Keigairengyoto, a traditional Japanese medicine, promotes bacterial clearance by activating innate immune cells in mouse cutaneous infection models

Abstract

Prompt elimination of pathogens including bacteria and dead cells prevents the expansion of secondary and prolonged inflammations and tissue damage. Keigairengyoto (KRT) is a traditional Japanese medicine prescribed for dermatoses such as purulent inflammations. Our aim is to clarify the actions of KRT in bacterial clearance and to examine the cell-kinetic profiles of phagocytes. In a mouse cutaneous infection model using living Staphylococcus aureus, KRT drastically reduced the number of bacteria in the infection sites. To evaluate the bacterial clearance, pseudo-infection was induced in mouse ears by intradermal injection of FITC-conjugated dead S. aureus. Biochemical and histological examinations revealed that KRT promoted bacterial clearance at 6 and 24 h post-injection. The numbers and phagocytic activities of neutrophils and macrophages in the ears were evaluated histologically using anti-Ly6G and F4/80 antibodies. KRT reduced bacterial deposition and increased the accumulation of F4/80 + resident macrophages around the lesion site. FACS analysis was performed on single cell suspensions dispersed enzymatically from skin lesions, followed by an investigation of CD11b + Ly6G + (neutrophils) and CD11b + Ly6G -(monocytes/macrophages) cells. KRT increased the mean fluorescent intensity of FITC in CD11b Ly6G-cells and the number of FITCpositive CD11b + Ly6G+ cells, while KRT did not change the numbers of these cells. To investigate the active constituents of KRT, phagocytosis assay using macrophages was performed, resulting in that some flavonoid glucuronides of KRT derivatives augmented phagocytosis. Collectively, KRT promoted bacterial clearance by enhancing the phagocytic capability of neutrophils and macrophages. KRT may exert unique properties in preventive and therapeutic strategies for skin infectious inflammation.

Pharmacokinetic Study of Bioactive Flavonoids in the Traditional Japanese Medicine Keigairengyoto Exerting Antibacterial Effects against Staphylococcus aureus

“…Second, KRT may exert antimicrobial effects by enhancing immune system activity, such as macrophage phagocytosis. We previously examined the effects of KRT in a pseudo-infection model using heat-killed S. aureus in order to eliminate the possibility of direct bactericidal effects and observed augmented bacteria clearance as evidenced by biochemical, histological, and immunological methods [ 18 ]. Moreover, macrophage activation was promoted by treatment with phytoestrogen flavonoids [ 23 , 24 , 25 , 26 , 27 , 28 , 29 ].…”

“…Glycyrrhetinic acid, the main metabolite of glycyrrhizic acid, was detected in the plasma after KRT administration. Our previous data showed that glycyrrhetinic acid has no macrophage phagocytosis-enhancing activity [ 18 ]. However, many pharmacological effects have been reported for glycyrrhetinic acid, including anti-inflammatory [ 37 ], anti-allergic [ 38 ], and analgesic effects [ 39 ], suggesting that glycyrrhetinic acid contributes to KRT efficacies.…”

“…Moreover, macrophage culture assays revealed that genistein 7- O -glucuronide increased phagocytosis, and that both β-glucuronidase and estrogen receptor signaling in macrophages were involved in this activation. Since genistein, liquiritigenin, and their glucuronides also appeared in the plasma of KRT-administered rats, these may be active ingredients in KRT as in Jumihaidokuto [ 18 ]. However, KRT includes more abundant flavonoid-rich crude drugs than Jumihaidokuto, such as Scutellariae radix (baicalin, baicalein, wogonin), Aurantii fructus immaturus (naringenin, hesperetin), Schizonepetae spica (apigenin, hesperetin, luteolin), and Glycyrrhizae radix (genistein, liquiritigenin).…”

“…In the present study, KRT was administrated orally to the experimental animals at 2 g/kg in distilled water. Although the dose of KRT was higher than the clinical dose used in humans, this dose was based on previous publications of experimental KRT studies [ 18 ] reporting the beneficial effects of KRT related to clinical efficacy. No adverse effects were observed in animals given 2 g/kg KRT in the present experiment.…”

“…The three-dimensional high-performance liquid chromatograph (3D-HPLC) profile of KRT provided by Tsumura & Co., shows that KRT includes various compounds, especially flavonoids, triterpenoids, and isoquinoline alkaloids ( Figure 1 ). We previously reported that oral administration of KRT suppressed microbe-induced dermatosis in mice and enhanced bacterial clearance through modulation of the immune system [ 18 ]. It is likely that bioactive flavonoids derived from KRT mediate the adjuvanticity against microbes.…”