Peimine impairs pro-inflammatory cytokine secretion through the inhibition of the activation of NF-κB and MAPK in LPS-induced RAW264.7 macrophages

Abstract

In the previous study, we found that peimine has good anti-inflammatory effects in vivo. However, the anti-inflammatory mechanism of peimine remains unclear. We, therefore, assessed the effects of peimine on inflammatory cytokines in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. We found that peimine (0–25?mg/L) significantly inhibited tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-1β, and increased IL-10 production. Furthermore, peimine significantly inhibited the phosphorylation of p38, ERK and c-jun N-terminal kinase (JNK) as well as decreased p65 and IκB. The present results indicate that peimine inhibits the production of inflammatory cytokines induced by LPS through blocking MAPKs and NF-κB signaling pathways.     

Chikusetsusaponin V inhibits inflammatory responses via NF-κB and MAPK signaling pathways in LPS-induced RAW 264.7 macrophages

Excessive activation of macrophages is implicated in various inflammation resulted injuries. Saponins from Panax japonicus (SPJ) have been shown to possess anti-inflammatory activities. However, whether Chikusetsusaponin V (CsV), the most abundant component of SPJ, can exert anti-inflammatory activities is unknown. The present study was aimed to investigate the anti-inflammatory effects of CsV in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells and the underlying mechanisms. Our data showed that CsV dose-dependently inhibited NO, iNOS, TNF-α and IL-1β expressions in LPS-stimulated RAW264.7 cells. Increased protein levels of nuclear NF-κB and elevated phosphorylation levels of ERK and JNK in LPS-stimulated RAW 264.7 cells were also found downregulated by CsV treatment. Furthermore, the increase of CD14 and TLR4 mRNA expression due to LPS stimulation were significantly reversed by CsV treatment. These results suggested that CsV attenuated LPS-induced inflammatory responses partly via TLR4/CD14-mediated NF-κB and MAPK pathways.     

EstA protein,a novel virulence factor of Streptococcus pneumoniae, induces nitric oxide and pro-inflammatory cytokine production in RAW 264.7 macrophages through NF-κB/MAPK

In the present study we characterized the molecular mechanism by which esterase A (EstA) protein, a novel virulence factor of Streptococcus pneumoniae induces inflammation. Stimulation of RAW 264.7 macrophages with purified EstA protein induced the expression of inducible nitrogen oxide synthase (iNOS) mRNA and nitrogen oxide (NO) production in a concentration-dependent manner. Inhibitors of iNOS, NF-κB, p38 and ERK 1/2 MAPK pathways significantly decreased (50–78%) EstA-induced NO production. Similarly, EstA induced TNF-α, IL-1β and IL-6 mRNA expression in RAW 264.7 macrophages in a dose-dependent manner, and pre-treatment of the cell cultures with specific NF-κB, p38 and ERK 1/2 MAPK pathway inhibitors significantly decreased EstA-induced TNF-α, IL-1β and IL-6 protein production. Furthermore, immunoblot analysis revealed the degradation of the inhibitory kappa B (IKB-α) in response to EstA stimulation. Taken together, our data suggests that EstA protein is a novel inducer of NO and pro-inflammatory cytokines by activating the NF-κB, p38 and ERK 1/2 MAPK pathways during inflammatory responses. Future studies on the upstream protein kinases of the MAPK/NF-κB pathways and the kinetics of cytokine production will provide further details into the mechanism of EstA-induced inflammatory response.     

Cryptotanshinone Suppressed Inflammatory Cytokines Secretion in RAW264.7 Macrophages through Inhibition of the NF-κB and MAPK Signaling Pathways

Cryptotanshinone (CTS), a major constituent extracted from the medicinal herb Salvia miltiorrhiza Bunge, has well-documented antioxidative and anti-inflammatory effects. In the present study, the pharmacological effects and underlying molecular mechanisms of CTS on lipopolysaccharide (LPS)-induced inflammatory responses were investigated. By enzyme-linked immunosorbent assay, we observed that CTS reduced significantly the production of proinflammatory mediators (tumor necrosis factor-α and interleukin-6) induced by LPS in murine macrophage-like RAW264.7 cells. Mechanistically, CTS inhibited markedly the phosphorylation of mitogen-activated protein kinases (MAPKs), including ERK1/2, p38MAPK, and JNK, which are crucially involved in regulation of proinflammatory mediator secretion. Moreover, immunofluorescence and western blot analysis indicated that CTS abolished completely LPS-triggered nuclear factor-κB (NF-κB) activation. Taken together, these data implied that NF-κB and MAPKs might be the potential molecular targets for clarifying the protective effects of CTS on LPS-induced inflammatory cytokine production in macrophages.     

Maspin inhibits macrophage phagocytosis and enhances inflammatory cytokine production via activation of NF-κB signaling

Maspin (mammary serine protease inhibitor) is a non-inhibitory member of the serine protease inhibitor superfamily and a tumor suppressor in several cancers due to its ability to inhibit cell invasion, angiogenesis, and promote apoptosis. However, its immunomodulatory function remains largely unexplored. Thus, we explored the potential link between Maspin and macrophage function, first evaluating the regulatory effects of conditioned medium (CM) of a Maspin-overexpressing CHO cell strain on mouse peritoneal macrophage phagocytosis and cytokine secretion. Next, we used a transwell co-culture system and recombinant Maspin (rMaspin) to confirm the effects of Maspin on macrophages, and attempted to clarify the underlying mechanisms. We found that irrespective of CM, rMaspin or co-culture of Maspin-overexpressing cells with macrophages impaired macrophages phagocytosing Saccharomyces cerevisiae. Furthermore, q-RT-PCR or ELISA confirmed increased IL-1β, TNF-α, IFN-γ, IL-6, IL-12, IL-10, and M1 marker iNOS production in macrophages after Maspin stimulation, but TGF-β and M2 marker Arg-1 production were suppressed. Western blot showed activated NF-κB signaling in Maspin-stimulated macrophages; upregulated cytokines were lowered, and impaired phagocytosis recovered after blocking NF-κB signaling with PDTC. Thus, Maspin mildly inhibited phagocytic activity, but markedly enhanced inflammatory cytokine production and likely skewed macrophages towards M1 polarization, partially due to activation of NF-κB signaling. These results reveal a novel biological function of Maspin in modulating macrophage activity and may open a new avenue for Maspin-based tumor therapy.     

Punicalagin Inhibits Inflammation in LPS-Induced RAW264.7 Macrophages via the Suppression of TLR4-Mediated MAPKs and NF-κB Activation

Punicalagin (2,3,hexahydroxydiphenoyl-gallagyl-d-glucose and referred to as PUN) is a bioactive ellagitannin isolated from pomegranate, which is widely used for the treatment of inflammatory bowel disease (IBD), diarrhea, and ulcers in Chinese traditional medicine. In this study, we detected the anti-inflammation potentials of PUN in lipopolysaccharide (LPS)-induced macrophages and tried to uncover the underlying mechanism. Results demonstrated that PUN (25, 50, or 100 μM) treatment could significantly decrease the LPS-induced production of nitric oxide), prostaglandin E₂ (PGE₂), interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α in RAW264.7 cells. Molecular research showed that PUN inhibited the activation of upstream mediator nuclear factor-κB by suppressing the phosphorylation of IκBα and p65. Results also indicated that PUN could suppress the phosphorylation of mitogen-activated protein kinase including p38, c-Jun N-terminal kinase, and extracellular signal-regulated kinase. In conclusion, we observed that PUN could inhibit LPS-induced inflammation, and it may be a potential choice for the treatment of inflammation diseases.     

7,8-Dihydroxyflavone exhibits anti-inflammatory properties by downregulating the NF-κB and MAPK signaling pathways in lipopolysaccharide-treated RAW264.7 cells

7,8-Dihydroxyflavone (7,8-DHF), a member of the flavonoid family, has received considerable attention as a selective tyrosine kinase receptor B agonist. Several studies have indicated that 7,8-DHF has neurotrophic and antioxidant activities. However, little is known about the cellular and molecular mechanisms underlying the anti-inflammatory activity of 7,8-DHF. Therefore, we investigated whether 7,8-DHF affects the expression of inflammatory mediators in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Our results indicated that 7,8-DHF significantly attenuated secretion of LPS-induced inflammatory mediators nitric oxide (NO), prostaglandin E? (PGE?) and interleukin-1β (IL-1β) in RAW264.7 cells. Additionally, LPS-induced expression of inducible NO synthase (iNOS), cyclooxygenase (COX)-2 and IL-1β was decreased by pre-treatment with 7,8-DHF. Our results also showed that 7,8-DHF reduces LPS-induced nuclear factor-κB (NF-κB) activity via the suppression of the nuclear translocation of NF-κB p65 and the degradation of inhibitor κB (lκB). In addition, 7,8-DHF inhibited the phosphorylation of mitogen-activated protein kinases (MAPKs) such as extracellular-signal-related kinase (ERK), p38, and c-Jun N-terminal kinase (JNK). These results suggest that the anti-inflammatory property of 7,8-DHF is related to the downregulation of iNOS, COX-2 and IL-1β, due to NF-κB inhibition as well as to the negative regulation of MAPK activation in RAW264.7 cells. Thus, 7,8-DHF may be a novel therapeutic agent for the prevention of various inflammatory diseases.     

Folic acid inhibits lipopolysaccharide-induced inflammatory response in RAW264.7 macrophages by suppressing MAPKs and NF-κB activation

Objective  

The aim of the present study was to investigate the effects of folic acid on the inflammatory responses to lipopolysaccharide (LPS) in RAW264.7 cells and the signal transduction pathways involved.     

Immune-enhancing effects of a polysaccharide PRG1-1 from Russula griseocarnosa on RAW264.7 macrophage cells via the MAPK and NF-κB signalling pathways

Polysaccharides are one of many bioactive compounds found in edible mushrooms. Edible mushrooms have become attractive as “health foods” and as source materials for immunomodulators. The aim of this project was to study the immunoregulatory effects of a purified polysaccharide derived from wild Russula griseocarnosa (PRG1-1) on macrophages. Our data showed that in RAW264.7 macrophage cells, PRG1-1 increased expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Furthermore, PRG1-1 increased the production of nitric oxide (NO) and cytokines, including interleukin 6 (IL-6) and tumour necrosis factor alpha (TNF-α). Western blotting demonstrated that the regulation of NO and cytokines was mediated through the nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signalling pathways. Therefore, PRG1-1 has the capacity to activate macrophages via the NF-κB and MAPK pathways. These findings helped to elucidate the immune-modulatory properties of the polysaccharide from R. griseocarnosa.     

Triphala herbal extract suppresses inflammatory responses in LPS-stimulated RAW 264.7 macrophages and adjuvant-induced arthritic rats via inhibition of NF-κB pathway

This study sought to explore the mechanism of anti-inflammatory effect of triphala in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages and in adjuvant-induced arthritic rats. In stimulated RAW 264.7 cells, triphala (100–300 μg/ml) significantly suppressed production of inflammatory mediators (e.g. TNFα, IL-1β, IL-6, MCP-1, VEGF, NO, PGE₂), intracellular free radicals and release of lysosomal enzymes (e.g. acid phosphatase, β-galactosidase, N-acetyl glucosamindase and cathepsin D) in a dose-related manner. With triphala, mRNA levels of genes for pro-inflammatory TNFα, IL-1β, IL-6 and MCP-1, inflammatory iNOS and COX-2 enzymes and NF-κBp65 were down-regulated in the stimulated cells; in contrast, there was up-regulation of heme oxygenase-1 (HO-1) expression. Western blot analyses revealed that triphala suppressed the protein expression of NF-κB p65 and p-NF-κB p65 in the stimulated cells, which subsequently reduced over-expression of TNFα, IL-17, iNOS and COX-2 in a manner similar to that observed with BAY 11-7082, an IκB kinase inhibitor. Immunofluorescence analysis revealed inhibition of p-NF-κB p65 nuclear translocation and COX-2 protein expression caused by triphala. Consistent with these findings, the animal studies presented confirmed that triphala exhibited anti-inflammatory effects in a rat adjuvant-induced arthritis model by reducing of inflammatory mediator (e.g. IL-17, COX-2 and RANKL) expression via inhibition of NF-κB activation. Taken together, the results here demonstrated that triphala has potential anti-inflammatory applications that could be used for the treatment of inflammatory disorders, including rheumatoid arthritis.     

Lycopene suppresses LPS-induced NO and IL-6 production by inhibiting the activation of ERK, p38MAPK, and NF-κB in macrophages

Background  

Lycopene has antioxidant, anticancer, and anti-inflammatory effects with molecular mechanisms not fully identified.     

Artemisinin inhibits inflammatory response via regulating NF-κB and MAPK signaling pathways

Artemisinin, isolated from the Chinese plant Artemisia annua, has been used for many years to treat different forms of malarial parasites. In this study, we explored the anti-inflammatory activity of artemisinin and the underlying mechanism of this action. We demonstrated that the anti-inflammatory effects of artemisinin in TPA-induced skin inflammation in mice. Then the artemisinin significantly inhibited the expression of NF-κB reporter gene induced by TNF-α in a dose-dependent manner. Artemisinin also inhibited TNF-α induced phosphorylation and degradation of IκBα, p65 nuclear translocation. Artemisinin also has an impact on upstream signaling of IKK through the inhibition of expression of adaptor proteins, TNF receptor-associated factor 2 (TRAF2) and receptor interacting protein 1 (RIP1). Furthermore, pretreatment of cells with artemisinin prevented the TNF-α-induced expression of NF-κB target genes, such as anti-apoptosis (c-IAP1, Bcl-2, and FLIP), proliferation (COX-2, cyclinD1), invasion (MMP-9), angiogenesis (VEGF), and major inflammatory cytokines (TNF-α, iNOS, and MCP1). We also proved that artemisinin potentiated TNF-α-induced apoptosis. Moreover, artemisinin significantly impaired the ROS production and phosphorylation of p38 and ERK, but did not affect the phosphorylation of JNK. Taken together, artemisinin may be a potentially useful therapeutic agent for inflammatory-related diseases.