Quantitative Proteomics Reveals Docosahexaenoic Acid-Mediated Neuroprotective Effects in Lipopolysaccharide-Stimulated Microglial Cells.
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ABSTRACT:
SUBMITTER: Yang B
PROVIDER: S-EPMC7282485 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Microglial cell activation has been linked to many neurodegenerative diseases. Upon stimulation by lipopolysaccharide (LPS), a number of proteins involved in inflammatory and oxidative pathways are activated. Production of nitric oxide has been regarded as a signature marker of inflammatory responses. Our recent studies demonstrated the effects of docosahexaenoic acid (DHA) to inhibit the LPS-induced inflammatory responses in BV-2 microglial cells. DHA also can upregulate the anti-oxidative pathway involving nuclear factor erythroid 2-Like 2 (Nrf2) and synthesis of heme oxygenase-1 (HO-1), a potent anti-oxidative enzyme. In order to further understand the proteins involved, this study used a label-free quantitative proteomics approach to examine effects of DHA and LPS on proteins and signaling pathways in microglial cells.
2020-04-20 | PXD013086 | Pride
Project description:Nitric oxide (NO) is a signaling molecule regulating numerous cellular functions in development and disease. In the brain, neuronal injury or neuroinflammation can lead to microglial activation, which induces NO production. NO can react with critical cysteine thiols of target proteins forming S-nitroso-proteins. This modification, known as S-nitrosylation, is an evolutionarily conserved redox-based post-translational modification (PTM) of specific proteins analogous to phosphorylation. In this study, we describe a protocol for analyzing S-nitrosylation of proteins using a gel-based proteomic approach and use it to investigate the modes of action of a botanical compound found in green tea, epigallocatechin-3-gallate (EGCG), on protein S-nitrosylation after microglial activation.To globally and quantitatively analyze NO-induced protein S-nitrosylation, the sensitive gel-based proteomic method, termed NitroDIGE, was developed by combining two-dimensional differential in-gel electrophoresis (2-D DIGE) with the modified biotin switch technique (BST) using fluorescence-tagged CyDye™ thiol reactive agents to label S-nitrosothiols. The NitroDIGE method showed high specificity and sensitivity in detecting S-nitrosylated proteins (SNO-proteins). Using this approach, we identified a subset of SNO-proteins ex vivo by exposing immortalized murine BV-2 microglial cells to a physiological NO donor, or in vivo by exposing BV-2 cells to endotoxin lipopolysaccharides (LPS) to induce a proinflammatory response. Moreover, EGCG was shown to attenuate S-nitrosylation of proteins after LPS-induced activation of microglial cells primarily by modulation of the nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated oxidative stress response.These results demonstrate that NitroDIGE is an effective proteomic strategy for "top-down" quantitative analysis of protein S-nitrosylation in multi-group samples in response to nitrosative stress due to excessive generation of NO in cells. Using this approach, we have revealed the ability of EGCG to down-regulate protein S-nitrosylation in LPS-stimulated BV-2 microglial cells, consistent with its known antioxidant effects.
| S-EPMC3922161 | biostudies-other
Project description:BackgroundMicroglial cells act as the sentinel of the central nervous system .They are involved in neuroprotection but are highly implicated in neurodegeneration of the aging brain. When over-activated, microglia release pro-inflammatory factors, such as nitric oxide (NO) and cytokines, which are critical in eliciting neuroinflammatory responses associated with neurodegenerative diseases. This study examined whether bromelain, the pineapple-derived extract, may exert an anti-inflammatory effect in primary microglia and may be neuroprotective by regulating microglial activation.MethodsFollowing the isolation of neonatal rat primary microglial cells, the activation profile of microglia was investigated by studying the effects of bromelain (5, 10, 20, and 30 µg/ml) on the levels of NO, inducible nitric oxide synthase (iNOS), and nuclear factor kappa B (NF-κB) in microglia treated with lipopolysaccharide (LPS) (1 µg/ml). Data were analyzed using Student's t-test. P values less than 0.05 were considered to be statistically significant, compared with the LPS-treated group without bromelain.ResultsResults showed that pretreatment of rat primary microglia with bromelain, decreased the production of NO induced by LPS (1 µg/ml) treatment in a dose-dependent manner. Bromelain (30 µg/ml) also significantly reduced the expression of iNOS at mRNA level and NF-κB at protein level. Moreover, the study of mitochondrial activity in microglia indicated that bromelain had no cytotoxicity at any of the applied doses, suggesting that the anti-inflammatory effects of bromelain are not due to cell death.ConclusionBromelain can be of potential use as an agent for alleviation of symptoms in neurodegenerative diseases.
| S-EPMC4689280 | biostudies-literature
Project description:Lipopolysaccharide (LPS), a glycolipid component of the outer membrane of Gram-negative bacteria, is a potent initiator of the innate immune response of the macrophage. LPS triggers downstream signaling by selectively recruiting and activating proteins in cholesterol-rich membrane microdomains called lipid rafts. We applied proteomics analysis to macrophage detergent-resistant membranes (DRMs) during an LPS exposure time course in an effort to identify and validate novel events occurring in macrophage rafts. Following metabolic incorporation in cell culture of heavy isotopes of amino acids arginine and lysine ([(13)C(6)]Arg and [(13)C(6)]Lys) or their light counterparts, a SILAC (stable isotope labeling with amino acids in cell culture)-based quantitative, liquid chromatography-tandem mass spectrometry proteomics approach was used to profile LPS-induced changes in the lipid raft proteome of RAW 264.7 macrophages. Unsupervised network analysis of the proteomics data set revealed a marked representation of the ubiquitin-proteasome system as well as changes in proteasome subunit composition following LPS challenge. Functional analysis of DRMs confirmed that LPS causes selective activation of the proteasome in macrophage rafts and proteasome inactivation outside of rafts. Given previous reports of an essential role for proteasomal degradation of IkappaB kinase-phosphorylated p105 in LPS activation of ERK mitogen-activated protein kinase, we tested for a role of rafts in compartmentalization of these events. Immunoblotting of DRMs revealed proteasome-dependent activation of MEK and ERK specifically occurring in lipid rafts as well as proteasomal activity upon raft-localized p105 that was enhanced by LPS. Cholesterol extraction from the intact macrophage with methyl-beta-cyclodextrin was sufficient to activate ERK, recapitulating the LPS-IkappaB kinase-p105-MEK-ERK cascade, whereas both it and the alternate raft-disrupting agent nystatin blocked subsequent LPS activation of the ERK cascade. Taken together, our findings indicate a critical, selective role for raft compartmentalization and regulation of proteasome activity in activation of the MEK-ERK pathway.
| S-EPMC2621002 | biostudies-literature
Project description:Lysine 2-hydroxyisobutyrylation (Khib) is a new type of posttranslational modifications (PTMs) extensively reported on eukaryotic cell histones. It is evolutionarily conserved and participates in diverse important biological processes, such as transcription and cell metabolism. Recently, it has been demonstrated that Khib can be regulated by p300 and Tip60. Although the specific Khib substrates mediated by p300 have been revealed, how Tip60 regulates diverse cellular processes through the Khib pathway and the different roles between Tip60 and p300 in regulating Khib remain largely unknown, which prevents us from understanding how this modification executes its biological functions. In this study, we report the first Khib proteome mediated by Tip60. In total, 3502 unique Khib sites from 1050 proteins were identified. Among them, 536 Khib sites from 406 proteins were present only in Tip60 overexpressing cells and 13 Khib sites increased more than 2-fold in response to Tip60 overexpression, indicating that Tip60 significantly affected global Khib. Notably, only 5 of the 549 Tip60-targeted Khib sites overlapped with the 149 known Khib sites targeted by p300, indicating the different Khib substrate preferences of Tip60 and p300. In addition, the Khib substrates regulated by Tip60 are deeply involved in processes such as nucleic acid metabolism and translation, and some are associated with Parkinson's and Prion diseases. In summary, our research reveals the Khib substrates targeted by Tip60, which elucidates the effect of Tip60 in regulating various cellular processes through the Khib pathway, and proposes novel views into the functional mechanism of Tip60.
| S-EPMC8847014 | biostudies-literature
Project description:Microglia-mediated neuroinflammation plays a vital role in the pathophysiological processes of multiple neurodegenerative diseases. Lipopolysaccharide (LPS) is an environmental poison that can induce inflammatory microglial activation. Matrix metalloproteinases (MMPs) are vital factors regulating microglial activation, and CD147 is a key MMP inducer, which can induce inflammation by inducing MMPs. However, whether it is involved in the regulation of microglial activation has not been reported. In this study, the role of CD147 in LPS-induced microglial inflammatory activation was investigated by establishing in vivo and in vitro models. The results suggested that LPS-induced microglial activation was accompanied by the induction of CD147 expression while the inhibition of CD147 expression could inhibit LPS-induced microglial inflammatory activation. In addition, the results also indicated that the role of CD147 in LPS-induced pro-inflammatory activation of microglia was related to its downstream MMP-3, MMP-8, and autophagy. Furthermore, the inhibition of MMP-3, MMP-8, and autophagy attenuated LPS-induced inflammatory activation of microglia. At the same time, there was a certain interaction between MMPs and autophagy, which is shown that inhibiting the expression of MMPs could inhibit autophagy, whereas inhibiting autophagy could inhibit the expression of MMPs. Taken together, we provided the first evidence that CD147/MMPs can be involved in LPS-induced inflammatory activation of microglia through an autophagy-dependent manner.
| S-EPMC9761036 | biostudies-literature
Project description:Non-alcoholic fatty liver disease (NAFLD) is a predominant contributor to end-stage liver disease in the forthcoming decades. Polygonum perfoliatum L. (PPL) is an herbal medicine with anti-lipid peroxidation and anti-inflammatory properties. However, detailed hepatoprotective effects of PPL against NAFLD and its underlying mechanisms are not fully understood. Here, we found that PPL protects against high fat diet (HFD)-induced hepatic steatosis, lipid peroxidation, and glucose-lipid metabolism dysfunction in NAFLD mice. We therefore performed a label-free quantitative proteomic profiling analysis to determine the effect of PPL treatment on liver tissue proteomics and identified that activated PPARs/CPT1A/CPT2-mediated hepatic fatty acid β-oxidation (FAO) process was significantly altered. In vitro treatment of hepatocytes with PPL confirmed this altered process and FAO inhibitor etomoxir (ETO) attenuated the lipid-lowering activity of PPL in hepatocytes. Ultra-high-performance liquid chromatography/Q Exactive-HFX (UPLC/QE-HFX) was used to determine the material basis of anti-NAFLD activity of PPL. Our results have demonstrated the efficacy and potential mechanisms of PPL as an effective pharmacological therapy of NAFLD.
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Project description:The objective of our present work was to explore the possible enhanced anti-neuroinflammatory ability of Aspergillus oryzae fermented hemp seed in lipopolysaccharide (LPS)-stimulated N9 microglial cells and elucidate its underlying mechanism. The water extract of hemp seed was fermented by Aspergillus oryzae. LPS-stimulated N9 microglial cells were employed for the inflammatory cell model. The release of nitric oxide (NO) was determined by Griess assay. The cytokines and inflammatory mediator expression were measured by qPCR and ELISA. The phosphorylated key signaling proteins, including nuclear factor-κB (NF-κB), mitogen-activated protein kinases (MAPKs), and phosphatidylinositol 3-kinase (PI3K/Akt), were quantified by western blot analysis. The production of intracellular reactive oxygen species (ROS) was measured by DCFH oxidation. Fermented hemp seed (FHS) reduced NO production by downregulating inducible nitric oxide synthase (iNOS) expression in LPS-stimulated N9 microglial cells. FHS treatment decreased LPS-stimulated expression of inflammatory cytokines either on mRNA or protein levels. Moreover, FHS inhibited LPS-stimulated phosphorylation of NF-κB, MAPKs, and PI3K/Akt signaling pathways. Furthermore, FHS significantly reduced the ROS production in the cells. It was concluded that FHS exerted its anti-neuroinflammatory activities by suppressing ROS production, thus inhibiting NF-κB, MAPKs, and PI3K/Akt activation, consequently decreasing the expression levels of inflammatory mediators and cytokines.
| S-EPMC9222285 | biostudies-literature
Project description:Celastrol, a natural substance isolated from plant extracts used in traditional Chinese medicine, has been extensively investigated as a possible drug for treatment of cancer, autoimmune diseases, and protein misfolding disorders. Although studies focusing on celastrol's effects in specific cellular pathways have revealed a considerable number of targets in a diverse array of in vitro models there is an essential need for investigations that can provide a global view of its effects. To assess cellular effects of celastrol and to identify target proteins as biomarkers for monitoring treatment regimes, we performed large-scale quantitative proteomics in cultured human lymphoblastoid cells, a cell type that can be readily prepared from human blood samples. Celastrol substantially modified the proteome composition and 158 of the close to 1800 proteins with robust quantitation showed at least a 1.5 fold change in protein levels. Up-regulated proteins play key roles in cytoprotection with a prominent group involved in quality control and processing of proteins traversing the endoplasmic reticulum. Increased levels of proteins essential for the cellular protection against oxidative stress including heme oxygenase 1, several peroxiredoxins and thioredoxins as well as proteins involved in the control of iron homeostasis were also observed. Specific analysis of the mitochondrial proteome strongly indicated that the mitochondrial association of certain antioxidant defense and apoptosis-regulating proteins increased in cells exposed to celastrol. Analysis of selected mRNA transcripts showed that celastrol activated several different stress response pathways and dose response studies furthermore showed that continuous exposure to sub-micromolar concentrations of celastrol is associated with reduced cellular viability and proliferation. The extensive catalog of regulated proteins presented here identifies numerous cellular effects of celastrol and constitutes a valuable biomarker tool for the development and monitoration of disease treatment strategies.
| S-EPMC3202559 | biostudies-literature
Project description:Epidemiological studies have suggested that coffee consumption is associated with a decrease in the risk of developing obesity and diabetes; however, the detailed mechanisms underlying these effects of coffee consumption remain poorly understood. In this study, we examined the effects of chlorogenic acid on energy metabolism in vitro. Hepatocellular carcinoma G2 (HepG2) cells were cultured in a medium containing chlorogenic acid. Chlorogenic acid increased the activity of mitochondrial enzymes, including citrate synthase, isocitrate dehydrogenase, and malate dehydrogenase (MDH), which are involved in the tricarboxylic acid (TCA) cycle. Proteome analysis using the isobaric tags for the relative and absolute quantitation (iTRAQ) method revealed the upregulation of proteins involved in the glycolytic system, electron transport system, and ATP synthesis in mitochondria. Therefore, we propose a notable mechanism whereby chlorogenic acid enhances energy metabolism, including the TCA cycle, glycolytic system, electron transport, and ATP synthesis. This mechanism provides important insights into understanding the beneficial effects of coffee consumption.
| S-EPMC9032979 | biostudies-literature