BackgroundBirch bark has a long lasting history as a traditional medicinal remedy to accelerate wound healing. Recently, the efficacy of birch bark preparations has also been proven clinically. As active principle pentacyclic triterpenes are generally accepted. Here, we report a comprehensive study on the underlying molecular mechanisms of the wound healing properties of a well-defined birch bark preparation named as TE (triterpene extract) as well as the isolated single triterpenes in human primary keratinocytes and porcine ex-vivo wound healing models.Methodology/Principal FindingsWe show positive wound healing effects of TE and betulin in scratch assay experiments with primary human keratinocytes and in a porcine ex-vivo wound healing model (WHM). Mechanistical studies elucidate that TE and betulin transiently upregulate pro-inflammatory cytokines, chemokines and cyclooxygenase-2 on gene and protein level. For COX-2 and IL-6 this increase of mRNA is due to an mRNA stabilizing effect of TE and betulin, a process in which p38 MAPK and HuR are involved. TE promotes keratinocyte migration, putatively by increasing the formation of actin filopodia, lamellipodia and stress fibers. Detailed analyses show that the TE components betulin, lupeol and erythrodiol exert this effect even in nanomolar concentrations. Targeting the actin cytoskeleton is dependent on the activation of Rho GTPases.Conclusion/SignificanceOur results provide insights to understand the molecular mechanism of the clinically proven wound healing effect of birch bark. TE and betulin address the inflammatory phase of wound healing by transient up-regulation of several pro-inflammatory mediators. Further, they enhance migration of keratinocytes, which is essential in the second phase of wound healing. Our results, together with the clinically proven efficacy, identify birch bark as the first medical plant with a high potential to improve wound healing, a field which urgently needs effective remedies.

The transmembrane protein claudin-1 is a major component of epidermal tight junctions (TJs), which create a dynamic paracellular barrier in the epidermis. Claudin-1 downregulation has been linked to atopic dermatitis (AD) pathogenesis but variable levels of claudin-1 have also been observed in healthy skin. To elucidate the impact of different levels of claudin-1 in healthy and diseased skin we determined claudin-1 levels in AD patients and controls and correlated them to TJ and skin barrier function. We observed a strikingly broad range of claudin-1 levels with stable TJ and overall skin barrier function in healthy and non-lesional skin. However, a significant decrease in TJ barrier function was detected in lesional AD skin where claudin-1 levels were further reduced. Investigations on reconstructed human epidermis expressing different levels of claudin-1 revealed that claudin-1 levels correlated with insideout and outside-in barrier function, with a higher coherence for smaller molecular tracers. Claudin-1 decrease induced keratinocyte-autonomous IL-1β expression and fostered inflammatory epidermal responses to non-pathogenic Staphylococci. In conclusion, claudin-1 decrease beyond a threshold level results in TJ and epidermal barrier function impairment and induces inflammation in human epidermis. Increasing claudin-1 levels might improve barrier function and decrease inflammation and therefore be a target for AD treatment. Tight junctions (TJs) are an important component of the complex epidermal barrier system. They are localized in the stratum granulosum (SG) of the epidermis and provide mechanical barrier function to ions and solutes of different molecular sizes 1-4. The transmembrane protein claudin-1 (Cldn-1) is a major component of TJs 5. It is also found outside of TJs in basal and suprabasal layers of the epidermis 2,5. Mice with a complete Cldn-1 knockout (KO) die at the first day of birth due to increased transepidermal water loss (TEWL) 5. They develop TJs leaky to a molecular tracer (Biotin-556) 5 , and a highly water permeable stratum corneum (SC) 6. Human subjects lacking Cldn-1 suffer from the Neonatal Ichthyosis-Sclerosing Cholangitis (NISCH) syndrome which includes an ichthyosiform skin phenotype 7. An archetypical disease of epidermal barrier dysfunction is atopic dermatitis (AD) 8. Cldn-1 single nucleotide polymorphisms were linked to AD in some cohorts 9-11 , but not in others 11,12. Using immunostaining-intensity measurements and western blot analyses, reduced Cldn-1 levels were found in lesional AD skin 13-16. For non-lesional skin, divergent observations were described. Some authors found decreased mRNA and immunointensity levels 10 , while others observed no alteration of Cldn-1 immunointensity and western-blot levels 14,16 .

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