Influence of silymarin components on keratinocytes and 3D reconstructed epidermis

Highlights

• Anti-inflammatory properties of Silymarin components was evaluated on NHEKs and RHE.

• Inflammation was induced after application of LPS on NHEKs and SDS on RHE.

• DHSB was the most effective to decrease the pro-inflammatory cytokines in NHEKs.

Abstract

Silymarin is a flavonoid complex isolated from the plant Silybum marianum which is well known for its antioxidant, hepatoprotective and immunomodulatory effects. Since little is known about its anti-inflammatory properties and healing effects, our study focused on whether or not silymarin components reduce inflammation and support epidermis regeneration. Lipopolysaccharides (LPS) and sodium dodecyl sulfate (SDS) were used to induce inflammation in normal human epidermal keratinocytes (NHEKs) and reconstructed epidermis (RHE), respectively. The expression of pro-inflammatory cytokines (IL-1, IL-6 and IL-8) in NHEKs and RHE was measured by enzyme – linked immunosorbent assay (ELISA). The expression of cytokeratin 14 and loricrin in RHE was detected by immunofluorescent analysis. Hematoxylin and eosin staining was used for the morphological evaluation of RHE. It was determined that 2, 3 – dehydrosilybin (DHSB) downregulated the production of selected pro-inflammatory cytokines produced by NHEKs. Although all layers of RHE displayed full thickness, when SDS was applied, cell detachment was seen in the stratum corneum and loricrin expression was diminished.

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).