Pathophysiological changes of dermal tissue in diabetic rats and their mechanisms

33 2 2012 4 JOURNAL OF TONGJI UNIVERSITY MEDICAL SCIENCE Vol. 33 No. 2 Apr. 2012 doi 10. 3969 /j. issn1008-0392. 2012. 02. 002 1 1 1 1 2 3 1. 200443 2. 200025 3. 200003 16 8 SD 8 8 12 GSP MDA GSH AGEs AGE-HSA AGEs ECV304 0. 016 ± 0. 007 mm 0. 037 ± 0. P < 0. 01 GSP MDA GSH P < 0. 01 AGEs AGEs S 4. 83 ± 2. 16 % 5. 01 ± 2. 47 % P > 0. 05 G 2 /M 0. 80 ± 0. 62 % 2. 86 ± 1. 10 % P < 0. 05 150 μg /ml AGE-HSA 48 h S G 2 /M P < 0. 05 ECV304 12. 5 25 50 μg /ml AGE-HSA 48 h 100 200 μg /ml AGE-HSA 6 12 24 48 h P < 0. 01 AGEs R 587 A 1008-0392 2012 02-0007-08 Pathophysiological changes of dermal tissue in diabetic rats and their mechanisms LIN Wei-dong 1 QIAN Xiong 1 JIANG Wan-li 1 LIU Wei-wei 1 LU Shu-liang 2 CHEN Xiang-fang 3 1. Dept. of Surgery Shanghai Fire Corps Hospital Chinese People's Armed Police Shanghai 200443 China 2. Shanghai Burns Institute Ruijin Hospital Shanghai Jiaotong University School of Medicine Shanghai 200025 China 3. Dept. of Endocrinology Changzheng Hospital Second Military Medical University Shanghai 200003 China 2011-10-09 81100585 08JC1407200 1973. E-mail linweidong5@ 163. com. E-mail cxf3918 @ 163. com 7

33 Abstract Objective To investigate pathophysiological changes of dermal tissue in diabetic rat and their mechanisms. Methods Sixteen 8-week-old male Sprague-Dawley SD rats were randomized into two groups rats in DM group were intraperitoneally injected with streptozotocin STZ at a dose of 65 mg /kg rats in NC group were injected with the same volume of citric acid buffer. All rats were sacrificed at week 12 after injection blood and dermal tissue samples were collected. The histological changes in dermal tissue were observed skin thickness and epidermal cell cycle were measured the skin glucose concentration and advanced glycation end products AGEs were determined. Plasma glycosylated protein GSP malondialdehyde MDA and glutathione GSH levels were evaluated. The cultured epidermal cells and ECV304 cells were exposed to AGE-modified human serum albumin AGE-HSA and the effects were evaluated. Results The diabetic rats exhibited changes in skin tissue including a decrease in thickness disappearance of the multilayer epithelium structure degeneration of collagen fibers and an increase in infiltration of inflammatory cells in addition to a significant increase in skin glucose and AGEs. Moreover diabetic rats had increased plasma GSP and MDA decreased plasma GSH levels. There were no differences in S phase percentage of epidermal cells between DM group 4. 83 ± 2. 16 % and NC group 5. 01 ± 2. 47 % P > 0. 05. In contrast there was a marked decrease in G 2 /M phase in DM group compared to control animals 0. 80 ±0. 62 % vs 2. 86 ±1. 10 % P < 0. 05. After exposure to 150 μg /ml AGE-HSA for 48 h the percentage of epidermal cell in both phases was decreased P <0. 05. Exposure of ECV304 cells to 100 or 200 μg /ml AGE-HSA for 6 12 24 or 48h led to a time-dependent and dose-dependent increase in apoptosis rate compared to the control group P < 0. 01. Conclusion The high glucose in the skin tissue coupled with the accumulation of toxic substances such as AGEs results in the dysfunction of dermal cells and/or the matrix. This might be the mechanism of diabetes-induced early-stage endogenous skin damage. Key words diabetes skin pathophysiology glycation end products 2007 2002 2003 2 300 2025 4 600 20 AGEs 10% 1 30% 1 2 1. 1 1. 1. 1 16 SPF SD 8 3 advanced glycation end products AGEs 200 ~ 220 g AGEs 1. 1. 2 streptozotocin STZ Sigma GSP GSH MDA AGEs 8

2 Trans Genic Inc One-touch GSP MDA GSH F-3010 HITACHI Beckman's 1. 2. 5 EPICS-XL Beckman 0. 5 cm 1 cm Axioskop2 plus Zeiss 4 Spectra 9. 8 cm 1 500 r /min 10 min MAX190 Molecular RNA Lysis Devices Eppendorf 5810R Hamburg 1. 2. 6 7 1. 2 0. 2 g 1. 2. 1 DM 16 SD 0. 24 mol /L Ba OH 2 2 8 1. 5 ml 14 cm 3 000 r /min 8 10 min Beckman's 24 h 65 mg /kg STZ mg /g = ml mmol /L 180 10-3 g < 1. 2. 7 AGEs 8 8. 9 mmol /L 11. 2 mmol /L F- 4 3010 EX 360 nm EM 420 nm 1. 2. 2 12 5 nm 3 AGEs 2. 5% 4% - 70 U /mg AGEs AGEs 1. 2. 3 5 μm 1. 2. 8 AGE-HSA H-E SD 5 40 H-E K-SFM 3 ~ 8 40 mm 5 3 10 5 / 4 ml K-SFM 6 37 5% CO 2 2. 5% 1% 24 h K-SFM AGE- 60 nm HSA 150 μg /ml3 HITACHI 500 AGE-HSA 48 h 1. 2. 4 3 1. 2. 9 AGE-HSA ECV304 Bouaicha 6 1 9

33 ECV304 10% FBS 100 U /ml 1 100 μg /ml DMEM /F12 2 ECV304 1 10 6 / 6 CO 2 24 h 24 h AGE-HSA 12. 5 25 50 100 200 μg /ml 1 3 Fig. 1 Comparison of normal rat and diabetic rat AGE-HSA 6 12 24 48 h 2. 2 Annexin V Fitc 3 1. 3 x ± s t SAS 6. 02 P < 0. 05 2 2 2. 1 3 10 2 H-E A D 40 B C E F 400 Fig. 2 Histological observation of skin tissues after hematoxylin and eosin staining H-E A D 40 B C E F 400 A B C D E F

2 AGEs AGEs 5 3 15 000 Fig. 3 Transmission electron microscopic imaging of ultrastructure of skin microvessels in normal and diabetic rats 15 000 A B 2. 3 GSP MDA GSH Fig. 4 P < 0. 01 1 Tab. 1 1 GSP 2 GSH MDA Changes of GSP MDA and GSH in plasma x ± s mmol L - 1 GSP GSH MDA 4 Comparison of cell cycle of keratinocytes from skin of diabetic and normal rats AGEs Tab. 2 Changes in skin glucose content and collagen-related fluorescence x ± s mg /g skin weight U /mg hydroxyproline 8 1. 2 ± 0. 32 0. 19 ± 0. 05 3. 5 ± 0. 72 6 1. 9 ± 0. 43 * 0. 11 ± 0. 03 * 5. 3 ± 1. 01 * * P < 0. 01 2 8 0. 85 ± 0. 29 22. 6 ± 5. 10 6 2. 77 ± 1. 12 * 33. 9 ± 4. 20 * * P < 0. 01 2 2. 4 0. 016 ± 0. 007 mm 0. 037 ± 0. 007 mm t = 3. 84 P < 0. 01 5 AGEs 400 2. 5 Fig. 5 Comparison of the expression of the AGEs protein S in the skin of diabetic and normal rats 400 4. 83 ± 2. 16 % 5. 01 ± 2. 47 % P > 0. 05 G 2 /M 0. 80 ± 0. 62 % 2. 86 ± 1. 10 % 0. 33 % 9. 01 ± 2. 74 % G 2 /M t = 2. 13 P < 14. 62 ± 0. 86 % 12. 23 ± 0. 47 % t = 1. 94 P < 0. 05 4 2. 6 AGEs 6A 150 μg /ml AGE-HSA 48 h r = 0. 58 P < 0. 01 6B 2 A B 2. 7 AGE-HSA S G 2 /M 11. 08 ± 0. 05 3 K-SFM 11

33 3 AGE-HSA 2. 8 AGE-HSA ECV304 Tab. 3 Comparison of cell cycle of primarily cultured ECV304 DMEM /F keratinocytes from AGEs and control groups 12 G 0 /G 1 /% S /% G 2 /M /% K-SFM 3 73. 16 ± 1. 21 14. 62 ± 0. 86 12. 23 ± 0. 47 AGE-HSA 3 78. 52 ± 4. 15 11. 08 ± 0. 33 * 9. 01 ± 2. 74 * 150 μg /ml * P < 0. 05 A K-SFM B 150 μg /ml AGE-HSA 200 7A 100 μg /ml AGE-HSA 48 h 7B Annexin V Fitc PI 7C ECV304 7D ECV304 12. 5 25 50 μg /ml AGE- HSA 48 h 100 200 μg /ml AGE-HSA 6 150 μg /ml AGE-HSA 48 h 6 12 24 48 h Fig. 6 Comparison of epidermal cells cultured in serum-free P < 0. 01 K-SFM and epidermal cells exposed to 150 μg /ml AGE-HSA 8 Fig. 7 7 100 μg /ml AGE-HSA 48 h ECV304 Cytomorphology observation of ECV304 cells exposed to 100 μg / ml AGE-HSA A DMEM /F 12 100 B 100 μg /ml AGE-HSA 100 C 100 μg /ml AGE-HSA ECV304 Annexin V Fitc PI D 100 μg /ml AGE-HSA ECV304 TEM 20 000 3 8 AGE-HSA ECV304 Fig. 8 Comparison of apoptotic rate of ECV304 cells cultured with AGE-HSA in different doses and durations G 2 /M 12

2 AGEs AGEs AGEs AGEs 9 AGEs 10 AGEs 11 12 13 AGEs RAGE G 2 /M 14 AGE-RAGE 15 18-19 AGEs RAGE ε- 20 15 AGEs AGEs AGEs 150 μg /ml AGE-HSA 16-17 S G 2 /M AGE-HSA DNA ECV304 AGE-HSA AGEs AGEs fibronectin FN laminin LN AGEs vitronectin VN AGEs NF-κB 20 AGEs GSP MDA GSH 15 13

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