Quassin alters the immunological patterns of murine macrophages through generation of nitric oxide to exert antileishmanial activity

The aim of this study was to characterize the in vitro antileishmanial activity of quassin, a traditional Chinese herbal medicine. The cytotoxic effect of quassin was studied in murine peritoneal macrophages at various concentrations using the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide method. The role of quassin as an antileishmanial agent was evaluated by microscopic counting of intracellular amastigotes in macrophages stained with Giemsa. To understand the effector mechanism of quassin-treated macrophages against leishmanial parasites, western blot and real-time PCR analysis of inducible nitric oxide (NO) synthase 2 (iNOS2) were done followed by measurement of NO generation by Griess reaction. The effect of quassin on the production of Th1 cytokines such as interleukin (IL)-12 and tumour necrosis factor (TNF)-alpha and Th2 cytokines such as IL-10 and transforming growth factor-beta was measured by ELISA, and the mRNA expression of these cytokines was analysed by real-time PCR. Quassin at a dose of 25 mu g/mL (64.36 mu M) showed less cytotoxicity to the host murine peritoneal macrophages but at the same dose was effective enough to control the intracellular parasitic load compared with higher doses of quassin. Leishmania donovani is known to exert its pathogenic effects mainly by the suppression of NO generation and subversion of the cellular inflammatory responses in the macrophages. Quassin was found to induce a potent host-protective immune response by enhancing NO generation and iNOS2 expression both at a protein and mRNA level and by up-regulating pro-inflammatory cytokines such as TNF-alpha and IL-12 in L. donovani-infected macrophages with concurrent inhibition of anti-inflammatory responses. These findings strongly support the effectiveness of quassin as a potent immunomodulatory tool for controlling the establishment of leishmanial parasite within the host macrophages.