Analysis of the brain transcriptome, microbiome and metabolome in ketogenic diet and experimental stroke.

The ketogenic diet (KD) has been shown to be effective in treating various brain pathologies. In this study, we conducted detailed transcriptomic and metabolomic profiling of rat brains after KD and ischemic stroke in order to investigate the effects of KD and its underlying mechanisms. We evaluated the effect of a two-month KD on gene expression in intact brain tissue and after middle cerebral artery occlusion (MCAO). We analyzed the effects of KD on gut microbiome composition and blood metabolic profile as well as investigated the correlation between severity of neurological deficits and KD-induced changes. We found transcriptional reprogramming in the brain after stroke and KD treatment. The KD altered the expression of genes involved in the regulation of glucose and fatty acid metabolism, mitochondrial function, the immune response, Wnt-associated signaling, stem cell development, and neurotransmission, both in intact rats and after MCAO. The KD led to a significant change in the composition of gut microbiome and the levels of amino acids, acylcarnitines, polyunsaturated fatty acids, and oxylipins in the blood. However, the KD slightly worsened the neurological functions after MCAO, so that the therapeutic effect of the diet remained unproven.

Plasma oxylipin profiles reflect Parkinson's disease stage.

Derivatives of polyunsaturated fatty acids (PUFAs), also known as oxylipins, are key participants in regulating inflammation. Neuroinflammation is involved in many neurodegenerative diseases, including Parkinson's disease. The development of ultra-high-performance liquid chromatography-mass spectrometry (UPLC-MS/MS) facilitated the study of oxylipins on a system level, i.e., the analysis of oxylipin profiles. We analyzed oxylipin profiles in the blood plasma of 36 healthy volunteers (HC) and 73 patients with Parkinson's disease (PD), divided into early (L\M, 29 patients) or advanced (H, 44 patients) stages based on the Hoehn and Yahr scale. Among the 40 oxylipins detected, we observed a decrease in the concentration of arachidonic acid (AA) and AA derivatives, including anandamide (AEA) and Leukotriene E4 (LTE4), and an increase in the concentration of hydroxyeicosatetraenoic acids 19-HETE and 12-HETE (PD vs HC). Correlation analysis of gender, age of PD onset, and disease stages revealed 20 compounds the concentration of which changed depending on disease stage. Comparison of the acquired oxylipin profiles to openly available PD patient brain transcriptome datasets showed that plasma oxylipins do not appear to directly reflect changes in brain metabolism at different disease stages. However, both the L\M and H stages are characterized by their own oxylipin profiles - in patients with the H stage oxylipin synthesis is increased, while in patients with L\M stages oxylipin synthesis decreases compared to HC. This suggests that different therapeutic approaches may be more effective for patients at early versus late stages of PD.

Blood Oxylipin Profiles as Markers of Oncological Diseases.

Oxylipins are signal lipid molecules formed from polyunsaturated fatty acids (PUFAs) in several multienzymatic metabolic pathways, such as cyclooxygenase (COX), lipoxygenase (LOX), epoxygenase (CYP), and anandamide pathways, as well as non-enzymatically. The pathways of PUFA transformation are activated in parallel, yielding a mixture of physiologically active substances. Although the association of oxylipins with carcinogenesis had been established a long time ago, only recently analytical methods have advanced to a degree allowing detection and quantification of oxylipins from different classes (oxylipin profiles). The review describes current approaches to the HPLC-MS/MS analysis of oxylipin profiles and compares oxylipin profiles from patients with oncological diseases (breast cancer, colorectal cancer, ovarian cancer, lung cancer, prostate cancer, liver cancer). The possibility of using blood oxylipin profiles as biomarkers in oncological diseases is discussed. Understanding the patterns of PUFA metabolism and physiological activity of combinations of oxylipins will improve early diagnostics of oncological diseases and evaluation of disease prognosis.

Anti-Inflammatory Properties of Metformin During Cultivation of Primary Rat Astrocytes in a Medium with High Glucose Concentration.

Investigation of the relationship between inflammation and energy metabolism is important for understanding biology of chronic noncommunicable diseases. Use of metformin, a drug for treatment of diabetes, is considered as a promising direction for treatment of neurodegenerative diseases and other neuropathologies with an inflammatory component. Astrocytes play an important role in the regulation of energy metabolism and neuroinflammation; therefore, we studied the effect of metformin on the cellular responses of primary rat astrocytes cultured in a medium with high glucose concentration (22.5 mM, 48-h incubation). Lipopolysaccharide (LPS) was used to stimulate inflammation. The effects of metformin were assessed by monitoring changes in the expression of proinflammatory cytokines and synthesis of oxylipins, assayed with ultra-high-performance liquid chromatography and tandem mass spectrometry (UPLC-MS/MS). Changes at the intracellular level were assessed by analyzing phosphorylation of ERK kinase and transcription factor STAT3, as well as enzymes mediating oxylipin synthesis, cyclooxygenase 1 and 2 (COX). It was found that, independent on glucose concentration, metformin reduced the LPS-stimulated release of cytokines IL-1ß and IL-6, decreased activity of the transcription factor STAT3, ERK kinase, synthesis of the derivatives of the cyclooxygenase branch of metabolism of oxylipins and anandamide, and did not affect formation of ROS. The study of energy phenotype of the cells showed that metformin activated glycolysis and inhibited mitochondrial respiration and oxidative phosphorylation, independent on LPS stimulation and cell cultivation at high glucose concentration. Thus, it has been shown that metformin exhibits anti-inflammatory effects, and its effect on the synthesis of cytokines, prostaglandins, and other lipid mediators could determine beneficial effects of metformin in models of neuropathology.

Comparative lipidomic analysis of inflammatory mediators in the aqueous humor and tear fluid of humans and rabbits.

INTRODUCTION: Ocular inflammation is a key pathogenic factor in most blindness-causing visual disorders. It can manifest in the aqueous humor (AH) and tear fluid (TF) as alterations in polyunsaturated fatty acids (PUFAs) and their metabolites, oxylipins, lipid mediators, which are biosynthesized via enzymatic pathways involving lipoxygenase, cyclooxygenase or cytochrome P450 monooxygenase and specifically regulate inflammation and resolution pathways. OBJECTIVES: This study aimed to establish the baseline patterns of PUFAs and oxylipins in AH and TF by their comprehensive lipidomic identification and profiling in humans in the absence of ocular inflammation and comparatively analyze these compounds in the eye liquids of rabbits, the species often employed in investigative ophthalmology. METHODS: Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used for qualitative and quantitative characterization of lipid compounds in the analyzed samples. RESULTS: A total of 28 lipid compounds were identified, including phospholipid derivatives and PUFAs, as well as 22 oxylipins. Whereas the PUFAs included arachidonic, docosahexaenoic and eicosapentaenoic acids, the oxylipins were derived mainly from arachidonic, linoleic and α-linolenic acids. Remarkably, although the concentration of oxylipins in AH was lower compared to TF, these liquids showed pronounced similarity in their lipid profiles, which additionally exhibited noticeable interspecies concordance. CONCLUSION: The revealed correlations confirm the feasibility of rabbit models for investigating pathogenesis and trialing therapies of human eye disorders. The identified metabolite patterns suggest enzymatic mechanisms of oxylipin generation in AH and TF and might be used as a reference in ocular inflammation studies.

Comparison of PPAR Ligands as Modulators of Resolution of Inflammation, via Their Influence on Cytokines and Oxylipins Release in Astrocytes.

Neuroinflammation is a key process of many neurodegenerative diseases and other brain disturbances, and astrocytes play an essential role in neuroinflammation. Therefore, the regulation of astrocyte responses for inflammatory stimuli, using small molecules, is a potential therapeutic strategy. We investigated the potency of peroxisome proliferator-activated receptor (PPAR) ligands to modulate the stimulating effect of lipopolysaccharide (LPS) in the primary rat astrocytes on (1) polyunsaturated fatty acid (PUFAs) derivative (oxylipins) synthesis; (2) cytokines TNFα and interleukin-10 (IL-10) release; (3) p38, JNK, ERK mitogen-activated protein kinase (MAPKs) phosphorylation. Astrocytes were exposed to LPS alone or in combination with the PPAR ligands: PPARα (fenofibrate, GW6471); PPARß (GW501516, GSK0660); PPARγ (rosiglitazone, GW9662). We detected 28 oxylipins with mass spectrometry (UPLC-MS/MS), classified according to their metabolic pathways: cyclooxygenase (COX), cytochrome P450 monooxygenases (CYP), lipoxygenase (LOX) and PUFAs: arachidonic (AA), docosahexaenoic (DHA), eicosapentaenoic (EPA). All tested PPAR ligands decrease COX-derived oxylipins; both PPARß ligands possessed the strongest effect. The PPARß agonist, GW501516 is a strong inducer of pro-resolution substances, derivatives of DHA: 4-HDoHE, 11-HDoHE, 17-HDoHE. All tested PPAR ligands decreased the release of the proinflammatory cytokine, TNFα. The PPARß agonist GW501516 and the PPARγ agonist, rosiglitazone induced the IL-10 release of the anti-inflammatory cytokine, IL-10; the cytokine index, (IL-10/TNFα) was more for GW501516. The PPARß ligands, GW501516 and GSK0660, are also the strongest inhibitors of LPS-induced phosphorylation of p38, JNK, ERK MAPKs. Overall, our data revealed that the PPARß ligands are a potential pro-resolution and anti-inflammatory drug for targeting glia-mediated neuroinflammation.

Inhibitor of Hyaluronic Acid Synthesis 4-Methylumbelliferone as an Anti-Inflammatory Modulator of LPS-Mediated Astrocyte Responses.

Astrocytes are glial cells that play an important role in neuroinflammation. Astrocytes respond to many pro-inflammatory stimuli, including lipopolysaccharide (LPS), an agonist of Toll-like receptor 4 (TLR4). Regulatory specificities of inflammatory signaling pathways are still largely unknown due to the ectodermal origin of astrocytes. Recently, we have shown that hyaluronic acid (HA) may form part of astrocyte inflammatory responses. Therefore, we tested 4-methylumbelliferone (4-MU), a specific inhibitor of HA synthesis, as a possible regulator of LPS-mediated responses. Rat primary astrocytes were treated with LPS with and without 4-MU and gene expression levels of inflammatory (interleukins 1ß, (IL-1ß), 6, (IL-6), tumor necrosis factor alpha TNFα,) and resolution interleukin 10 (IL-10) markers were evaluated via real-time PCR and western blot. The release of cytokines and HA was determined by ELISA. Oxylipin profiles were measured by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis. Our data show that 4-MU (i) has anti-inflammatory effects in the course of TLR4 activation, decreasing the cytokines level TNFα, IL-6 and IL-1ß and increasing IL-10, (ii) downregulates prostaglandin synthesis but not via cyclooxygenases COX-1 and COX-2 pathways, (iii) modulates HA synthesis and decreases LPS-induced HA synthase mRNA expression (HAS-1, HAS-2) but does not have an influence on HAS-3, HYAL1 and HYAL2 mRNAs; (iv) the effects of 4-MU are predominantly revealed via JNK but not p38, ERK mitogen-activated protein kinases (MAPKs) or nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) pathways. For the first time, it is shown that 4-MU possesses the useful potential to regulate an inflammatory astrocyte response.

Oxylipin Profiles as Functional Characteristics of Acute Inflammatory Responses in Astrocytes Pre-Treated with IL-4, IL-10, or LPS.

Functional phenotypes, which cells can acquire depending on the microenvironment, are currently the focus of investigations into new anti-inflammatory therapeutic approaches. Glial cells, microglia, and astrocytes are major participants in neuroinflammation, but their roles differ, as microglia are cells of mesodermal origin, while astrocytes are cells of ectodermal origin. The inflammatory phenotype of cells can be modulated by ω-6- and ω-3-polyunsaturated fatty acid-derived oxylipins, although data on changes in oxylipin profiles in different cell adaptations to pro- and anti-inflammatory stimuli are scarce. Our study aimed to compare UPLC-MS/MS-measured oxylipin profiles in various rat astrocyte adaptation states. We used cells treated for 24 h with lipopolysaccharide (LPS) for classical pro-inflammatory adaptation and with interleukin 4 (IL-4) or 10 (IL-10) for alternative anti-inflammatory adaptation, with the resulting phenotypes characterized by quantitative real-time PCR (RT-PCR). We also tested long-term, low-concentration LPS treatment (endotoxin treatment) as a model of astrocyte adaptations. The functional response of astrocytes was estimated by acute (4 h) LPS-induced cell reactivity, measured by gene expression markers and oxylipin synthesis. We discovered that, as well as gene markers, oxylipin profiles can serve as markers of pro- (A1-like) or anti-inflammatory (A2-like) adaptations. We observed predominant involvement of ω-6 polyunsaturated fatty acid (PUFA) and the cyclooxygenase branch for classical (LPS) pro-inflammatory adaptations and ω-3 PUFA and the lipoxygenase branch for alternative (IL-4) anti-inflammatory adaptations. Treatment with IL-4, but not IL-10, primes the ability of astrocytes to activate the innate immunity signaling pathways in response to LPS. Endotoxin-treated astrocytes provide an alternative anti-inflammatory adaptation, which makes cells less sensitive to acute LPS stimulation than the IL-4 induced adaptation. Taken together, the data reveal that oxylipin profiles associate with different states of polarization to generate a pro-inflammatory or anti-inflammatory phenotype. This association manifests itself both in native cells and in their responses to a pro-inflammatory stimulus.

Mechanisms and Treatment of Light-Induced Retinal Degeneration-Associated Inflammation: Insights from Biochemical Profiling of the Aqueous Humor.

Ocular inflammation contributes to the pathogenesis of blind-causing retinal degenerative diseases, such as age-related macular degeneration (AMD) or photic maculopathy. Here, we report on inflammatory mechanisms that are associated with retinal degeneration induced by bright visible light, which were revealed while using a rabbit model. Histologically and electrophysiologically noticeable degeneration of the retina is preceded and accompanied by oxidative stress and inflammation, as evidenced by granulocyte infiltration and edema in this tissue, as well as the upregulation of total protein, pro-inflammatory cytokines, and oxidative stress markers in aqueous humor (AH). Consistently, quantitative lipidomic studies of AH elucidated increase in the concentration of arachidonic (AA) and docosahexaenoic (DHA) acids and lyso-platelet activating factor (lyso-PAF), together with pronounced oxidative and inflammatory alterations in content of lipid mediators oxylipins. These alterations include long-term elevation of prostaglandins, which are synthesized from AA via cyclooxygenase-dependent pathways, as well as a short burst of linoleic acid derivatives that can be produced by both enzymatic and non-enzymatic free radical-dependent mechanisms. The upregulation of all oxylipins is inhibited by the premedication of the eyes while using mitochondria-targeted antioxidant SkQ1, whereas the accumulation of prostaglandins and lyso-PAF can be specifically suppressed by topical treatment with cyclooxygenase inhibitor Nepafenac. Interestingly, the most prominent antioxidant and anti-inflammatory benefits and overall retinal protective effects are achieved by simultaneous administrating of both drugs indicating their synergistic action. Taken together, these findings provide a rationale for using a combination of mitochondria-targeted antioxidant and cyclooxygenase inhibitor for the treatment of inflammatory components of retinal degenerative diseases.

High and Low Molecular Weight Hyaluronic Acid Differentially Influences Oxylipins Synthesis in Course of Neuroinflammation.

Hyaluronic acid (HA), a major glycosaminoglycan of the extracellular matrix, has cell signaling functions that are dependent on its molecular weight. Anti-inflammatory effects for high-molecular-weight (HMW) HA and pro-inflammatory effects for low-molecular-weight (LMW) HA effects were found for various myeloid cells, including microglia. Astrocytes are cells of ectodermal origin that play a pivotal role in brain inflammation, but the link between HA with different molecular weights and an inflammatory response in these cells is not clear. We tested the effects of LMW and HMW HA in rat primary astrocytes, stimulated with Poly:IC (PIC, TLR3 agonist) and lipopolysaccharide (LPS, TLR4 agonist). Oxylipin profiles were measured by the UPLC-MS/MS analysis and metabolites HDoHEs (from docosahexaenoic acid), -HETEs, prostaglandins (from arachidonic acid), DiHOMEs and HODEs (from linoleic acid) were detected. Both, HMW and LMW HA downregulated the cyclooxygenase-mediated polyunsaturated fatty acids metabolism, LMW also reduced lipoxygenase-mediated fatty acid metabolism. Taken together, the data show that both LMW and HMW (i) influence themselves on cytokines (TNFα, IL-6, IL-10), enzymes iNOS, COX-2, and oxylipin levels in extracellular medium of cultured astrocytes, (ii) induced cellular adaptations in long-term applications, (iii) modulate TLR4- and TLR3-signaling pathways. The effects of HMW and LMW HA are predominantly revealed in TLR4- and TLR3- mediated responses, respectively.

Astrocytes synthesize primary and cyclopentenone prostaglandins that are negative regulators of their proliferation.

Recently, the modulation of cellular inflammatory responses via endogenous regulators became a major focus of medically relevant investigations. Prostaglandins (PGs) are attractive regulatory molecules, but their synthesis and mechanisms of action in brain cells are still unclear. Astrocytes are involved in manifestation of neuropathology and their proliferation is an important part of astrogliosis, a cellular neuroinflammatory response. The aims of our study were to measure synthesis of PGs by astrocytes, and evaluate their influence on proliferation in combination with addition of inflammatory pathway inhibitors. With UPLC-MS/MS analysis we detected primary PGs (1410 ±â€¯36 pg/mg PGE2, 344 ±â€¯24 PGD2) and cyclopentenone PGs (cyPGs) (87 ±â€¯17 15d-PGJ2, 308 ±â€¯23 PGA2) in the extracellular medium after 24-h lipopolysaccharide (LPS) stimulation of astrocytes. PGs reduced astrocytic proliferation with the following order of potencies (measured as inhibition at 20 µM): most potent 15d-PGJ2 (90%) and PGA2 (80%), > PGD2 (40%) > 15d-PGA2 (20%) > PGE2 (5%), the least potent. However, PGF2α and 2-cyclopenten-1-one, and ciglitazone and rosiglitazone (synthetic agonists of PPARγ) had no effect. Combinations of cyPGs with SC-560 or NS-398 (specific anti-inflammatory inhibitors of cyclooxygenase-1 and -2, respectively) were not effective; while GW9662 (PPARγ antagonist) or MK-741 (inhibitor of multidrug resistance protein-1, MRP1, and CysLT1 receptors) amplified the inhibitory effect of PGA2 and 15d-PGJ2. Although concentrations of individual PGs and cyPGs are low, all of them, as well as primary PGs suppress proliferation. Thus, the effects are potentially additive, and activated PGs synthesis suppresses proliferation in astrocytes.

Resolution of inflammation and mood disorders.

Relationship between mood disorders and inflammation is now well-documented, although molecular mechanisms are not understood. Previously mostly pro-inflammatory cytokines of immune system (IL-6, TNF, etc.) were taken into account. However, recent understanding of resolution of inflammation as an active process drew attention to mediators of resolution, which include both proteins and ω-3 and ω-6 polyunsaturated fatty acids derivatives (resolvins, cyclopentenone prostaglandins, etc.). This review takes into account new data on resolution of inflammation and action of mediators of resolution in models of depression. New facts and ideas about mechanisms of chronic inflammation onset are considered in relation to mood disorders. Basic control mechanisms of inflammation at the cellular level and the role of resolution substances in regulation of depression and other mood disorders are discussed. Signaling systems of innate immunity located in non-immune cells and their ability to generate substances that affect an onset of depression are reviewed. A novel hypothesis of depression as a type of abnormal resolution is proposed.

Sex-Mediated Differences in LPS Induced Alterations of TNFα, IL-10 Expression, and Prostaglandin Synthesis in Primary Astrocytes.

Although many neurological and psychiatric disorders reveal clear sex-dependent variations, the molecular mechanism of this process is not clear enough. Astrocytes are involved in the response of neural tissue to injury and inflammation, produce steroid hormones, and sense steroid presence. To explore the hypothesis that astrocytes may participate in sex-mediated differences of inflammatory responses, we have examined whether male and female primary rat astrocytes show different responses to lipopolysaccharide (LPS) as a toll-like receptor 4 (TLR4) agonist. Levels of mRNA and proteins of tumor necrosis factor alpha (TNFα), interleukin-10 (IL-10), and cyclooxygenase (COX)-2 were assessed using qPCR, immunoblotting, and ELISA. UPLC-MS/MS was used to detect prostaglandins (PGs). LPS stimulation resulted in different levels of cytokine production; more TNFα and less IL-10 were produced in female cells compared with male astrocytes. Although the levels of the COX-2 expression were not altered, LPS significantly induced the synthesis of PGs with notable sex-related differences. PGE2 and PGD2 were less and 6-keto-PGF1α was more upregulated in female astrocytes, and TXB2 had similar levels in cells obtained from males and females. Trilostane, an inhibitor of 3ß-Hydroxysteroid dehydrogenase (3ß-HSD), inhibited the LPS-induced TNFα production and the release of PGE2, PGD2, and 6-keto-PGF1α in female astrocytes. Thus, male and female astrocytes differentially respond to inflammatory challenges on the level of production of cytokines and steroid hormones. Sex-mediated differences in pro- and anti-inflammatory responses should be taken into consideration for the effective treatment of disorders with neuroinflammation.

Rosiglitazone as a Modulator of TLR4 and TLR3 Signaling Pathways in Rat Primary Neurons and Astrocytes.

An antidiabetic drug of the thiazolidinedione class, rosiglitazone (RG) demonstrates anti-inflammatory properties in various brain pathologies. The mechanism of RG action in brain cells is not fully known. To unravel mechanisms of RG modulation of toll-like receptor (TLR) signaling pathways, we compare primary rat neuron and astrocyte cultures stimulated with the TLR4 agonist lipopolysaccharide (LPS) and the TLR3 agonist poly I:C (PIC). Both TLR agonists induced tumor necrosis factor (TNFα) release in astrocytes, but not in neurons. Neurons and astrocytes released interleukin-10 (IL-10) and prostaglandin E2 (PGE2) in response to LPS and PIC. RG decreased TLR-stimulated TNFα release in astrocytes as well as potentiated IL-10 and PGE2 release in both astrocytes and neurons. RG induced phosphorylation of p38 and JNK MAPK (mitogen-activated protein kinase) in neurons. The results reveal new role of RG as a modulator of resolution of neuroinflammation.

Deuterated Arachidonic Acids Library for Regulation of Inflammation and Controlled Synthesis of Eicosanoids: An In Vitro Study.

The synthesis of signal lipids, including eicosanoids, is not fully understood, although it is key to the modulation of various inflammatory states. Recently, isotopologues of essential polyunsaturated fatty acids (PUFAs) deuterated at bis-allylic positions (D-PUFAs) have been proposed as inhibitors of non-enzymatic lipid peroxidation (LPO) in various disease models. Arachidonic acid (AA, 20:4 n-6) is the main precursor to several classes of eicosanoids, which are produced by cyclooxygenases (COX) and lipoxygenases (LOX). In this study we analyzed the relative activity of human recombinant enzymes COX-2, 5-LOX, and 15-LOX-2 using a library of arachidonic acids variably deuterated at the bis-allylic (C7, C10, and C13) positions. Kinetic parameters (KM, Vmax) and isotope effects calculated from kH/kD for seven deuterated arachidonic acid derivatives were obtained. Spectroscopic methods have shown that deuteration at the 13th position dramatically affects the kinetic parameters of COX-2 and 15-LOX-2. The activity of 5-LOX was evaluated by measuring hydroxyeicosatetraenoic acids (8-HETE and 5-HETE) using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Deuteration at the seventh and 10th positions affects the performance of the 5-LOX enzyme. A flowchart is proposed suggesting how to modulate the synthesis of selected eicosanoids using the library of deuterated isotopologues to potentially fine-tune various inflammation stages.

Regulation of peroxisome proliferator-activated receptors (PPAR) α and -γ of rat brain astrocytes in the course of activation by toll-like receptor agonists.

Peroxisome proliferator-activated receptors (PPAR)-α and -γ in astrocytes play important roles in inflammatory brain pathologies. Understanding the regulation of both activity and expression levels of PPARs is an important neuroscience issue. Toll-like receptor (TLR) agonists are inflammatory stimuli that could modulate PPAR, but the mechanisms of their control in astrocytes are poorly understood. In the present study, we report that lipopolysaccharide, peptidoglycan, and flagellin, which are agonists of TLR4, TLR1/2, and TLR5, respectively, exert time- and nuclear factor kappa-light-chain-enhancer of activated B cells-dependent suppression of mRNA, protein and activity of PPARα and PPARγ. In naïve astrocytes, PPARα and PPARγ mRNA have short turnover time (half-life about 30 min for PPARα, 75 min for PPARγ) with a nearly two-fold stabilization after TLR-activation. p38 inhibition abolished TLR-induced stabilization. The levels of PPARα and PPARγ mRNA, and protein and DNA-binding activity could be modified using c-Jun N-terminal Kinase and p38 inhibitors. In addition, the expression levels of both PPARα and PPARγ isotypes were induced after inhibition of protein synthesis. This induction signifies participation of additional regulatory proteins with short life-time. They are p38-sensitive for PPARα and c-Jun N-terminal Kinase-sensitive for PPARγ. Thus, PPARα and PPARγ are regulated in astrocytes on mRNA and protein levels, mRNA stability, and DNA-binding activity during TLR-mediated responses. Astrocytes have the triad of PPARα, PPARß/δ, and PPARγ in regulation of proinflammatory responses. Activation of Toll-like receptors (TLR) leads to PPARß/δ overexpression, PPARα and PPARγ suppression via TLR/NF-κB pathway on mRNA, protein and activity levels. Mitogen-activated protein kinases (MAPK) p38 and JNK are involved in regulation of PPAR expression. p38 MAPK plays a special role in stabilization of PPAR mRNA.

Regulation of peroxisome proliferator-activated receptor ß/δ expression and activity levels by toll-like receptor agonists and MAP kinase inhibitors in rat astrocytes.

Peroxisome proliferator-activated receptor ß/δ (PPARß/δ) is a potential regulator of neuroinflammation. Toll-like receptors (TLR) are innate immunity-related receptors of inflammatory stimuli. In the present report, we evaluate the molecular mechanisms of regulation of mRNA, protein, and transcriptional activity levels of PPARß/δ by agonists of TLR4, TLR1/2, and TLR5, using lipopolysaccharide (LPS), peptidoglycan, and flagellin, respectively. We found that these stimuli increase the PPARß/δ levels in astrocytes. Expression and activity of PPARß/δ are separately regulated by inhibitors of p38, MEK1/2, extracellular signal-regulated kinases 1/2, and c-Jun N-terminal Kinase mitogen-activated protein kinases. The LPS-induced kinetics of PPARß/δ expression is similar to that of the proinflammatory gene cyclooxygenase 2. Moreover, for both genes the expression depends on nuclear factor kappa-light-chain-enhancer of activated B cells and p38, and is induced after inhibition of protein synthesis. The up-regulation of the expression after inhibition of protein synthesis signifies the participation of a labile protein in regulation of PPARß/δ expression. In contrast to cyclooxygenase 2, the cycloheximide-sensitive PPARß/δ expression was not responsive to nuclear factor kappa-light-chain-enhancer of activated B cells inhibition. Measurements of PPARß/δ mRNA stability showed that the PPARß/δ mRNA levels are regulated post-transcriptionally. We found that in LPS-stimulated astrocytes, the half-life of PPARß/δ mRNA was 50 min. Thus, we demonstrate that PPARß/δ expression and activity are regulated in TLR agonist-stimulated astrocytes by mechanisms that are widely used for regulation of proinflammatory genes. Protein expression level of nuclear receptor PPARß/δ is important for functions of this transcription factor. We investigate the regulatory mechanisms of PPARß/δ in rat primary astrocytes stimulated by agonists of toll-like receptors (TLR): TLR4, TLR1/2, and TLR5. Expression, activity, mRNA stability, and superinduction of PPARß/δ were up-regulated after TLR stimulation. These processes are sensitive to MAPKs and NF-kB inhibitors. Superinduction is up-regulation of mRNA expression after inhibition of protein synthesis.

TLR3-mediated Astrocyte Responses in High and Normal Glucose Adaptation Differently Regulated by Metformin.

Toll-like receptors 3 (TLR3) are innate immune receptors expressed on a wide range of cell types, including glial cells. Inflammatory responses altered by hyperglycemia highlight the need to explore the molecular underpinnings of these changes in cellular models. Therefore, here we estimated TLR3-mediated response of astrocytes cultured at normal (NG, 5 mM) and high (HG, 22.5 mM) glucose concentrations for 48 h before stimulation with polyinosinic:polycytidylic acid Poly(I:C) (PIC) for 6 h. Seahorse Extracellular Flux Analyzer (Seahorse XFp) was used to estimate the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR). Although adaptation to HG affected ECAR and OCR, the stimulation of cells with PIC had no effect on ECAR. PIC reduced maximal OCR, but this effect disappeared upon adaptation to HG. PIC-stimulated release of cytokines IL-1ß, IL-10 was reduced, and that of IL-6 and iNOS was increased in the HG model. Adaptation to HG reduced PIC-stimulated synthesis of COX-derived oxylipins measured by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Adaptation to HG did not alter PIC-stimulated p38 activity, ERK mitogen-activated protein kinase, STAT3 and ROS production. Metformin exhibited anti-inflammatory activity, reducing PIC-stimulated synthesis of cytokines and oxylipins. Cell adaptation to high glucose concentration altered the sensitivity of astrocytes to TLR3 receptor activation, and the hypoglycemic drug metformin may exert anti-inflammatory effects under these conditions.

Arachidonic acid: reconciling the dichotomy of its oxidative cascade through specific deuteration.

A new approach to attenuating pathological inflammatory reactions by buffering the eicosanoid pathways with oxidation-resistant hexadeuterated arachidonic acid (D-ARA) is discussed. Enzymatic processing of ARA, released by phospholipase A2, by lipoxygenases, cyclooxygenases, and cytochromes yields a wide range of bioactive eicosanoids, including pro-inflammation, pro-angiogenesis and pro-thrombosis species that, when produced in excess, are an underlying cause of pathology. Conversely, some products of ARA oxidation possess pro-resolving properties. Non-enzymatic free radical oxidation of ARA generates another large group of products such as isoprostanes and their metabolites, associated with inflammation, ischemia-reperfusion stress, and atherosclerosis. A separate group comprises reactive carbonyl derivatives that irreversibly damage diverse biomolecules. Being resistant to both enzymatic and non-enzymatic oxidation pathways due to large kinetic isotope effects, D-ARA may play a role in mitigating inflammation-related disorders and conditions, including inflammaging.

Investigation of the Role of PUFA Metabolism in Breast Cancer Using a Rank-Based Random Forest Algorithm.

Polyunsaturated fatty acid (PUFA) metabolism is currently a focus in cancer research due to PUFAs functioning as structural components of the membrane matrix, as fuel sources for energy production, and as sources of secondary messengers, so called oxylipins, important players of inflammatory processes. Although breast cancer (BC) is the leading cause of cancer death among women worldwide, no systematic study of PUFA metabolism as a system of interrelated processes in this disease has been carried out. Here, we implemented a Boruta-based feature selection algorithm to determine the list of most important PUFA metabolism genes altered in breast cancer tissues compared with in normal tissues. A rank-based Random Forest (RF) model was built on the selected gene list (33 genes) and applied to predict the cancer phenotype to ascertain the PUFA genes involved in cancerogenesis. It showed high-performance of dichotomic classification (balanced accuracy of 0.94, ROC AUC 0.99) We also retrieved a list of the important PUFA genes (46 genes) that differed between molecular subtypes at the level of breast cancer molecular subtypes. The balanced accuracy of the classification model built on the specified genes was 0.82, while the ROC AUC for the sensitivity analysis was 0.85. Specific patterns of PUFA metabolic changes were obtained for each molecular subtype of breast cancer. These results show evidence that (1) PUFA metabolism genes are critical for the pathogenesis of breast cancer; (2) BC subtypes differ in PUFA metabolism genes expression; and (3) the lists of genes selected in the models are enriched with genes involved in the metabolism of signaling lipids.