Inhibition of microglial activation by elderberry extracts and its phenolic components.

AIMS: Elderberry (Sambucus spp.) is one of the oldest medicinal plants noted for its cardiovascular, anti-inflammatory, and immune-stimulatory properties. In this study, we investigated the anti-inflammatory and anti-oxidant effects of the American elderberry (Sambucus nigra subsp. canadensis) pomace as well as some of the anthocyanins (cyanidin chloride and cyanidin 3-O-glucoside) and flavonols (quercetin and rutin) in bv-2 mouse microglial cells. MAIN METHODS: The bv-2 cells were pretreated with elderberry pomace (extracted with ethanol or ethyl acetate) or its anthocyanins and flavonols and stimulated by either lipopolysaccharide (LPS) or interferon-γ (IFNγ). Reactive oxygen species (ROS) and nitric oxide (NO) production (indicating oxidative stress and inflammatory response) were measured using the ROS detection reagent DCF-DA and the Griess reaction, respectively. KEY FINDINGS: Analysis of total monomeric anthocyanin (as cyanidin 3-O-glucoside equivalents) indicated five-fold higher amount in the freeze-dried ethanol extract as compared to that of the oven-dried extract; anthocyanin was not detected in the ethyl acetate extracts. Elderberry ethanol extracts (freeze-dried or oven-dried) showed higher anti-oxidant activities and better ability to inhibit LPS or IFNγ-induced NO production as compared with the ethyl acetate extracts. The phenolic compounds strongly inhibited LPS or IFNγ-induced ROS production, but except for quercetin, they were relatively poor in inhibiting NO production. SIGNIFICANCE: These results demonstrated differences in anti-oxidative and anti-inflammatory effects of elderberry extracts depending on solvents used. Results further identified quercetin as the most active component in suppressing oxidative stress and inflammatory responses on microglial cells.

Sutherlandia frutescens ethanol extracts inhibit oxidative stress and inflammatory responses in neurons and microglial cells.

Sutherlandia frutescens (L.) R.Br. (SF) is a medicinal plant indigenous to southern Africa and used in folk and contemporary remedies for stress, chronic diseases, cancer, and HIV/AIDS. While previous studies have focused on physiological effects of SF on cellular and systemic abnormalities associated with these diseases, little is known about its effects in the brain and immune cells in the central nervous system. Results of this study indicate that ethanol extracts of SF (SF-E) suppress NMDA-induced reactive oxygen species (ROS) production in neurons, and LPS- and IFNγ-induced ROS and nitric oxide (NO) production in microglial cells. SF-E's action on microglial cells appears to be mediated through inhibition of the IFNγ-induced p-ERK1/2 signaling pathway which is central to regulating a number of intracellular metabolic processes including enhancing STAT1α phosphorylation and filopodia formation. The involvement of SF in these pathways suggests the potential for novel therapeutics for stress and prevention, and/or treatment of HIV/AIDS as well as other inflammatory diseases in the brain.

Role of cytosolic phospholipase A2 in oxidative and inflammatory signaling pathways in different cell types in the central nervous system.

Phospholipases A(2) (PLA(2)s) are important enzymes for the metabolism of fatty acids in membrane phospholipids. Among the three major classes of PLA(2)s in the mammalian system, the group IV calcium-dependent cytosolic PLA(2) alpha (cPLA(2)α) has received the most attention because it is widely expressed in nearly all mammalian cells and its active participation in cell metabolism. Besides Ca(2+) binding to its C2 domain, this enzyme can undergo a number of cell-specific post-translational modifications, including phosphorylation by protein kinases, S-nitrosylation through interaction with nitric oxide (NO), as well as interaction with other proteins and lipid molecules. Hydrolysis of phospholipids by cPLA(2) yields two important lipid mediators, arachidonic acid (AA) and lysophospholipids. While AA is known to serve as a substrate for cyclooxygenases and lipoxygenases, which are enzymes for the synthesis of eicosanoids and leukotrienes, lysophospholipids are known to possess detergent-like properties capable of altering microdomains of cell membranes. An important feature of cPLA(2) is its link to cell surface receptors that stimulate signaling pathways associated with activation of protein kinases and production of reactive oxygen species (ROS). In the central nervous system (CNS), cPLA(2) activation has been implicated in neuronal excitation, synaptic secretion, apoptosis, cell-cell interaction, cognitive and behavioral function, oxidative-nitrosative stress, and inflammatory responses that underline the pathogenesis of a number of neurodegenerative diseases. However, the types of extracellular agonists that target intracellular signaling pathways leading to cPLA(2) activation among different cell types and under different physiological and pathological conditions have not been investigated in detail. In this review, special emphasis is given to metabolic events linking cPLA(2) to activation in neurons, astrocytes, microglial cells, and cerebrovascular cells. Understanding the molecular mechanism(s) for regulation of this enzyme is deemed important in the development of new therapeutic targets for the treatment and prevention of neurodegenerative diseases.

Magnolia polyphenols attenuate oxidative and inflammatory responses in neurons and microglial cells.

BACKGROUND: The bark of magnolia has been used in Oriental medicine to treat a variety of remedies, including some neurological disorders. Magnolol (Mag) and honokiol (Hon) are isomers of polyphenolic compounds from the bark of Magnolia officinalis, and have been identified as major active components exhibiting anti-oxidative, anti-inflammatory, and neuroprotective effects. In this study, we investigate the ability of these isomers to suppress oxidative stress in neurons stimulated by the ionotropic glutamate receptor agonist N-methyl-D-aspartate (NMDA) and oxidative and inflammatory responses in microglial cells activated by interferon-γ (IFNγ) and lipopolysaccharide (LPS). We also attempt to elucidate the mechanism and signaling pathways involved in cytokine-induced production of reactive oxygen species (ROS) in microglial cells. METHODS: Dihydroethidium (DHE) was used to assay superoxide production in neurons, while CM-H2DCF-DA was used to test for ROS production in murine (BV-2) and rat (HAPI) immortalized microglial cells. NADPH oxidase inhibitors (for example, diphenyleneiodonium (DPI), AEBSF, and apocynin) and immunocytochemistry targeting p47phox and gp91phox were used to assess the involvement of NADPH oxidase. Western blotting was used to assess iNOS and ERK1/2 expression, and the Griess reaction protocol was employed to determine nitric oxide (NO) concentration. RESULTS: Exposure of Hon and Mag (1-10 µM) to neurons for 24 h did not alter neuronal viability, but both compounds (10 µM) inhibited NMDA-stimulated superoxide production, a pathway known to involve NADPH oxidase. In microglial cells, Hon and Mag inhibited IFNγ±LPS-induced iNOS expression, NO, and ROS production. Studies with inhibitors and immunocytochemical assay further demonstrated the important role of IFNγ activating the NADPH oxidase through the p-ERK-dependent pathway. Hon and, to a lesser extent, Mag inhibited IFNγ-induced p-ERK1/2 and its downstream pathway for ROS and NO production. CONCLUSION: This study highlights the important role of NADPH oxidase in mediating oxidative stress in neurons and microglial cells and has unveiled the role of IFNγ in stimulating the MAPK/ERK1/2 signaling pathway for activation of NADPH oxidase in microglial cells. Hon and Mag offer anti-oxidative or anti-inflammatory effects, at least in part, through suppressing IFNγ-induced p-ERK1/2 and its downstream pathway.

Pro-inflammatory cytokines and lipopolysaccharide induce changes in cell morphology, and upregulation of ERK1/2, iNOS and sPLA2-IIA expression in astrocytes and microglia.

BACKGROUND: Activation of glial cells, including astrocytes and microglia, has been implicated in the inflammatory responses underlying brain injury and neurodegenerative diseases including Alzheimer's and Parkinson's diseases. Although cultured astrocytes and microglia are capable of responding to pro-inflammatory cytokines and lipopolysaccharide (LPS) in the induction and release of inflammatory factors, no detailed analysis has been carried out to compare the induction of iNOS and sPLA2-IIA. In this study, we investigated the effects of cytokines (TNF-alpha, IL-1beta, and IFN-gamma) and LPS + IFN-gamma to induce temporal changes in cell morphology and induction of p-ERK1/2, iNOS and sPLA2-IIA expression in immortalized rat (HAPI) and mouse (BV-2) microglial cells, immortalized rat astrocytes (DITNC), and primary microglia and astrocytes. METHODS/RESULTS: Cytokines (TNF-alpha, IL-1beta, and IFN-gamma) and LPS + IFN-gamma induced a time-dependent increase in fine processes (filopodia) in microglial cells but not in astrocytes. Filopodia production was attributed to IFN-gamma and was dependent on ERK1/2 activation. Cytokines induced an early (15 min) and a delayed phase (1 ~ 4 h) increase in p-ERK1/2 expression in microglial cells, and the delayed phase increase corresponded to the increase in filopodia production. In general, microglial cells are more active in responding to cytokines and LPS than astrocytes in the induction of NO. Although IFN-gamma and LPS could individually induce NO, additive production was observed when IFN-gamma was added together with LPS. On the other hand, while TNF-alpha, IL-1beta, and LPS could individually induce sPLA2-IIA mRNA and protein expression, this induction process does not require IFN-gamma. Interestingly, neither rat immortalized nor primary microglial cells were capable of responding to cytokines and LPS in the induction of sPLA2-IIA expression. CONCLUSION: These results demonstrated the utility of BV-2 and HAPI cells as models for investigation on cytokine and LPS induction of iNOS, and DITNC astrocytes for induction of sPLA2-IIA. In addition, results further demonstrated that cytokine-induced sPLA2-IIA is attributed mainly to astrocytes and not microglial cells.

Inhibition of Stat3 expression induces apoptosis and suppresses proliferation in human leukemia HL-60 cells.

Acute myeloid leukemia (AML) is the most common myeloid leukemia. It is highly malignant, thus, most patients with AML will relapse and die after traditional treatment. Stat3, a member of the signal transducer and activator of transcription (Stat) family, is involved in the development and progression of many tumors. The purpose of the study was to investigate whether the down-regulation of Stat3 expression by RNA interference is effective against human leukemia HL-60 cells. The results indicated that constitutively expressed Stat3 is present in human leukemia HL-60 cells, and the down-regulation of Stat3 expression caused significant induction of apoptosis as well as inhibition of proliferation in HL-60 cells. These data further demonstrated that Stat3 plays a critical role in human leukemia HL-60 cell apoptosis and proliferation. Thus, targeting Stat3 may be a useful adjunctive treatment strategy in AML.

Reciprocal effects of conditioned medium on cultured glioma cells and neural stem cells.

Malignant gliomas are among the most intractable brain cancers. Neural stem cells (NSC) are tissue-specific stem cells with self-renewal capacity and the potential to differentiate into glia and neurons. It has been proposed that NSC could serve as a therapeutic vehicle for the treatment of gliomas. Previous studies showed that NSC, after being implanted into the brain, could migrate to the invading tumor border and target infiltrating tumor cells. These findings suggested that NSC and gliomas could interact, although the mechanism is still not well understood. Here we report that the stem-cell state of NSC is disrupted and NSCs become differentiated when they are co-cultured in vitro with a medium in which glioma cells have been cultured (conditioned medium). The ratio of neurons in these differentiated cells is significantly higher than that in the controls (NSC cultured in regular medium). Conditioned medium in which primary NSC have been grown can inhibit proliferation of glioma cells, an effect that was greater with NSC conditioned medium of embryonic mice than neonatal mice. These results suggest that glioma cells and NSC can interact at the niche or micro-environment level, potentially leading to proliferation and differentiation of NSC and suppression of proliferation of glioma cells. These findings may shed new light on the development of novel strategies for the treatment of malignant gliomas.