Recombinant Photolyase-Thymine Alleviated UVB-Induced Photodamage in Mice by Repairing CPD Photoproducts and Ameliorating Oxidative Stress

Abstract

Cyclobutane pyrimidine dimers (CPDs) are the main mutagenic DNA photoproducts caused by ultraviolet B (UVB) radiation and represent the major cause of photoaging and skin carcinogenesis. CPD photolyase can efficiently and rapidly repair CPD products. Therefore, they are candidates for the prevention of photodamage. However, these photolyases are not present in placental mammals. In this study, we produced a recombinant photolyase-thymine (rPHO) from Thermus thermophilus (T. thermophilus). The rPHO displayed CPD photorepair activity. It prevented UVB-induced DNA damage by repairing CPD photoproducts to pyrimidine monomers. Furthermore, it inhibited UVB-induced ROS production, lipid peroxidation, inflammatory responses, and apoptosis. UVB-induced wrinkle formation, epidermal hyperplasia, and collagen degradation in mice skin was significantly inhibited when the photolyase was applied topically to the skin. These results demonstrated that rPHO has promising protective effects against UVB-induced photodamage and may contribute to the development of anti-UVB skin photodamage drugs and cosmetic products.

Keywords: DNA damage; oxidative stress; photorepair; recombinant photolyase.

Figure 1

Recombinant photolyase-thymine protein (rPHO) from T. thermophilus displayed CPD photorepair activity. (A) Mechanism underlying photolyase-mediated repair of UVB-induced DNA damage. (B) Structure of recombinant photolyase-thymine (rPHO). (C) The purified protein was separated by SDS-PAGE; arrow shows the target protein (rPHO). (D) Enzymatic activity assay was used to examine the photorepair activity of rPHO. CPD photoproducts were formed by exposure of Oligo (dT)16 to UVB; then, the CPD photoproducts were incubated with rPHO (50 μg/mL) for 0, 5, 10, and 20 min, followed by the detection of the OD at 260 nm. * p < 0.05.

Figure 2

rPHO was taken up into cells and absorbed by the keratinocytes of the skin epidermis. (A) HaCaT cells were treated with different concentrations of rPHO-FITC. (B) FITC-labeled rPHO carbomer gels were applied to the dorsal skin for 24 h. Vertical skin sections with a thickness of 10 µm were extracted with cryotome and observed under a fluorescence microscope for skin-associated fluorescence. Scale bar = 20 µm.

Figure 3

rPHO significantly reduced UVB-induced death of HaCaT cells. (A) UVB irradiation experimental design. (B) Images of cells were obtained under a bright field by a microscope after rPHO treatment. Scale bar = 150 μm. (C) Cell survival was measured. (D) Cells were stained with calcein-AM/PI; dead cells were stained red and live cells were stained green. Scale bar = 100 μm. Data are presented as means ± SD (n = 3). ** p < 0.01, *** p < 0.001.

Figure 4

rPHO prevented UVB-induced photoaging in mice. (A) Schematic diagram of UVB irradiation and rPHO administration. (B) Representative images of the morphological changes of the dorsal skin in each group after rPHO treatment for 15 days. (C) Representative images of HE staining after rPHO treatment for 15 days. Scale bar = 100 μm. (D) The thickness of the epidermis was measured using ImageJ. Data are presented as means ± SD (n = 5). *** p < 0.001.

Figure 5

Photolyase inhibited UVB-induced reduction in collagen. (A) Masson staining; blue represents collagen fibers. (B) Representative picrosirius red staining of the dorsal skin. Images were obtained by polarized light microscopy. Type-I collagen: strong orange-yellow or bright red; type-III collagen: green. Scale bar = 200 μm. (C) The percentages of type-I and type-III collagen were calculated using ImageJ. (D) Hydroxyproline (HYP) in the dorsal skin was quantified. Data are shown as means ± SD (n = 5). ** p < 0.01, *** p < 0.001.

Figure 6

Photolyase significantly inhibited UVB-induced oxidative stress and inflammatory responses in mice. (A) Lipid peroxidation in the dorsal skin in each group after rPHO treatment was measured by quantifying malondialdehyde (MDA). (B) SOD activity in the dorsal skin after rPHO treatment. (C) GSH-Px activity in the dorsal skin after rPHO treatment. (D–F) Expression of IL-6, IL-1β, and TNF-α in skin was measured. mRNA relative levels were normalized to Gapdh levels. (G) Expression of caspase 3, Bcl-2, and Bax protein examined by western blotting. (H) Semi-quantification analysis of (G) by ImageJ. Data are expressed as means ± SD (n = 5). * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 7

rPHO reduced UVB-induced DNA damage in HaCaT cells. (A) DNA damage measured by H2AX phosphorylation (γ-H2AX). HaCaT cells were treated with rPHO for 24 h after UVB irradiation (2 mJ/cm2), and immunostaining of γ-H2AX in HaCaT cells was performed. Blue: DAPI; green: γ-H2AX. (B) Representative alkaline comet assay images. Scale bar = 50 μm. (C,D) Tail DNA and DNA tail length were evaluated using ImageJ. Data are expressed as means ± SD (n = 3). * p < 0.05, ** p <0.01, *** p < 0.001.

Figure 8

Photolyase inhibited UVB-induced ROS production and the release of inflammatory factors. (A) ROS production was analyzed by DCFH-DA staining under a fluorescence microscope. Scale bar = 50 μm. (B) ROS production was measured by flow cytometry. (C) Quantification of intracellular ROS levels. (D–F) The expression levels of IL-6, IL-1β, and TNF-α in HaCaT cells were measured. Relative levels of mRNA were normalized against levels of GAPDH. Data are expressed as means ± SD (n = 3). * p < 0.05, ** p < 0.01.

Figure 9

Photolyase inhibited UVB-induced apoptosis in HaCaT cells. (A) Release of cytochrome c in HaCaT cells treated with rPHO by immunofluorescence staining. Cytochrome c and nuclei are indicated by green and blue fluorescence, respectively. Scale bars = 100 μm. (B) Expression of caspase 3, Bcl-2, and Bax in HaCaT cells examined by western blotting. (C,D) Statistical analysis of protein expression in (B). (E) Apoptosis was measured by flow cytometry. (F) Statistical analysis of the apoptosis rates. Data are presented as means ± SD (n = 3). * p < 0.05, ** p < 0.01.

Figure 10

Mechanisms involved in protective effects of rPHO.