Thymoquinone is a known inhibitor of neuroinflammation. However, the mechanism(s) involved in its action remain largely unknown. In this study, we investigated the roles of cellular reactive oxygen species (ROS), 5′ AMP-activated protein kinase (AMPK) and sirtuin 1 (SIRT1) in the anti-neuroinflammatory activity of thymoquinone. We investigated effects of the compound on ROS generation in LPS-activated microglia using the fluorescent 2′,7′-dichlorofluorescin diacetate (DCFDA)-cellular ROS detection. Immunoblotting was used to detect protein levels of p40phox, gp91phox, AMPK, LKB1 and SIRT1. Additionally, ELISA and immunofluorescence were used to detect nuclear accumulation of SIRT1. NAD+/NADH assay was also performed. The roles of AMPK and SIRT1 in anti-inflammatory activity of thymoquinone were investigated using RNAi and pharmacological inhibition. Our results show that thymoquinone reduced cellular ROS generation, possibly through inhibition of p40phox and gp91phox protein. Treatment of BV2 microglia with thymoquinone also resulted in elevation in the levels of LKB1 and phospho-AMPK proteins. We further observed that thymoquinone reduced cytoplasmic levels and increased nuclear accumulation of SIRT1 protein and increased levels of NAD+. Results also show that the anti-inflammatory activity of thymoquinone was abolished when the expressions of AMPK and SIRT1 were suppressed by RNAi or pharmacological antagonists. Pharmacological antagonism of AMPK reversed thymoquinone-induced increase in SIRT1. Taken together, we propose that thymoquinone inhibits cellular ROS generation in LPS-activated BV2 microglia. It is also suggested that activation of both AMPK and NAD+/SIRT1 may contribute to the anti-inflammatory, but not antioxidant activity of the compound in BV2 microglia.
Objectives The effects of a root extract of Zanthoxylum zanthoxyloides on neuroinflammation in BV-2 microglia stimulated with LPS and hemozoin were investigated. Methods ELISA, enzyme immunoassay and Griess assay were used to evaluate levels of cytokines, PGE2 and NO in culture supernatants, respectively. Microglia-mediated neurotoxicity was evaluated using a BV-2 microglia-HT-22 neuron transwell co-culture. Key findings Treatment with Z. zanthoxyloides caused reduced elevated levels of TNFα, IL-6, IL-1β, NO and PGE2, while increasing the levels of IL-10. In addition, there were reduced levels of iNOS and COX-2 proteins. This was accompanied by a prevention of microglia-mediated damage to HT-22 mouse hippocampal neurons. Z. zanthoxyloides reduced elevated levels of phospho-IκB and phospho-p65, while preventing degradation of IκB protein and DNA binding of p65. Further mechanistic studies revealed that Z. zanthoxyloides reduced the levels of pro-IL-1β and IL-1β in hemozoin-activated BV-2 microglia. This was accompanied by a reduction in caspase-1 activity and NLRP3 protein expression. Bioassay-guided fractionation resulted in the isolation of skimmianine as an anti-inflammatory compound in Z. zanthoxyloides. Conclusion This is the first report showing the inhibition of neuroinflammation in LPS- and hemozoin-activated BV-2 microglia by the root extract of Z. zanthoxyloides by targeting the activation of both NF-κB and NLRP3 inflammasome.
Skimmianine is a furoquinoline alkaloid which is found in the Zanthoxylum genus and also in other plants of the Rutaceae family. This study evaluated the effects of skimmianine on the production of pro-inflammatory mediators in LPS-activated BV-2 microglia. Cultured BV-2 cells were treated with skimmianine (10, 20 and 30 μM), followed by stimulation with LPS (100 ng/mL). Levels of TNFα and IL-6 in cell supernatants were measured using ELISA, while NO and PGE2 levels were evaluated with Griess assay and EIA, respectively. Western blotting was used to determine the protein expression of iNOS, COX-2, phospho-p65 and phospho-IκBα. Results showed that Skimmianine reduced LPS-induced elevated the secretion of TNFα, IL-6, NO, and PGE2, as well as the increased protein expression of iNOS and COX-2. Experiments to elucidate the mechanisms of the anti-neuroinflammatory activity of skimmianine revealed the significant inhibition of LPS-induced increased NF-κB-mediated luciferase activity. Pre-treatment with skimmianine also reduced LPS-induced the increased phosphorylation of NF-κB/p65 and IκBα proteins. Furthermore, skimmianine interfered with the binding capacity of NF-κB to consensus sites. Skimmianine pre-treatment protected HT-22 cells from toxicity induced by microglia-conditioned media, as well as increasing MAP-2 expression. The results of this study suggest that skimmianine inhibits neuroinflammation in LPS-activated microglia by targeting the NF-κB activation pathway. Skimmianine also produced neuroprotection against neurotoxicity induced by microglia-conditioned media.
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