stabilized tissues were treated or not with 25 μl of HA5 and allowed
to recover for different lengths of time (16 or 24 hours). The epidermis and dermis were physically separated and independently processed for gene expression analysis using real-time quantitative reverse transcription PCR (qRT-PCR). Figure
2 shows the quantification of keratinocytes differentiation markers: fillagrin (FLG), involucrin (INV), and loricrin (LOR). It is important to note that the major function of the epidermis
is to provide a barrier between the external environment and the internal organs of the body.82 To fulfill this function, keratinocytes undergo a complex pathway of differentiation, which culminates in cornification and in the formation of extracellular,
lipid-enriched lamellar membranes in the stratum corneum (SC).82,83Â This process is controlled by a cluster of genes present in chromosome 1q21, named epidermal differentiation
complex (EDC). Alteration in the expression of EDC genes are linked to common skin disorders such as ichthyosis vulgaris, atopic dermatitis, and psoriasis. A decrease in the expression of these proteins is also associated with intrinsic and extrinsic aging. Our results demonstrated that treatment with HA5 results in enhanced expression of FLG, INV, and LOR (Figure 2 A, B, and C).
Another important factor involved in preserving the functionality
of the epidermal barrier and prevention of TEWL is the formation of tight junctions between keratinocytes, which occurs
at the stratum granulosum level. Claudin-1 (CLDN1) and claudin-4 (CLDN4) are key factors in the formation of these structures, and deficiency in CLDN1 is linked to increased TEWL and abnormal SC formation.84 We demonstrated that treatment with HA5 triggers an increase in CLDN1 expression (Figure 2D) which, in addition to increased epidermal differentiation, may improve epidermal health and barrier formation/function.
An appropriated antioxidant capacity is a key factor involved in epidermal protection and homeostasis as this not only maintains the proper redox balance necessary for the normal metabolism but also neutralizes the deleterious effects of ROS. HA5 contains a SkinMedica proprietary blend of antioxidants derived from flower stem cells, in addition to the blend of 5 different HAs, which may also act as free radicals scavengers. We evaluated the antioxidant capacity in HA5 by radiating the tissues with UVB light. In brief, tissues were treated or not with 25 μl of HA5 10 minutes before UVB radiation (220 mJ/cm2) and allowed to recover for 24 hours. As expected, this dose of UVB radiation (equivalent to 5 MED) triggered a significant increase in the number of sunburn cells (SBCs). However, pre-treatment with HA5 significantly prevented formulation
of SBCs (Figure 3, black arrows), indicating excellent antioxidant protection.
As discussed before, epidermal homeostasis is deeply linked to the presence of balanced levels of HA in this skin
compartment. Adequate HA levels not only promote skin hydration
but also play a critical role in controlling proliferation/differentiation/migration of keratinocytes, inflammation, and oxidative stress and promoting cell survival. Restoration of epidermal
HA can only be accomplished by boosting endogenous HA production in the skin because topically applied HAs, including
nano-sized hydrolyzed HA, are too large to penetrate the SC and/or are degraded rapidly. We observed that a single treatment with HA5 significantly increased in the expression of epidermal HAS while decreasing HYAL expression (Figure 4A), suggesting a boosting of the endogenous epidermal production
of HA. Furthermore, we observed an enhanced staining
