Effects of the Chinese traditional medicine mao-bushi-saishin-to on therapeutic efficacy of a new benzoxazinorifamycin, KRM-1648, against Mycobacterium avium infection in mice

Abstract

The Chinese traditional medicine mao-bushi-saishin-to (MBST), which has anti-inflammatory effects and has been used to treat the common cold and nasal allergy in Japan, was examined for its effects on the therapeutic activity of a new benzoxazinorifamycin, KRM-1648 (KRM), against Mycobacterium avium complex (MAC) infection in mice. In addition, we examined the effects of MBST on the anti-MAC activity of murine peritoneal macrophages (M phi s). First, MBST significantly increased the anti-MAC therapeutic activity of KRM when given to mice in combination with KRM, although MBST alone did not exhibit such effects. Second, MBST treatment of M phi s significantly enhanced the KRM-mediated killing of MAC bacteria residing in M phi s, although MBST alone did not potentiate the M phi anti-MAC activity. MBST-treated M phi s showed decreased levels of reactive nitrogen intermediate (RNI) release, suggesting that RNIs are not decisive in the expression of the anti-MAC activity of such M phi populations. MBST partially blocked the interleukin-10 (IL-10) production of MAC-infected M phi s without affecting their transforming growth factor beta (TGF-beta)-producing activity. Reverse transcription-PCR analysis of the lung tissues of MAC-infected mice at weeks 4 and 8 after infection revealed a marked increase in the levels of tumor necrosis factor alpha, gamma interferon (IFN-gamma), IL-10, and TGF-beta mRNAs. KRM treatment of infected mice tended to decrease the levels of the test cytokine mRNAs, except that it increased TGF-beta mRNA expression at week 4. MBST treatment did not affect the levels of any cytokine mRNAs at week 8, while it down-regulated cytokine mRNA expression at week 4. At week 8, treatment of mice with a combination of KRM and MBST caused a marked decrease in the levels of the test cytokines mRNAs, especially IL-10 and IFN-gamma mRNAs, although such effects were obscure at week 4. These findings suggest that down-regulation of the expression of IL-10 and TGF-beta is related to the combined therapeutic effects of KRM and MBST against MAC infection.

FIG. 1

Effects of MBST on Mφ anti-MAC activity. Mφs were preincubated in culture medium in the absence or presence of 1-, 10-, or 100-μg/ml MBST for 2 days, infected with MAC for 2 h, and further cultured in the same medium with or without the addition of MBST at corresponding doses (1, 10, or 100 μg/ml, respectively), in the absence (A) or the presence (B) of 1-μg/ml KRM, for up to 7 days. Symbols in panel A: ○, without MBST treatment; ●, ▴, ■, treated with 1-, 10-, or 100-μg/ml MBST, respectively. Symbols in panel B: ○, no drug added; ▵, KRM alone; ▴, ■, treatment with KRM plus MBST at 10 and 100 μg/ml, respectively. Each symbol indicates the mean ± the standard error of the mean (n = 3). The asterisk indicates a value significantly smaller than that of Mφs treated with KRM alone (P < 0.05 by Bonferroni’s multiple t test).

FIG. 2

Effects of MBST on the expression of TNF-α, IFN-γ, IL-10, and TGF-β mRNAs in the lungs of MAC-infected mice at week 4 after infection. Induction of MAC infection and subsequent drug treatment of infected mice were as described in Table 1. RT-PCR analysis of the lungs of mice was done at week 4. The relative intensities of the RT-PCR bands of individual cytokines were calculated by normalizing to the intensity of the β-actin band. The values in parentheses are cytokine band/β-actin band ratios (the mean of six mice). The standard error of the mean ranged from 5 to 28% (TNF-α), from 14 to 29% (IFN-γ), from 11 to 28% (IL-10), and from 3 to 10% (TGF-β).

FIG. 3

Effects of MBST on the expression of TNF-α, IFN-γ, IL-10, and TGF-β mRNAs in the lungs of MAC-infected mice at week 8 after infection. Induction of MAC infection and subsequent drug treatment of infected mice were as described in Table 2. The other details are the same as those described in the legend to Fig. 2, except that mice were sacrificed at week 8.