Maryam Bahrami - Department of Immunology & Microbiology, School of Medicine, Arak University of Medical Sciences, Arak, Iran.
Ali Ghazavi - Traditional and Complementary Medicine Research Center (TCMRC), Department of Microbiology and Immunology, School of Medicine, Arak University of Medical Sciences, Arak, Iran.
Ali Ganji - Molecular and Medicine Research Center, Department of Microbiology and Immunology, School of Medicine, Arak University of Medical Sciences, Arak, Iran.
Ghasem Mosayebi - Molecular and Medicine Research Center, Department of Microbiology and Immunology, School of Medicine, Arak University of Medical Sciences, Arak, Iran.

Background: Nigella sativa (N. sativa) and Silybum marianum (S. marianum) are used to regulate macrophage polarization in lipopolysaccharide-induced RAW ۲۶۴.۷ cells and thioglycollate-elicited peritoneal inflammation. Methods: Cytotoxicity assays and acute toxicity tests were performed to investigate the safe dose and toxicity of the prepared extracts. Also, nitric oxide production was determined by Griess assay on RAW۲۶۴.۷ and peritoneal macrophage supernatants. After RNA extraction from macrophages, real-time PCR was performed to measure the relative gene expression of tumor necrosis factor (TNF)-α, interleukin (IL)-۶, transforming growth factor (TGF)-β, and IL-۱۰. Finally, regulatory T cells (Treg cells) were counted by flow cytometry. Results: S. marianum methanolic extract (SME), N. sativa ethanolic extract (NEE), and their mixture (SME+NEE) decreased NO levels significantly in RAW۲۶۴.۷ and peritoneal murine macrophages. N. sativa ethanolic extract significantly increased IL-۱۰ gene expression and significantly decreased IL-۶ and TNF-α expression in RAW۲۶۴.۷ cells. In mixture-treated peritoneal macrophages, IL-۱۰ and TGF-β expression were significantly increased, while IL-۶ and TNF-α were significantly decreased. Also, the percentage of Treg cells was significantly greater in the mixture-treated cells than in controls. Conclusions: These results suggest that an SME and NEE mixture has anti-inflammatory and immunomodulatory activities and may be useful in the treatment of diseases of immunopathologic origin characterized by macrophage hyperactivation.

Cytokine, Inflammation, Nigella sativa, Nitric oxide, Silybum marianum.