糖尿病小鼠皮肤组织中晚期糖基化终末产物的表达及其对胶原纤维的影响

Expressions of advanced glycosylation end products in skin of diabetic mice and their influence on collagen fibers

目的 观察不同病程糖尿病小鼠皮肤组织晚期糖基化终末产物（AGE）的表达及其对胶原纤维的影响.方法 健康8周龄C57BL/6J雄性小鼠采用小剂量链佐星（50 mg/kg）多次注射法建立糖尿病模型,分别在造模后第4周和12周处死小鼠,取背部中央皮肤,HE染色观察组织学结构,样本碱水解法测定羟脯氨酸水平反映胶原总量,限制性胃蛋白酶降解法测定胶原交联程度,实时定量PCR法测定Ⅰ型胶原和Ⅲ型胶原基因表达,荧光分光光度法和Western印迹检测晚期糖基化终末产物水平,硫代巴比妥酸法测定丙二醛含量.统计学分析采用t检验.结果 镜下可见糖尿病小鼠皮肤组织明显变薄,胶原呈进行性减少.第4周和12周时,糖尿病组小鼠皮肤组织羟脯氨酸含量与正常对照组比较显著降低[（684.5±76.7）比（787.7±87.7） mg/g,（558.1±73.1）比（757.8±75.3） mg/g,均P＜ 0.01],皮肤AGE水平显著上升[（37.47±10.65）比（26.39±3.74）AUF/mg羟脯氨酸,（47.70±5.66）比（29.91±6.50） AUF/mg羟脯氨酸,均P＜ 0.01],丙二醛含量显著增加[（6.62±0.47）比（4.82±0.56） μmol/L,（8.63±0.36）比（5.15±0.46）μmol/L,均P＜ 0.01],交联胶原以及Ⅰ、Ⅲ型胶原mRNA表达均下降（P＜0.01或0.05）,糖尿病12周组非交联胶原亦明显下降（P＜0.05）;与糖尿病4周组相比,糖尿病12周组小鼠皮肤羟脯氨酸含量、Ⅰ型胶原表达进一步减少（P＜0.05）,AGE和丙二醛水平进一步增加（P＜0.01）.结论 糖尿病小鼠皮肤组织胶原纤维性状发生改变,可能与皮肤组织中晚期糖基化终末产物水平上升及氧化损伤加剧有关.

Objective To investigate the expressions of advanced glycosylation end products （AGE） in skin of mice with diabetes mellitus （DM） for different durations,and to evaluate their influence on collagen fibers.Methods Forty healthy 8-week-old male C57BL/6J mice were divided into DM group （n =20） and control group （n =20） to receive multiple intraperitoneal injections of low dose streptozotocin （50 mg/kg） and citric acid buffer （0.1 mol/L）,respectively,for 5 consecutive days.Ten mice were sacrificed in each group on week 4 and 12 respectively after the last intraperitoneal injection,and full-thickness skin tissue samples were harvested from the middorsal region of each mouse.Then,hematoxylin-eosin （HE） staining was performed to observe histological changes,and total collagen content was estimated according to hydroxyproline content measured by an alkalinehydrolysis method.The cross-linking degree of collagen was determined by Edman degradation method using pepsin,the mRNA expression level of collagen type Ⅰ and Ⅲ by real-time quantitative PCR,the content of AGE by fluorospectrophotometry and Western blotting,and the level of malondialdehyde （MDA） by using a thiobarbituric acid method.Statistical analysis was carried out by t test.Results As light microscopy showed,the skin became obviously thinner in the diabetic mice with a progressive decrease in the number of collagen fibers in comparison with the control mice.On week 4 and 12 after the last injection,the diabetic mice exhibited a significant reduction in the content of hydroxyproline （（684.5 ± 76.7） vs.（787.7 ± 87.7） rg/g,（558.1 ± 73.1） vs.（757.8 ± 75.3） mg/g,both P 〈 0.01） and in the levels of cross-linked collagen as well as mRNA expressions of collagen Ⅰ and Ⅲ （P 〈 0.01 or 0.05）,but a significant increase in the content of AGE （（37.47 ± 10.65） vs.（26.39 ± 3.74） AUF/mg hydroxyproline,（47.70 ± 5.66） vs.（29.91 ± 6.50） AUF/mg hydroxyproline,both P 〈 0.01） and MDA （（6.62 ± 0.47） vs.（4.82 ± 0.56） μmol/L,（8.63 ± 0.36） vs.（5.15 ± 0.46） μmol/L,both P〈 0.01） in skin tissue,compared with the control mice.The level of non-cross-linked collagen in skin tissue was also lower in the diabetic mice than in the control mice on week 12 （P 〈 0.05）.Moreover,the contents of hydroxyproline and the expression levels of collagen I in skin were significantly lower （P 〈 0.05）,but the levels of AGE and MDA were significantly higher （P 〈 0.01） in the diabetic mice on week 12 than in those on week 4.Conclusions The characteristics of collagen fibers in skin are altered in diabetic mice when compared with normal control mice,which may be associated with increased AGE content and oxidative injury in skin.