Rosmarinic acid inhibits poly(I:C)-induced inflammatory reaction of epidermal keratinocytes
Introduction
Skin is the largest organ, encompassing the entire body and demarcating the organism and environment. Its main function is to protect the body against harmful external insults [1]. The outmost layer epidermis is comprised of several cell types including keratinocytes, melanocytes and Langerhans cells. Among those, keratinocytes are the predominant cells and exert their role for physical barrier against external stimuli. In addition, keratinocytes contribute to innate immune surveillance through expressing various Toll-like receptors (TLRs), kinds of important pattern recognition receptors (PRRs). The specific recognition of pathogen-associated molecular patterns (PAMPs) by keratinocytes results in activation of inflammation-related intracellular signaling and production of inflammatory cytokines [2], [3].
Psoriasis is an immune-mediated inflammatory skin disorder characterized by hyperplasia of epidermal keratinocytes and infiltration of inflammatory cells [4]. It can be provoked or exacerbated by risk factors affecting the skin barrier function such as infections and mild trauma (scratching, piercings), suggesting that keratinocytes may be the potential origin cells for psoriasis. The cross-talk between innate and adaptive immunity is the fundamental mechanistic basis for psoriasis, in which many cytokines are functionally involved [5]. Especially, stimulation of keratinocytes with various PAMPs results in activation of innate immunity of keratinocytes, leading to the production of inflammatory cytokines related with psoriasis, including IL-1β and TNF-α [6]. Considering the importance of this initial innate immune response, blocking the inflammatory reaction of keratinocytes can be a good target for treating psoriasis.
Polyinosinic:polycytidylic acid (poly(I:C)) is a synthetic analogue of viral double-stranded RNA (dsRNA) which can induce innate immune response in a TLR3-dependent manner [7]. Keratinocytes express high level of TLR3, and stimulation of this receptor with its synthetic ligand poly(I:C) provokes inflammatory reaction in epidermal keratinocytes [8], [9]. In an attempt to find the therapeutics for psoriasis, we found that rosmarinic acid (RA) can be a possible candidate for drug development. It has been reported that RA possesses antioxidant and anti-inflammatory properties in macrophages [10]. However, the effect of RA on epidermal keratinocytes remains to be elucidated. In this study, we demonstrated that RA has an inhibitory potential on poly(I:C)-induced inflammatory reaction of epidermal keratinocytes.
Section snippets
Cell culture and drug preparations
Human skin tissues were obtained under the written informed consent of donors, in accordance with the ethical committee approval process of the Institutional Review Board of Chungnam National University Hospital (IRB file number: 2015-05-013). Primary keratinocytes were cultured according to the method previously described. Primary keratinocytes were isolated from epidermis, and then immortalized using the recombinant retrovirus expressing simian virus 40 T antigen (SV40Tag) [11].
Cytotoxicity of rosmarinic acid (RA) on keratinocytes
We tried to seek potential therapeutics for psoriasis, and found that rosmarinic acid (RA) (Fig. 1A) has inhibitory potential on inflammatory reaction of human keratinocytes. At first, we determined the cytotoxicity of RA on SV-HEKs. The MTT assay showed that RA did not induce cell death up to the dose of 100 μM (Fig. 1B). In LDH assay, the doses over 50 μM resulted in increased secretion of LDH from SV-HEKs (Fig. 1C). These results implicated that RA caused some damage of plasma membrane.
Discussion
Epidermal keratinocytes are innate immune cells that respond to TLR3 stimulation with high production of pro-inflammatory cytokines and chemokines such as IL-1β, TNFα, IL-6, IL-8 and CCL20 [13]. Psoriasis is an immune-mediated inflammatory skin disorder, and inflammatory cytokines secreted by psoriatic keratinocytes have important roles to recruit and activate immune cells such as neutrophils and activated T cells [14]. It has been described that psoriatic keratinocytes show increased
Conclusion
This study demonstrated that RA inhibited poly(I:C)-induced inflammatory reaction of epidermal keratinocytes, suggesting that RA could hold promise for the therapeutics of chronic inflammatory skin diseases such as psoriasis.
Conflict of interest statement
The authors declare that they have no conflict of interest.
Acknowledgments
This research was supported by Chungnam National University Hospital Research Fund 2015, and a grant of the Korean Health Technology R&D Project (grant number: HN12C0062, Ministry of Health & Welfare, Republic of Korea).
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