5-aminolaevulinic acid-based photodynamic therapy inhibits ultraviolet B-induced skin photodamage

Abstract

To evaluate the photoprotective effect of 5-aminolaevulinic acid-based photodynamic therapy (ALA-PDT) on ultraviolet B (UVB)-induced skin photodamage. In vivo experiments, the dorsal skin of hairless mice were treated with ALA-PDT or saline-PDT, and then exposed to 180 mJ/m² UVB. Results showed that the number of sunburn cells and apoptotic cells in the epidermis of ALA-PDT-treated groups at 24 h after UVB irradiation were significantly decreased compared with those in the UVB groups. And the removal rate of CPDs was obviously higher in ALA-PDT-treated groups. At 48 h, the number of Ki67 positive nuclei in ALA-PDT-UVB group was significantly fewer than that in UVB group. Further in vitro experiments, human keratinocyte cell line (HaCaT) cells of two groups (one treated with ALA-PDT, the other untreated), were exposed to 60 mJ/m² UVB irradiation. We found 0.5 mmol/L of ALA and 3 J/cm² of red light did not affect the vitality of cells, and could reduce UVB induced apoptosis, accelerate the clearance of CPDs, inhibit proliferation and activate p53. Thus, our data demonstrate that ALA-PDT pretreatment can induce a protective DNA damage response that protects skin cells from UVB-induced photodamages.

Keywords: CPDs; DNA damage; p53; photodamage; photodynamic therapy; ultraviolet radiation.

Figure 1

ALA-PDT accelerated the removal of CPDs in the dorsal skin of hairless mice and HaCaT cells after UVB irradiation. (A) CPDs positive cells in the epidermis of each group, stained by immunofluorescence staining and detected by fluorescence microscopy with × 400 magnifications. (B) CPDs positive cells in HaCaT cells of each group, stained by immunofluorescence staining and detected by fluorescence microscopy with × 400 magnifications. (C) The number of CPDs positive cells in the epidermis of each group. The number of CPDs positive cells in ALA-PDT-UVB groups was significantly decreased compared with UVB group at 6, 24 and 48 h after UVB irradiation (P < 0.05). (D) The number of CPDs positive cells in HaCaT cells of each group. The number of CPDs positive cells in ALA-PDT-UVB groups was significantly decreased compared with UVB group at 6, 24 and 48 h after UVB irradiation (P < 0.05). The symbol (*) indicates a significant difference (P < 0.05). Bar= 50μm, N= 3.

Figure 2

ALA-PDT suppressed UVB-induced apoptosis. (A) Evaluation of apoptosis of HaCaT cells by using annexin V/PI staining and flow cytometry. Annexin-V(-)/PI(-) cells are live cells, annexin-V(+)/PI(-) cells are early apoptotic cells and annexin-V(+)/PI(+) cells are late apoptotic or necrotic cells. The rate of apoptosis in ALA-PDT-UVB group was lower than that in UVB group (P < 0.05).(B) Sunburn cells in the epidermis of each group, stained by hematoxylin and eosin (H&E) staining and detected by light microscopy with × 200 magnifications. The number of sunburn cells in the epidermis of ALA-PDT-UVB group at 24 h after UVB irradiation was decreased compared with UVB group. (C) Apoptotic cells in the epidermis of each group, stained by TUNEL staining and detected by fluorescence microscope with × 400 magnifications. Compared with UVB group, the number of apoptotic cells in ALA-PDT-UVB group was significantly decreased (P < 0.05). The symbol (*) indicates a significant difference (P < 0.05). Bar= 100μm, N= 3.

Figure 3

ALA-PDT pretreatment inhibited UVB-induced hyperproliferation in hairless mice skin. (A) Ki67 protein expression in the epidermis of each group at 6, 24, and 48 h after UVB irradiation, stained by immunohistochemical staining and detected by light microscopy with × 200 magnifications. (B) When compared with the UVB group, the number of Ki67 positive cells decreased in ALA-PDT-UVB group at 48 h after UVB irradiation (P < 0.05). The symbol (*) indicates a significant difference (P < 0.05). Bar= 100μm, N= 3.

Figure 4

ALA-PDT protected HaCaT cells from UV-induced cell viability impairment. (A) Inverted phase contrast microscope observation of HaCaT cells. Cells of control group had the same shapes, clear boundaries, close arrangement. UVB group showed decreased cell number, decreased cell adhesion, increased intercellular space, rounded or irregular shapes, cell debris, and a large number of suspended cells. However, the cell morphology in ALA-PDT-UVB group was significantly normalized compared with UVB group. (B) Cell viability was analyzed by CCK-8. ALA (0.5 mmol/L) and low level of red light (3 J/cm²) had no effect on cell viability (P > 0.05). Cell viability of ALA-PDT-UVB group was higher than that of UVB group (P < 0.05). The symbol (*) indicates a significant difference (P < 0.05).

Figure 5

ALA-PDT up-regulated and activated p53 in HaCaT cells. (A) Western blotting for detection of p53^total and p53^ser-15 protein in HaCaT cells of each group at 6, 24, and 48 h. Con: control group, N: UVB group, T: ALA-PDT-UVB group. (B) Expression of p53^total in HaCaT cells at 24 h after ALA-PDT treatment (0.5 mmol/L + 3 J/cm²). (C) (D) Protein expressions were quantified by β-actin as a control. The ratio of gray scale values represents the ratio of the amount of protein to interest/actin. The symbol (*) indicates a significant difference (P < 0.05). N= 3.