Astaxanthin exerts anti-inflammatory and antioxidant effects in macrophages in NRF2-dependent and independent manners

Although anti-inflammatory effects of astaxanthin (ASTX) have been suggested, the underlying mechanisms have not been fully understood. Particularly, the modulatory action of ASTX in the interplay between nuclear factor E2-related factor 2 (NRF2) and nuclear factor κB (NFκB) to exert its anti-inflammatory effect in macrophages is unknown. The effect of ASTX on mRNA and protein expression of pro-inflammatory and antioxidant genes and/or cellular reactive oxygen species (ROS) accumulation were determined in RAW 264.7 macrophages, bone marrow-derived macrophages (BMDM) from wild-type (WT) and Nrf2-deficient mice, and/or splenocytes and peritoneal macrophages of obese mice fed ASTX. The effect of ASTX on M1 and M2 macrophage polarization was evaluated in BMDM. ASTX significantly decreased LPS-induced mRNA expression of interleukin 6 (Il-6) and Il-1β by inhibiting nuclear translocation of NFκB p65; and attenuated LPS-induced ROS with an increase in NRF2 nuclear translocation, concomitantly decreasing NADPH oxidase 2 expression in RAW 264.7 macrophages. In BMDM of WT and Nrf2-deficient mice, ASTX decreased basal and LPS-induced ROS accumulation. The induction of Il-6 mRNA by LPS was repressed by ASTX in both types of BMDM while Il-1β mRNA was decreased only in WT BMDM. Furthermore, ASTX consumption lowered LPS sensitivity of splenocytes in obese mice. ASTX decreased M1 polarization of BMDM while increasing M2 polarization. ASTX exerts its anti-inflammatory effect by inhibiting nuclear translocation of NFκB p65 and by preventing ROS accumulation in NRF2-dependent and -independent mechanisms. Thus, ASTX is an agent with anti-inflammatory and antioxidant properties that may be used for the prevention of inflammatory conditions.     

CREB1 regulates glucose transport of glioma cell line U87 by targeting GLUT1

Chemical modification of chitosan is a promising method for the improvement of biological activity. In this study, chitosan-caffeic acid (CCA) was prepared and its in vitro hepatoprotective ability against hydrogen peroxide-induced hepatic damage in liver cells was evaluated. Treatment with CCA (50–400 µg/mL) did not show cytotoxicity and also significantly (p < 0.05) recovered cell viability against 650 µM hydrogen peroxide-induced hepatotoxicity. CCA treatment attenuated reactive oxygen species generation and lipid peroxidation in addition to increasing cellular glutathione level in cultured hepatocytes. To validate the underlying mechanism, antioxidant and phase II detoxifying enzyme expressions, which are mediated by NF-E2-related factor 2 (Nrf2) activation, were analyzed and CCA treatment was found to increase the expression of superoxide dismutase-1 (SOD-1), glutathione reductase (GR), heme oxygenase-1 (HO-1), and NAD(P)H:quinine oxidoreductase 1 (NQO1). CCA treatment resulted in increased Nrf2 nuclear translocation. The phosphorylation of extracellular regulated kinase (ERK), c-Jun NH₂-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) by CCA treatment contributed to Nrf2 activation. Pharmacological blockade of ERK, JNK, and p38 MAPK revealed that SP600125 (JNK inhibitor) and PD98059 (ERK inhibitor) treatment reduced Nrf2 translocation into the nucleus while SB203580 (p38 inhibitor) exhibited weak inhibition. Collectively, CCA protects liver cells against hydrogen peroxide-induced injury and this ability is attributed to the induction of antioxidants and phase II detoxifying enzymes that are mediated by Nrf2 translocation via JNK/ERK signaling.     

Blocking of tripartite motif 8 protects against lipopolysaccharide (LPS)-induced acute lung injury by regulating AMPKα activity

Acute lung injury (ALI) and its more serious form, respiratory distress syndrome (ARDS), are considered as an acute and severe inflammatory process existing in lungs, and still remain high mortality rates. Tripartite motif 8 (TRIM8) contains an N-terminal RING finger, which is followed by two B-boxes and a coiled-coil domain, belonging to the TRIM/RBCC family and playing significant role in meditating inflammation, oxidative stress and apoptosis. In the study, we investigated the role of TRIM8 in ALI induced by lipopolysaccharide (LPS) and the underlying molecular mechanisms. The in vitro results indicated that LPS time-dependently enhanced TRIM8 expression in lung epithelial cells. Suppressing TRIM8 markedly ameliorated LPS-elicited inflammatory response, as evidenced by the down-regulated mRNA levels of interleukin-1β (IL-1β), IL-6 and tumor necrosis factor-α (TNF-α) in cells mainly through inactivating nuclear factor-kappa B (NF-κB) signaling pathway; however, over-expressing TRIM8 markedly promoted inflammation in LPS-challenged cells. In addition, LPS-induced oxidative stress was accelerated by TRIM8 over-expression, while being alleviated by TRIM8 knockdown by regulating Nrf2 signaling. Importantly, TRIM8 could negatively meditate AMP-activated protein kinase-α (AMPKα) activation to modulate LPS-triggered inflammatory response and ROS generation in vitro. Additionally, our in vivo findings suggested that TRIM8 knockdown effectively attenuated LPS-induced lung injury nu decrease of lung wet/dry (W/T) ratio, protein concentrations, neutrophil infiltration, myeloperoxidase (MPO) activity, reactive oxygen species (ROS) production and superoxide dismutase (SOD) depletion. Meanwhile, the loss of TRIM8 markedly lessened IL-1β, IL-6 and TNF-α expression in lung tissues of LPS-challenged mice, and reduced NF-κB phosphorylation. Furthermore, TRIM8 knockdown evidently improved nuclear factor-erythroid 2 related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) expressions in lung of LPS-treated mice. The anti-inflammation and anti-oxidant role of TRIM8-silence might be associated with AMPKα phosphorylation. Together, our study firstly provided a support that TRIM8 knockdown effectively protected LPS-induced ALI against inflammation and oxidative stress largely dependent on the promotion of AMPKα pathway.     

Enhanced Keap1-Nrf2/Trx-1 axis by daphnetin protects against oxidative stress-driven hepatotoxicity via inhibiting ASK1/JNK and Txnip/NLRP3 inflammasome activation

BackgroundOxidative stress-triggered fatal hepatotoxicity is an essential pathogenic factor in acute liver failure (ALF).AimsTo investigate the protective effect of daphnetin (Daph) on tert-butyl hydroperoxide (t-BHP) and acetaminophen (APAP)-induced hepatotoxicity through altering Nrf2/Trx-1 pathway activation.Materials and methodsIn vivo, male C57BL/6 mice with Wild-type (WT) and Nrf2^−/− were divided into five groups and acute liver injury model were established by APAP or LPS/GalN after injection with Daph (20, 40, or 80 mg/kg), seperately. Then, liver tissue and serum were collected for biochemical determination, TUNEL and H & E staining, and western blot analysis. In vitro, HepG2 cells were used to investigate the protective effect and mechanism of daphnetin against ROS and apoptosis induced by t-BHP via apoptosis detection, western blot, immunofluorescence analysis, and sgRNA transfection.ResultsOur results indicated that Daph efficiently inhibited t-BHP-stimulated hepatotoxicity, and modulated Trx-1 expression and Nrf2 activation which decreased Keap1-overexpression in HepG2 cells. Moreover, Daph inhibited t-BHP-excited hepatotoxicity and enhanced Trx-1 expression, which was reversed in Nrf2^−/− HepG2 cells. In vivo, a survival rate analysis first suggested that Daph significantly reduced the lethality induced by APAP or GalN/LPS in a Nrf2-dependent or -independent manner by using Nrf2^−/− mice, respectively. Next, further results implicated that Daph not only effectively alleviated APAP-induced an increase of ALT and AST levels, histopathological changes, ROS overproduction, malondialdehyde (MDA) formation and GSH/GSSG reduction, but it also relieved hepatic apoptosis by strengthening the suppression of cleaved-caspase-3 and expression of P53 protein. Additionally, Daph attenuated mitochondrial dysfunction by suppressing ASK1/JNK activation and decreasing apoptosis-inducing factor (AIF) and Cytochrome c release and Bax mitochondrial translocation. Daph inhibited inflammatory responses by inactivating the thioredoxin-interacting protein (Txnip)/NLRP3 inflammasome. Furthermore, Daph efficiently enhanced Nrf2 nuclear translocation and Trx-1 expression. However, these effects in WT mice were eliminated in Nrf2^−/− mice.ConclusionsThese investigations demonstrated that Daph treatment has protective potential against oxidative stress-driven hepatotoxicity by inhibition of ASK1/JNK and Txnip/NLRP3 activation, which may be strongly related to the Nrf2/Trx-1 upregulation.     

Negative regulation of LPS-stimulated expression of inducible nitric oxide synthase by AP-1 in macrophage cell line J774A.1.

The level of NOS II mRNA was markedly increased during 24 h lipopolysaccharide (LPS) stimulation, but showed no further increase thereafter. On the other hand, the level of NOS II mRNA in J774A.1 cells transfected with an expression vector containing the rat csk cDNA (J.Csk) was significantly increased during 3 h LPS stimulation, but rather decreased thereafter. Although no significant difference was observed in the activation of NF-kappaB by LPS among parental J774A.1, J774A.1 transfected with promoterless vector (J.pBK), and J.Csk cells, activity of c-Jun N-terminal kinase (JNK) and nuclear translocation of nuclear factor activator protein-1 (AP-1) were markedly upregulated in the J.Csk cells. Then luciferase reporter vectors containing NOS II promoter with mutations in two AP-1-like sites (U site, -1126 approximately -1120; L site, -524 approximately -518) were transiently transfected in J774A.1 cells. The promoter activity following LPS stimulation for 24 h was significantly increased by mutation at the L site, but not by mutation at the U site, suggesting that NOS II expression is negatively regulated, at least in part, through the AP-1-like L site in response to LPS.     

Shizukaol A exerts anti-inflammatory effect by regulating HMGB1/Nrf2/HO-1 pathway

BackgroundSarcandra glabra (Thunb.) Makino (Chloranthaceae) has a long history of being used in Traditional Chinese medicines (TCMs) to treat painful joints, fractures, arthritis, and other diseases caused by inflammation. It has been reported that lindenane-type sesquiterpenoid dimers are main anti-inflammatory ingredient of S. glabra. Meanwhile, shizukaol A, the precursor of these sesquiterpene dimers, possesses a good inhibitory effect on nitric oxide (NO) in our previous study. But its anti-inflammatory mechanism is still unclear.PurposeThis study aimed to explore the possible anti-inflammatory mechanism and potential targets of shizukaol A in lipopolysaccharide (LPS)-induced RAW 264.7 cells.MethodsThe release of NO and inflammatory cytokines in LPS-stimulated RAW 264.7 cells were measured by Griess reagent and ELISA, respectively. The relevant proteins including inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), nuclear factor kappa B (NF-κB) p65, High mobility group box 1 (HMGB1) were detected by western blot. Nuclear translocation of p65, HMGB1 and nuclear factor E2-related factor 2 (Nrf2) were examined by immunofluorescence. The level of reactive oxygen species (ROS) was tested by flow cytometry. The target of shizukaol A was investigated by molecular docking and Drug Affinity Responsive Target Stability (DARTS).ResultsShizukaol A had a good inhibitory effect on NO with half maximal inhibitory concentration (IC₅₀) of 13.79 ± 1.11 μM. Shizukaol A could down-regulate the expression of iNOS and COX-2. Further studies demonstrated that shizukaol A can significantly inhibit phosphorylation and nuclear translocation of NF-κB. Meanwhile, shizukaol A decreased the level of ROS and enhanced the expression of heme oxygenase-1 (HO-1) and NAD(P)H: quinone oxidoreductase 1 (NQO1). Furthermore, shizukaol A up-regulated the expression of Nrf2 and its nuclear translocation. More importantly, shizukaol A could inhibit activation of HMGB1 by targeting HMGB1.ConclusionShizukaol A inhibited inflammation by targeting HMGB1 to regulate the Nrf2/HO-1 signaling pathway. Thus, shizukaol A may be an attractive therapeutic candidate for inflammatory diseases.     

Anti-neuroinflammatory Effect of Emodin in LPS-Stimulated Microglia: Involvement of AMPK/Nrf2 Activation

AMPK/Nrf2 signaling regulates multiple antioxidative factors and exerts neuroprotective effects. Emodin is one of the main bioactive components extracted from Polygonum multiflorum, a plant possessing important activities for human health and for treating a variety of diseases. This study examined whether emodin can activate AMPK/Nrf2 signaling and induce the expression of genes targeted by this pathway. In addition, the anti-neuroinflammatory properties of emodin in lipopolysaccharide (LPS)-stimulated microglia were examined. In microglia, the emodin treatment increased the levels of LKB1, CaMKII, and AMPK phosphorylation. Emodin increased the translocation and transactivity of Nrf2 and enhanced the levels of HO-1 and NQO1. In addition, the emodin-mediated expression of HO-1 and NQO1 was attenuated completely by an AMPK inhibitor (compound C). Moreover, emodin decreased dramatically the LPS-induced production of NO and PGE₂ as well as the protein expression and promoter activity of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). In addition, emodin effectively inhibited the production of pro-inflammatory cytokines, TNF-α and IL-6, and reduced the level of IκBα phosphorylation, leading to the suppression of the nuclear translocation, phosphorylation, and transactivity of NF-κB. Emodin also suppressed the LPS-stimulated activation of STATs, JNK, and p38 MAPK. The anti-inflammatory effects of emodin were reversed by transfection with Nrf-2 and HO-1 siRNA and by a co-treatment with an AMPK inhibitor. These results suggest that emodin isolated from P. multiflorum can be used as a natural anti-neuroinflammatory agent that exerts its effects by inducing HO-1 and NQO1 via AMPK/Nrf2 signaling in microglia.     

LL202 ameliorates colitis against oxidative stress of macrophage by activation of the Nrf2/HO-1 pathway

LL202, a newly synthesized flavonoid derivative, has been confirmed to inhibit the mitogen-activated protein kinase pathway and activation protein-1 activation in monocytes; however, the anti-inflammatory mechanism has not been clearly studied. Uncontrolled overproduction of reactive oxygen species (ROS) has involved in oxidative damage of inflammatory bowel disease. In this study, we investigated that LL202 reduced lipopolysaccharide (LPS)-induced ROS production and malondialdehyde levels and increased superoxide dismutase, glutathione, and total antioxidant capacity in RAW264.7 cells. Mechanically, LL202 could upregulate heme oxygenase-1 (HO-1) via promoting nuclear translocation of nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2) to regulate LPS-induced oxidative stress in macrophages. In vivo, we validated the role of LL202 in dextran sulfate sodium- and TNBS-induced colitis models, respectively. The results showed that LL202 decreased the proinflammatory cytokine expression and regulated colonic oxidative stress by activating the Nrf2/HO-1 pathway. In conclusion, our study showed that LL202 exerts an anti-inflammatory effect by enhancing the antioxidant capacity of the Nrf2/HO-1 pathway to macrophages.     

Tanshinone IIA suppresses cholesterol accumulation in human macrophages: role of heme oxygenase-1

Accumulation of foam cells in the neointima represents a key event in atherosclerosis. We previously demonstrated that Tanshinone IIA (Tan), a lipophilic bioactive compound extracted from Salvia miltiorrhiza Bunge, inhibits experimental atherogenesis, yet the detailed mechanisms are not fully understood. In this study, we sought to explore the potential effects of Tan on lipid accumulation in macrophage foam cells and the underlying molecular mechanisms. Our data indicate that Tan treatment reduced the content of macrophages, cholesterol accumulation, and the development of atherosclerotic plaque in apolipoprotein E-deficient mice. In human macrophages, Tan ameliorated oxidized low density lipoporotein (oxLDL)-elicited foam cell formation by inhibiting oxLDL uptake and promoting cholesterol efflux. Mechanistically, Tan markedly reduced the expression of scavenger receptor class A and increased the expression of ATP-binding cassette transporter A1 (ABCA1) and ABCG1 in lipid-laden macrophages via activation of the extracellular signal-regulated kinase (ERK)/nuclear factor-erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway. Tan treatment induced the phosphorylation and nuclear translocation of Nrf2 and subsequently increased the expression of HO-1, and these effects were abolished by the specific ERK inhibitors, PD98059 and U0126. Moreover, HO-1 small interfering RNA or zinc protoporphyrin (a HO-1 inhibitor) abrogated Tan-mediated suppression of lipid accumulation in macrophages. Our current findings demonstrate that a novel HO-1-dependent mechanism is involved in the regulation of cholesterol balance by Tan.