Topical Hesperidin Enhances Epidermal Function in an Aged Murine Model

Experimental protocols and functional studies: All animal procedures were approved by the Animal Studies Subcommittee (IACUC) of the San Francisco Veterans Administration Medical Center and performed in accordance with their guidelines. Since hesperidin is not soluble in 100% ethanol, 70% ethanol was used as vehicle. Since the turnover time for hairless is about 8-9.5 days in normal young mice (Potten et al., 1987), we chose to treat aged mice for 9 days. Both flanks of 12-15 month old mice were treated topically with 60 lof 2% hesperidin or 70% ethanol twice daily for 9 days. Basal epidermal permeability barrier function was assessed by measuring transepidermal water loss (TEWL) using TM300 connected to MPA5 (C&K, Cologne, Germany)(Mao-Qiang et al., 2004; Man et al., 2012). For barrier recovery, TEWL was measured using an electrolytic water analyzer (Meeco, Warrington, PA) at 0, 2 and 4 hours after tape stripping (10-fold increase in TEWL), and percent barrier recovery was calculated as Keratinocyte Culture: Second-passage keratinocytes isolated from adult human (donor aged 60-65 year old) were cultured in serum-free keratinocyte growth medium containing 0.07 mM calcium (Clonetics, San Diego, California). Cells at 60%–70% confluence were switched to a medium containing 1.2 mM calcium and treated with either 0.02% hesperidin or vehicle alone (0.02% ethanol). After 24 and 48 hrs of treatment, keratinocytes were collected for Q-PCR analysis (Hou et al., 2013). Immunohistochemistry: Immunohistochemical staining for assessing changes in epidermal differentiation was performed as described earlier (Hou et al., 2013; Mao-Qiang et al., 2004). Briefly, 5 μm paraffin sections were incubated with the primary antibodies (Covance, Emeryville, CA) overnight at 4°C. After washes ×3, sections were incubated with the secondary antibody for