Silibinin reverses CEES-induced skin injury and inflammatory biomarkers in SKH-1 hairless mouse skin.

<p>Dorsal skin of mice was exposed topically to either 200 µL of acetone or CEES (2 mg) in 200 µL acetone, or treated with either silibinin alone or with 0.5 or 1 mg silibinin 30 min after CEES exposure. After 9, 12 and 24 h of the indicated treatments, skin bi-fold thickness was measured using a digital caliper (<b>A</b>) as detailed under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046149#s2" target="_blank">Materials and Methods</a>. Mice were sacrificed and dorsal skin tissue samples were collected at 24 h following the above exposures and treatment. The skin sections (5 µM) were processed, H&E stained and analyzed for histopathological changes [representative pictures show changes in the epidermal thickness, epidermal necrosis, increase in blood vessels (<b>A</b>) and neutrophil infiltration (<b>B</b>)] and quantified for epidermal thickness (<b>B</b>; 400× magnification). Apototic cell death was assessed and quantified in the skin sections subjected to DeadEnd Colorimetric TUNEL assay (<b>C</b>) as detailed under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046149#s2" target="_blank">Materials and Methods</a>. The epidermal thickness (µm) and the apoptotic index (positive cells ×100/total number of cells) were calculated after their quantification in 10 randomly selected fields at 400× magnification under a microscope. To determine the increase in neutrophil infiltration observed in the H&E stained skin sections (representative pictures of H&E stained dermis demonstrated an increase in the neutrophil infiltration <b>D</b>, top panels), treated skin tissue samples were used for lysate preparation and MPO activity was determined by a fluorescence assay (<b>D</b>; bottom graph) as detailed under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046149#s2" target="_blank">Materials and Methods</a>. Data presented are mean ± SEM of 5 animals in each treatment group. *, p<0.05 as compared to CEES exposed group. VC, vehicle (acetone); SB, silibinin; CEES+0.5 SB, 0.5 mg silibinin treatment 30 min after CEES exposure; CEES+1SB, 1 mg silibinin treatment 30 min after CEES exposure; e, epidermis; d, dermis; black arrow, epidermal necrosis; green arrows, blood vessels; red arrowheads, TUNEL positive brown cells; red arrows, neutrophils.