Resveratrol Inhibits Inflammatory Responses via the Mammalian Target of Rapamycin Signaling Pathway in Cultured LPS-Stimulated Microglial Cells

Resveratrol have been known to possess many pharmacological properties including antioxidant, cardioprotective and anticancer effects. Although current studies indicate that resveratrol produces neuroprotection against neurological disorders, the precise mechanisms for its beneficial effects are still not fully understood. We investigate the effect of anti-inflammatory and mechamisms of resveratrol by using lipopolysaccharide (LPS)-stimulated murine microglial BV-2 cells.BV-2 cells were treated with resveratrol (25, 50, and 100 µM) and/or LPS (1 µg/ml). Nitric oxide (NO) and prostaglandin E2 (PGE2) were measured by Griess reagent and ELISA. The mRNA and protein levels of proinflammatory proteins and cytokines were analysed by RT-PCR and double immunofluorescence labeling, respectively. Phosphorylation levels of PTEN (phosphatase and tensin homolog deleted on chromosome 10), Akt, mammalian target of rapamycin (mTOR), mitogen-activated protein kinases (MAPKs) cascades, inhibitor κB-α (IκB-α) and cyclic AMP-responsive element-binding protein (CREB) were measured by western blot. Resveratrol significantly attenuated the LPS-induced expression of NO, PGE2, inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and nuclear factor-κB (NF-κB) in BV-2 cells. Resveratrol increased PTEN, Akt and mTOR phosphorylation in a dose-dependent manner or a time-dependent manner. Rapamycin (10 nM), a specific mTOR inhibitor, blocked the effects of resveratrol on LPS-induced microglial activation. In addition, mTOR inhibition partially abolished the inhibitory effect of resveratrol on the phosphorylation of IκB-α, CREB, extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal protein kinase (JNK), and p38 mitogen-activated protein kinase (p38 MAPK).This study indicates that resveratrol inhibited LPS-induced proinflammatory enzymes and proinflammatory cytokines via down-regulation phosphorylation of NF-κB, CREB and MAPKs family in a mTOR-dependent manner. These findings reveal, in part, the molecular basis underlying the anti-inflammatory properties of resveratrol

Gastrodin Inhibits Expression of Inducible NO Synthase, Cyclooxygenase-2 and Proinflammatory Cytokines in Cultured LPS-Stimulated Microglia via MAPK Pathways

Microglial activation plays an important role in neurodegenerative diseases by producing several proinflammatory enzymes and proinflammatory cytokines. The phenolic glucoside gastrodin, a main constituent of a Chinese herbal medicine, has been known to display anti-inflammatory properties. The current study investigates the potential mechanisms whereby gastrodin affects the expression of potentially pro-inflammatory proteins by cultured murine microglial BV-2 cells stimulated with lipopolysaccharide (LPS).BV-2 cells were pretreated with gastrodin (30, 40, and 60 µM) for 1 h and then stimulated with LPS (1 µg/ml) for another 4 h. The effects on proinflammatory enzymes, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), and proinflammatory cytokines, tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β), are analysed by double-immunofluorescence labeling and RT-PCR assay. To reveal the mechanisms of action of gastrodin we investigated the involvement of mitogen-activated protein kinases (MAPKs) cascades and their downstream transcription factors, nuclear factor-κB (NF-κB) and cyclic AMP-responsive element (CRE)-binding protein (CREB). Gastrodin significantly reduced the LPS-induced protein and mRNA expression levels of iNOS, COX-2, TNF-α, IL-1β and NF-κB. LPS (1 µg/ml, 30 min)-induced phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal protein kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK) and this was inhibited by pretreatment of BV-2 cells with different concentrations of gastrodin (30, 40, and 60 µM). In addition, gastrodin blocked LPS-induced phosphorylation of inhibitor κB-α (IκB-α) (and hence the activation of NF-κB) and of CREB, respectively.This study indicates that gastrodin significantly attenuate levels of neurotoxic proinflammatory mediators and proinflammatory cytokines by inhibition of the NF-κB signaling pathway and phosphorylation of MAPKs in LPS-stimulated microglial cells. Arising from the above, we suggest that gastrodin has a potential as an anti-inflammatory drug candidate in neurodegenerative diseases

Balancing the immune response in the brain: IL-10 and its regulation

Background: The inflammatory response is critical to fight insults, such as pathogen invasion or tissue damage, but if not resolved often becomes detrimental to the host. A growing body of evidence places non-resolved inflammation at the core of various pathologies, from cancer to neurodegenerative diseases. It is therefore not surprising that the immune system has evolved several regulatory mechanisms to achieve maximum protection in the absence of pathology. Main body: The production of the anti-inflammatory cytokine interleukin (IL)-10 is one of the most important mechanisms evolved by many immune cells to counteract damage driven by excessive inflammation. Innate immune cells of the central nervous system, notably microglia, are no exception and produce IL-10 downstream of pattern recognition receptors activation. However, whereas the molecular mechanisms regulating IL-10 expression by innate and acquired immune cells of the periphery have been extensively addressed, our knowledge on the modulation of IL-10 expression by central nervous cells is much scattered. This review addresses the current understanding on the molecular mechanisms regulating IL-10 expression by innate immune cells of the brain and the implications of IL-10 modulation in neurodegenerative disorders. Conclusion: The regulation of IL-10 production by central nervous cells remains a challenging field. Answering the many remaining outstanding questions will contribute to the design of targeted approaches aiming at controlling deleterious inflammation in the brain.We acknowledge the Portuguese Foundation for Science and Technology (FCT) for providing a PhD grant to DLS (SFRH/BD/88081/2012) and a post-doctoral fellowship to SR (SFRH/BPD/72710/2010). DS, AGC and SR were funded by FEDER through the Competitiveness Factors Operational Programme (COMPETE) and National Funds through FCT under the scope of the project POCI-01-0145-FEDER007038; and by the project NORTE-01-0145-FEDER-000013, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). The MS lab was financed by Fundo Europeu de Desenvolvimento Regional (FEDER) funds through the COMPETE 2020—Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by Portuguese funds through FCT in the framework of the project “Institute for Research and Innovation in Health Sciences ” (POCI-01-0145-FEDER-007274). MS is a FCT Associate Investigator. The funding body had no role in the design of the study and collection, analysis, and interpretation of the data and in writing the manuscript

Food for mood: relevance of nutritional omega-3 fatty acids for depression and anxiety

The central nervous system (CNS) has the highest concentration of lipids in the organism after adipose tissue. Among these lipids, the brain is particularly enriched with polyunsaturated fatty acids (PUFAs) represented by the omega-6 (omega 6) and omega-3 (omega 3) series. These PUFAs include arachidonic acid (AA) and docosahexaenoic acid (DHA), respectively. PUFAs have received substantial attention as being relevant to many brain diseases, including anxiety and depression. This review addresses an important question in the area of nutritional neuroscience regarding the importance of omega 3 PUFAs in the prevention and/or treatment of neuropsychiatric diseases, mainly depression and anxiety. In particular, it focuses on clinical and experimental data linking dietary intake of omega 3 PUFAs and depression or anxiety. In particular, we will discuss recent experimental data highlighting how omega 3 PUFAs can modulate neurobiological processes involved in the pathophysiology of anxiety and depression. Potential mechanisms involved in the neuroprotective and corrective activity of omega 3 PUFAs in the brain are discussed, in particular the sensing activity of free fatty acid receptors and the activity of the PUFAs-derived endocannabinoid system and the hypothalamic-pituitary-adrenal axis

Prenatal N-acetyl-cysteine prevents social anxiety and modulates hippocampal inflammatory-and plasticity-related genes in adolescent mice prenatally exposed to a high-fat diet

High-fat diet (HFD) consumption during pregnancy is associated with increased oxidative stress (OS) and low-grade chronic inflammation, and may affect fetal brain development, setting the stage for increased vulnerability to mood disorders later in life [1,2]. However, the biological mechanisms underlying the negative long-term effects of maternal HFD are poorly understood. N-acetyl-cysteine (NAC) is a promising antioxidant compound [3] that has revealed beneficial effects in the treatment of psychopathology. The aim of this study was to investigate inflammation, OS and hypothalamic-pituitary-adrenal (HPA) axis reactivity in a mouse model of maternal HFD as potential mechanisms affecting brain development and emotional behavior in the offspring. The prenatal NAC treatment was tested to prevent the negative effects of maternal HFD on adolescent offspring, an age of main vulnerability for the onset of psychopathologies. Female C57BL/6N mice were fed either HFD (energy 5.56 kcal/g, fat 58%, carbohydrate 25.5% and protein 16.4%) or control diet (CD, energy 4.07 kcal/g, fat 10.5%, carbohydrate 73.1% and protein 16.4%) before and during pregnancy (13 weeks); after 5 weeks on diets, half of them received NAC (1g/kg) for 8 weeks, until delivery. Emotionality and social behavior of male and female adolescent offspring (35-45 days) were assessed through the elevated plus maze (EPM) and the social interaction test (SIT); HPA axis functionality was assessed measuring plasma corticosterone levels by ELISA under basal conditions and following an acute stress. Gene expression levels of CD68, Bdnf and Nrf2 were measured in hippocampus as markers of microglial activation, brain plasticity and antioxidant capacity respectively by RealTime PCR. Data were analyzed using parametric analysis of variance (ANOVA) with diet (HFD vs. CD), treatment (NAC vs. WATER), sex (females vs. males) as between subjects factors. Post hoc comparisons were performed using the Tukey’s test. Prenatal exposure to HFD affected sociability reducing social behaviors (p<0.01, post hoc HFD-WATER vs. CD-WATER p<0.05) in the SIT and reduced exploration in the EPM (frequency of crossings p<0.01; head dipping p=0.0292; wall-rearing p=0.0255). As for the HPA axis functionality, reduced levels of basal corticosterone were found in HFD males (p<0.01, post hoc HFD-WATER vs. CD-WATER p<0.05). Moreover, prenatal HFD decreased hippocampal Bdnf levels in females (p<0.01, post hoc HFD-WATER vs. CD-WATER p<0.05), while males showed increased CD68 expression (p<0.01). Prenatal NAC administration prevented social anxiety, restored HPA axis basal activity in males and Bdnf levels in females (p<0.01, post hoc HFD-WATER vs. HFD-NAC p<0.05). In addition, hippocampal levels of Nrf2 resulted increased in both males and females (p<0.01), suggesting that NAC may act, at least in part, through an upregulation of this important regulator of brain antioxidant defenses. Overall, these data showed that maternal HFD induces long-term negative effects on the adolescent offspring, affecting brain, neuroendocrine system and emotional/social behavior. These effects are partially prevented by prenatal administration of NAC suggesting that immune and OS pathways may play an important role in fetal programming of mental disorders. Funding: ERANET-NEURON-JTC-2018 Project EMBE

The matrix metalloproteinase inhibitor marimastat promotes neural progenitor cell differentiation into neurons by gelatinase-independent TIMP-2-dependent mechanisms.

Metalloproteinases (MMPs) and their endogenous inhibitors (TIMPs), produced in the brain by cells of non-neural and neural origin, including neural progenitors (NPs), are emerging as regulators of nervous system development and adult brain functions. In the present study, we explored whether MMP-2, MMP-9, and TIMP-2, abundantly produced in the brain, modulate NP developmental properties. We found that treatment of NPs, isolated from the murine fetal cerebral cortex or adult subventricular zone, with the clinically tested broad-spectrum MMP inhibitor Marimastat profoundly affected the NP differentiation fate. Marimastat treatment allowed for an enrichment of our cultures in neuronal cells, inducing NPs to generate higher percentage of neurons and a lower percentage of astrocytes, possibly affecting NP commitment. Consistently with its proneurogenic effect, Marimastat early downregulated the expression of Notch target genes, such as Hes1 and Hes5. MMP-2 and MMP-9 profiling on proliferating and differentiating NPs revealed that MMP-9 was not expressed under these conditions, whereas MMP-2 increased in the medium as pro-MMP-2 (72 kDa) during differentiation; its active form (62 kDa) was not detectable by gel zymography. MMP-2 silencing or administration of recombinant active MMP-2 demonstrated that MMP-2 does not affect NP neuronal differentiation, nor it is involved in the Marimastat proneurogenic effect. We also found that TIMP-2 is expressed in NPs and increases during late differentiation, mainly as a consequence of astrocyte generation. Endogenous TIMP-2 did not modulate NP neurogenic potential; however, the proneurogenic action of Marimastat was mediated by TIMP-2, as demonstrated by silencing experiments. In conclusion, our data exclude a major involvement of MMP-2 and MMP-9 in the regulation of basal NP differentiation, but highlight the ability of TIMP-2 to act as key effector of the proneurogenic response to an inducing stimulus such as Marimastat

Striatal 6-Ohda Lesion In Mice: Investigating Early Neurochemical Changes Underlying Parkinson's Disease

Early phases of Parkinson's disease (PD) are characterized by a mild reduction of dopamine (DA) in striatum and by emergence of psychiatric disturbances that precede overt motor symptoms. in order to characterize the neurochemical re-arrangements induced by such striatal impairment, we used a mouse model in which a low dose of 6-hydroxydopamine (6-OHDA) was bilaterally injected into the dorsal striatum. These mice showed a DA reduction of about 40% that remained stable up to 12 weeks after injection. This reduction was accompanied by changes in DA metabolite levels, such as HVA, transiently reduced at 4 weeks, and DOPAC, decreased at 12 weeks. No change in the 5-hydroxytryptamine (5-HT) levels was found but the 5-hydroxyindoleacetic acid (5-HIAA)/5-HT ratio was increased at 4 weeks. In addition, at the same time-point, the levels of 15-F(2t)-IsoP, an index of oxidative stress, and of PGE2, a major product of cyclooxygenase-2, were decreased in different brain areas while BDNF levels were increased. These neurochemical changes were accompanied by altered behavioral responses concerning the emotional reactivity. Overall, the present findings suggest that a change of 5-HT metabolism and a modification of oxidative stress levels may play a role in the early PD degeneration phases

Modulatory effects following subchronic stimulation of brain 5-HT7-R system in mice and rats.

The serotonin receptor 7 (5-HT7-R), is very abundant in the thalamus, hypothalamus, dorsal raphe nucleus and hippocampus; at lower levels it is found also in the cortex, striatum and amygdala. This neurochemical receptor system plays important functional roles in learning and memory, in regulation of mood and circadian rhythmicity. Recently, many agonist drugs selective for this receptor have been developed and their effects reported. Presently, we review some recent studies we have conducted aimed to evaluate the modulatory effect of a subchronic regimen with LP-211, a new selective agonist compound belonging to 1-arylpiperazine category, on behavioural and molecular parameters. At low dose (0.25 mg/kg, i.p.), LP-211 induced in adult mice a six-hour anticipated wake up with no temporal landmark (constant light) acting as non-photic stimulus for “internal clock” resetting. In standard 12:12h L/D cycle, we observed a subchronic effect (5-6 days at 0.25 mg/kg, once/day), on delay of wake up associated with a reduction of peak locomotor activity and any evidence for a real cellular proliferation after ex vivo analysis. The other studies, conduct on rats, were aimed to investigate long-term effects of subchronic LP-211 administration into adulthood development. Subchronic LP-211 (0.125 mg/kg, i.p. once/day) during the prepuberal phase reduced L-Glutamate, NMDAR1 and DAT, within the ventral striatum. Moreover, we observed a clear reduction of the immunoreactivity of serotonin transporter (SERT) and dopaminergic D2 receptors at dorsal striatum. These results represent a starting point to explore the neurophysiology of 5-HT7-R: subchronic LP-211 in rats and mice appears a suitable tool for studying the role of 5-HT7-R in sleep disorders, emotional and motivational regulations, attentive processes and executive functions