The effects of conifer polyprenol nanoemulsions on skin cell culture proliferation rate and gene expression levels of structural proteins, growth factor and inflammatory cytokines

[Speaker] Ilona Vanaga:1,2,3
[Co-author] Janis Ancans:4, Ilze Blake:4, Ugis Kletnieks:2,3, Jerzy Gubernator:5
1:Dept. of Pharmacology, University of Latvia, Latvia, 2:Silv EXPO, SIA, Latvia, 3:JSC Biolat, Latvia, 4:Faculty of Biology, University of Latvia, Latvia, 5:Faculty of Biotechnology, University of Wroclaw, Poland

Background: Conifer needle polyprenols are known to improve muscle strength in rats and also act protectively in patients suffering from statin-induced myopathies (Jansone et al., 2016, Latkovskis et al., 2016), but little is known about polyprenols in relation to gene expression in vitro.
Objective: the objective of the study was to assess polyprenol nanoemulsion effects on the human cell culture proliferation rate and the expression of human skin structural protein (ELN, COL1A1, K10), growth factor (FGF7) and inflammatory cytokine (TGF-beta1, TNF-alpha, MMP1, IL5, IL6, IL8) associated genes.
Methods: Abies Sibirica (80% purity) and Picea abies (95% purity) polyprenols were acquired from JSC Biolat. Polyprenols and corn oil (control) were used to prepare nanoemulsions with droplet size range of 120-160 nm. Immortalized human melanoma cells FM55, human keratinocyte line (HaCaT) and dermal fibroblasts (HDF) were exposed to control solution (2.25% glycerol) and polyprenol (80% and 95% purity) nanoemulsions of different final concentrations (0.1% - 20% v/v). Kinetics of the cell growth was monitored using the IncuCyte integrated confluence algorithm. RT-PCR was done using Maxima SYBR Green/ROX qPCR Master Mix (2X) kit (Thermo-scientific) and gene primers. Data analysis was done using relative fold changes and quantified by comparative 2(-Delta Delta C(T)) method (Livak and Schmittgen, 2001).
Results: 95% polyprenol nanoemulsion did not influence cell proliferation rate for HaCaT and HDF cultures at the concentrations of up to 5% (v/v), while 80% polyprenol nanoemulsion showed reduction in cell density starting at 0.5% and 1% (v/v). In HDF, expression level of ELN increased more than 3-fold after treatment with 1% polyprenol emulsion following further increase to 8-fold for 5% emulsion compared to control cells. Significant expression level increases were observed also for MMP1 (more than 3-fold increase) when treated with 1% and 5% polyprenol emulsions. The most significant decrease of expression level was observed in HaCaT with TGF-beta1 and IL6 gene expression level reduced 3-fold for 5% polyprenol emulsion compared to control. Results are summarized in Table 1.
Conclusions: Picea abies 95% polyprenol nanoemulsions showed excellent safety profile and reduction of inflammatory cytokine expression, indicating a potential application in skin tissue regeneration formulations.

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