Anti-inflammatory effects of thymoquinone in activated BV-2 microglial cells

Abstract

Thymoquinone (TQ), the main pharmacological active ingredient within the black cumin seed (Nigella sativa) is believed to be responsible for the therapeutic effects on chronic inflammatory conditions such as arthritis, asthma and neurodegeneration. In this study, we evaluated the potential anti-inflammatory role of TQ in lipopolysaccharide (LPS)-stimulated BV-2 murine microglia cells. The results obtained indicate that TQ was effective in reducing NO2(-) with an IC50 of 5.04μM, relative to selective iNOS inhibitor LNIL-l-N6-(1-iminoethyl)lysine (IC50 4.09μM). TQ mediated reduction in NO2(-) was found to parallel the decline of iNOS protein expression as confirmed by immunocytochemistry. In addition, we evaluated the anti-inflammatory effects of TQ on ninety-six (96) cytokines using a RayBio AAM-CYT-3 and 4 cytokine antibody protein array. Data obtained establish a baseline protein expression profile characteristic of resting BV-2 cells in the order of osteopontin>MIP-1alpha>MIP-1g>IGF-1 and MCP-I. In the presence of LPS [1ug/ml], activated BV-2 cells produced a sharp rise in specific pro-inflammatory cytokines/chemokine's IL-6, IL-12p40/70, CCL12 /MCP-5, CCL2/MCP-1, and G-CSF which were attenuated by the addition of TQ (10μM). The TQ mediated attenuation of MCP-5, MCP-1 and IL-6 protein in supernatants from activated BV-2 cells were corroborated by independent ELISA. Moreover, the data obtained from the RT(2) PCR demonstrated a similar pattern where the LPS mediated elevation of mRNA for IL-6, CCL12/MCP-5, CCL2/MCP-1 were significantly attenuated by TQ (10μM). Also, in this study, consistent data were obtained for both protein antibody array densitometry and ELISA assays. In addition, TQ was found to reduce LPS mediated elevation in gene expression of Cxcl10 and a number of other cytokines in the panel. These findings demonstrate the significant anti-inflammatory properties of TQ in LPS activated microglial cells. Therefore, the obtained results might indicate the usefulness of TQ in delaying the onset of inflammation-mediated neurodegenerative disorders involving activated microglia cells.

Keywords: Black cumin seed; Cytokines; Microglial cells, Parkinson's, neurodegeneration; Nitric oxide; Thymoquinone.