Quercitrin Stimulates Hair Growth with Enhanced Expression of Growth Factors via Activation of MAPK/CREB Signaling Pathway

Abstract

The present study aimed to investigate the molecular mechanism of quercitrin, a major constituent of Hottuynia cordata extract, for its hair growth stimulating activities in cultured human dermal papilla cells (hDPCs). Quercitrin enhanced the cell viability and cellular energy metabolism in cultured hDPCs by stimulating the production of NAD(P)H and mitochondrial membrane potential (ΔΨ). The expression of Bcl2, an essential marker for anagen hair follicle and cell survival, was increased by quercitrin treatment. Quercitrin also increased the cell proliferation marker Ki67. The expression of growth factors-such as bFGF, KGF, PDGF-AA, and VEGF-were increased by quercitrin both in mRNA and protein levels. In addition, quercitrin was found to increase the phosphorylation of Akt, Erk, and CREB in cultured hDPCs, while inhibitors of MAPKs reversed the effects of quercitrin. Finally, quercitrin stimulated hair shaft growth in cultured human hair follicles. Our data obtained from present study are in line with those previously reported and demonstrate that quercitrin is (one of) the active compound(s) of Hottuynia cordata extract which showed hair growth promoting effects. It is strongly suggested that the hair growth stimulating activity of quercitrin was exerted by enhancing the cellular energy metabolism, increasing the production of growth factors via activation of MAPK/CREB signaling pathway.

Keywords: CREB; MAPK; growth factors; hair growth; human DPCs; quercitrin.

Figure 1

Quercitrin enhanced cell viability in cultured hDPCs. (a) Quercitrin chemical structure. (b) Cell viability was assessed using CCK-8 assay kit after quercitrin treatment (0.1, 1, 10, 100 nM and 1 µM) for 24 h. The value of non-treated control was taken to be 100%. N.T, non-treated control; MNX, minoxidil. Significantly different compared with N.T (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 2

Effect of quercitrin on cellular energy metabolism in cultured hDPCs. (a) Mitochondrial membrane potential was measured after quercitrin treatment (1, 10, and 100 nM) for 24 h. The 100 nM minoxidil was used as a positive control. (b) JC-1 monomer form was seen as green and aggregate form as red by fluorescent microscopy. N.T, non-treated control; MNX, minoxidil. Significantly different compared with N.T (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 3

Effect of quercitrin on mRNA expression levels of proliferative/apoptotic genes and protein level of Bcl2 in cultured hDPCs. The cultured DPCs were harvested after quercitrin treatment (1, 10, and 100 nM) for 24 h. (a) The mRNA expression levels of Bcl2, Bad, Bax and Ki67 genes in cultured hDPCs were measured by real-time PCR. (b) Whole cell lysates (50 µg protein) from DPCs were analyzed by immunoblotting to determine the levels of Bcl2, and (c) the band intensity was quantitated. N.T, non-treated control; MNX, minoxidil. Significantly different compared with N.T (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 4

Effect of quercitrin on mRNA expression levels of growth factor genes in cultured hDPCs. The cells were harvested after quercitrin treatment (1, 10, 100 nM and 1 µM) for 24 h. The mRNA expression levels of eight genes in cultured hDPCs were measured by real-time PCR. N.T, non-treated control; MNX, minoxidil. Significantly different compared with N.T (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 5

Effect of quercitrin on the expression of growth factors in cultured hDPCs. The DPCs were treated with quercitrin (0, 100 nM and 1 µM) for 24 h, and then collected. Cells cultured with vehicle medium were used as non-treated control. Total of 41 types of human growth factors were analyzed. (a) The 12 types of growth factors and receptors were displayed and (b) the band intensity was quantitated. N.T, non-treated control. Positive, biotin-conjugated IgG. Significantly different compared with N.T (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 6

Effect of quercitrin on the protein expression of bFGF and KGF in cultured hDPCs. The DPCs were treated with quercitrin at concentrations of 1, 10, 100 nM and 1 µM for 24 h. Whole cell lysates (a,c) and culture medium (b,d) of each cultured DPCs were analyzed by ELISA to determine the levels of bFGF (a,b) and KGF (c,d). N.T, non-treated control. Significantly different compared with N.T (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 7

Effect of quercitrin on Akt, Erk, and CREB phosphorylation in cultured hDPCs. Quercitrin was treated for different times (0, 1, 2, 5, 10, and 20 min). (a) Whole cell lysates were analyzed by immunoblotting to determine the levels of Akt, phospho-Akt, Erk, phospho-Erk, CREB and phospho-CREB. As an internal control, GAPDH were used. (b) The ratio of pAkt/Akt, pErk/Erk, and pCREB/CREB was calculated. N.T, non-treated control. The data represent the means of five independent samples. Significantly different compared with N.T (Quercitrin * p < 0.05, ** p < 0.01, *** p < 0.001; Minoxidil # p < 0.05, ## p < 0.01, ### p < 0.001).

Figure 8

Effect of Akt inhibitor API-2 and Erk inhibitor U0126 on quercitrin stimulated gene expression. The cultured DPCs were harvested after 100 nM quercitrin treatment with inhibitors (API-2 and U0125) for 24 h. The mRNA expression levels of bFGF, KGF, Bcl2, and Ki67 were measured by real-time PCR. The data represent the means of six independent samples. Significantly different compared with 100 nM quercitrin treatment (API-2 * p < 0.05, ** p < 0.01, *** p < 0.001; U0126 # p < 0.05, ## p < 0.01, ### p < 0.001).

Figure 9

Quercitrin stimulated the receptor tyrosine kinases and non-receptor tyrosine kinases in cultured hDPCs. 100 nM of quercitrin was treated for appropriate times (0, 0.5, 1 min). Whole cell lysates were analyzed by immunoblotting to determine the level of phospho-tyrosine following manufacturer’s instruction. Total of 71 types of tyrosine kinase were analyzed. The 8 receptor tyrosine kinases and 10 non-receptors tyrosine kinases were displayed (Significantly different compared with 0 min, p < 0.05). Positive, biotin-conjugated IgG.

Figure 10

Effect of quercitrin on hair growth in human hair follicle organ culture. In order to evaluate the effect of quercitrin, the anagen human hair follicle were prepared and cultured for 6 days. Quercitrin was treated at concentrations of 5, 10 µM. (a) At day 4 and 6, 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 means of sixteen follicles. Significantly different compared with N.T (* p < 0.05, ** p < 0.01, *** p < 0.001).