Niu Yi-wen, Miao Ming-yuan, Dong Wei, Dong Jiao-yun, Cao Xiao-zan, Lu Shu-liang
Shanghai Institute of Burns, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
Zhonghua Shao Shang Za Zhi. 2012 Feb;28(1):32-5.
To investigate the accumulation of advanced glycation end products (AGE) and the inflammatory response of skin and wound in diabetic patients, and to analyze their relationship in vitro.
Histological staining and immunohistochemical staining was respectively performed on skin and wound tissue specimens collected from 10 patients with Type II diabetes mellitus (diabetes group) and 12 non-diabetic patients with skin injury (control group) to observe the arrangement of collagen and the distribution of inflammatory cells, and to determine the expression levels of AGE and its receptor (RAGE). Malondialdehyde (MDA) levels in skin and wound tissue homogenates were assayed by enzyme-linked immunosorbent assay. In vitro, human neutrophils were isolated and treated with RPMI-1640 culture medium or that containing AGE-human serum albumin in the concentration of 0.315, 0.625, 1.250 mg/mL, and they were identified as normal control (NC) group, low concentration (L) group, moderate concentration (M) group, and high concentration (H) group. Cell viability in each group was determined by MTT colorimetric assay, and the reactive oxygen species (ROS) in cell was measured with 2', 7'-dichlorofluorescein-diacetate. Data were processed with t test.
Compared with those of skin in control group, collagens of skin tissues in diabetes group atrophied and disorderly arranged. Inflammatory cells in wounds in diabetes group were dispersed, in which collagens arranged loosely and irregularly, as compared with those of wounds in control group. Expression levels of AGE and RAGE of skin in diabetes group were higher than those in control group. In diabetes and control groups, especially in diabetes group, the numbers of RAGE-positive cells in wound tissue were more than those in skin tissue. Large amount of inflammatory cells with positive expression of RAGE were observed in diabetes group. MDA level of skin and wound tissue in diabetes group was respectively (6.3 ± 1.0), (7.1 ± 2.4) nmol per milligram protein, which were obviously higher than those in control group [(2.9 ± 1.0), (3.6 ± 1.4) nmol per milligram protein, with t value respectively 8.017, 4.349, P < 0.05 or P < 0.01]. Cell viability and ROS levels in neutrophils were increased in L, M, and H groups [(59 ± 8)%, (77 ± 5)%, (67 ± 6)% and 1.67 ± 0.14, 2.13 ± 0.17, 3.48 ± 0.48] as compared with those in NC group [(34 ± 5)% and 0.58 ± 0.06, with t value respectively 7.195, 14.890, 11.130 and 20.195, 24.905, 16.864, P < 0.05 or P < 0.01].
Abnormal oxidative stress in diabetic skin leads to an atypical origin of wound repair. AGE-RAGE effect is a critical mediator for oxidative stress in diabetic wound tissue during wound healing.
研究糖尿病患者皮肤及伤口中晚期糖基化终末产物（AGE）的蓄积及炎症反应，并在体外分析它们之间的关系。
分别对10例2型糖尿病患者（糖尿病组）和12例非糖尿病皮肤损伤患者（对照组）的皮肤及伤口组织标本进行组织学染色和免疫组化染色，观察胶原排列及炎症细胞分布，检测AGE及其受体（RAGE）的表达水平。采用酶联免疫吸附测定法检测皮肤及伤口组织匀浆中丙二醛（MDA）水平。体外分离人中性粒细胞，分别用RPMI-1640培养基或含浓度为0.315、0.625、1.250mg/mL AGE-人血清白蛋白的培养基处理，分别设为正常对照组（NC组）、低浓度组（L组）、中浓度组（M组）和高浓度组（H组）。采用MTT比色法测定各组细胞活力，用2',7'-二氯荧光素二乙酸酯检测细胞内活性氧（ROS）。数据采用t检验处理。
与对照组皮肤相比，糖尿病组皮肤组织中的胶原萎缩且排列紊乱。与对照组伤口相比，糖尿病组伤口中的炎症细胞分散，其中胶原排列松散且不规则。糖尿病组皮肤中AGE和RAGE的表达水平高于对照组。在糖尿病组和对照组中，尤其是糖尿病组，伤口组织中RAGE阳性细胞数多于皮肤组织。糖尿病组可见大量RAGE阳性表达的炎症细胞。糖尿病组皮肤和伤口组织的MDA水平分别为（6.3±1.0）、（7.1±2.4）nmol/mg蛋白，明显高于对照组[（2.9±1.0）、（3.6±1.4）nmol/mg蛋白，t值分别为8.017、4.349，P<0.05或P<0.01]。与NC组相比，L、M、H组中性粒细胞的细胞活力和ROS水平升高[（59±8）%、（77±5）%、（67±6）%和1.67±0.14、2.13±0.17、3.48±0.48]，而NC组为[（34±5）%和0.58±0.06，t值分别为7.195、14.890、11.130和20.195、24.905、16.864，P<0.05或P<0.01]。
糖尿病皮肤中异常的氧化应激导致伤口修复的异常起始。AGE-RAGE效应是糖尿病伤口愈合过程中糖尿病伤口组织氧化应激的关键介质。
