Pirfenidone Protects from UVB-Induced Photodamage in Hairless Mice

Abstract

Background: Ultraviolet radiation (UV) is the main environmental factor that causes histological degenerative changes of the skin giving rise to a chronic process called photodamage. Non-melanoma skin cancer induced by UVB radiation is a result of a cascade of molecular events caused by DNA damage in epidermis cells, including persistent inflammation, oxidative stress, and suppression of T cell-mediated immunity. Retinoids such as tretinoin have been widely used in skin to treat photoaging and photodamage, though its secondary adverse effects have been recognized. Pirfenidone (PFD) has emerged as an antifibrogenic, anti-inflammatory and antioxidant agent, and in this work its efficacy was evaluated in a model of UVB-induced photodamage.

Methods: Epidermal, dermal, and inflammatory changes were measured by histomorphometric parameters. In addition, gene, and protein expression of key molecules in these processes were evaluated.

Results: Our results revealed an anti-photodamage effect of topical PFD with absence of inflammatory skin lesions determined by dermoscopy. In addition, PFD reduced elastosis, improved organization, arrangement, and deposition of dermal collagens, downregulated several pro-inflammatory markers such as NF-kB, IL-1, IL-6 and TNFα, and decreased keratinocyte damage.

Conclusion: Topical pirfenidone represents a promising agent for the treatment of cell photodamage in humans. Clinical trials need to be carried out to explore this premise.

Keywords: photodamage; pirfenidone; pro-inflammatory markers; skin; ultraviolet radiation.

Figure 1

(A) Control group mice, animals not exposed to UVB radiation. (B) UVB group, mice exposed to UVB radiation, exploration of the dorsal area shows severe inflammatory lesions (→). (C) UVB + TRE12 group, mice treated with TRE (0.05% Retin-A^® cream) after UVB irradiation, tumor lesions (*) and inflammatory lesions (→) are observed. (D) UVB + PFD group, mice treated with PFD (8% gel) after UVB irradiation, only mild inflammation is observed.

Figure 2

Dermoscopic analysis of skin lesions. (A) Group of control mice, without exposure to UVB irradiation. (B) UVB group, mice exposed to UVB irradiation, examination of the dorsal area revealed inflammatory skin lesions such as macules (*) and ulceration (↑); and non-inflammatory skin lesions such as scales (^). (C) UVB + TRE12 group, mice treated with TRE (0.05% Retin-A^® cream) inflammatory skin lesions such as macules (*) and ulceration (↑) are also observed; and non-inflammatory skin lesions such as scales (^). (D) UVB + PFD group, mice treated with PFD (8% gel), in which only few, non-inflammatory, scale-like skin lesions were developed (^).

Figure 3

Evaluation of photodamage. (A) Histological analysis of photodamaged skin. Analysis included Hematoxylin and Eosin (H&E), Masson´s trichrome and Modified Verhoeff staining. Staining showed that pirfenidone treated groups display mild acanthosis and noninflammatory cells in dermis, as well as normal organization of collagen fibers (yellow arrows). (B) Epidermal thickness increased significantly (** p ≤ 0.005) in the TRE group compared to untreated mice. (C) Collagen fibers staining decreased in the PFD group. (D) PFD treatment reduced elastosis, (E) PFD significantly reduced PCNA quantification (p ≤ 0.005). (F) NF-kB expression was highly reduced in UVB + PFD12 mice (p ≤ 0.05). (G) Control mice reached minimal score, while tretinoin and untreated mice displayed the major scores. (H) Mast cells were increased in UVB + TRE12 compared to untreated UVB mice (*** p ≤ 0.0005) and UVB + PFD12 mice. Data are expressed as the mean ± SEM. * p ≤ 0.05; ** p ≤ 0.005; **** p ≤ 0.0001.

Figure 4

Gene expression of molecules implicated in photodamage. Pro-inflammatory cytokines (A) Il1b-1, (B) Il6, and (C) Tnf decreased after PDF treatment. (D) Nfkb1 and (E) Col1a1 were downregulated by PFD. (F) Col3a1 mRNA levels are downregulated in UVB + TRE12 and UVB + PDF groups. (G) Tgfb1 is a well-known target of pirfenidone that showed decreased expression. Data are expressed as the mean ± SEM. * p ≤ 0.05; **p ≤ 0.005; *** p ≤ 0.0005; **** p ≤ 0.0001.