Influence of silymarin components on keratinocytes and 3D reconstructed epidermis
Introduction
Animal models have, in the past, been commonly used in preclinical studies and safety testing. However, animal models are now recognised as poorly predicting the human response due to differences in skin morphology and immune response (Kazem et al., 2019). Together with the principles of the 3Rs (Replacement, Reduction and Refinement) (Törnqvist et al., 2014), this has resulted in a transition from animal testing to alternative models and many 3Rs have been developed in toxicological research, as well as at legislative levels. Thus, human skin implants and reconstructed human skin or epidermis have begun to be widely used for risk assessments of drugs, corrosion, toxicity and sensitization to several compounds (De Vuyst et al., 2016; Catarino et al., 2018). The recent development of epidermis and skin equivalents provides a new system for the monitoring of pharmacological properties and is also of interest for the management of large skin defects and deep wounds (Kim et al., 2002).
The activation of pro-inflammatory parameters (cytokines) plays a key role in the inflammatory response (Wang et al., 2016). One of the major activators may be bacterial lipopolysaccharides that cause the release of pro-inflammatory cytokines such as IL-1, IL-6, IL-8 and TNF-α (Kulshrestha et al., 2019). However, although inflammation is a part of the wound healing process, uncontrolled inflammation can cause chronic inflammation conditions e.g. non-healing wounds. Suppression of excessive inflammation leads to rapid wound healing.
Silymarin (SM) is extracted from the fruit of Silybum marianum (milk thistle), which contains flavonolignan components with the dominant active compounds silybin (Soleimani et al., 2019) (SB, silybin A and silybin B), isosilybin A, isosilybin B, silychristin, silydianin, isosilychristin and taxifolin (Esmail et al., 2017). 2, 3-dehydrosilybin (DHSB) is also present in small amounts. SM, SB, quercetin (QE) and DHSB are well known as antioxidant, immunomodulatory and hepatoprotective agents (Amin and Arbid, 2015; Kosina et al., 2019). It has been demonstrated that they can inhibit the production of pro-inflammatory cytokines and increase the production of anti-inflammatory cytokines. Several studies indicate that SM also has anti-inflammatory potential via the suppression of transcription factor NF-κB, which leads to a reduction in pro-inflammatory (tumor necrosis factor alpha and interleukin 1 beta) cytokines and prostaglandins levels (Esmail et al., 2017). The flavonolignan QE exhibits similar properties to SM and can modulate downstream signalling pathways, including MAPK, NF- κB and Nrf-2 (Chen et al., 2017). Due to its lipophilic properties, DHSB better inhibits lipid peroxidation than SB. It is also a greater inhibitor of MMP-2 and MMP-9, corroborating the with antioxidant and anti-inflammatory activity of DHSB. Vostalova et al. also found that pure SM (SB and DHSB) flavonolignans have regenerative potential for UVA damage to the HaCaT cell line by affecting skin enzymes (Vostalová et al., 2019).
Our study focused on evaluating the anti-inflammatory properties of SB, QE and DHSB in comparison to the anti-inflammatory drug indomethacin (IND) on normal human epidermal keratinocytes (NHEK) and reconstructed epidermis (RHE).
Section snippets
Tested compounds
SB and DHSB were provided by the Laboratory of Biotransformation, Institute of Microbiology of the Czech Academy of Science, Prague, Czech Republic. The isolation of SB from crude extracts of milk thistle fruit (silymarin) as well as its oxidation, which gives rise to the derivative DHSB, has been described in detail previously (Gažák et al., 2004; Křenek et al., 2014). QE and IND were purchased from Sigma-Aldrich (Czech Republic). Millimolar concentrations of stock compound solutions were
Production of IL-6, IL-8 and IL-1α by keratinocytes and RHE
The production of pro-inflammatory cytokines has a key role during the inflammatory process. IL-1α is released from keratinocytes and takes part in skin inflammation, which consequently induces other cytokines, such as TNF – α, IL-6 and IL-8. In our study, we focused on clarifying the influence of the active compound silymarin on inflammation cytokines (IL-1α, IL-6 and IL-8) increased by LPS on NHEKs or SDS on RHE (Fig. 1, Fig. 2). Our data showed that LPS and SDS intensify the levels of
Discussion
Skin is the largest organ of the human body and can be divided into epidermis, dermis and deeper subcutaneous tissue. The epidermis is 95% made up of keratinocytes and is composed of 5 layers (stratum basale, stratum spinosum, stratum granulosum, stratum lucidum and stratum corneum) (Ponec et al., 2002). Keratinocytes are not only the dominant cells of the epidermis but also the first cells to encounter chemicals penetrating through the stratum corneum (Saito et al., 2013). Keratinocytes
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgement
The authors are thankful to I. Travnickova for her help with histological analysis and Dr. Buhumil Zalesak from the Department of Plastic and Aesthetic Surgery, Faculty Hospital Olomouc for his help with the provision of skin samples. This paper was supported by the following grants: RVO 61989592, Palacky University Olomouc, Czech Republic; Ministry of Education, Youth and Sports of the Czech Republic [LO1304] and the Czech Science Foundation [18-00132S].
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