Glycogen Phosphorylase Inhibitor Promotes Hair Growth via Protecting from Oxidative-Stress and Regulating Glycogen Breakdown in Human Hair follicles

Abstract

Hair growth cycles are mainly regulated by human dermal papilla cells (hDPCs) and human outer root sheath cells (hORSCs). Protecting hDPCs from excessive oxidative stress and hORSCs from glycogen phosphorylase (PYGL) is crucial to maintaining the hair growth phase, anagen. In this study, we developed a new PYGL inhibitor, Hydroxytrimethylpyridinyl Methylindolecarboxamide (HTPI) and assessed its potential to prevent hair loss. HTPI reduced oxidative damage, preventing cell death and restored decreased level of anagen marker ALP and its related genes induced by hydrogen peroxide in hDPCs. Moreover, HTPI inhibited glycogen degradation and induced cell survival under glucose starvation in hORSCs. In ex-vivo culture, HTPI significantly enhanced hair growth compared to the control with minoxidil showing comparable results. Overall, these findings suggest that HTPI has significant potential as a therapeutic agent for the prevention and treatment of hair loss.

Keywords: Anagen; Dermal papillia cells; Glycogen phosphorylase; Hair loss; Outer root sheath cells; Oxidative stress.

Fig. 1

The chemical structure of HTPI.

Fig. 2

hDPCs viability in the presence of various concentrations of HTPI. (A) HTPI was treated with hDPCs for 24 h. (B, C) HTPI was pre-treated for 2 h in hDPCs and treated with H2O2 for 24 h. *p<0.05; **p<0.01; ***p<0.001 vs. control.

Fig. 3

Predicted binding mode of PYGL and HTPI complex. (A)Two identical subunits of PYGL are depicted in pale cyan and pink colors. Two HTPI molecules are bound allosterically at the dimeric interface of PYGL and are depicted as sticks in green. (B) Major binding interactions in the PYGL and HTPI complex. Hydrogen bonds, cation-p and p-p stacking interactions are shown as dotted lines in yellow, green and cyan colors, respectively.

Fig. 4

HTPI reduces ROS levels induced by H2O2. The ROS production of hDPCs was measured. (A) Intracellular ROS content was measured after HTPI pre-treatment for 2 h and H2O2 treatment for 4 h using DCFDA. (B) Mitochondrial ROS production was analyzed using the JC-1 assay. *p<0.05; **p<0.01; ***p<0.001 vs. control.

Fig. 5

HTPI increased the level of ALP activity, which was reduced by H2O2 treatment in hDPCs. After pre-treatment with HTPI for 2 h, hDPCs were treated with H2O for 24 h. ***p<0.001 vs. control.

Fig. 6

Effect of HTPI on hair growth in human hair follicle organ culture. In order to evaluate the effect of HTPI, the anagen human hair follicle were prepared and cultured for 8 days. HTPI was treated at concentrations of 1 and 2 µM. (A) At day 4, 6 and 8, the cultured hair follicles were photo-documented. (B) The hair shaft growth was analyzed. Minoxidil was used as a positive control. The data represent the mean±SD of eighteen follicles. p-values were obtained by Mann-Whitney U test. Significantly different compared with control. *p<0.05; **p<0.01 vs. control.

Fig. 7

Effect of HTPI on hORSCs in the presence or absence of glucose. hORSCs were pre-treated with HTPI for 2 h, and then hORSCs were incubated with glucose-free medium for 24 h. (A) Glycogen in hORSCs. (B) Cell viability was measured. *p<0.05; **p<0.01; ***p<0.001 vs. control.