Derinat Protects Skin against Ultraviolet-B (UVB)-Induced Cellular Damage

Wen-Li Hsu, Jian-He Lu, Mami Noda, Ching-Ying Wu, Jia-Dai Liu, Manabu Sakakibara, Ming-Hsien Tsai, Hsin-Su Yu, Ming-Wei Lin, Yaw-Bin Huang, Shian-Jang Yan, Tohru Yoshioka, Wen-Li Hsu, Jian-He Lu, Mami Noda, Ching-Ying Wu, Jia-Dai Liu, Manabu Sakakibara, Ming-Hsien Tsai, Hsin-Su Yu, Ming-Wei Lin, Yaw-Bin Huang, Shian-Jang Yan, Tohru Yoshioka

Abstract

Ultraviolet-B (UVB) is one of the most cytotoxic and mutagenic stresses that contribute to skin damage and aging through increasing intracellular Ca(2+) and reactive oxygen species (ROS). Derinat (sodium deoxyribonucleate) has been utilized as an immunomodulator for the treatment of ROS-associated diseases in clinics. However, the molecular mechanism by which Derinat protects skin cells from UVB-induced damage is poorly understood. Here, we show that Derinat significantly attenuated UVB-induced intracellular ROS production and decreased DNA damage in primary skin cells. Furthermore, Derinat reduced intracellular ROS, cyclooxygenase-2 (COX-2) expression and DNA damage in the skin of the BALB/c-nu mice exposed to UVB for seven days in vivo. Importantly, Derinat blocked the transient receptor potential canonical (TRPC) channels (TRPCs), as demonstrated by calcium imaging. Together, our results indicate that Derinat acts as a TRPCs blocker to reduce intracellular ROS production and DNA damage upon UVB irradiation. This mechanism provides a potential new application of Derinat for the protection against UVB-induced skin damage and aging.

Keywords: Derinat; ROS; TRPCs; UVB; calcium.

Conflict of interest statement

The authors state no conflict of interest.

Figures

Figure 1
Figure 1
Derinat protected skin cells from UVB damage. The effect of UVB irradiation on cell viability in (A) keratinocytes (KCs) and (B) human dermal fibroblasts (HDF) (***, p < 0.001). The cell survival probability for cells pretreated with different concentrations of Derinat for 24 h and irradiated with or without UVB-exposure in (C) KCs and (D) HDF (*, p < 0.05; ***, p < 0.001). Further confirming the result of (C,D), in (E) KCs and (F) HDF were pretreated with 15 μg and 150 μg of Derinat and irradiated with 50 mJ/cm2 and 100 mJ/cm2 UVB, respectively. The cell survival probability was analyzed using an MTT assay after 24 h of UVB-exposure (*, p <0.05; ***, p <0.001).
Figure 2
Figure 2
The effect of Derinat on the intracellular Ca2+ concentration of UVB-exposed skin cells. (A) Experimental design for the treatment of each group: control, UVB, EDTA, UVB+D and Derinat only; (B) Ca2+ calibration curve; (C,D), Mean value of intracellular Ca2+ for control, UVB-exposed, UVB-exposed with different concentrations of EDTA, UVB-exposure with Derinat and Derinat in KCs and HDF cultured in BSS for 30 min (*, p < 0.05; ***, p < 0.001); (E) KCs and (F) HDF were pretreated with or without the Derinat for 24 h and exposed to UVB. The intracellular Ca2+ concentration was then measured after 24 h of incubation in normal medium (***, p < 0.001).
Figure 3
Figure 3
Derinat reduced UVB activated Ca2+ signaling in skin cells. Ca2+ imaging analysis of the thapsigargin (TG)-induced Ca2+ response after the application of 20 nM TG (small black bars) in (A) and KCs and 100 nM TG in (B) HDF (n = 3). After 30 min of pretreatment with Derinat (D), SKF96365 (SKF, 20 μM in KCs and 50 μM in HDF), 2-aminoethoxydiphenyl borate (2-APB, 50 μM in KCs and 100 μM in HDF) and SKF + D; OAG at 100 μM and 200 μM was applied to (C) KCs and (D) HDF, respectively, to stimulate the Ca2+ responses of TRPCs after 30 min of UVB exposure (n = 3). (E,F), quantification of the peak of the intracellular Ca2+ responses shown in (C) and (D) (*, p < 0.05; **, p < 0.01; ***, p < 0.001). Ca2+ imaging analysis of the adenosine triphosphate (ATP)-induced Ca2+ response. The application of 100 μM ATP (small black bars) in (G) KCs and 200 μM ATP in (H) HDF (n = 3). (I,J), quantification of the peak of the intracellular Ca2+ responses shown in (G,H) (*, p < 0.05; ***, p < 0.001). CaCl2 was extracellularly applied (large black bar) to increase Ca2+ from 0 to 2 mM and open the TRPCs after the application of ATP (small black bars) in (K) KCs and in (L) HDF in Ca2+-free BSS solution (open bar) (n = 3). (M,N) quantification of the peak of the intracellular Ca2+ responses shown in (K,L) after applying CaCl2 (*, p < 0.05; **, p < 0.01; ***, p < 0.001). The Ca2+ signals represent the mean value of 20 cells.
Figure 4
Figure 4
Derinat significantly inhibited UVB-induced increase of TRPC7 expression in skin cells. The cells were exposed to UVB irradiation after Derinat and 2-APB pretreatment for 30 min. After radiation of UVB for 24 h, expressions of TRPC1, TRPC4, TRPC6 and TRPC7 mRNA, which were detected from total RNA extracts of (A) KCs and (B) HDF, were measured using a qRT–PCR (*, p < 0.05; **, p < 0.01; ***, p < 0.001).
Figure 5
Figure 5
The effect of Derinat on UVB induced intracellular ROS production in skin cells. (A) KCs and (B) HDF were pretreated with Derinat and 0.5 mM NAC for 24 h and then irradiated with UVB. EDTA (1 mM) was applied after UVB exposure. The ROS generation was measured after 30 min of UVB irradiation by using 5 μM dihydroethidium (DHE) staining. The intracellular ROS production level with an average fluorescence intensity of more than 150 cells from (A) and (B) is quantified in (C) KCs and (D) HDF (***, p < 0.001; *, p < 0.01). To further investigate the intracellular ROS generation in (E) KCs and (F) HDF, the cells were cultured in normal medium for 24 h after UVB-exposure, and the intracellular ROS production was detected with a confocal microscope using DHE staining. The quantification of intracellular ROS production from (E,F) is shown in (G,H) (***, p <0.001).
Figure 6
Figure 6
The effect of Derinat on the UVB-induced DNA damage in skin cells. 8-oxodG was detected to determine the DNA damage resulting from UVB-exposure in cells pretreated with Derinat and NAC: (A) KCs and (C) HDF. The relative values of DNA damage from 8-oxodG are quantified in (B,D) (**, p < 0.01; ***, p < 0.001). The data shown represent the average of three independent experiments.
Figure 7
Figure 7
The effect of Derinat on UVB induced skin damage in BALB/c-nu mice. (A) BALB/c-nu mice were covered with Derinat hydrogel or pure hydrogel for 3 h and then irradiated with 360 mJ/cm2 UVB. After seven days, the images of animals presented UVB-induced desquamation in the dorsal areas but no effect on the abdominal areas. The middle panel shows the amplified images from the dorsal areas of UVB-induced desquamation; (B) Derinat protected BALB/c-nu mice skin from UVB-induced epidermal proliferation, DNA damage and cyclooxygenase (COX)-2 expression. The skins of normal and UVB irradiated mice covered with Derinat hydrogel or hydrogel were stained with DHE and H & E (hematoxylin and eosin), and the oxidative DNA damage 8-oxodG was analyzed via an immunohistochemical assay with a specific anti-8-oxodG mouse monoclonal antibody. The expression of COX-2 in the epidermis was detected with an anti-COX-2 rabbit monoclonal antibody; (C,D), the quantification of intracellular ROS production and COX-2 level from (B) shown in arroew indicated region includes both epidermis and dermis without sebaceous glands (*, p < 0.05; **, p < 0.01; ***, p < 0.001). Derinat decreased the UVB-induced level of TRPC7 (E) and DNA damage 8-oxodG (F) in BALB/c-nu mouse skin based on qRT–PCR and ELISA assay, respectively (*, p < 0.05; **, p < 0.01).

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