Abstract:

Xu Xinya, Xu Qingfang, Zheng Yue, Huang Yunfen, Lai Wei, Gong Zijian, Lu Chun Department of Dermatology and Venereology, Third Affiliated Hospital, Sun Yat?sen University, Guangzhou 510630, China Corresponding author: Xu Qingfang, Email: xqf69@163.com 【Abstract】 Objective To investigate the effects of advanced glycation end products （AGE） on the s and activity of cathepsin D （CatD） in ultraviolet A（UVA）?irradiated human dermal fibroblasts. Methods Human dermal fibroblasts were isolated and harvested from the circumcised foreskin of children, and subjected to a primary culture. CCK?8 assay was performed to screen non?cytotoxic concentrations of AGE?bovine serum albumin （BSA）. Some fibroblasts were incubated with 50, 100 and 300 mg/L AGE?BSA separately for 24 hours, with untreated cells as the control group. Then, reverse transcription （RT）?PCR, Western?blot analysis and a fluorimetric assay were performed to measure the mRNA and protein s as well as activity of CatD, respectively. Some fibroblasts were classified into six groups: control group receiving no treatment, AGE?BSA group and BSA group treated with the highest non?cytotoxic concentration of AGE?BSA and the same concentration of BSA respectively for 24 hours, UVA group irradiated by 10 J/cm2 UVA, UVA?AGE?BSA group and UVA?BSA group treated with AGE?BSA and BSA at the above non?cytotoxic concentration respectively for 24 hours both before and after UVA radiation at 10 J/cm2. After the treatments, RT?PCR, Western?blot analysis and a fluorimetric assay were conducted to detect mRNA and protein s and activity of CatD respectively. Results AGE?BSA of 50 - 200 mg/L exhibited no obvious influence on cellular proliferation of fibroblasts. The fibroblasts incubated with AGE?BSA of 50, 100 and 200 mg/L showed a significant increase in the mRNA （0.267 ± 0.007, 0.348 ± 0.007, and 0.418 ± 0.006 respectively）, protein （1.403 ± 0.181, 2.233 ± 0.090 and 2.477 ± 0.111 respectively）, and activity （1.760 ± 0.080, 2.330 ± 0.060 and 2.890 ± 0.080 respectively） of CatD compared with the control group（mRNA: 0.161 ± 0.006; protein: 0.903 ± 0.200; activity: 1.100 ± 0.090, all P < 0.05）. AGE?BSA increased CatD s and activity in a dose?dependent manner. The mRNA and protein s as well as activity of CatD were significantly higher in the UVA group than in the control group （mRNA : 0.480 ± 0.005 vs. 0.155 ± 0.005; protein : 2.583 ± 0.199 vs. 0.920 ± 0.235; activity: 2.970 ± 0.110 vs. 1.110 ± 0.040, all P < 0.05）, but significantly lower in the UVA?AGE?BSA group than in the UVA group （mRNA : 0.394 ± 0.008 vs. 0.480 ± 0.005; protein : 2.070 ± 0.125 vs. 2.583 ± 0.199; activity: 2.560 ± 0.060 vs. 2.970 ± 0.110, all P < 0.05）. Conclusion AGEs could increase CatD s and activity in human dermal fibroblasts not receiving UVA irradiation, but inhibit their increase in UVA?induced human dermal fibroblasts.