Amelioration of Androgenetic Alopecia by Algal Oligosaccharides Prepared by Deep-Sea Bacterium Biodegradation

Abstract

Androgenetic alopecia (AGA) is a dihydrotestosterone (DHT)-mediated hair loss disorder characterized by shortened anagen hair cycle. Oligosaccharides derived from seaweeds possess diverse biological functions. However, little is known about their effects on AGA. In this study, algal oligosaccharide (AOS) was characterized for its mitigation effects on key features involved in AGA pathogenesis, such as DHT- mediated cellular signaling and shortened anagen hair cycle. AOS with varying degrees of polymerization (DP), namely, AOS (DP2), AOS (DP4-6), and AOS (DP8-12), were prepared by agar biodegradation with Flammeovirga pacifica WPAGA1, an agarolytic bacterium isolated from deep-sea sediments. In vitro results showed that AOS with varying DPs significantly ameliorated the DHT-induced alterations of regulatory factors in human hair follicle dermal papilla cells in a dose- and DP-dependent manner, as revealed by the normalization of several hair-growth-stimulating or inhibitory factors. In vivo studies showed that AOS (DP2) extended the anagen phase and thereby delayed catagen progression in mice. Furthermore, AOS (DP2) stimulated dorsal hair growth in mice by increasing hair length, density, and thickness. Therefore, our findings indicated that AOS antagonized key factors involved in AGA pathogenesis, suggesting the potential application of AOS in the prevention and the treatment of AGA.

Keywords: algal oligosaccharides; anagen; androgenetic alopecia; deep-sea bacterium; dihydrotestosterone.

FIGURE 1

Preparation of algal oligosaccharide (AOS) with varying DP from agar. (A) Preparation of AOS (DP2) by enzymatic degradation of AOS (DP4–6) with Aga2660. (B) Preparation of AOS (DP4–6) by enzymatic degradation of agar with Aga4383. This picture is adapted from the study of Hou et al. (2015). (C) Preparation of AOS (DP8–12) by enzymatic degradation of agar with Aga2133. M, agaroligosaccharide markers; NA2, neoagarobiose; NA4, neoagarotetraose; NA6, neoagarohexaose; NA8, neoagarooctaose; NA10, neoagarodecaose; NA12, neoagarododecaose. The hydrolysate indicated with an arrow in each thin-layer chromatography plate was purified and used in the following experiments.

FIGURE 2

Proliferation of cultured human hair follicle dermal papilla cells (hHFDPCs) treated with algal oligosaccharide (AOS) of varying doses. Different concentrations of AOS were added into cultured hHFDPCs, and cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. As a positive control, 4% dimethyl sulfoxide was included. Columns represent the mean of triplicate assays. Results that are significantly different from those in the blank control group are shown by asterisks (^∗p < 0.05, ^∗∗p < 0.01).

FIGURE 3

Effects of algal oligosaccharide (AOS) with varying degrees of polymerization (DPs) on vascular endothelial growth factor (VEGF) secretion and hair-growth-related gene expression. (A) Influences of dihydrotestosterone (DHT) treatment on the growth of human hair follicle dermal papilla cells (hHFDPCs). Different concentrations of DHT were added into hHFDPCs. After 24 h, cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. (B) Effects of AOS with varying DPs on the VEGF secretion of hHFDPCs. Different concentrations of AOS were added into hHFDPCs, and the concentration of extracellular VEGF was determined by enzyme-linked immunosorbent assay. (C) Effects of AOS with varying DPs on hair-growth-related gene expression. GAPDH was used as the reference gene for internal control. For all figures, the control group was treated with culture medium. The final concentrations of DHT and minoxidil were 800 nM and 500 μM, respectively. Columns represent the mean of triplicate assays. Significant differences between DHT and control groups are indicated with double hash symbols (^##p < 0.01). Results that are significantly different from those in the DHT group are shown by asterisks (^∗p < 0.05, ^∗∗p < 0.01).

FIGURE 4

Effects of algal oligosaccharide (AOS) on hair growth of mice. (A) In vitro transdermal permeation of different AOS penetration-enhancing formula. The abdomen skin of Sprague–Dawley rats was fixed in a Franz diffusion pool. Five hundred microliters of different AOS penetration-enhancing formulas was adopted as the donor solution. The amount of AOS (DP2) in the receiving tank was determined by the dinitrosalicylic acid method. The cumulative permeation per area of the skin (Q_n, μg/cm²) was calculated. The experiment was repeated three times. (B) Scheme of the animal experiment. The back skin of C57BL/6 mice (n = 6) was treated with vehicle [2% glycerol and 1% polyethylene glycol (PEG) 400], AOS alone (1% AOS DP2), or AOS formula (1% AOS DP2 with 2% glycerol and 1% PEG 400) at anagen stage for 10 days. (C) Effects of AOS on hair follicle growth cycle and hair density. Representative morphological (top) and histological (bottom) images of skin tissue at day 21 are shown. (D) Effects of AOS on hair length. The length of randomly plucked hairs (n = 30) was measured. (E) Calculation of the hair cycle scores. Fifty hair follicles identified on sections were scored for each mouse, and five mice were used for each group. Each stage of the hair cycle has been scored as follows: anagen V–VI = 100, early catagen = 200, mid catagen = 300, late catagen = 400. The value indicates the mean hair cycle score per group (^∗p < 0.05).