Takahiro Funatsu

Effects of Hangeshashinto on Growth of Oral Microorganisms.

Oral mucositis (OM) in cancer patients induced by chemotherapy or radiotherapy has a significant impact on quality of life, and causes considerable morbidity. Oral microorganisms are likely to intensify the inflammatory process and aggravate the formation of ulcers. Hangeshashinto (HST), a Japanese kampo medicine, has been reported to be effective when used as a gargle for the treatment of OM. To clarify the effects of HST on oral microorganisms, we assessed its antimicrobial activity against 27 microbial species, including 19 oral bacteria and one fungus. HST extract inhibited the growth of Gram-negative bacteria, including Fusobacterium nucleatum, Porphyromonas gingivalis, Porphyromonas endodontalis, Prevotella intermedia, Prevotella melaninogenica, Tannerella forsythia, Treponema denticola, and Porphyromonas asaccharolytica, though inhibitory effects were less pronounced for Gram-positive bacteria and the fungal strain. We then investigated the effects of antibacterial activities on 15 purified ingredients of HST and determined that baicalein, berberine, coptisine, [6]-shogaol, and homogentisic acid actively inhibited the growth of these bacteria. These findings showed that HST inhibits the growth of specific Gram-negative periodontopathogenic bacteria, which are significant pathogens in OM, without disturbing the normal oral flora. Our data suggest that HST may be a useful treatment for OM in patients undergoing anticancer treatment.

Microarray and gene co-expression analysis reveals that melatonin attenuates immune responses and modulates actin rearrangement in macrophages

Melatonin produced by the pineal gland suppresses inflammatory responses in innate immune cells. However, the mechanism of how melatonin affects inflammatory gene regulation remains unclear. Here we performed comprehensive microarray analysis combined with transcription factor binding site (TFBS) analysis using LPS-induced mouse macrophages to investigate the effect of melatonin treatment. The results showed that melatonin preferentially downregulated interferon regulatory factors (IRFs) and signal transducers and activators of transcription (STATs) related signaling. The results also showed that melatonin strongly suppressed virus infection related gene expression. Furthermore, TFBS analysis implicated that melatonin downregulated the binding activity of hypoxia inducible factors (HIFs), following destabilizing actin cytoskeleton which are indispensable for induction of the TRIF-dependent signaling pathway. Indeed, it was demonstrated that melatonin treatment caused impaired phagocytosis in macrophages. Thus, melatonin regulates inflammatory responses by inhibiting specific subsets of transcription factors (TFs) by disrupting actin dynamics in the macrophage.

The inhibition of malignant melanoma cell invasion of bone by the TLR7 agonist R848 is dependent upon pro-inflammatory cytokines produced by bone marrow macrophages

Distant metastasis remarkably worsens the prognoses of malignant melanoma patients. Toll-like receptors (TLRs) recognize molecules derived from many types of pathogens and activate the innate intravital immune system. In this study, we examined the effects of R848, a TLR7 ligand, on bone invasion by malignant melanoma cells. Mice underwent transplantation with cells of a malignant melanoma cell line B16F10, and were also administered R848 every three days. Hindlimbs were obtained 13 days after transplantation and invasion of bone marrow by B16F10 cells was evaluated. ELISA was used to determine the concentrations of cytokines in mouse serum and in the culture medium from bone marrow macrophages (BMMs) in the presence or absence of R848. In addition, MTS assays were used to examine the effects of media from BMM cultures on the proliferation of B16F10 cells. The rate of infiltration by B16F10 cells and the area of invasion were significantly reduced with R848 administration. Furthermore, serum levels of IL-6, IL-12, and IFN-γ were significantly increased in mice administered R848, with the same trend observed in the culture medium of BMMs treated with R848. In addition, B16F10 cell proliferation was suppressed by the addition of medium from cultured BMMs treated with R848. Neutralization by antibodies against IL-6, IL-12, and IFN-γ abrogated the suppression of proliferation of B16F10 cells by culture medium from BMMs treated with R848. Our results suggest that R848 drives the production of IL-6, IL-12, and IFN-γ in BMMs, which reduces proliferation and bone invasion by B16F10 cells.