Abstract:

Zhang Lichao, Zhang Hairong, Zhou Bingrong, Luo Dan, Ma Liwen, Zhang Jia′an, Wang Shen, Yi Fei, Maya Valeska Gozali, Liu Juan. Department of Dermatovenereology, First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China Corresponding authors: Luo Dan, Email: daniluo2013@njmu.edu.cn; Zhou Bingrong, Email: bingrong.2002@163.com 【Abstract】 Objective To study the effect of aminolevulinic acid-based photodynamic therapy （ALA-PDT） on oxidative damage to and apoptosis of prematurely senescent fibroblasts induced by ultraviolet B stress （UVB-SIPS-FB）. Methods Both normal fibroblasts and UVB-SIPS-FB were divided into 2-hour and 6-hour groups with the duration of incubation with ALA away from light being 2 and 6 hours respectively, and each group was divided into 7 subgroups: control subgroup receiving no treatment, ALA subgroup treated with ALA alone, red laser group treated with 100 J/cm2 red laser alone, 3 ALA-PDT subgroups pretreated with ALA followed by red laser radiation at 25, 50 and 100 J/cm2 respectively, NAC + ALA-PDT subgroup sequentially pretreated with ALA and NAC （5 mmol/L） followed by red laser radiation at 50 J/cm2. The wavelength and power density of red laser was 635 nm and 50 mW/cm2 respectively in this study. Fluorescence microscopy and flow cytometry were performed to determine the levels of reactive oxygen species （ROS） and mitochondrial membrane potentials （MMPs）, and Hoechst staining and flow cytometry to detect cell apoptosis. Statistical analysis was carried out with the software SPSS 13.0 by one-way analysis of variance （ANOVA） and q test. Results The apoptosis rate of UVB-SIPS-FB was significantly higher in the 25-, 50-, 100-J/cm2 ALA-PDT subgroups （2-hour group: 7.34% ± 0.87%, 8.39% ± 1.16% and 17.03% ± 1.29% vs. 3.81% ± 0.59%, F = 102.70, P < 0.05; 6-hour group: 13.85% ± 1.71%, 23.40% ± 2.14% and 41.02% ± 2.73% vs. 5.09% ± 1.64%, F = 106.00, P < 0.05） than in the control subgroups, but lower in the NAC + ALA-PDT subgroups （2-hour group: 5.35% ± 0.58%, 6-hour group: 9.97% ± 3.23%, both P < 0.05） than in the 50-J/cm2 ALA-PDT subgroups. There was no significant difference in the apoptosis rate of UVB-SIPS-FB between the ALA subgroups or red laser subgroups and control subgroups. ALA-PDT subgroups showed significantly increased ROS level and MMP, and the degree of increase was dependent on the duration of incubation with ALA and dose of red laser, while the pretreatment with the antioxidant NAC could reduce the ALA-PDT induced increase in ROS level and MMP, which was consistent with the changes of apoptosis rate following the treatment. Conclusion ALA-PDT can induce marked apoptosis of UVB-SIPS-FB, likely through the oxidative damage to cells.