Apoptosis in diabetic foot ulcers and human fibroblast cells treated with AGEs
10.3969/j.issn.1000-4718.2014.08.002
- VernacularTitle:糖尿病足伤口皮肤细胞凋亡情况及 AGEs对人皮肤成纤维细胞凋亡的影响
- Author:
Guojuan LAO
;
Meng REN
;
Yanrui HUANG
;
Chuan YANG
;
Xiaoyi WANG
;
Li YAN
- Publication Type:Journal Article
- Keywords:
Diabeticfoot;
Woundhealing;
Apoptosis;
Advancedglycosylationendproducts
- From:
Chinese Journal of Pathophysiology
2014;(8):1351-1356
- CountryChina
- Language:Chinese
-
Abstract:
[ABSTRACT]AIM:Toinvestigatecellapoptosisindiabeticfootulcersandtheeffectofadvancedglycosylation end products (AGEs) on apoptosis in human fibroblast cells.METHODS: Diabetic foot patients (n=18) and 18 age-matched non-diabetic controls were recruited .The clinical and biochemical features were compared by statistics methods . Skin biopsies were obtained from foot .Cleaved caspase-3 was measured by immunohistochemistry using the technique of streptavidin-biotin complex ( SABC ) staining.Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling ( TUNEL) technique was used to detect apoptosis of the skin tissues .Human primary foreskin fibroblasts were isolated and cultured in the presence of 5.6 mmo/L glucose, 25 mmo/L glucose, fluctuant glucose ( changing the glucose from 5.6 mmo/L to 25 mmo/L every 8 h) or AGEs (150 mg/L, containing 5.6 mmo/L glucose).After 72 h treatment, Western blotting was used to determine the levels of the apoptotic protein cleaved-caspase-3.Other cells were trypsinized , washed with cold PBS and incubated with PI and Annexin V-FITC, then analyzed by flow cytometry to detect cell apoptosis .RE-SULTS:Diabetic patients had higher levels of fasting blood glucose (FBG), 2-hour postprandial blood glucose (2 h PBG) and glycosylated hemoglobin A1c (HbA1c), and longer wound duration.The protein level of cleaved caspase-3 was signifi-cantly higher in diabetic group , suggesting that apoptosis was increased in diabetic skin tissues .TUNEL analysis showed that apoptotic index was higher in diabetic group compared with that in non-diabetic group (8.4%±1.5% vs 3.8%± 0.8%) , which further confirmed that cell apoptosis was increased in diabetic foot tissues .In human fibroblasts , the levels of cleaved caspase-3 in normal group , sustained high glucose group , fluctuant high glucose group and AGEs group were 0.80 ±0.13, 1.22 ±0.18, 1.46 ±0.32 and 1.83 ±0.25, respectively.The apoptotic rates detected by flow cytometry were 2.43%±0.19%, 2.89%±0.51%, 3.99% ±0.24% and 6.83% ±0.36%, respectively.Both the level of cleaved caspase-3 and the apoptotic rate in AGEs group were higher than those in normal glucose group and sustained high glucose group .CONCLUSION:Increased apoptosis in diabetic foot ulcers is one of the most important reasons for im-paired wound healing .As compared to sustained high glucose and glucose fluctuations , AGEs induce greater apoptosis in human fibroblast cells .
