Oligomeric Amyloid-ß and Tau Alter Cell Adhesion Properties and Induce Inflammatory Responses in Cerebral Endothelial Cells Through the RhoA/ROCK Pathway.

Dysfunction of cerebral endothelial cells (CECs) has been implicated in the pathology of Alzheimer's disease (AD). Despite evidence showing cytotoxic effects of oligomeric amyloid-ß (oAß) and Tau (oTau) in the central nervous system, their direct effects on CECs have not been fully investigated. In this study, we examined the direct effects of oAß, oTau, and their combination on cell adhesion properties and inflammatory responses in CECs. We found that both oAß and oTau increased cell stiffness, as well as the p-selectin/Sialyl-LewisX (sLeX) bonding-mediated membrane tether force and probability of adhesion in CECs. Consistent with these biomechanical alterations, treatments with oAß or oTau also increased actin polymerization and the expression of p-selectin at the cell surface. These toxic oligomeric peptides also triggered inflammatory responses, including upregulations of p-NF-kB p65, IL-1ß, and TNF-α. In addition, they rapidly activated the RhoA/ROCK pathway. These biochemical and biomechanical changes were further enhanced by the treatment with the combination of oAß and oTau, which were significantly suppressed by Fasudil, a specific inhibitor for the RhoA/ROCK pathway. In conclusion, our data suggest that oAß, oTau, and their combination triggered subcellular mechanical alterations and inflammatory responses in CECs through the RhoA/ROCK pathway.

COVID-19, Blood Lipid Changes, and Thrombosis.

Although there is increasing evidence that oxidative stress and inflammation induced by COVID-19 may contribute to increased risk and severity of thromboses, the underlying mechanism(s) remain to be understood. The purpose of this review is to highlight the role of blood lipids in association with thrombosis events observed in COVID-19 patients. Among different types of phospholipases A2 that target cell membrane phospholipids, there is increasing focus on the inflammatory secretory phospholipase A2 IIA (sPLA2-IIA), which is associated with the severity of COVID-19. Analysis indicates increased sPLA2-IIA levels together with eicosanoids in the sera of COVID patients. sPLA2 could metabolise phospholipids in platelets, erythrocytes, and endothelial cells to produce arachidonic acid (ARA) and lysophospholipids. Arachidonic acid in platelets is metabolised to prostaglandin H2 and thromboxane A2, known for their pro-coagulation and vasoconstrictive properties. Lysophospholipids, such as lysophosphatidylcholine, could be metabolised by autotaxin (ATX) and further converted to lysophosphatidic acid (LPA). Increased ATX has been found in the serum of patients with COVID-19, and LPA has recently been found to induce NETosis, a clotting mechanism triggered by the release of extracellular fibres from neutrophils and a key feature of the COVID-19 hypercoagulable state. PLA2 could also catalyse the formation of platelet activating factor (PAF) from membrane ether phospholipids. Many of the above lipid mediators are increased in the blood of patients with COVID-19. Together, findings from analyses of blood lipids in COVID-19 patients suggest an important role for metabolites of sPLA2-IIA in COVID-19-associated coagulopathy (CAC).

Microglia Signaling Pathway Reporters Unveiled Manganese Activation of the Interferon/STAT1 Pathway and Its Mitigation by Flavonoids.

Neuroinflammatory responses to neurotoxic manganese (Mn) in CNS have been associated with the Mn-induced Parkinson-like syndromes. However, the framework of molecular mechanisms contributing to manganism is still unclear. Using an in vitro neuroinflammation model based on the insulated signaling pathway reporter transposon constructs stably transfected into a murine BV-2 microglia line, we tested effects of manganese (II) together with a set of 12 metal salts on the transcriptional activities of the NF-κB, activator protein-1 (AP-1), signal transducer and activator of transcription 1 (STAT1), STAT1/STAT2, STAT3, Nrf2, and metal-responsive transcription factor-1 (MTF-1) via luciferase assay, while concatenated destabilized green fluorescent protein expression provided for simultaneous evaluation of cellular viability. This experiment revealed specific and strong responses to manganese (II) in reporters of the type I and type II interferon-induced signaling pathways, while weaker activation of the NF-κB in the microglia was detected upon treatment of cells with Mn(II) and Ba(II). There was a similarity between Mn(II) and interferon-γ in the temporal STAT1 activation profile and in their antagonism to bacterial LPS. Sixty-four natural and synthetic flavonoids differentially affected both cytotoxicity and the pro-inflammatory activity of Mn (II) in the microglia. Whereas flavan-3-ols, flavanones, flavones, and flavonols were cytoprotective, isoflavones enhanced the cytotoxicity of Mn(II). Furthermore, about half of the tested flavonoids at 10-50 µM could attenuate both basal and 100-200 µM Mn(II)-induced activity at the gamma-interferon activated DNA sequence (GAS) in the cells, suggesting no critical roles for the metal chelation or antioxidant activity in the protective potential of flavonoids against manganese in microglia. In summary, results of the study identified Mn as a specific elicitor of the interferon-dependent pathways that can be mitigated by dietary polyphenols.

Anti-Inflammatory Effects of Phytochemical Components of Clinacanthus nutans.

Recent studies on the ethnomedicinal use of Clinacanthus nutans suggest promising anti-inflammatory, anti-tumorigenic, and antiviral properties for this plant. Extraction of the leaves with polar and nonpolar solvents has yielded many C-glycosyl flavones, including schaftoside, isoorientin, orientin, isovitexin, and vitexin. Aside from studies with different extracts, there is increasing interest to understand the properties of these components, especially regarding their ability to exert anti-inflammatory effects on cells and tissues. A major focus for this review is to obtain information on the effects of C. nutans extracts and its phytochemical components on inflammatory signaling pathways in the peripheral and central nervous system. Particular emphasis is placed on their role to target the Toll-like receptor 4 (TLR4)-NF-kB pathway and pro-inflammatory cytokines, the antioxidant defense pathway involving nuclear factor erythroid-2-related factor 2 (NRF2) and heme oxygenase 1 (HO-1); and the phospholipase A2 (PLA2) pathway linking to cyclooxygenase-2 (COX-2) and production of eicosanoids. The ability to provide a better understanding of the molecular targets and mechanism of action of C. nutans extracts and their phytochemical components should encourage future studies to develop new therapeutic strategies for better use of this herb to combat inflammatory diseases.

Neuroprotective effects of DHA-derived peroxidation product 4(RS)-4-F4t-neuroprostane on microglia.

The abundance of docosahexaenoic acid (DHA) in brain membrane phospholipids has stimulated studies to explore its role in neurological functions. Upon released from phospholipids, DHA undergoes enzymatic reactions resulting in synthesis of bioactive docosanoids and prostanoids. However, these phospholipids are also prone to non-enzymatic reactions leading to more complex pattern of metabolites. A non-enzymatic oxidized product of DHA, 4(RS)-4-F4t-Neuroprostane (44FNP), has been identified in cardiac and brain tissues. In this study, we examined effects of the 44FNP on oxidative and inflammatory responses in microglial cells treated with lipopolysaccharide (LPS). The 44FNP attenuated LPS-induced production of reactive oxygen species (ROS) in both primary and immortalized microglia (BV2). It also attenuated LPS-induced inflammation through suppressing NFκB-p65 and levels of iNOS and TNFα. In addition, 44FNP also suppressed LPS-induced mitochondrial dysfunction and upregulated the Nrf2/HO-1 antioxidative pathway. In sum, these findings with microglial cells demonstrated neuroprotective effects of this 44FNP and shed light into the potential of nutraceutical therapy for neurodegenerative diseases.

Long-Term Effects of Low-Intensity Blast Non-Inertial Brain Injury on Anxiety-Like Behaviors in Mice: Home-Cage Monitoring Assessments.

Mild traumatic brain injury induced by low-intensity blast (LIB) exposure poses concerns in military personnel. Using an open-field, non-inertial blast model and assessments by conventional behavioral tests, our previous studies revealed early-phase anxiety-like behaviors in LIB-exposed mice. However, the impact of LIB upon long-term anxiety-like behaviors requires clarification. This study applied a highly sensitive automated home-cage monitoring (HCM) system, which minimized human intervention and environmental changes, to assess anxiety-like responses in mice 3 months after LIB exposure. Initial assessment of 72-h spontaneous activities in a natural cage condition over multiple light and dark phases showed altered sheltering behaviors. LIB-exposed mice exhibited a subtle, but significantly decreased, duration of short shelter visits as compared to sham controls. Other measured responses between LIB-exposed mice and sham controls were insignificant. When behavioral assessments were performed in a challenged condition using an aversive spotlight, LIB-exposed mice demonstrated a significantly higher frequency of movements of shorter distance and duration per movement. Taken together, these findings demonstrated the presence of chronic anxiety-like behaviors assessed by the HCM system under both natural and challenged conditions in mice occurring post-LIB exposure. This model thus provides a platform to test for screening and interventions on anxiety disorders occurring after LIB non-inertial brain injury.

Bidirectional Responses of Eight Neuroinflammation-Related Transcriptional Factors to 64 Flavonoids in Astrocytes with Transposable Insulated Signaling Pathway Reporters.

Neuroinflammation is implicated in a variety of pathologies and is mechanistically linked to hyperactivation of glial cells in the central nervous system (CNS), predominantly in response to external stimuli. Multiple dietary factors were reported to alter neuroinflammation, but their actions on the relevant transcription factors in glia are not sufficiently understood. Here, an in vitro protocol employing cultured astroglial cells, which carry reporters of multiple signaling pathways associated with inflammation, was developed for screening environmental factors and synthetic drugs. Immortalized rat astrocyte line DI TNC1 was stably transfected with piggyBac transposon vectors containing a series of insulated reporters for the transcriptional activity of NF-κB, AP-1, signal transducer and activator of transcription 1 (STAT1), signal transducer and activator of transcription 3 (STAT3), aromatic hydrocarbon receptor (AhR), Nrf2, peroxisome proliferator-activated receptor γ (PPARγ), and HIF-1α, which is quantified via luciferase assay. Concatenated green fluorescent protein (GFP) expression was employed for simultaneous evaluation of cellular viability. Responses to a set of 64 natural and synthetic monomeric flavonoids representing six main structural classes (flavan-3-ols, flavanones, flavones, flavonols, isoflavones, and anthocyan(id)ins) were obtained at 10 and 50 µM concentrations. Except for HIF-1α, the activity of NF-κB and other transcription factors (TFs) in astrocytes was predominantly inhibited by flavan-3-ols and anthocyan(id)ins, while flavones and isoflavones generally activated these TFs. In addition, we obtained dose-response profiles for 11 flavonoids (apigenin, baicalein, catechin, cyanidin, epigallocatechin gallate, genistein, hesperetin, kaempferol, luteolin, naringenin, and quercetin) within the 1-100 µM range and in the presence of immune-stimulants and immune-suppressors. The flavonoid concentration profiles for TF-activation reveal biphasic response curves from the astrocytes. Apart from epigallocatechin gallate (EGCG), flavonoids failed to inhibit the NF-κB activation by proinflammatory agents [lipopolysaccharide (LPS), cytokines], but most of the tested polyphenols synergized with STAT3 inhibitors (stattic, ruxolitinib) against the activation of this TF in the astrocytes. We conclude that transposable insulated reporters of transcriptional activation represent a convenient neurochemistry tool in screening for activators/inhibitors of signaling pathways.

Dynamic Role of Phospholipases A2 in Health and Diseases in the Central Nervous System.

Phospholipids are major components in the lipid bilayer of cell membranes. These molecules are comprised of two acyl or alkyl groups and different phospho-base groups linked to the glycerol backbone. Over the years, substantial interest has focused on metabolism of phospholipids by phospholipases and the role of their metabolic products in mediating cell functions. The high levels of polyunsaturated fatty acids (PUFA) in the central nervous system (CNS) have led to studies centered on phospholipases A2 (PLA2s), enzymes responsible for cleaving the acyl groups at the sn-2 position of the phospholipids and resulting in production of PUFA and lysophospholipids. Among the many subtypes of PLA2s, studies have centered on three major types of PLA2s, namely, the calcium-dependent cytosolic cPLA2, the calcium-independent iPLA2 and the secretory sPLA2. These PLA2s are different in their molecular structures, cellular localization and, thus, production of lipid mediators with diverse functions. In the past, studies on specific role of PLA2 on cells in the CNS are limited, partly because of the complex cellular make-up of the nervous tissue. However, understanding of the molecular actions of these PLA2s have improved with recent advances in techniques for separation and isolation of specific cell types in the brain tissue as well as development of sensitive molecular tools for analyses of proteins and lipids. A major goal here is to summarize recent studies on the characteristics and dynamic roles of the three major types of PLA2s and their oxidative products towards brain health and neurological disorders.

Glial Cell Line-Derived Neurotrophic Factor and Focal Ischemic Stroke.

Focal ischemic stroke (FIS) is a leading cause of human debilitation and death. Following the onset of a FIS, the brain experiences a series of spatiotemporal changes which are exemplified in different pathological processes. One prominent feature of FIS is the development of reactive astrogliosis and glial scar formation in the peri-infarct region (PIR). During the subacute phase, astrocytes in PIR are activated, referred to as reactive astrocytes (RAs), exhibit changes in morphology (hypotrophy), show an increased proliferation capacity, and altered gene expression profile, a phenomenon known as reactive astrogliosis. Subsequently, the morphology of RAs remains stable, and proliferation starts to decline together with the formation of glial scars. Reactive astrogliosis and glial scar formation eventually cause substantial tissue remodeling and changes in permanent structure around the PIR. Glial cell line-derived neurotrophic factor (GDNF) was originally isolated from a rat glioma cell-line and regarded as a potent survival neurotrophic factor. Under normal conditions, GDNF is expressed in neurons but is upregulated in RAs after FIS. This review briefly describes properties of GDNF, its receptor-mediated signaling pathways, as well as recent studies regarding the role of RAs-derived GDNF in neuronal protection and brain recovery. These results provide evidence suggesting an important role of RA-derived GDNF in intrinsic brain repair and recovery after FIS, and thus targeting GDNF in RAs may be effective for stroke therapy.

Docosahexaenoic Acid (DHA) Supplementation Alters Phospholipid Species and Lipid Peroxidation Products in Adult Mouse Brain, Heart, and Plasma.

The abundance of docosahexaenoic acid (DHA) in phospholipids in the brain and retina has generated interest to search for its role in mediating neurological functions. Besides the source of many oxylipins with pro-resolving properties, DHA also undergoes peroxidation, producing 4-hydroxyhexenal (4-HHE), although its function remains elusive. Despite wide dietary consumption, whether supplementation of DHA may alter the peroxidation products and their relationship to phospholipid species in brain and other body organs have not been explored sufficiently. In this study, adult mice were administered a control or DHA-enriched diet for 3 weeks, and phospholipid species and peroxidation products were examined in brain, heart, and plasma. Results demonstrated that this dietary regimen increased (n-3) and decreased (n-6) species to different extent in all major phospholipid classes (PC, dPE, PE-pl, PI and PS) examined. Besides changes in phospholipid species, DHA-enriched diet also showed substantial increases in 4-HHE in brain, heart, and plasma. Among different brain regions, the hippocampus responded to the DHA-enriched diet showing significant increase in 4-HHE. Considering the pro- and anti-inflammatory pathways mediated by the (n-6) and (n-3) polyunsaturated fatty acids, unveiling the ability for DHA-enriched diet to alter phospholipid species and lipid peroxidation products in the brain and in different body organs may be an important step forward towards understanding the mechanism(s) for this (n-3) fatty acid on health and diseases.

Clinacanthus nutans Mitigates Neuronal Death and Reduces Ischemic Brain Injury: Role of NF-κB-driven IL-1ß Transcription.

Neuroinflammation has been shown to exacerbate ischemic brain injury, and is considered as a prime target for the development of stroke therapies. Clinacanthus nutans Lindau (C. nutans) is widely used in traditional medicine for treating insect bites, viral infection and cancer, due largely to its anti-oxidative and anti-inflammatory properties. Recently, we reported that an ethanol extract from the leaf of C. nutans could protect the brain against ischemia-triggered neuronal death and infarction. In order to further understand the molecular mechanism(s) for its beneficial effects, two experimental paradigms, namely, in vitro primary cortical neurons subjected to oxygen-glucose deprivation (OGD) and in vivo rat middle cerebral artery (MCA) occlusion, were used to dissect the anti-inflammatory effects of C. nutans extract. Using promoter assays, immunofluorescence staining, and loss-of-function (siRNA) approaches, we demonstrated that transient OGD led to marked induction of IL-1ß, IL-6 and TNFα, while pretreatment with C. nutans suppressed production of inflammatory cytokines in primary neurons. C. nutans inhibited IL-1ß transcription via preventing NF-κB/p65 nuclear translocation, and siRNA knockdown of either p65 or IL-1ß mitigated OGD-mediated neuronal death. Correspondingly, post-ischemic treatment of C. nutans attenuated IκBα degradation and decreased IL-1ß, IL-6 and TNFα production in the ischemic brain. Furthermore, IL-1ß siRNA post-ischemic treatment reduced cerebral infarct, thus mimicking the beneficial effects of C. nutans. In summary, our findings demonstrated the ability for C. nutans to suppress NF-κB nuclear translocation and inhibit IL-1ß transcription in ischemic models. Results further suggest the possibility for using C. nutans to prevent and treat stroke patients.

Recent Insights on the Role of PPAR-ß/δ in Neuroinflammation and Neurodegeneration, and Its Potential Target for Therapy.

Peroxisome proliferator-activated receptor (PPAR) ß/δ belongs to the family of hormone and lipid-activated nuclear receptors, which are involved in metabolism of long-chain fatty acids, cholesterol, and sphingolipids. Similar to PPAR-α and PPAR-γ, PPAR-ß/δ also acts as a transcription factor activated by dietary lipids and endogenous ligands, such as long-chain saturated and polyunsaturated fatty acids, and selected lipid metabolic products, such as eicosanoids, leukotrienes, lipoxins, and hydroxyeicosatetraenoic acids. Together with other PPARs, PPAR-ß/δ displays transcriptional activity through interaction with retinoid X receptor (RXR). In general, PPARs have been shown to regulate cell differentiation, proliferation, and development and significantly modulate glucose, lipid metabolism, mitochondrial function, and biogenesis. PPAR-ß/δ appears to play a special role in inflammatory processes and due to its proangiogenic and anti-/pro-carcinogenic properties, this receptor has been considered as a therapeutic target for treating metabolic syndrome, dyslipidemia, carcinogenesis, and diabetes. Until now, most studies were carried out in the peripheral organs, and despite of its presence in brain cells and in different brain regions, its role in neurodegeneration and neuroinflammation remains poorly understood. This review is intended to describe recent insights on the impact of PPAR-ß/δ and its novel agonists on neuroinflammation and neurodegenerative disorders, including Alzheimer's and Parkinson's, Huntington's diseases, multiple sclerosis, stroke, and traumatic injury. An important goal is to obtain new insights to better understand the dietary and pharmacological regulations of PPAR-ß/δ and to find promising therapeutic strategies that could mitigate these neurological disorders.

Maternal Immune Activation Induces Neuroinflammation and Cortical Synaptic Deficits in the Adolescent Rat Offspring.

Maternal immune activation (MIA), induced by infection during pregnancy, is an important risk factor for neuro-developmental disorders, such as autism. Abnormal maternal cytokine signaling may affect fetal brain development and contribute to neurobiological and behavioral changes in the offspring. Here, we examined the effect of lipopolysaccharide-induced MIA on neuro-inflammatory changes, as well as synaptic morphology and key synaptic protein level in cerebral cortex of adolescent male rat offspring. Adolescent MIA offspring showed elevated blood cytokine levels, microglial activation, increased pro-inflammatory cytokines expression and increased oxidative stress in the cerebral cortex. Moreover, pathological changes in synaptic ultrastructure of MIA offspring was detected, along with presynaptic protein deficits and down-regulation of postsynaptic scaffolding proteins. Consequently, ability to unveil MIA-induced long-term alterations in synapses structure and protein level may have consequences on postnatal behavioral changes, associated with, and predisposed to, the development of neuropsychiatric disorders.

Quantitative Proteomics Reveals Docosahexaenoic Acid-Mediated Neuroprotective Effects in Lipopolysaccharide-Stimulated Microglial Cells.

The high levels of docosahexaenoic acid (DHA) in cell membranes within the brain have led to a number of studies exploring its function. These studies have shown that DHA can reduce inflammatory responses in microglial cells. However, the method of action is poorly understood. Here, we report the effects of DHA on microglial cells stimulated with lipopolysaccharides (LPSs). Data were acquired using the parallel accumulation serial fragmentation method in a hybrid trapped ion mobility-quadrupole time-of-flight mass spectrometer. Over 2800 proteins are identified using label-free quantitative proteomics. Cells exposed to LPSs and/or DHA resulted in changes in cell morphology and expression of 49 proteins with differential abundance (greater than 1.5-fold change). The data provide details about pathways that are influenced in this system including the nuclear factor κ-light-chain-enhancer of the activated B cells (NF-κB) pathway. Western blots and enzyme-linked immunosorbent assay studies are used to help confirm the proteomic results. The MS data are available at ProteomeXchange.

Neddylation inactivation represses androgen receptor transcription and inhibits growth, survival and invasion of prostate cancer cells.

Androgen receptor (AR) and its constitutively active variants (AR-Vs) have been extensively implicated in the progression and recurrence of prostate cancer, making them attractive targets in the treatment of this disease. Whether and how neddylation modification regulates AR, and the therapeutic implications of this potential regulation, are relatively unexplored areas of investigation. Here we report that neddylation inactivation by the pharmacological inhibitor MLN4924 or Lenti-shRNA-based genetic knockdown of neddylation activating enzyme (NAE) selectively suppressed growth and survival of prostate cancer cells with minor, if any, effect on normal prostate epithelial cells. MLN4924 also significantly suppressed the invasive capacity of prostate cancer cells. Furthermore, compared to monotherapy, the combination of MLN4924 with AR antagonist or castration significantly enhanced growth suppression of prostate cancer cells in vitro, and tumor growth in an in vivo xenograft model. Mechanistically, MLN4924 repressed the transcription of AR/AR-V7 and its downstream targets, and blocked MMP2 and MMP9 expression. Taken together, our study reveals that the neddylation pathway positively regulates AR/AR-V7 transcription, and that the neddylation inhibitor MLN4924 has therapeutic potential for the treatment of aggressive prostate cancers.

Bioactive components from garlic on brain resiliency against neuroinflammation and neurodegeneration.

Garlic (Allium sativum) has been widely used for culinary and medicinal purposes. Aged garlic extract (AGE) and sulfur-containing compounds, including S-allylcysteine (SAC) are well documented botanical active components of garlic. AGE is prepared by the prolonged extraction of fresh garlic with aqueous ethanol and is considered a nutritional supplement with potential to promote human health. SAC is a water-soluble organosulfur compound and the most abundant component of AGE. Studies have demonstrated that both AGE and SAC can exert neuroprotective effects against neuroinflammation and neurodegeneration. Another bioactive component in AGE is N-α-(1-deoxy-D-fructos-1-yl)-L-arginine (FruArg) although less is known about the metabolic activity of this compound. The main aim of this review was to provide an undated overview of the neuroprotective perspectives of these active garlic components (AGE, SAC and FruArg). Of interest, our studies and those of others indicate that both AGE and FruArg are involved in the regulation of gene transcription and protein expression. AGE has been shown to reverse 67% of the transcriptome alteration induced by endotoxins-lipopolysaccharide (LPS), and FruArg has been shown to account for the protective effects by reversing 55% of genes altered in a cell-based neuroinflammation paradigm stimulated by LPS in murine BV-2 microglial cells. AGE and FruArg can alleviate neuroinflammatory responses through a variety of signaling pathways, such as Toll-like receptor and interleukin (IL)-6 signaling, as well as by upregulating the nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated oxidative stress pathways known to promote microglial resiliency against neuroinflammation and neurodegeneration. The capability of FruArg to pass through the blood-brain barrier further supports its potential as a therapeutic compound. In summary, these experimental results provide new insight into the understanding of the neuroprotective effects of garlic components in promoting brain resiliency for health benefits.

Harpagophytum procumbens Extract Ameliorates Allodynia and Modulates Oxidative and Antioxidant Stress Pathways in a Rat Model of Spinal Cord Injury.

Spinal cord injury (SCI) is a deliberating disorder with impairments in locomotor deficits and incapacitating sensory abnormalities. Harpagophytum procumbens (Hp) is a botanical widely used for treating inflammation and pain related to various inflammatory and musculoskeletal conditions. Using a modified rodent contusion model of SCI, we explored the effects of this botanical on locomotor function and responses to mechanical stimuli, and examined possible neurochemical changes associated with SCI-induced allodynia. Following spinal cord contusion at T10 level, Hp (300 mg/kg, p.o.) or vehicle (water) was administered daily starting 24 h post-surgery, and behavioral measurements made every-other day until sacrifice (Day 21). Hp treatment markedly ameliorated the contusion-induced decrease in locomotor function and increased sensitivity to mechanical stimuli. Determination of Iba1 expression in spinal cord tissues indicated microglial infiltration starting 3 days post-injury. SCI results in increased levels of 4-hydroxynonenal, an oxidative stress product and proalgesic, which was diminished at 7 days by treatment with Hp. SCI also enhanced antioxidant heme oxygenase-1 (HO-1) expression. Concurrent studies of cultured murine BV-2 microglial cells revealed that Hp suppressed oxidative/nitrosative stress and inflammatory responses, including production of nitric oxide and reactive oxygen species, phosphorylation of cytosolic phospholipases A2, and upregulation of the antioxidative stress pathway involving the nuclear factor erythroid 2-related factor 2 and HO-1. These results support the use of Hp for management of allodynia by providing resilience against the neuroinflammation and pain associated with SCI and other neuropathological conditions.

Effects of Docosahexaenoic Acid and Its Peroxidation Product on Amyloid-ß Peptide-Stimulated Microglia.

Growing evidence suggests that docosahexaenoic acid (DHA) exerts neuroprotective effects, although the mechanism(s) underlying these beneficial effects are not fully understood. Here we demonstrate that DHA, but not arachidonic acid (ARA), suppressed oligomeric amyloid-ß peptide (oAß)-induced reactive oxygen species (ROS) production in primary mouse microglia and immortalized mouse microglia (BV2). Similarly, DHA but not ARA suppressed oAß-induced increases in phosphorylated cytosolic phospholipase A2 (p-cPLA2), inducible nitric oxide synthase (iNOS), and tumor necrosis factor-α (TNF-α) in BV2 cells. LC-MS/MS assay indicated the ability for DHA to cause an increase in 4-hydroxyhexenal (4-HHE) and suppress oAß-induced increase in 4-hydroxynonenal (4-HNE). Although oAß did not alter the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway, exogenous DHA, ARA as well as low concentrations of 4-HHE and 4-HNE upregulated this pathway and increased production of heme oxygenase-1 (HO-1) in microglial cells. These results suggest that DHA modulates ARA metabolism in oAß-stimulated microglia through suppressing oxidative and inflammatory pathways and upregulating the antioxidative stress pathway involving Nrf2/HO-1. Understanding the mechanism(s) underlying the beneficial effects of DHA on microglia should shed light into nutraceutical therapy for the prevention and treatment of Alzheimer's disease (AD).

Yin-Yang Mechanisms Regulating Lipid Peroxidation of Docosahexaenoic Acid and Arachidonic Acid in the Central Nervous System.

Phospholipids in the central nervous system (CNS) are rich in polyunsaturated fatty acids (PUFAs), particularly arachidonic acid (ARA) and docosahexaenoic acid (DHA). Besides providing physical properties to cell membranes, these PUFAs are metabolically active and undergo turnover through the "deacylation-reacylation (Land's) cycle". Recent studies suggest a Yin-Yang mechanism for metabolism of ARA and DHA, largely due to different phospholipases A2 (PLA2s) mediating their release. ARA and DHA are substrates of cyclooxygenases and lipoxygenases resulting in an array of lipid mediators, which are pro-inflammatory and pro-resolving. The PUFAs are susceptible to peroxidation by oxygen free radicals, resulting in the production of 4-hydroxynonenal (4-HNE) from ARA and 4-hydroxyhexenal (4-HHE) from DHA. These alkenal electrophiles are reactive and capable of forming adducts with proteins, phospholipids and nucleic acids. The perceived cytotoxic and hormetic effects of these hydroxyl-alkenals have impacted cell signaling pathways, glucose metabolism and mitochondrial functions in chronic and inflammatory diseases. Due to the high levels of DHA and ARA in brain phospholipids, this review is aimed at providing information on the Yin-Yang mechanisms for regulating these PUFAs and their lipid peroxidation products in the CNS, and implications of their roles in neurological disorders.

Maternal Dietary Docosahexaenoic Acid Alters Lipid Peroxidation Products and (n-3)/(n-6) Fatty Acid Balance in Offspring Mice.

The abundance of docosahexaenoic acid (DHA) in the mammalian brain has generated substantial interest in the search for its roles in regulating brain functions. Our recent study with a gene/stress mouse model provided evidence to support the ability for the maternal supplement of DHA to alleviate autism-associated behavior in the offspring. DHA and arachidonic acid (ARA) are substrates of enzymatic and non-enzymatic reactions, and lipid peroxidation results in the production of 4-hydroxyhexenal (4-HHE) and 4-hydroxynonenal (4-HNE), respectively. In this study, we examine whether a maternal DHA-supplemented diet alters fatty acids (FAs), as well as lipid peroxidation products in the pup brain, heart and plasma by a targeted metabolite approach. Pups in the maternal DHA-supplemented diet group showed an increase in DHA and a concomitant decrease in ARA in all brain regions examined. However, significant increases in 4-HHE, and not 4-HNE, were found mainly in the cerebral cortex and hippocampus. Analysis of heart and plasma showed large increases in DHA and 4-HHE, but a significant decrease in 4-HNE levels only in plasma. Taken together, the DHA-supplemented maternal diet alters the (n-3)/(n-6) FA ratio, and increases 4-HHE levels in pup brain, heart and plasma. These effects may contribute to the beneficial effects of DHA on neurodevelopment, as well as functional changes in other body organs.