Representative species, skin diseases and Kampo medicines(Chemical & Pharmacological study)

We examined the relationship between the Propionibacterium species and Kampo formulations, between the Staphylococcus species and Shiunko, or between acne vulgaris and Kampo formulations. Jumihaidokuto, Unseiin and Shiunko suppressed Propionibacterium species lipase, and in correlation with the lipase activity, their clinical effect was indicated. Among Shiunko, white petrolatum and 3.5% salt water, Shiunko strongly inhibited the growth of the Staphylococcus species isolated from skin lesions of atopic dermatitis. Also, Kampo formulations were effective for inflammatory acne rash as well as incidental symptoms in acne patients. Especially, the effects of Kampo formulations on the incidental symptoms were remarkably higher than those of antimicrobial agents. These effects might be due to the various actions of Kampo crude drugs composed of Kampo formulations or Shiunko

Involvement of MIF in basement membrane damage in chronically UVB-exposed skin in mice.

Solar ultraviolet (UV) B radiation is known to induce matrix metalloproteinases (MMPs) that degrade collagen in the basement membrane. Macrophage migration inhibitory factor (MIF) is a pluripotent cytokine that plays an essential role in the pathophysiology of skin inflammation induced by UV irradiation. This study examined the effects of MIF on basement membrane damage following chronic UVB irradiation in mice. The back skin of MIF transgenic (Tg) and wild-type (WT) mice was exposed to UVB three times a week for 10 weeks. There was a decrease in intact protein levels of type IV collagen and increased basement membrane damage in the exposed skin of the MIF Tg mice compared to that observed in the WT mice. Moreover, the skin of the MIF Tg mice exhibited higher MIF, MMP-2 and MMP-9 expression and protein levels than those observed in the WT mice. We also found that chronic UVB exposure in MIF Tg mice resulted in higher levels of neutrophil infiltration in the dermis compared with that observed in the WT mice. In vitro experiments revealed that MIF induced increases in the MMPs expression, including that of MMP-9 in keratinocytes and MMP-2 in fibroblasts. Cultured neutrophils also secreted MMP-9 stimulated by MIF. Therefore, MIF-mediated basement membrane damage occurs primarily through MMPs activation and neutrophil influx in murine skin following chronic UVB irradiation

Enhanced expression and production of MIF in the MIF Tg mouse skin following chronic UVB exposure.

<p>The total RNA and protein were isolated at 0, 4 and 10(300 mJ/cm²×3/week). (A) The expression of MIF mRNA was examined by RT-PCR. (B) A Western blot analysis was performed, and β-actin was used to normalize each expression level. A densitometric analysis was also performed to quantify the intensity of each band. (Each experiment was repeated two times with similar results).</p

Enhanced production of MIF, MMP-9 and MMP-2 in the MIF Tg mouse skin following chronic UVB exposure.

<p>(A) Western blot analysis of MIF, MMP-2 and MMP-9 was conducted to evaluate the effects of UVB (300 mJ/cm²×3/week) on the expressions of these compounds in comparison with those observed in WT mice. The band of β-actin was used to normalize each expression level. Each band shows a representative result of the Western blotting analyses, which were repeated five times. A densitometric analysis was also performed to evaluate the intensity of each band. (B) Zymography, using the total skin specimens of each indicative sample, is shown. (These experiments were repeated two times with similar results).</p

Effects of MIF on the acute UVB-induced expression and production of MMP-9 in neutrophils.

<p>Neutrophils (approximately 90% purified) were pretreated with recombinant MIF at different concentrations (10, 50 and 100 μM) for six or 24 hours. (A) Cells were analyzed with RT-PCR of MMP-9. (B) Western blot analysis of the MMP-9 protein expression was then performed. The bands were quantified densitometrically and normalized to the level of β-actin. These experiments were repeated three times with similar results.</p

Effects of an MIF inhibitor (ISO-1) or MIF si RNA on the expression and production of MMP-9 and MMP-2 following UVB radiation.

<p>(A) The cells were treated with ISO-1 at 50 μM for 24 hours or MIF siRNA (30 pmol/ml) for 48 hours. Next, the expression of MIF mRNA and MIF protein was examined. (B) The expression levels of MMP-9 and MMP-2 mRNA were examined. Cells were pretreated with ISO-1 at 50 μM for 24 hours or MIF siRNA (30 pmol/ml) for 48 hours, and then were exposed to UVB light (30 mJ/cm²). Total RNA was isolated six hours after UVB exposure and analyzed by RT-PCR. (C) Cells were pretreated with ISO-1 at 50 μM for 24 hours or MIF siRNA (30 pmol/ml) for 48 hours, and were then exposed to UVB light (30 mJ/cm²). The Western blot analysis for the MMP-9 and MMP-2 proteins (30 μg of protein for each group) was performed 24 hours after UVB irradiation. The band of β-actin was used to normalize the expression level. A densitometric analysis was also performed to evaluate the intensity of each band. The data shown are representative of three independent experiments.</p

Changes in the levels of basement membrane collagen and basement membrane ultrastructure in the MIF Tg mouse skin following chronic UVB exposure.

<p>(A) Western blot analysis of type IV collagen was conducted to evaluate the effects of UVB (300 mJ/cm²×3/week) on the expression of this compound. The band of β-actin was used to normalize the expression level. Each band shows a representative result of the Western blotting analyses, which were repeated five times. A densitometric analysis was also performed to evaluate the intensity of each band. *P<0.001. The values reported here represent the means ± SD as determined by a one-way ANOVA. (B) Murine skin was irradiated with UVB (300 mJ/cm²×3/week) for 10 weeks. The disruption areas of basal lamina were counted under electron microscope at a magnification of ×12000 and expressed as the mean number in ten random fields (two sections per mouse, five mice per group). *P<0.005. The values reported here represent the means ± SD analyzed by Student's <i>t-</i>test. (C) An ultrastructure of junctional cleavages with disruptions of basal lamina in WT mice (a) and MIF Tg mice (b) after 10 weeks of UVB irradiation. An ultrastructure of the normal basal lamina in non-irradiated WT mice (c) and MIF Tg mice (d). Arrowheads indicate the disruptions of basal lamina. Scale bars  = 0.5 μm. (These experiments were repeated two times with similar results).</p

Effects of chronic UVB irradiation on murine skin neutrophils and mast cells.

<p>Murine skin was irradiated with UVB (300 mJ/cm²×3/week) for 10 weeks. Staining of neutrophils and mast cells in the UV irradiated murine skin sections using the H&E and toluidine blue methods. (A) A quantitative analysis of the number of neutrophils in the UV irradiated (0 and 10 weeks) skin sections was analyzed by a one-way ANOVA (n = 5 in each section). (B) UV irradiated skin (H&E, WT and MIF Tg mouse, 10 weeks). Arrowheads point to neutrophils. Scale bars  = 100 μm. (C) A quantitative analysis of the number of mast cells in the UV irradiated (0 and 10 weeks) skin sections was analyzed by a one-way ANOVA (n = 5 in each section). And UV irradiated skin (Toluidine blue, WT and MIF Tg mouse, 10 weeks). Scale bars  = 200 μm. (The experiments were repeated two times and similar results were obtained).</p

Enhanced expression and production of MMP-9 and MMP-2 in the keratinocytes and fibroblasts of the MIF Tg mice following acute UVB exposure.

<p>(A) The expressions of MIF, MMP-2 and MMP-9 mRNA were examined. Total RNA was isolated at six hours after UVB irradiation (30 mJ/cm²) and analyzed with RT-PCR (n = 5). (B) Western blot analysis of MIF, MMP-2 and MMP-9 was performed 24 hours after UVB (30 mJ/cm²) on the production of these compounds. The band of β-actin was used to normalize the expression level. A densitometric analysis was also performed to evaluate the intensity of each band. The data shown are representative of three independent experiments.</p