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. 2016 Aug 3:6:31169.
doi: 10.1038/srep31169.

Anti-photoaging properties of the phosphodiesterase 3 inhibitor cilostazol in ultraviolet B-irradiated hairless mice

Ha Neui Kim  1   2 , Chan Hee Gil  1 , Yu Ri Kim  1   2 , Hwa Kyoung Shin  1   2   3 , Byung Tae Choi  1   2   3
Affiliations

Anti-photoaging properties of the phosphodiesterase 3 inhibitor cilostazol in ultraviolet B-irradiated hairless mice

Ha Neui Kim et al. Sci Rep. .

Abstract

We investigated whether cilostazol, an activator of cyclic adenosine monophosphate (cAMP)-dependent intracellular signaling, could inhibit ultraviolet B (UVB) irradiation-induced photoaging in HR-1 hairless mice. Cilostazol decreased wrinkle formation and skin thickness in UVB-irradiated mice, as well as increased staining of collagen fibers and inhibition of reactive oxygen species (ROS) formation in the skin. Moreover, the proteolytic activities of gelatinase matrix metalloproteinase (MMP)-9 and collagenase MMP-3 were significantly decreased in UVB-irradiated mice treated with cilostazol. Western blotting showed that UVB-induced activation of p38 mitogen-activated protein kinases (MAPK) and nuclear factor (NF)-κB was significantly inhibited by cilostazol, whereas the activation of Akt was significantly enhanced by cilostazol. Confirmation of localized protein expression in the skin revealed marked p38 MAPK and NF-κB activation that was mainly detected in the dermis. Marked Akt activation was mainly detected in the epidermis. Our results suggest that cilostazol may have anti-photoaging effects on UVB-induced wrinkle formation by maintaining the extracellular matrix density in the dermis, which occurs via regulation of ROS and related p38 MAPK and NF-κB signaling, and subsequent down-regulation of MMPs. Therefore, cilostazol may protect against photoaging-induced wrinkle formation.

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Figures

Figure 1
Figure 1. The effect of cilostazol on visible skin condition and wrinkle formation in the dorsal skin of UVB-irradiated mice.
(A) Photographs and replicas of the mouse dorsal skin. (B,C) Histogram of replica analysis. Treatment with cilostazol improved visible skin condition and reduced the percentage of wrinkles per unit area and the mean depth of wrinkles in UVB-irradiated mice. **P < 0.01 and ***P < 0.001 vs. control mice; ###P < 0.001 vs. UVB-irradiated mice.
Figure 2
Figure 2. The effect of cilostazol on epidermal and dermal thickness and density of collagen fibers in the dorsal skin of UVB-irradiated mice.
(A) Hematoxylin and eosin staining and (B) Masson’s trichrome staining. Scale bar = 200 μm. Scale bar in rectangular box = 50 μm. (C,D) Histogram of hematoxylin and eosin staining. (E) Histogram of Masson’s trichrome staining. Treatment with cilostazol significantly suppressed the UVB irradiation-induced increase in epidermal and dermal thickness and prevented UVB-induced loss of collagen fibers. **P < 0.01 and ***P < 0.001 vs. control mice; #P < 0.05, ##P < 0.01, and ###P < 0.001 vs. UVB-irradiated mice.
Figure 3
Figure 3. The effect of cilostazol on the production of ROS in the dorsal skin of UVB-irradiated mice.
(A) Histogram of total ROS assay. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. control mice; #P < 0.05 vs. UVB-irradiated mice. (B) Immunohistochemical staining of ROS. Scale bar = 200 μm. The increased ROS free radical activity was significantly inhibited by cilostazol treatment and was confirmed by fluorescence expression (red) in the dorsal skin.
Figure 4
Figure 4. The effect of cilostazol on gelatinase and MMP-9 activity in the dorsal skin of UVB-irradiated mice.
(A) Images of the in situ zymographic analysis of gelatinase and MMP-9, and (B) histogram of the IOD measurement. Treatment with cilostazol significantly suppressed gelatinase activity and MMP-9 expression in the dorsal skin of UVB-irradiated mice. ***P < 0.001 vs. control mice and ###P < 0.001 vs. UVB-irradiated mice. Scale bar = 200 μm.
Figure 5
Figure 5. The effect of cilostazol on MAPK and PI3K/Akt expression in the dorsal skin of UVB-irradiated mice.
(A) Western blotting and (B,C) relative densities of p38 MAPK and Akt. UVB irradiation-induced upregulation of the expression of phosphorylated p38 was significantly decreased by cilostazol treatment, whereas phosphorylated Akt expression was increased by cilostazol treatment. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. control mice; #P < 0.05 and ##P < 0.01 vs. UVB-irradiated mice.
Figure 6
Figure 6. The effect of cilostazol on the expression of the transcription factors AP-1 and NF-κB with MMP-9 in the dorsal skin of UVB-irradiated mice.
(A) Western blotting and (B,C) relative densities of NF-κB and MMP-9. UVB irradiation significantly increased the expression of phosphorylated NF-κB and MMP-9, which was decreased by cilostazol treatment. *P < 0.05 vs. control mice and #P < 0.05 vs. UVB-irradiated mice.
Figure 7
Figure 7. Immunohistochemical analysis of the expression of p38 MAPK, Akt, and NF-κB in the dorsal skin of UVB-irradiated mice.
(A) Images of immunohistochemically stained skin sections. The expression of phosphorylated Akt was mainly detected in the epidermis (black rectangular box) while phosphorylated p38 MAPK and NF-κB were mainly detected in the dermis (white rectangular box). Scale bar = 100 μm. (B–D) Histogram analysis of the IOD measurement of (B) phosphorylated p38 MAPK, (C) Akt, and (D) NF-κB. The significant increase in phosphorylated p38 MAPK and NF-κB by UVB irradiation was attenuated by cilostazol treatment. *P < 0.05 and ***P < 0.001 vs. control mice; #P < 0.05, ##P < 0.01, and ###P < 0.001 vs. UVB-irradiated mice.
Figure 8
Figure 8. The protective effects of cilostazol against skin damage in UVB-irradiated mice.
(A) Immunohis-tochemical staining of ROS. Increased ROS activity was inhibited by cilostazol treatment. Scale bar = 200 μm. (B) Images of the in situ zymographic analysis of gelatinase and MMP-9 and (C) the related histogram analysis. Skin treated with cilostazol showed significant suppression of gelatinase activity and MMP-9 expression. *P < 0.05 vs. non-treated skin with cilostazol. Scale bar = 200 μm. (D) Immunohistochemical images of NF-κB and (E) the related histogram analysis. The significant increase in phosphorylated p38 MAPK and NF-κB by UVB irradiation was attenuated by cilostazol treatment. ***P < 0.001 vs. non-treated skin with cilostazol. Scale bar = 100 μm.
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