Seven-week-old female C57BL/6 mice (18–20 g), with natural hair loss in the telogen phase (pink color), and normal mice were purchased from Samtako Experimental Animal Center (Ossan, Gyeonggi-do, Korea). Animals were divided into four groups: Normal, normal group without depilation; Vehicle, control group with depilation; A IV (1 μM), 1 μM of Astragaloside IV-treated group; and A IV (100 μM), 100 μM of Astragaloside IV-treated group (n = 7, respectively). Animals were given an unlimited amount of water and food and kept at a constant temperature (23°C) and humidity (55%) with a 12-h light/dark cycle during the experiment. All experiments were conducted according to the guidelines of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by Committee on Care and Use of Laboratory Animals of the Kyung Hee University (Permit Number: KHUASP(SE)-13-046).

One-hundred microliters of 1 and 100 μM Astragaloside IV (PubChem CID: 13943297; Sigma, St. Louis, MO, USA) were applied topically on the upper back with depilation. Vehicle mice were topically applied 100 μL of vehicle (100% pure methanol) on the depilated skin. Normal mice did not receive any treatment. These procedures were performed once per day for 2 weeks. Following challenge for 15 days, photographs were taken and mice were sacrificed. Dorsal skins from mice were fixed in 4% formaldehyde or collected and frozen until use.

Dorsal skin were fixed in formaldehyde for 24 h and transferred to phosphate-buffered saline (PBS). Fixed skins were hydrated with ethanol and PBS. Paraffin-embedded sections were cut every 4 μm. Hematoxylin and eosin (H&E) were applied to the section to monitor hair regrowth. Digital images were obtained using the Leica Application Suite (LAS) Microscope Software (Leica Microsystems Inc., IL, USA). The magnifications used were ×100, ×200 and ×400.

Sections were boiled in 10 mM sodium citrate buffer for antigen unmasking. Slides were treated with 3% H₂O₂ for 30 min to reduce endogenous peroxidase and with normal goat serum to minimize non-specific binding. To detect apoptotic cells, commercially available terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) kit (Millipore, Billerica, MA) was used. After incubation with biotin-dUTP and terminal deoxynucleotidyl transferase (TdT), TUNEL-positive cells were visualized by anti-digoxigenin conjugate. To detect caspase-3-positive cells, the slide was treated by anti-mouse caspase-3 IgG (BD Pharmingen, San Jose, CA, USA) overnight at 4°C, and then biotinylated anti-rabbit antibody (BD Pharmingen) was applied for 1 h. Sections were then stained using the avidin/biotinylated enzyme complex (ABC) kit (Vector Laboratories, Burlingame, CA) and developed using the 3,3-diaminobenzidine (DAB; Dako, Glostrup, Denmark) substrate. Digital images were obtained using Leica Application Suite (LAS) Microscope Software (Leica Microsystems Inc., Buffalo Grove, IL, USA). The magnification used was ×100.

Whole-tissue protein fractions from skin tissues (100 mg, n = 7 per group) were obtained by homogenization in 2 mL T-PER tissue protein extraction reagent (Pierce, Rockford, IL, USA) containing a protease inhibitor cocktail (Roche, Indianapolis, IN, USA) on ice. Nuclear and cytoplasmic protein fractions from skin tissues (100 mg, n = 7 per group) were obtained by homogenization in cytoplasmic buffer (10 mM HEPES [pH 7.9], 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.15% Nonidet P-40, 50 mM β-glycerophosphate, 10 mM NaF, 5 mM Na3VO4), and the pellets were suspended in nuclear buffer (20 mM HEPES [pH 7.9], 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.50% Nonidet P-40, 50 mM β-glycerophosphate, 10 mM NaF, 5 mM Na3VO4) containing protease inhibitor cocktail on ice for 15 min. The resulting homogenate was centrifuged at 12,000×g for 30 min at 4°C and supernatants were labeled whole-tissue, cytoplasmic or nuclear protein.

The activation of β-actin, p21waf, keratinocyte growth factor (KGF), p53, extracellular signal-regulated kinase (ERK), phospho (p)-ERK, stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (SAPK/JNK), p-SAPK/JNK, p38, p-p38, caspases-3, -8, -9, p53, Bcl-2, Bcl-xL and Bax was investigated using the whole-tissue protein fraction. The nuclear protein fraction was used for assessment of the activation of nuclear factor (NF)-κB and the cytoplasmic protein fraction was used to determine IκB-α and p-IκB-α levels. Thirty micrograms of protein were electrophoresed in a 10% SDS-PAGE gel and transferred onto a PVDF membrane (Bio-Rad, Hercules, CA, USA). Each primary antibody (diluted 1∶1000 in 3% bovine serum albumin (BSA); Cell Signaling, USA) was then added to the membrane. Anti-rabbit alkaline phosphatase-conjugated secondary antibody (diluted 1∶2000 in 3% BSA; Santa Cruz, CA, USA) was used to detect the target proteins. After incubation for 2 h at room temperature, bound antibodies were visualized using an enhanced chemiluminescence (ECL) detection reagent (Amersham Pharmacia, Piscataway, NJ, USA). Relative band densities were determined using a computerized densitometry system.

Protein assay reagent (Bio-Rad, Hercules, CA, USA) was used to quantify protein concentrations under identical conditions. Tissue cytokine concentrations were quantified using a mouse tumor necrosis factor (TNF)-α, interleukin (IL)-1β ELISA kit (BD Bioscience, San Jose, CA, USA) according to the manufacturer's instructions. Cytokine levels were calculated as pg per mg total protein. The optical density was read at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader (Molecular Devices, Downingtown, PA).

Significance was determined by one-way analysis of variance (ANOVA) and nonparametric tests. In all analyses, P<0.05 was taken to indicate statistical significance.

Topical application of Astragaloside IV resulted in near-complete recovery to normal levels in all mice, while the back of depilated mice retained a gray color in spontaneous catagen conditions (fig.1A). In addition, cutaneous color changes, indicating the inhibition of catagen and entry into anagen, occurred with Astragaloside IV.

In histology, spontaneous catagen with depilation induced dystrophic follicles along with fragmented hair shafts. Broken hair shafts did not emerge from hair follicles on the skin surface. Miniaturized hair follicles contained no hair fibers. Treatment with Astragaloside IV led to a recovery of tapering hair shaft length and broken hair follicles. New hairs on the surface of the epidermis appeared to be straight after Astragaloside IV treatment. Reversed miniaturized hair follicles showed hair fibers (fig.1B).

The cell death pattern of hair follicles in catagen was indicated by TUNEL-positive cells with miniaturized follicles in the region of hair loss. In depilated mice, TUNEL-positive cells were seen in the epithelial cells surrounding the dermal papilla, but not in the dermal papilla of follicles (fig.2A). In Astragaloside IV-treated skin, few TUNEL-positive cells were seen in the hair follicles matrix as well as in dermal papilla (DP). Caspase-3-positive cells were also seen in hair follicles in the depilated region (fig.3A). Treatment with Astragaloside IV markedly decreased the number of caspase-3-positive cells with recovery of the hair shaft.

To explore the possible molecular mechanisms of inhibition of catagen by treatment with Astragaloside IV, apoptosis-associated protein expression was confirmed by Western blotting. The expression of caspases-3, -8 and -9 was decreased by 1 and 100 μM Astragaloside IV, but not in depilated mice, and there was a notable decrease in the level of Bax and p53 after treatment with Astragaloside IV in apoptotic catagen-affected mice (fig.2B, 2C and 3B).

The levels of Bcl-2, Bcl-xL and Bax expression were analyzed to evaluate effects on caspase activation. Astragaloside IV treatment reduced the expression of the proapoptotic factor Bax compared to control (fig.2C). Additionally, there were notable decreases in the levels of Bcl-2 and Bcl-xL. These results showed that the Bcl-2/Bax ratio was increased by Astragaloside IV, while it was decreased in catagen-affected skin.

NF-κB and p-IκB-α expression levels in the skin of Astragaloside IV-treated mice were decreased, indicating that Astragaloside IV blocked the translocation of NF-κB to the nucleus and the phosphorylation of IκB-α (fig.4A). In addition, the levels of ERK, SAPK/JNK, and p38 were assessed in catagen-affected skin to investigate whether Astragaloside IV inhibits the phosphorylation of these three MAP kinases (fig.4B, 4C and 4D). Depilated skin contained high levels of ERK, SAPK/JNK and p38; these were decreased by Astragaloside IV. These decreases in MAP kinases were accompanied by dose-dependent reductions in the level of c-Jun.

Levels of keratinocyte premature differentiation markers, which indicate catagen, were evaluated in depilated skin. Figure 5A shows the protein expression of KGF, p21. Astragaloside IV induced KGF expression. Assessment of the level of p21 expression revealed decreases in treated mice.

In addition, we evaluated the protein levels of cytokines known to modulate hair growth and regression using whole-tissue lysates of dorsal skin tissue. Levels of TNF-α and IL-1β were increased in the depilated group (fig.5B). Astragaloside IV significantly reduced the level of IL-1β in a dose-dependent manner, compared to the depilated group. The level of TNF-α was decreased by Astragaloside IV treatment.