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Comparative Study of the Effect of 5,6-Dihydroergosterol and 3-epi-5,6-dihydroergosterol on Chemokine Expression in Human Keratinocytes

5,6-Dihydroergosterol-glucose is an organic synthetic derivative of spinasterol-glucose, which has potent anti-inflammatory activity. We previously synthesized alpha and beta anomers of DHE-glycosides and compared their inhibitory activity on CCL17 and CCL22 mRNA expression induced by TNF-α/IFN-γ in activated HaCaTs. Recently, we synthesized a new type of DHE-glycosides, 3-epi-5,6-dihydroergosterol(3-epi-DHE)-glycosides, and compared its inhibitory activity on mRNA expression levels of CCL17 and CCL22 in TNF-α/IFN-γ-induced HaCaT. DHE-Xly did not affect TNF-α/IFN-γ induced CCL17 and CCL22 mRNA expression in HaCaTs, however, 3-epi-DHE-Xly strongly inhibited TNF-α/IFN-γ induced CCL17 and CCL22 mRNA expression levels in human keratinocytes. These results provide important clues for development of chronic dermatitis treatment via inhibition of chemokine expression using DHE derivatives.


Introduction
In general, structural changes according to epimerization in several synthetic compounds are known to present different biological activities. In the case of vitamin D, the epimeric compound has traditionally been reported to be inactive, but a recent study has shown that C3-epi 25 (OH) D3 affects growth and bone mineral density. It has also been reported that C3-epi 25 (OH) D3 has distinctly different biological activities and that further studies are needed for verification [1,2]. 5,6-Dihydroergosterol-glucose (DHE-Glc) is an organic synthetic derivative of spinasterol-glucose, which is a phytosterol isolated from the Srewarita Koreana leaf extract [3]. In several studies, spinasterol-Glc was found to have various physiological activities as well as potent anti-inflammatory activity, and our previous study confirmed that its derivative DHE-Glc also had various biological activities [4,5]. In particular, it was confirmed that DHE-Glc strongly inhibits the mRNA and protein expression levels of pro-inflammatory cytokines and chemokines in human keratinocytes (HaCaTs) activated by TNF-α/IFN-γ. In addition, DHE-Glc was found to have potent anti-inflammatory activity in the 2,4-dinitrochlorobenzene (DNCB)-induced chronic dermatitis mouse animal model [5].
Chemokines are cytokines with chemotaxis and cytokine or signal transduction proteins secreted from various cell types including endothelial cells, epithelial cells, and immune cells after stimulation by external and/or internal stimuli [6]. Chemokines are classified into four main subfamilies-CXC, CC, CX3C, and XC-depending on the important four cysteine residues in the conserved position. These proteins are known to exert biological activities by interacting with specific binding to G protein-linked coupled receptors (GPCRs), which are selectively found on the surface of each of their target cells [7]. In several recent studies, thymus-and activation-regulated chemokine (TARC/CCL17) and monocyte-derived chemokine (MDC/CCL22) belonging to CC-chemokine are reported as important biomarkers in chronic dermatitis [8,9]. It has been reported that levels of CCL17 and CCL22 are increased in the blood and dermatitis skin tissues of many chronic dermatitis patients, causing continuous immune cell activation [10].
We previously have reported that alpha and beta anomers of DHE-glycosides are produced during the synthetic process of DHE-glycoside compounds. Also, we compared the inhibitory activity of these compounds on CCL17 and CCL22 mRNA expression in HaCaTs activated by TNF-α/IFN-γ and it confirmed that the beta anomer of DHE glycoside has a greater effect than the alpha anomer of DHE-glycoside on the expression levels of CCL17 and CCL22 in TNF-α/IFN-γ -induced HaCaT cells. This result was confirmed to be probably due to the stability of both compounds [4]. In this study, we synthesized a new type of DHE-glycosides, such as 3-epi-5,6-dihydroergosterol(3-epi-DHE)-glycosides, and compared the inhibitory activity of mRNA expression levels of CCL17 and CCL22 in TNF-α/IFN-γ-induced HaCaT. These results provide important clues for development of chronic dermatitis treatment via inhibition of chemokine expression using DHE derivatives.

Scheme 2. Synthesis of 3-epi-DHE 2 via Mistunobu reaction and its glycosides by Schmidt glycosylation.
We examined the effects of the epi-DHE-glycoside on CCL17 and CCL22 mRNA expression in TNF-α/IFN-γ-induced HaCaT cells.
The cell cytotoxicity of HaCaT cells treated with 3-epi-DHE-glycoside (4, 6, and 8) was determined with MTT assays (data not shown). The cell viability of the other synthetic DHE glycoside compounds did not have a cytotoxic effect up to 20 µM in our previous studies [3,4].
We tested the mRNA expression levels of CCL17 and CCL22 using an RT-PCR assay in TNF-α/IFN-γ-treated HaCaT cells. Furthermore, we confirmed mRNA expression levels of CCL17 and CCL22 by real-time PCR assay ( Figure 1). Analysis of the mRNA expression of CCL17 and CCL22, it was confirmed that the positive control group treated with TNF-A/IFN-Γ (10 ng/mL) increased about 3-to 4-folds compared to that of the negative control group treated with media only. Examining the mRNA expression of CCL17, it was confirmed that only the DHE-Glc 3 (20 µM) had a strong inhibitory activity, as in the previous results [3,4]. On the other hand, the 3-epi-DHE glycosides including 3-epi-DHE 2, 3-epi-DHE-Glc 4, 3-epi-DHE-Gal 6, and 3-epi-DHE-Xyl 8 increased the activity in all compounds (20 µM). As shown in quantitative PCR ( Figure 1B), we could confirm that the change of structure of DHE-Xyl 7 on the DHE-glycoside affects both CCL17 and CCL22 mRNA expression levels. DHE-Xly 7 did not affect on TNF-α/IFN-γ-induced CCL17 and CCL22 mRNA expression in HaCaTs, however, 3-epi-DHE-Xly 8 inhibited TNF-α/IFN-γ induced CCL17 and CCL22 mRNA expression levels in human keratinocyte. Prediction of the stable structure of DHE-glycoside and its epimer, 3-epi-DHE-glycoside, by DFT calculation showed obvious differences. As shown in Figure 2, DHE-xylose 7 has a relatively linear shape, while 3-epi-DHE-xylose 8 has a curved shape. In addition, the calculated energy difference between the two epimers is only 0.70 kcal/mol. Therefore, the structural difference between the two epimers likely influenced the change of bioactivity.

Quantitative Real-Time PCR
RNA samples and cDNA were obtained using the same methods as for RT-PCR analysis. Reverse transcription reaction was carried out by using AccuPower RT Premix (Bioneer, Daejeon, Korea) according to the manufacturer's recommendations. Real-time PCR was conducted in a Lightcycler Nano system (Roche, Mannheim, Germany) using LightCycler DNA Master SYBER GREEN I (Roche, Mannheim, Germany). The mRNA expression levels were normalized to GAPDH by calculating delta Ct values. The specific primers as follows: CCL17 (forward) 5 -AGG GAC CTG CAC ACA GAG AC-3 , (reverse) 5 -CTC GAG CTG CGT GGA TGT GC-3 ; CCL22 (forward) 5 -ATG GCT CGC CTA CAG ACT GCA CTC-3 (reverse) 5 -CAC GGC AGC AGA CGC TGT CTT CCA-3 .

Statistical Analysis
Unless otherwise stated, all experiments were performed with triplicate samples and repeated at least three times. Data are presented as means ± S.D. and statistical comparisons between groups were performed using one-way ANOVA followed by a Student's t-test.

Conflicts of Interest:
The authors declare no conflicts of interest.