Accelerated Barrier Repair in Human Skin Explants Induced with a Plant-Derived PPAR-α Activating Complex via Cooperative Interactions

Abstract

Background: Peroxisome proliferator-activated receptors (PPARs) govern epidermal lipid synthesis and metabolism. In skin, PPAR activation has been shown to regulate genes responsible for permeability barrier homeostasis, epidermal differentiation, lipid biosynthesis, and inflammation.

Objective: Given the known dermatologic benefits of PPARs, we set out to discover a naturally derived, multi-molecule complex that would be superior to the more commonly formulated conjugated linoleic acids (CLAs). We hypothesized that a complex may be capable of modulating PPAR-α by cooperative or multi-ligand binding interactions to accelerate skin barrier repair.

Methods: To achieve this, we assembled a novel PPAR-α agonist complex, referred to as RFV3, from a combination of small molecules routinely used in Ayurvedic medicine and accepted in cosmetic and topical over-the-counter dermatologic products. We tested RFV3's potential as a PPAR-α agonist by evaluating its transcriptional response, ligand binding affinity to PPAR-α, gene expression profiles and barrier repair properties in human skin explant models.

Results: We assembled RFV3 by solubilizing two standardized plant extracts in a suitable solvent and induced a significant transcriptional response in PPAR-α luciferase reporter assay. Furthermore, transcriptome profiling of RFV3-treated epidermal substitutes revealed expressed genes consistent with known targets of PPAR-α, including those involved in epidermal barrier repair. In addition, in silico modeling demonstrated differential co-binding affinities of RFV3 to PPAR-α compared with those of the endogenous ligands (CLAs) and a synthetic PPAR-α agonist. Lastly, delipidated skin explant models confirmed accelerated barrier repair activity with significant increases in ceramides, filaggrin and transglutaminase-1 after treatment.

Conclusion: These findings suggest that the RFV3 complex successfully mimics a PPAR-α agonist and induces synthesis of skin barrier lipids and proteins consistent with known PPAR pathways.

Keywords: PPAR-α; ceramides; cooperative binding; epidermal barrier; explants.

Figure 1

Degree of PPAR-α transcription activation induced by test materials depicted by the Reporter Assay System. Data are presented as mean value ± SEM, n=13 for water, n= 3 for test samples; *p-value < 0.05, **p-value < 0.01, n.s. = not significant, † = cytotoxic conditions observed.

Figure 2

Graphic representation of connectivity in genes modulated by RFV3 (2%) in epidermal tissue substitutes vs glycerin control (in silico analysis with StringDB software).

Figure 3

Interacting amino acid residues and conformations within the LBD of PPAR-α. From left to right: (A) ximenynic acid + glyceryl linolenate at distance criterion of 2.5 Ȧ, (B) RFV3 at distance criterion of 2.5 Ȧ and (C) RFV3 at distance criterion of 6 Ȧ.

Figure 4

Microscopic evaluation of tissue and cell morphology of day 0 and day 1 explants. No alterations observed in the epidermis and dermis across all samples.

Figure 5

Immunostaining of ceramides in the stratum corneum with day 1 explants. Starting top left samples: Untreated control, delipidated control, delipidated excipient treated, delipidated 0.5% RFV3 treated, delipidated 0.3% RFV3 treated, delipidated 5% RFV3 treated.

Figure 6

Image analysis for % surface positive immunostaining of ceramides in the stratum corneum of day 1 explants. Error bars represent SD. Explants analyzed: 18 (6 batches, 3 explants per batch). Image analyses n=9 (3 images per explant). Treated samples vs U(C): § for p < 0.05 and §§ for p < 0.01. Treated vs D(C): †† for p < 0.01. Treated samples vs D(E) ** for p < 0.01.

Figure 7

Immunostaining of filaggrin at the bottom of the stratum corneum with day 1 explants. Starting top left samples: Untreated control, delipidated control, delipidated excipient treated, delipidated 0.5% RFV3 treated, delipidated 0.3% RFV3 treated, delipidated 5% RFV3 treated.

Figure 8

Image analysis for % surface positive immunostaining of filaggrin at the bottom of the stratum corneum with day 1 explants. Error bars represent SD. Explants analyzed: 18 (6 batches, 3 explants per batch). Image analyses n=9 (3 images per explant). Treated samples vs U(C): § for p < 0.05. Treated vs D(C): †† for p < 0.01. Treated samples vs D(E) ** for p < 0.01.

Figure 9

Immunostaining of transglutaminase-1 in the epidermis granular layer with day 1 explants. Starting top left samples: Untreated control, delipidated control, delipidated excipient treated, delipidated 0.5% RFV3 treated, delipidated 0.3% RFV3 treated, delipidated 5% RFV3 treated.

Figure 10

Image analysis for % surface positive immunostaining of transglutaminase-1 in the epidermis granular layer with day 1 explants. Error bars represent SD. Explants analyzed: 18 (6 batches, 3 explants per batch). Image analyses n=9 (3 images per explant). Treated samples vs U(C): §§ for p < 0.01. Treated vs D(C): † for p < 0.05 and †† for p < 0.01. Treated samples vs D(E) ** for p < 0.01.