Anti-inflammatory activity of myricetin from Diospyros lotus through suppression of NF-κB and STAT1 activation and Nrf2-mediated HO-1 induction in lipopolysaccharide-stimulated RAW264.7 macrophages

Diospyros lotus is traditionally used for the treatment of diabetes, diarrhea, tumor, and hypertension. The purpose of this study was to investigate the anti-inflammatory effect and underlying molecular mechanisms of myricetin in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Myricetin dose-dependently suppressed the production of pro-inflammatory mediators (NO, iNOS, PGE₂, and COX-2) in LPS-stimulated RAW264.7 macrophages. Myricetin administration decreased the production of NO, iNOS, TNF-α, IL-6, and IL-12 in mice. Myricetin decreased NF-κB activation by suppressing the degradation of IκBα, nuclear translocation of p65 subunit of NF-κB, and NF-κB DNA binding activity in LPS-stimulated RAW264.7 macrophages. Moreover, myricetin attenuated the phosphorylation of STAT1 and the production of IFN-β in LPS-stimulated RAW264.7 macrophages. Furthermore, myricetin induced the expression of HO-1 through Nrf2 translocation. In conclusion, these results suggest that myricetin inhibits the production of pro-inflammatory mediators through the suppression of NF-κB and STAT1 activation and induction of Nrf2-mediated HO-1 expression in LPS-stimulated RAW264.7 macrophages.     

MicroRNA-146 protects A549 and H1975 cells from LPS-induced apoptosis and inflammation injury

Pneumonia is an inflammatory condition affecting the lungs, in which pro-inflammatory cytokines are secreted. It has been shown that microRNA-146 (miR-146) is involved in the regulation of immune and inflammatory responses. The present study explored the protective effects of miR-146 overexpression on lipopolysaccharide (LPS)-mediated injury in A549 and H1975 cells. In this study, A549 and H1975 cells were transfected with miR-146 mimic or inhibitor, and then were subjected with LPS. Thereafter, cell viability, colony formation capacity, apoptosis, the release of proinflammatory factors, Sirt1 expression, and the expression of NF-κB and Notch pathway proteins were respectively assessed. As a result, miR-146 overexpression exerted protective functions on LPS-damaged A549 and H1975 cells, as evidenced by the increases in cell viability and colony number, the decrease in apoptotic cell rate, as well as the down-regulations of IL-1, IL-6, and TNF-α. Sirt1 can be positively regulated by miR-146. Furthermore, miR-146 overexpression blocked NF-κB and Notch pathways, while these blocking effects were abolished when Sirt1 was silenced. The findings in the current study indicated that miR-146 protected A549 and H1975 cells from LPS-induced apoptosis and inflammation injury. miR-146 exerted protective functions might be via up-regulation of Sirt1 and thereby blocking NF-κB and Notch pathways.     

miR-520a-3p调控宫颈癌细胞因子分泌的信号通路*

目的: 探讨miR-520a-3p调控宫颈癌细胞因子分泌的分子机制。方法: 通过TargetScanHuman分析miR-520a-3p与NF-κB复合体亚基RELA的匹配情况,然后通过荧光素酶报告系统检测miR-520a-3p是否靶向NF-κB复合体亚基RELA;使用LPS刺激宫颈癌HELA细胞后,将miR-520a-3p mimics与转染试剂混合后滴入HELA细胞中,此为过表达组;将miR-520a-3p inhibitor与转染试剂混合后滴入HELA细胞中,此为敲低组,通过酶联免疫吸附试验检测过表达组和敲低组GCSF, GM-CSF, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12 p40, IL-12 p70, IL-13, IL-17, IFN-γ, MCP-1, MCP-5, RANTES, TNFα的表达水平。每次实验重复3次。结果: miR-520a-3p靶向RELA的3’UTR;LPS激活NF-kB信号通路后,宫颈癌HELA细胞分泌的细胞因子GCSF, GM-CSF, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12 p40, IL-12 p70, IL-13, IL-17, IFN-γ, MCP-1, MCP-5, RANTES, TNFα的蛋白表达水平上升(P＜0.05);过表达组中NF-κB复合体亚基RELA的蛋白表达水平下降,宫颈癌HELA细胞分泌的上述细胞因子的蛋白表达水平下降(P＜0.05);敲低组中NF-κB复合体亚基RELA的蛋白表达水平上升,宫颈癌HELA细胞分泌的上述细胞因子的蛋白表达水平上升(P＜0.05)。结论: miR-520a-3p通过靶向NF-κB信号通路的关键分子RELA抑制宫颈癌HELA细胞的细胞因子分泌。     

γ-Tocotrienol inhibits lipopolysaccharide-induced interlukin-6 and granulocyte colony-stimulating factor by suppressing C/EBPβ and NF-κB in macrophages

Cytokines generated from macrophages contribute to pathogenesis of inflammation-associated diseases. Here we show that γ-tocotrienol (γ-TE), a natural vitamin E form, inhibits lipopolysaccharide (LPS)-induced interleukin (IL)-6 production without affecting tumor necrosis factor α (TNF-α), IL-10 or cyclooxygenase-2 (COX-2) up-regulation in murine RAW264.7 macrophages. Mechanistic studies indicate that nuclear factor κB (NF-κB), but not c-Jun NH(2)-terminal protein kinase, p38 or extracellular signal-regulated kinase mitogen-activated protein kinases (MAPKs), is important to IL-6 production and that γ-TE treatment blocks NF-κB activation. In contrast, COX-2 appears to be regulated by p38 MAPK in RAW cells, but γ-TE has no effect on LPS-stimulated p38 phosphorylation. Despite necessary for IL-6, NF-κB activation by TNF-α or other cytokines is not sufficient for IL-6 induction with exception of LPS. CCAAT/enhancer-binding protein (C/EBP) β appears to be involved in IL-6 formation because LPS induces C/EBPβ up-regulation, which parallels IL-6 production, and knockdown of C/EBPβ with small interfering RNA results in diminished IL-6. LPS but not individual cytokines is capable of stimulating C/EBPβ and IL-6 in macrophages. Consistent with its dampening effect on IL-6, γ-TE blunts LPS-induced up-regulation of C/EBPβ without affecting C/EBPδ. γ-TE also decreases LPS-stimulated granulocyte colony-stimulating factor (G-CSF), a C/EBPβ target gene. Compared with RAW264.7 cells, γ-TE shows similar or stronger inhibitory effects on LPS-triggered activation of NF-κB, C/EPBβ and C/EBPδ and more potently suppresses IL-6 and G-CSF in bone marrow-derived macrophages. Our study demonstrates that γ-TE has antiinflammatory activities by inhibition of NF-κB and C/EBPs activation in macrophages.     

Long noncoding RNA ANRIL contributes to the development of ulcerative colitis by miR-323b-5p/TLR4/MyD88/NF-κB pathway

The aim of this study was to investigate the role and possible mechanism of long noncoding RNA ANRIL in the development of ulcerative colitis (UC). The expression of ANRIL in colonic mucosa tissues collected from the sigmoid colon of UC patients and healthy control was determined. Subsequently, fetal human cells (FHCs) were treated with lipopolysaccharide (LPS) to stimulate UC-caused inflammatory injury, followed by detection of the effects of suppression of ANRIL on cell viability, apoptosis and cytokines production in LPS-stimulated FHCs. Moreover, the regulatory relationship between ANRIL and miR-323b-5p as well as the target relationship between miR-323b-5p and TLR4 were investigated. Furthermore, the effects of ANRIL/miR-323b-5p axis on the activation of TLR4/MyD88/NF-κB pathway in LPS-stimulated FHCs were investigated. LncRNA ANRIL was highly expressed in colonic mucosa tissues of UC patients. In addition, LPS markedly induced cell injury in FHC cells (inhibited cell viability and promoted cell apoptosis and cytokine production). Suppression of ANRIL alleviated LPS-induced injury in FHC cells, which was achieved by negatively regulating miR-323b-5p. Moreover, miR-323b-5p negatively regulated TLR4 expression and TLR4 was a target of miR-323b-5p. Knockdown of TLR4 reversed the effects of miR-323b-5p suppression on LPS-induced injury in LPS-stimulated FHCs. Furthermore, the effects of ANRIL on LPS-induced cell injury were achieved by TLR4/MyD88/NF-κB pathway. Our data indicate that suppression of ANRIL may inhibit the development of UC by regulating miR-323b-5p/TLR4/MyD88/NF-κB pathway. ANRIL/miR-323b-5p/TLR4/MyD88/NF-κB pathway may provide a new strategy for UC therapy.     

Crocin Upregulates CX3CR1 Expression by Suppressing NF-κB/YY1 Signaling and Inhibiting Lipopolysaccharide-Induced Microglial Activation

Glaucoma is a group of neurodegenerative diseases characterized by the progressive loss of retinal ganglion cells (RGCs) and optic nerve fibers. Microglial activation has been shown to be deleterious to RGCs and may participate in the progression of glaucoma. Crocin, one of the major active ingredients in saffron, has been found to inhibit microglial activation. However, the mechanism remains unclear. The aim of this study was to investigate whether crocin can inhibit lipopolysaccharide (LPS)-induced microglial activation and to clarify the mechanisms involved. The influence of crocin on primary RGCs and LPS-stimulated BV2 microglial cells survival was determined by the MTT and lactate dehydrogenase assays, or by flow cytometry. BV2 cells were pretreated with various concentrations of crocin for 2 h followed by 1 μg/mL LPS stimulation. Microglial markers and pro-inflammatory mediators were assessed by real-time PCR, western blot and ELISA. Furthermore, CX3CR1 expression was detected and the underlying mechanism was examined. The concentrations of crocin ranged from 0.1 to 1 μM, and did not show any cytotoxicity in RGC and BV2 cells. After crocin pretreatment, the expression of microglial markers (CD11b and Iba-1) and pro-inflammatory mediators (iNOS, COX-2, IL-1β, and TNF-α) induced by LPS were significantly decreased in a dose-dependent manner. Additionally, CX3CR1 expression was remarkably increased by crocin via the suppression of NF-κB/Yin Yang 1 (YY1) signaling in BV2 cells. In conclusion, crocin effectively suppresses microglial activation and upregulates CX3CR1 expression by suppressing NF-κB/YY1 signaling.     

miR-29b-3p protects cardiomyocytes against endotoxin-induced apoptosis and inflammatory response through targeting FOXO3A

Cardiac dysfunction represents a main component of death induced by sepsis in critical care units. And microRNAs (miRNAs) have been reported as important modulators or biomarkers of sepsis. However, the molecular detail of miRNAs involved in septic cardiac dysfunction remains unclear. Here we showed that endotoxin (lipopolysaccharide, LPS) significantly down-regulated expression of miR-29b-3p in heart. Increased expression of miR-29b-3p by lentivirus improved cardiac function and attenuated damage of cardiac induced by LPS in mice. Furthermore, overexpression or knockdown of miR-29b-3p showed its crucial roles on regulation of apoptosis and production of pro-inflammatory cytokines in NRCMs through directly targeting FOXO3A. miR-29b-3p ameliorates inflammatory damage likely via reducing activation of MAPKs and nuclear-translocation of NF-κB to block LPS-activated NF-κB signaling. Notably, miR-29b is also down-regulated in septic patients' plasma compared with normal subjects, indicating a potential clinical relevance of miR-29b. Taken together, our findings demonstrate that upregulation of miR-29b-3p can attenuate myocardial injury induced by sepsis via regulating FOXO3A, which provide a potential therapy target for interference of septic cardiac dysfunction.     

The Suppressive Effect of β-Glucan on the Production of Tumor Necrosis Factor-Alpha in BV2 Microglial Cells

β-Glucan was recently shown to have the ability to enhance and stimulate the immune system in humans, but little is known about its the anti-inflammatory effects. We investigated the effect of β-glucan on the production of tumor necrosis factor-alpha (TNF-α), a major pro-inflammatory mediator, in lipopolysaccharide (LPS)-stimulated BV2 microglial cells. β-Glucan decreased the production and expression of TNF-α. In addition, it blocked LPS-stimulated activation of nuclear factor kappa B (NF-κB). Hence β-glucan might suppress LPS-stimulated TNF-α production by inhibiting NF-κB in BV2 microglial cells.     

Ursolic acid inhibits nuclear factor-κB signaling in intestinal epithelial cells and macrophages,and attenuates experimental colitis in mice

Aims

Ursolic acid (UA), a natural pentacyclic triterpenoid acid, has been reported to show immunomodulatory activity. This study investigated the effects of UA on nuclear factor-kappa B (NF-κB) signaling in cells and experimental murine colitis.

Main methods

Human intestinal epithelial cells (IECs) COLO 205 and peritoneal macrophages from IL-10-deficient (IL-10^−/−) mice were pretreated with UA and then stimulated with tumor necrosis factor-α (TNF-α) and lipopolysaccharide (LPS), respectively. The expression of pro-inflammatory cytokines was determined by real-time RT-PCR and ELISA. The effect of UA on NF-κB signaling was examined by immunoblot analysis to detect IκBα phosphorylation/degradation and electrophoretic mobility shift assay to assess the DNA binding activity of NF-κB. For in vivo studies, dextran sulfate sodium (DSS)-induced acute colitis in C57BL/6 wild-type mice and chronic colitis in IL-10^−/− mice were treated with or without UA. Colitis was quantified by histopathologic evaluation. Immunohistochemical staining for phosphorylated IκBα was performed in the colonic tissue.

Key findings

UA significantly inhibited the production of pro-inflammatory cytokines, IκBα phosphorylation/degradation and NF-κB DNA binding activity in both IEC and IL-10^−/− peritoneal macrophages stimulated with TNF-α and LPS, respectively. UA significantly reduced the severity of DSS-induced murine colitis, as assessed by the disease activity index, colon length, and histopathology. UA also significantly ameliorated the severity of colitis in IL-10^−/− mice. Furthermore, UA suppressed IκBα phosphorylation in the colonic tissue.

Significance

UA inhibits NF-κB activation in both IECs and macrophages, and attenuates experimental murine colitis. These results suggest that UA is a potential therapeutic agent for inflammatory bowel disease.     

Recombinant HBsAg,an apoptotic-like lipoprotein,interferes with the LPS-induced activation of ERK-1/2 and JNK-1/2 in monocytes

Yeast expressed Hepatitis B surface antigen (rHBsAg) binds to monocytes through interaction with the LPS binding protein (LBP) and the LPS receptor CD14. Charged phospholipids of rHBsAg determine the interaction with these proteins. Although attachment of rHBsAg resembles the pro-inflammatory binding of LPS to CD14, rHBsAg does not activate monocytes and even reduces the expression of pro-inflammatory cytokines by LPS-stimulated monocytes. It is reported here that addition of rHBsAg to LPS-stimulated PBMC often results in increased secretion of IL-10, suggesting a similarity between the interaction of monocytes with apoptotic cells and rHBsAg. Using THP-1 cells, it is shown that IL-10 is not necessary to reduce TNFalpha protein levels. Addition of rHBsAg to LPS-stimulated cells reduces TNFalpha mRNA levels, but does not affect phosphorylation of p65 NF-kappaB and p38 MAP kinase. Instead, a reduced phosphorylation of ERK-1/2 and JNK-1/2 MAP kinases is observed.     

Artemisinin attenuates lipopolysaccharide-stimulated proinflammatory responses by inhibiting NF-κB pathway in microglia cells

Microglial activation plays an important role in neuroinflammation, which contributes to neuronal damage, and inhibition of microglial activation may have therapeutic benefits that could alleviate the progression of neurodegeneration. Recent studies have indicated that the antimalarial agent artemisinin has the ability to inhibit NF-κB activation. In this study, the inhibitory effects of artemisinin on the production of proinflammatory mediators were investigated in lipopolysaccharide (LPS)-stimulated primary microglia. Our results show that artemisinin significantly inhibited LPS-induced production of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), monocyte chemotactic protein-1 (MCP-1) and nitric oxide (NO). Artemisinin significantly decreased both the mRNA and the protein levels of these pro-inflammatory cytokines and inducible nitric oxide synthase (iNOS) and increased the protein levels of IκB-α, which forms a cytoplasmic inactive complex with the p65-p50 heterodimeric complex. Artemisinin treatment significantly inhibited basal and LPS-induced migration of BV-2 microglia. Electrophoretic mobility shift assays revealed increased NF-κB binding activity in LPS-stimulated primary microglia, and this increase could be prevented by artemisinin. The inhibitory effects of artemisinin on LPS-stimulated microglia were blocked after IκB-α was silenced with IκB-α siRNA. Our results suggest that artemisinin is able to inhibit neuroinflammation by interfering with NF-κB signaling. The data provide direct evidence of the potential application of artemisinin for the treatment of neuroinflammatory diseases.     

A20 inhibits the release of inflammatory cytokines by suppressing the activation of the nuclear factor-kappa B pathway in osteoarthritic fibroblast-like synoviocytes

A growing number of studies suggest that synovitis plays an important role in the pathogenesis and progression of osteoarthritis (OA). As a negative mediator of the nuclear factor-kappa B (NF-κB) signaling pathway, the zinc finger protein A20 has significant anti-inflammatory properties. In this study, the differential expression of A20 was investigated at the mRNA and protein levels in human normal OA fibroblast-like synoviocytes (FLSs) and normal FLSs pretreated with TNF-α. We then measured the activation of the NF-κB pathway and expression of pro-inflammatory cytokines in the above three groups by western blotting, a human cytokine array and ELISA. We found that TNF-α activated the NF-κB pathway, increased the expression of the pro-inflammatory cytokines IL-6 and IL-8, and A20 expression in human normal FLSs. However, the role of A20 in FLSs was unclear. To clarify this, we investigated the effect of A20 overexpression in human normal FLSs. The results indicate that A20 inhibits the NF-κB signaling pathway activation and OA-associated pro-inflammatory cytokines release. The results of this study indicate that A20 has anti-inflammatory effects in FLSs, which makes it a potential target for OA synovitis treatment.     

Protopine reduces the inflammatory activity of lipopolysaccharide-stimulated murine macrophages

Protopine is an isoquinoline alkaloid contained in plants in northeast Asia. In this study, we investigated whether protopine derived from Hypecoum erectum L could suppress lipopolysaccharide (LPS)-induced inflammatory responses in murine macrophages (Raw 264.7 cells). Protopine was found to reduce nitric oxide (NO), cyclooxygenase-2 (COX-2), and prostaglandin E(2) (PGE(2)) production by LPS-stimulated Raw 264.7 cells, without a cytotoxic effect. Pre-treatment of Raw 264.7 cells with protopine reduced the production of pro-inflammatory cytokines. These inhibitory effects were caused by blocking phosphorylation of mitogen-activated protein kinases (MAP kinases) and also blocking activation of a nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB).