Suppression of Propionibacterium acnes-Induced Dermatitis by a Traditional Japanese Medicine, Jumihaidokuto, Modifying Macrophage Functions

Abstract

Purpose. Macrophages serve as sweepers of microbes and inflammation-derived wastes and regulators of inflammation. Some traditional Japanese medicines are reported to have adjuvant effects by modifying macrophages. Our aim was to characterize the actions of jumihaidokuto (JHT) for treatment of skin inflammations including acne vulgaris, in which Propionibacterium acnes has pathogenic roles. Methods. Dermatitis was induced in rat ears by intradermal injection of P. acnes. JHT or prednisolone (PDN) was given orally, and ear thickness and histology were evaluated. The effects of constituents and metabolites of JHT on monocytes were tested by cell-based assays using the human monocytic THP-1 cell. Results. JHT and PDN suppressed the ear thickness induced by P. acnes injection. Histological examinations revealed that JHT, but not PDN, promoted macrophage accumulation at 24 h after the injection. PDN suppressed the macrophage chemokine MCP-1 in the inflamed ears, while JHT did not affect it. The JHT constituents liquiritigenin and isoliquiritin increased expression of CD86 (type-1 macrophage marker) and CD192 (MCP-1 receptor) and enhanced phagocytosis by THP-1. Conclusions. JHT suppressed dermatitis, probably by enhancing type-1 macrophage functions, with an action different from PDN. JHT may be a beneficial drug in treatment of skin inflammation induced by P. acnes.

Figure 1

Suppression of P. acnes-induced dermatitis in rats by jumihaidokuto administration. Dermatitis was induced by intradermal injection of heat-killed P. acnes bacteria suspended in saline (0.14 mg/50 μL/ear) into the pinna. The thickness of the ears was measured at 0, 2, 4, 6, and 24 h and 2, 3, 4, 5, 6, and 7 d after the injection. Jumihaidokuto (JHT) suspended in distilled water (DW) was given orally to rats at doses of 0.1, 0.5, 1, or 2 g/10 mL/kg, at 1 h before 6, 24 h, 2, 3, 4, 5, and 6 d after the injection. Prednisolone (PDN, 10 mg/10 mL/kg) was used as a reference drug. Data represent the relative ear thickness, which was standardized against the previous values before the injection. N = 10 (a) or 6-7 (b). ^## P < 0.01 versus saline + DW group, ^{∗, ∗∗} P < 0.05, 0.01 significant versus P. acne + DW group (two-way repeated measures ANOVA + Bonferroni test).

Figure 2

HE staining of pinna 2 h after P. acnes injection. The pinnae were excised at 2 h after the P. acnes injection and evaluated histologically by hematoxylin and eosin (HE) staining. Representative images are shown for saline + DW ((a) and (b)), P. acnes + DW ((c) and (d)), P. acnes + 0.5 g/kg jumihaidokuto (JHT) ((e) and (f)), and P. acnes + 10 mg/kg prednisolone (PDN) ((g) and (h)). Yellow boxes indicate the sites of each magnification. Scale bars: 100 μm.

Figure 3

HE staining of pinna 24 h after P. acnes injection. The pinnae were excised at 24 h after the P. acnes injection and evaluated histologically by hematoxylin and eosin (HE) staining. Representative images of low-power (upper panels) and high-power (middle and lower panels) fields in pinna sections are shown for saline + DW ((a), (b), and (c)), P. acnes + DW ((d), (e), and (f)), P. acnes + 0.5 g/kg jumihaidokuto (JHT) ((g), (h), and (i)), and P. acnes + 10 mg/kg prednisolone (PDN) ((j), (k), and (l)). Yellow boxes indicate the sites of each magnification, and arrows indicate macrophage-like cells. Scale bars: 100 μm.

Figure 4

Immunofluorescent staining of macrophages 24 h after P. acnes injection. Pinnae sectioned at 4 μm were incubated with a PE-labeled anti-rat macrophage subset monoclonal antibody (clone: HIS36) and then mounted with antifade reagent with DAPI. Representative images merged with DAPI are shown for saline + DW (a), P. acnes + DW ((b) and (c)), P. acnes + 0.5 g/kg jumihaidokuto (JHT) ((d) and (e)), and P. acnes + 10 mg/kg prednisolone (PDN) ((f) and (g)). Panels (b), (d), and (f) are images of the inside of abscesses, and panels (c), (e), and (g) are the outside of abscess. Arrowheads indicate macrophages, which stained double positively with PE and DAPI. PE-positive and DAPI-negative cells found in the images are thought to be red blood cells and regarded as false positives for analysis of macrophages. Scale bars: 20 μm.

Figure 5

Jumihaidokuto promoted macrophage accumulation in inflamed ears. Macrophages were stained by the procedures described in Figure 4. Accumulation of macrophages in the inflamed ears was evaluated relative to areas of PE-positive cells per unit area inside or outside the abscess using Biorevo BZ-9000 microscope and BZ-II software (Keyence, Osaka, Japan). N = 5. ^∗∗ P < 0.01 (Dunnett's test).

Figure 6

Jumihaidokuto had no effect on chemokine concentrations in inflamed ears. The pinnae were collected at 2 and 24 h after injection of P. acnes. Each sample was homogenized in cold PBS supplemented with proteinase inhibitors and centrifuged. CINC-1 (a) and MCP-1 (b) concentrations in the supernatants were determined by ELISA. Jumihaidokuto (JHT, 0.5 g/kg), prednisolone (PDN, 10 mg/kg), or distilled water (DW) was administered orally 1 h before and 6 h after bacterial injection. N = 10 (2 h), 4-5 (24 h). ^## P < 0.01 significant versus saline + DW group (Student's t-test), ^{∗, ∗∗} P < 0.05 and 0.01, respectively, and significant versus P. acnes + DW group (Dunnett's test).