Amelioration of atopic-like skin conditions in NC/Tnd mice by topical application with distilled Alpinia intermedia Gagnep extracts

Abstract

Alpinia intermedia, a perennial plant that belongs to the Zingiberaceae family, has been used in folk medicine for a long time in the southern region of Japan. Because skin care is an effective approach that enables patients to manage their atopic dermatitis (AD), various herbal ingredients with few adverse effects have been evaluated for use in AD patients in recent years. In this study, we examined whether distilled extracts obtained from A. intermedia were beneficial for AD-like skin conditions in NC/Tnd mice. Topical application with the A. intermedia extracts significantly reduced the severity of AD, transepidermal water loss and scratching behavior in the mice. Supplementation of the extracts to cell cultures suppressed the expression of Tslp mRNA in PAM212 keratinocytes, degranulation in bone marrow-derived cultured mast cells (BMCMC), and neurite outgrowth in PC12 cells and dorsal root ganglia. In addition, the component analysis revealed that β-pinene was a major constituent of the A. intermedia extracts. The inhibitory effects of β-pinene both in vivo and in vitro were also demonstrated. These results indicate that topical application with the A. intermedia extract to the skin of NC/Tnd mice improved the condition of the skin by suppressing multiple inflammatory responses. The extracts may become novel skin-care remedies for AD patients.

Keywords: Alpinia intermedia; NC/Tnd mouse; atopic dermatitis; skin care; β-pinene.

Figure 1

Effects of Alpinia intermedia extracts on the development of atopic dermatitis and scratching behavior in NC/Tnd mice. (a) The clinical skin severity scores of the mice. Data represent the means and the standard errors of the means (SE) from the results from seven mice in each group. *P < 0.05 compared with the vehicle group by using Welch's t‐test. (b) Representative photographs of a mouse in each treatment group. (c) Transepidermal water loss (TEWL) analysis. Data represent the means and SE of the results from seven mice in each group. **P < 0.01 compared with the vehicle group by using Welch's t‐test. (d) The scratching frequencies and (e) the total scratching durations are represented. Both sets of data were based on 30‐min evaluations of the mice that were treated with the vehicle or the A. intermedia extracts for 2–6 weeks. *P < 0.05 for the scratching frequency and **P < 0.01 for the total scratching durations compared with the vehicle group by using Welch's t‐test. (f) The effects of diluted extracts application on the development of atopic dermatitis (AD) and scratching behavior in NC/Tnd mice. Either undiluted (100%) or diluted (25%) extracts were applied to the skin of NC/Tnd mice for 2 weeks, and the number of scratching behavior was counted. Each point represents the mean and SE of the accumulated number of scratching behaviors of four mice in each group. *P < 0.05 compared with the control by using Dunnett's test. (g,h) Microscopic features of the skin after treatment with either vehicle or A. intermedia extracts for 6 weeks (original magnification ×200; scale bar, 100 μm. The arrowheads indicate the mast cells. (h) Each column represents the means ± SE of the mast cell numbers from five randomly selected microscopic fields. **P < 0.01 compared with the vehicle group by using Welch's t‐test. (i) Immunohistochemical analysis of the skin after treatment with either vehicle or A. intermedia extracts for 6 weeks. The tissues were incubated with an anti‐PGP9.5 antibody, followed by incubation with the Alexa Fluor 488‐conjugated anti‐rabbit immunoglobulin (Ig)G antibody and 4′,6‐diamidino‐2‐phenylindole (DNP) (original magnification ×200; scale bar, 50 μm). The arrowheads indicate the PGP9.5‐positive neurites. (j) The average fold change ± SE of PGP9.5 staining intensity from isotype control‐stained sections is indicated (n = 7, respectively). *P < 0.05 compared with the vehicle group by using Welch's t‐test. H‐E, hematoxylin–eosin.

Figure 2

Inhibitory effects of the Alpinia intermedia extracts on the inflammatory responses. (a) Tslp mRNA production in PAM212 keratinocytes. The cells were serum‐starved for 12 h, stimulated with 1 mg/mL lipoteichoic acid (LTA) for 12 h in the presence or absence of the A. intermedia extracts, and the real‐time reverse transcription polymerase chain reaction was carried out. Data represent the means and the standard errors (SE) from three independent experiments. **P < 0.01 compared with the LTA treatment group by using Dunnett's test. (b,c) Degranulation responses of the bone marrow‐derived cultured mast cells (BMCMC) from (b) an NC/Tnd mouse and (c) a C57BL/6J mouse. The cells were treated with anti‐dinitrophenyl (DNP) immunoglobulin (Ig)E and DNP‐bovine serum albumin following treatment with the A. intermedia extracts for 2 h. Data represent the means and the SE from three independent experiments. **P < 0.01 compared with the control by using Dunnett's test. (d,e) Effects of the A. intermedia extracts on neurite outgrowth in PC12 cells. The nerve growth factor (NGF)‐primed cells were incubated with 50 ng/mL NGF and the A. intermedia extracts for 48 h, and the (d) number of neurite‐bearing cells as well as the (e) number of neurites per cell were counted. Data represent the means and SE from three independent experiments. **P < 0.01 compared with the control by using Dunnett's test.

Figure 3

Gas chromatography mass spectrometry (GC‐MS) chromatograms of the Alpinia intermedia extracts. Peak 1, camphene; peak 2, β‐pinene; peak 3, m‐cymene; and peak 4, limonene.

Figure 4

Effects of β‐pinene on the development of atopic dermatitis and the scratching behavior in NC/Tnd mice. (a) The clinical skin severity score data from the mice. Data represent the means and standard errors (SE) from the results from seven mice in each group. *P < 0.05 compared with the control mice by using Dunnett's test. (b) The scratching frequencies and (c) the total scratching durations are presented. Both sets of data were based on 30‐min evaluation of the mice that were treated with the vehicle or β‐pinene extracts for 1–2 weeks. *P < 0.05 compared with the control group by using Dunnett's test. (d) Tslp mRNA production in the PAM212 keratinocytes. The cells were serum‐starved for 12 h, stimulated with 1 mg/mL lipoteichoic acid (LTA) for 12 h in the presence or absence of β‐pinene, and then the real‐time reverse transcription polymerase chain reaction was carried out. Data represent the means and SE from three independent experiments. **P < 0.01 compared with the LTA treatment group by using Dunnett's test. (e) Degranulation responses of bone marrow‐derived cultured mast cells (BMCMC) derived from NC/Tnd mice. The cells were treated with anti‐dinitrophenyl (DNP) immunoglobulin (Ig)E and DNP‐bovine serum albumin after being treated with β‐pinene for 2 h. Data represent the means and SE from three independent experiments. **P < 0.01 compared with the control group by using Dunnett's test. (f) Effects of β‐pinene on neurite outgrowth in PC12 cells. Nerve growth factor (NGF)‐primed cells were incubated with 50 ng/mL NGF and β‐pinene for 48 h, and the number of neurite‐bearing cells was counted. Data represent the means and SE from three independent experiments. **P < 0.01 compared with the control by using Dunnett's test.

Figure 5

Effects of Alpinia intermedia extracts and β‐pinene on neurite outgrowth in NC/Tnd mice‐derived dorsal root ganglia (DRG). (a) Cells were incubated with either the extracts or β‐pinene for 72 h, and the number of neurite‐bearing cells was counted. Data represent the means and standard errors (SE) from three independent experiments. *P < 0.05 and **P < 0.01 compared with the control by using Dunnett's test, respectively. (b) Representative photographs of the neurites cultured for 3 days. Cells were stained with anti‐PGP9.5 antibody and Alexa Fluor 488‐conjugated anti‐rabbit immunoglobulin (Ig)G antibody (original magnification ×200; scale bar, 100 μm). The arrowheads indicate ganglion cells. Possible signaling pathways involved in the suppressive effects of A. intermedia extracts and β‐pinene. (c) Western blot analysis of PAM212 cells and the quantification of the ratio of the phosphorylated : total amounts of Stat3. The cells were serum‐starved for 12 h, stimulated with 1 mg/mL lipoteichoic acid (LTA) for 12 h in the presence or absence of the A. intermedia extracts or β‐pinene. The relative mean pStat3 : Stat3 ratios ± SE from three independent experiments are shown. **P < 0.01 compared with treatment with LTA treatment by using Dunnett's test. (d) Western blot analysis of the PC12 cells and the quantification of the ratio of the phosphorylated : total amounts of Stat6. NGF‐primed PC12 cells were incubated with 50 ng/mL NGF and either extracts of A. intermedia or β‐pinene for 48 h. The relative mean pStat6 : Stat6 ratios ± SE from three independent experiments are shown. *P < 0.05 and **P < 0.01, compared with treatment with NGF alone by using Dunnett's test. Med, medium alone; Ext., 5% A. intermedia extracts; Pin., 0.025% β‐pinene. (e) Western blot analysis of DRG and the quantification of the ratio of the phosphorylated : total amounts of Stat6. DRG were incubated with either extracts of A. intermedia or β‐pinene for 24 h. The relative mean pStat6 : Stat6 ratios ± SE from three independent experiments are shown. **P < 0.01 compared with treatment with medium alone by using Dunnett's test. Med, medium alone; Ext., 5% A. intermedia extracts; Pin., 0.025% β‐pinene.