Immunopotentiator from Pantoea agglomerans 1 (IP-PA1) Promotes Murine Hair Growth and Human Dermal Papilla Cell Gene Expression

Materials and Methods

Sample preparation. Wheat fermentation extract (immunopotentiator from Pantoea agglomerans 1: containing IP-PA1 6.8 mg/g; lot.130222) was purchased from Macrophi Inc. (Shikoku, Japan). A small amount of the extract (0.5 g) was dissolved in 100 ml distilled water to prepare a sample for oral administration (IP-PA1-PO). Five grams of wheat fermentation extract was dissolved in 100 ml distilled water to prepare a sample for skin application (IP-PA1-SC). For the minoxidil skin application, a commercially available product, RiUP X plus 5%; lot.055D1, was purchased from Taisho Pharmaceutical Co., Ltd. (Tokyo, Japan).

For cell experiments, an IP-PA1 purified product, lot.M2, was purchased from Macrophi Inc. and dissolved in dimethyl sulfoxide (DMSO) (Nacalai tesque Inc., Kyoto, Japan) to prepare a 500 mg/ml stock solution. Adenosine from Wako Inc. (Tokyo, Japan) was dissolved in DMSO to a final concentration of 100 mM and minoxidil from Sigma-Aldrich Japan (Tokyo, Japan) was dissolved in 50% ethanol to a final concentration of 15 mM to prepare stock solutions, which were used as positive controls.

Dermal papilla cell growth media from TOYOBO Inc. (Osaka, Japan), dermal papilla cell subculturing unit (TOYOBO Inc.) and live cell counting reagent SF from Nakalai tesque Inc. were used for cell cultures .

Forty-eight-well Cellmatrix Type I-C (Nitta-gelatin Inc., Osaka, Japan) culture plates were used. FastLane Cell® cDNA Kit (QIAGEN Co., Ltd., Venlo, Netherlands), SYBER® Premix Ex Taq 5 ml (Takara Inc., Shiga, Japan), oligonucleotides (primers; FASMAC Inc., Kanagawa, Japan) (Table I) were purchased for use in quantitative real-time PCR (qPCR).

Murine hair growth test. Seven-week-old C3H/HeN male mice were purchased from Charles River Japan, Inc. (Kanagawa, Japan). They were housed in individual cages under conventional conditions consisting of 12-h light-dark cycle, 23±1°C temperature and 55±15% humidity.

The back of the mice were consistently shaved using an electronic shaver PHILIPS Co Ltd., (Amsterdam, Netherlands) and animals divided into four groups of eight individuals each as follows: the group allowed to freely intake IP-PA1-PO from a water bottle; two groups that received a skin application of 250 μl IP-PA1-SC or minoxidil (RiUP X plus 5%) to their backs with a glass bar every day, respectively; and a no treatment (control). These mice were either administered the sample drug or given a skin application. Photographs were taken 14 days later to digitize the state of hair growth as shown in Table II.

At the end of the experiment, animals were sacrificed and skin was obtained. The skin tissues were fixed in 10% neutral-buffered formalin, samples were embedded in paraffin and thin sections (5 μm) were cut. Sections were stained with hematoxylin and eosin (H&E) solution. Skin lesions were histologically examined using a light microscope (Olympus AX70; Olympus, Tokyo, Japan) with 40× and 100× objective lenses.

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Figure 1.

Changing hair growth score in oral or topical application of IP-PA1. The back hair of mice was shaved and, after 14 days, hair growth efficiency was evaluated. A) Oral administration of IP-PA1-PO sample (0.5% free access). B) Topical application of minoxidil (RiUP X plus 5%) or IP-PA1-SC (5%) samples. Data are shown as means±SD. Significant differences (*p<0.05, **p<0.01) were obtained between control and samples on topical application examination. M=Median.

The present investigation conforms to the Guiding Principles for the Care and Use of Experimental Animal of Hokkaido Pharmaceutical University (published 1998, revised in 2001 and 2007). The protocol approval number was H27-009.

In vitro cell viability test. Human dermal papilla cells were obtained from the calvaria of a 63-year-old Caucasian male (Code No. CA602t05a; TOYOBO Inc.). Cells were cultivated using designated culture media and plates.

For the cytotoxicity test, a control (final concentration of 0.1% DMSO) and IP-PA1 (final concentration of 0.1% DMSO) were adjusted to seven different concentrations. These samples were added to cells that had reached confluence (n=3). Cells were further incubated for 48 h and viability was measured using SF reagent (OD 450 nm).

cDNA preparation. FastLane Cell® cDNA Kit was used to extract total RNA and 2 μl gDNA Wipeout Buffer, 1 μl Fast Lane Lysate, as well as 1 μl RNase-Free Water were added to a PCR tube, followed by incubation at 42°C for 5 min. Then, 6 μl reverse transcription master mix solution (Quantiscript Reverse Transcriptase 1 μl, Quantiscript RT Buffer 4 μl, RT Primer Mix 1 μl) was added and incubated at 42°C for 30 min. Finally, the mixture was incubated at 95°C for 3 min to inactivate the reverse transcriptase. The product was used as synthesized cDNA for the qPCR analysis.

Quantitative real-time polymerase chain reaction. qPCR was performed using SYBER® Premix Ex Taq. Previously reported primers were used (Table I). PCR conditions were set at 95°C for 10 s and 60°C for 30 s. Relative quantification was performed by normalizing target expression to the housekeeping gene GAPDH. Data were expressed as change (n-fold) in FGF-7 and VEGF mRNA expression compared with human dermal papilla cells incubated without samples before co-culture.

Statistical analysis. Results are expressed as means±S.E. One-way analysis of variance followed by Tukey's honestly significant difference test was used for comparing differences among multiple groups. Differences were considered significant at **p<0.01 and *p<0.05.