相点创面组织中磷酸化Akt蛋白的表达量均明显高于糖尿病组（t值为4．091～20．555，P〈0．05或P〈0．01）。伤后3～21d，非糖尿病组大鼠创面组织与未致伤皮肤组织中ILK蛋白表达量相近（F=2．522，P〉0．05），创面组织中磷酸化Akt蛋白表达量明显增高（F=117．329，P〈0．001）；糖尿病组大鼠创面组织与未致伤皮肤组织中ILK蛋白表达量相近（F=1．337，P〉0．05），创面组织中磷酸化Akt蛋白表达量明显增高（F=184．120，P〈0．001）。结论糖尿病皮肤病变可能与ILK信号通路中ILK、Akt和磷酸化Akt的表达下降有关，糖尿病大鼠创面愈合缓慢可能与磷酸化Akt的表达下降有关。

Objective To investigate the role of integrin-linked kinase (ILK) signaling pathway in the skin lesions and wound healing in diabetic rats.Methods Thirty-six SD rats were divided into diabetic wound group (D) and non-diabetic wound group (N) according to the random number table,with 18 rats in each group.10 g/L streptozocin (60 mg/kg) was intraperitoneally injected in rats in group D,while the rats in group N were given same quantity of sodium citrate buffer.Two weeks after successful reproduction of diabetic model of rats in group D,two full-thickness skin of an area of 2 cm × 2 cm was resected on both sides of back of rats in the two groups.Wounds of three rats of each group were photographed and examined on post injury day (PID) 1,3,7,10,14,and 21,and the wound healing rates were calculated.The noninjured skin and wound tissue (central part) on back of three rats of the rest 15 rats in the two groups were harvested on PID 3,7,10,14,and 21,respectively.Morphology of the non-injured skin tissue was observed with HE staining,and the thickness of full-thickness skin and epidermis were measured.The mRNA expression levels of ILK,protein kinase B (Akt),and glycogen synthase kinase-3β (GSK-3β) in non-injured skin tissue were determined with real-time fluorescent quantitative RT-PCR.The protein expression levels of ILK,Akt,phosphorylated Akt,GSK-3β,and phosphorylated GSK-3β in non-injured skin tissue,and ILK,phosphorylated Akt in wound tissue were assessed with Western blotting.Data were processed with two independent-sample t test,one-way analysis of variance,SNK test and analysis of variance of factorial design.Results (1) After injury,the wound scabs of rats in group N were dry,and red granulation tissue with no excretion were seen when the scabs fell off,and the wound healed fast.After injury,excretion under the wound scabs of rats in group D was seen,and the scabs easily fell off with exposure of pink granulation tissue with much excretion,and the wounds healed slowly.Except for PID 3,the wound healing rate of rats in group D was significantly lower than that in group N on other PIDs (with t values from 3.858 to 13.738,P ＜ 0.05 or P ＜ 0.01).(2) On PID 3,the hair follicles and blood vessels in the non-injured skin tissue of rats in group N were rich,and the epidermis was composed of stratified cells in form of basal cells and keratinocyte,and the hair follicles and blood vessels in the non-injured skin tissue of rats in group D were scarce,and the epidermis was nearly composed of one-layer of cells.The thickness of full-thickness skin and epidermis of non-injured skin tissue of rats in group N was similar from PID 3 to 21,and the thickness of full-thickness skin and epidermis of non-injured skin tissue of rats in group D on PID 3 was respectively (1 074 ±66) and (15.1 ±3.8) μm,and they gradually thinned out to (785 ± 122) and (9.7 ±2.1) μm on PID 21,respectively.The thickness of full-thickness skin and epidermis of non-injured skin tissue of rats in group N were significantly thicker than those in group D on each PID (with t values from 4.620 to 23.549,P values below 0.001).(3) From PID 3 to 21,the mRNA expression levels of ILK and Akt in non-injured skin tissue of rats in group D were significantly lower than those in group N (with t values respectively 4.779 and 3.440,P values below 0.05),the mRNA expression levels of GSK-3 β in non-injured skin tissue of rats were similar in two groups (t =0.363,P ＞0.05).(4) From PID 3 to21,the protein expression levels of ILK,Akt and phosphorylated Akt in non-injured skin tissue of rats in group D were significantly lower than those in group N (with t values from 2.630 to 6.209,P ＜ 0.05 or P ＜ 0.01);the protein expression levels of GSK-3β in non-injured skin tissue of rats in two groups were similar (t =0.652,P ＞ 0.05);the protein expression level of phosphorylated GSK-3β in non-injured skin tissue of rats in group D was significantly higher than that in group N (t =4.131,P ＜0.001).The protein expression levels of ILK in wound tissue of rats in two groups were similar on each PID (with t values from 0.381 to 2.440,P values above 0.05).Except for PID 3,the protein expression levels of phosphorylated Akt in wound tissue of rats in group N were significantly higher than that in group D on other PIDs (with t values from 4.091 to 20.555,P ＜0.05 or P ＜0.01).From PID 3 to 21,the protein expression levels of ILK in wound tissue and non-injured skin tissue of rats in group N were similar (F =2.522,P ＞ 0.05),and the protein expression level of phosphorylated Akt in wound tissue was significantly higher than that in non-injured skin tissue (F =117.329,P ＜ 0.001);the protein expression levels of ILK in wound tissue and noninjured skin tissue of rats in group D were similar (F =1.337,P ＞ 0.05),and the protein expression level of phosphorylated Akt in wound tissue was significantly higher than that in non-injured skin tissue (F =184.120,P ＜ 0.001).Conclusions The skin lesion of diabetic rats may be related to the declined expression levels of ILK,Akt and phosphorylated Akt in the ILK signaling pathway.The refractory healing of wound in diabetic rats may be related to the declined expression level of phosphorylated Akt.