Neuroinflammation and microglial activation are pathological markers of a number of central nervous system (CNS) diseases. Chronic activation of microglia induces the release of excessive amounts of reactive oxygen species (ROS) and pro-inflammatory cytokines. Additionally, chronic microglial activation has been implicated in several neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease. Thymoquinone (TQ) has been identified as one of the major active components of the natural product Nigella sativa seed oil. TQ has been shown to exhibit anti-inflammatory, anti-oxidative, and neuroprotective effects. In this study, lipopolysaccharide (LPS) and interferon gamma (IFNγ) activated BV-2 microglial cells were treated with TQ (12.5 μM for 24 h). We performed quantitative proteomic analysis using Orbitrap/Q-Exactive Proteomic LC-MS/ MS (Liquid chromatography-mass spectrometry) to globally assess changes in protein expression between the treatment groups. Furthermore, we evaluated the ability of TQ to suppress the inflammatory response using ELISArray™ for Inflammatory Cytokines. We also assessed TQ's effect on the gene expression of NFκB signaling targets by profiling 84 key genes via real-time reverse transcription (RT2) PCR array. Our results indicated that TQ treatment of LPS/IFNγ-activated microglial cells significantly increased the expression of 4 antioxidant, neuroprotective proteins: glutaredoxin-3 (21 fold; p < 0.001), biliverdin reductase A (15 fold; p < 0.0001), 3-mercaptopyruvate sulfurtransferase (11 fold; p < 0.01), and mitochondrial lon protease (> 8 fold; p < 0.001) compared to the untreated, activated cells. Furthermore, TQ treatment significantly (P < 0.0001) reduced the expression of inflammatory cytokines, IL-2 = 38%, IL-4 = 19%, IL-6 = 83%, IL-10 = 237%, and IL-17a = 29%, in the activated microglia compared to the untreated, activated which expression levels were significantly elevated compared to the control microglia: IL-2 = 127%, IL-4 = 151%, IL-6 = 670%, IL-10 = 133%, IL-17a = 127%. Upon assessing the gene expression of NFκB signaling targets, this study also demonstrated that TQ treatment of activated microglia resulted in > 7 fold down-regulation of several NFκB signaling targets genes, including interleukin 6 (IL6), complement factor B (CFB), chemokine (C–C motif) ligand 3 (CXCL3), chemokine (C–C) motif ligand 5 (CCL5) compared to the untreated, activated microglia. This modulation in gene expression counteracts the > 10-fold upregulation of these same genes observed in the activated microglia compared to the controls. Our results show that TQ treatment of LPS/IFNγ-activated BV-2 microglial cells induce a significant increase in expression of neuroprotective proteins, a significant decrease in expression inflammatory cytokines, and a decrease in the expression of signaling target genes of the NFκB pathway. Our findings are the first to show that TQ treatment increased the expression of these neuroprotective proteins (biliverdin reductase-A, 3-mercaptopyruvate sulfu...

Alzheimer’s and Parkinson’s diseases are the two most common forms of neurodegenerative diseases. The exact etiology of these disorders is not well known; however, environmental, molecular, and genetic influences play a major role in the pathogenesis of these diseases. Using Alzheimer’s disease (AD) as the archetype, the pathological findings include the aggregation of Amyloid Beta (Aβ) peptides, mitochondrial dysfunction, synaptic degradation caused by inflammation, elevated reactive oxygen species (ROS), and cerebrovascular dysregulation. This review highlights the neuroinflammatory and neuroprotective role of epigallocatechin-3-gallate (EGCG): the medicinal component of green tea, a known nutraceutical that has shown promise in modulating AD progression due to its antioxidant, anti-inflammatory, and anti-aging abilities. This report also re-examines the current literature and provides innovative approaches for EGCG to be used as a preventive measure to alleviate AD and other neurodegenerative disorders.

Thymoquinone (TQ), the main pharmacological active ingredient within the black cumin seed (Nigella sativa) is believed to be responsible for 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 indicated that TQ was effective in reducing NO2- with an IC50 of 5.04 μM, relative to selective iNOS inhibitor LNIL- L-N6-(1-Iminoethyl)lysine (IC50 4.09 μM). TQ mediated reduction in NO2- was found to parallel the decline of iNOS protein expression as confirmed by immunocytochemistry. In the next study, 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 and mRNA expression profiling using RT2 Profiler PCR cytokine arrays. Moreover, the data obtained from the RT2 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.

Diallyl trisulfide (DATS) is a garlic organosulfide that is toxic to cancer cells, however, little is known about its effect in the initiation phase of carcinogenesis. We sought to determine whether DATS could inhibit the carcinogen, benzo(a)pyrene (BaP), from inducing precancerous activity, in vitro. MCF-10A cells were either pre-treated (PreTx) or concurrently treated (CoTx) with 1 μM BaP, and 6 or 60 μM DATS for up to 24 hours. The DATS 6 and 60 μM CoTx inhibited BaP-induced cell proliferation by an average of 71.1 and 120.8%, respectively, at 6 hours. The 60 μM DATS pretreatment decreased BaP-induced G2/M cell cycle transition by 127 %, and reduced the increase in cells in the S-phase by 42%; whereas 60 μM DATS CoTx induced a 177% increase in cells in G1. DATS effectively inhibited (P<0.001) BaP-induced peroxide formation by at least 54%, which may have prevented the formation of BaP-induced DNA strand breaks. In this study, we reveal mechanisms involved in DATS inhibition of BaP-induced carcinogenesis, including inhibition of cell proliferation, regulation of cell cycle, attenuation of ROS formation, and inhibition of DNA damage. At the doses evaluated, DATS appears to be an effective attenuator of BaP-induced breast carcinogenesis, in vitro.

TNFα receptors are constitutively overexpressed in tumor cells, correlating to sustain elevated NFκB and monocyte chemotactic protein-1 (MCP-1/CCL2) expression. The elevation of CCL2 evokes aggressive forms of malignant tumors marked by tumor associated macrophage (TAM) recruitment, cell proliferation, invasion and angiogenesis. Previously, we have shown that the organo-sulfur compound diallyl disulfide (DADS) found in garlic (Allium sativum) attenuates TNFα induced CCL2 production in MDA-MB-231 cells. In the current study, we explored the signaling pathways responsible for DADS suppressive effect on TNFα mediated CCL2 release using PCR Arrays, RT-PCR and western blots. The data in this study show that TNFα initiates a rise in NFκB mRNA, which is not reversed by DADS. However, TNFα induced heightened expression of IKKε and phosphorylated ERK. The expression of these proteins corresponds to increased CCL2 release that can be attenuated by DADS. CCL2 induction by TNFα was also lessened by inhibitors of p38 (SB202190) and MEK (U0126) but not JNK (SP 600125), all of which were suppressed by DADS. In conclusion, the obtained results indicate that DADS down regulates TNFα invoked CCL2 production primarily through reduction of IKKε and phosphorylated-ERK, thereby impairing MAPK/ERK, and NFκB pathway signaling. Future research will be required to evaluate the effects of DADS on the function and expression of TNFα surface receptors.