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.     

FK506 Attenuates the Inflammation in Rat Spinal Cord Injury by Inhibiting the Activation of NF-κB in Microglia Cells

FK-506 (Tacrolimus) is a very commonly used immunomodulatory agent that plays important roles in modulating the calcium-dependent phosphoserine–phosphothreonine protein phosphatase calcineurin and thus inhibits calcineurin-mediated secondary neuronal damage. The biological function of FK-506 in the spinal cord has not been fully elucidated. To clarify the anti-inflammatory action of FK-506 in spinal cord injury (SCI), we performed an acute spinal cord contusion injury model in adult rats and hypoxia-treated primary spinal cord microglia cultures. This work studied the activation of NF-κB and proinflammatory cytokine (TNF-a, IL-1b, and IL-6) expression. ELISA and q-PCR analysis revealed that TNF-a, IL-1b, and IL-6 levels significantly increased 3 days after spinal cord contusion and decreased after 14 days, accompanied by the increased activation of NF-κB. This increase was reversed by an FK-506 treatment. Double immunofluorescence labeling suggested that NF-κB activation was especially prominent in microglia. Immunohistochemistry confirmed no alteration in the number of microglia. Moreover, the results in hypoxia-treated primary spinal cord microglia confirmed the effect of FK-506 on TNF-a, IL-1b, and IL-6 expression and NF-κB activation. These findings suggest that FK-506 may be involved in microglial activation after SCI.     

4-O-carboxymethylascochlorin protected against microglial-mediated neurotoxicity in SH-SY5Y and BV2 cocultured cells from LPS–induced neuroinflammation and death by inhibiting MAPK,NF-κB,and Akt pathways

In our previous studies, structurally similar compounds of ascochlorin and ascofuranone exhibited anti-inflammatory activity. Neural inflammation plays a significant role in the commence and advancement of neurodegenerative diseases. It is not known whether 4-O-carboxymethylascochlorin (AS-6) regulates the initial stage of inflammatory responses at the cellular level in BV2 microglia cells. We here investigated the anti-inflammatory effects of AS-6 treatment in microglia cells with the microglial protection in neurons. We found that the lipopolysaccharide (LPS)-stimulated production of nitric oxide, a main regulator of inflammation, is suppressed by AS-6 in BV2 microglial cells. In addition, AS-6 dose-dependently suppressed the increase in COX-2 protein and messenger RNA levels in LPS-stimulated BV2 cells. Moreover, AS-6 inhibited the expression and secretion of proinflammatory cytokines in BV2 microglial cells. At the intracellular level, AS-6 inhibited LPS-activated nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in BV2 microglial cells. AS-6 negatively affected mitogen-activated protein kinases (MAPK) and Akt phosphorylation: Phosphorylated forms of ERK, JNK, p38, and Akt decreased. To check whether AS-6 protects against inflammatory inducer-mediated neurotoxicity, neuronal SH-SY5Y cells were coincubated with BV2 cells in conditioned medium. AS-6 exerted a neuroprotective effect by suppressing microglial activation by LPS or amyloid-β peptide. AS-6 is a promising suppressor of inflammatory responses in LPS-induced BV2 cells by attenuating NF-κB and MAPKs signaling. AS-6 protected against microglial-mediated neurotoxicity in SH-SY5Y and BV2 cocultured cells from LPS–induced neuroinflammation and death via inhibiting MAPK, NF-κB, and Akt pathways.     

Curcumin protects against acute liver damage in the rat by inhibiting NF-κB,proinflammatory cytokines production and oxidative stress

Curcumin, an anti-inflammatory and antioxidant compound, was evaluated for its ability to suppress acute carbon tetrachloride-induced liver damage. Acute hepatotoxicity was induced by oral administration of CCl₄ (4 g/kg, p.o.). Curcumin treatment (200 mg/kg, p.o.) was given before and 2 h after CCl₄ administration. Indicators of necrosis (alanine aminotransferase) and cholestasis (γ-glutamyl transpeptidase and bilirubins) resulted in significant increases after CCl₄ intoxication, but these effects were prevented by curcumin treatment. As an indicator of oxidative stress, GSH was oxidized and the GSH/GSSG ratio decreased significantly by CCl₄, but was preserved within normal values by curcumin. In addition to its antioxidants properties, curcumin is capable of preventing NF-κB activation and therefore to prevent the secretion of proinflammatory cytokines. Therefore, in this study we determined the concentrations of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) mRNA, and NF-κB activation. CCl₄-administered rats depicted significant increases in TNF-α, IL-1β, and IL-6 production, while curcumin remarkably suppressed these mediators of inflammation in liver damage. These results were confirmed by measuring TNF-α, and IL-1β protein production using Western Blot analysis. Accordingly, these proteins were increased by CCl₄ and this effect was abolished by curcumin. Administration of CCl₄ induced the translocation of NF-κB to the nucleus; CCl₄ induced NF-κB DNA binding activity was blocked by curcumin treatment. These findings suggest that curcumin prevents acute liver damage by at least two mechanisms: acting as an antioxidant and by inhibiting NF-κB activation and thus production of proinflammatory cytokines.     

CD36 participates in PrP(106-126)-induced activation of microglia

Microglial activation is a characteristic feature of the pathogenesis of prion diseases. The molecular mechanisms that underlie prion-induced microglial activation are not very well understood. In the present study, we investigated the role of the class B scavenger receptor CD36 in microglial activation induced by neurotoxic prion protein (PrP) fragment 106-126 (PrP(106-126)). We first examined the time course of CD36 mRNA expression upon exposure to PrP(106-126) in BV2 microglia. We then analyzed different parameters of microglial activation in PrP(106-126)-treated cells in the presence or not of anti-CD36 monoclonal antibody (mAb). The cells were first incubated for 1 h with CD36 monoclonal antibody to block the CD36 receptor, and were then treated with neurotoxic prion peptides PrP(106-126). The results showed that PrP(106-126) treatment led to a rapid yet transitory increase in the mRNA expression of CD36, upregulated mRNA and protein levels of proinflammatory cytokines (IL-1β, IL-6 and TNF-α), increased iNOS expression and nitric oxide (NO) production, stimulated the activation of NF-κB and caspase-1, and elevated Fyn activity. The blockade of CD36 had no effect on PrP(106-126)-stimulated NF-κB activation and TNF-α protein release, abrogated the PrP(106-126)-induced iNOS stimulation, downregulated IL-1β and IL-6 expression at both mRNA and protein levels as well as TNF-α mRNA expression, decreased NO production and Fyn phosphorylation, reduced caspase-1 cleavage induced by moderate PrP(106-126)-treatment, but had no effect on caspase-1 activation after treatment with a high concentration of PrP(106-126). Together, these results suggest that CD36 is involved in PrP(106-126)-induced microglial activation and that the participation of CD36 in the interaction between PrP(106-126) and microglia may be mediated by Src tyrosine kinases. Our findings provide new insights into the mechanisms underlying the activation of microglia by neurotoxic prion peptides and open perspectives for new therapeutic strategies for prion diseases by modulation of CD36 signaling.     

Extracellular purine metabolism and signaling of CD73-derived adenosine in murine Treg and Teff cells

CD73-derived adenosine acts as potent inhibitor of inflammation, and regulatory T cells (Treg) have been shown to express CD73 as a novel marker. This study explored the role of endogenously formed adenosine in modulating NF-κB activity and cytokine/chemokine release from murine Treg and effector T cells (Teff) including key enzymes/purinergic receptors of extracellular ATP catabolism. Stimulating murine splenocytes and CD4(+) T cells with anti-CD3/anti-CD28 significantly upregulated activated NF-κB in CD73(-/-) T cells (wild type: 4.36 ± 0.21; CD73(-/-): 6.58 ± 0.75; n = 4; P = 0.029). This was associated with an augmented release of proinflammatory cytokines IL-2, TNF-α, and IFN-γ. Similar changes were observed with the CD73 inhibitor APCP (50 μM) on NF-κB and IFN-γ in wild-type CD4(+) T-cells. Treatment of stimulated CD4(+) T-cells with adenosine (25 μM) potently reduced IFN-γ release which is mediated by adenosine A2a receptors (A2aR). AMP (50 μM) also reduced cytokine release which was not inhibited by APCP. In Teff, A2aR activation (CGS21680) potently inhibited the release of IL-1, IL-2, IL-3, IL-4, IL-12, IL-13, IFN-γ, TNF-α, granulocyte-macrophage colony-stimulating factor (GM-CSF), CCL3, and CCL4. However, in Treg, CGS21680 did not alter cytokine/chemokine release. In summary, CD73-derived adenosine tonically inhibits active NF-κB in CD4(+) T-cells, thereby modulating the release of a broad spectrum of proinflammatory cytokines and chemokines. Downregulation of P2X7 and upregulation of CD73 in Treg after antigenic stimulation may be an important mechanism to maintain the ability of Treg to generate immunosuppressive adenosine.     

Histone methyltransferase NSD2/MMSET mediates constitutive NF-κB signaling for cancer cell proliferation, survival, and tumor growth via a feed-forward loop

Constitutive NF-κB activation by proinflammatory cytokines plays a major role in cancer progression. However, the underlying mechanism is still unclear. We report here that histone methyltransferase NSD2 (also known as MMSET or WHSC1), a target of bromodomain protein ANCCA/ATAD2, acts as a strong coactivator of NF-κB by directly interacting with NF-κB for activation of target genes, including those for interleukin-6 (IL-6), IL-8, vascular endothelial growth factor A (VEGFA), cyclin D, Bcl-2, and survivin, in castration-resistant prostate cancer (CRPC) cells. NSD2 is recruited to the target gene promoters upon induction and mediates NF-κB activation-associated elevation of histone H3K36me2 and H3K36me3 marks at the promoter, which involves its methylase activity. Interestingly, we found that NSD2 is also critical for cytokine-induced recruitment of NF-κB and acetyltransferase p300 and histone hyperacetylation. Importantly, NSD2 is overexpressed in prostate cancer tumors, and its overexpression correlates with NF-κB activation. Furthermore, NSD2 expression is strongly induced by tumor necrosis factor alpha (TNF-α) and IL-6 via NF-κB and plays a crucial role in tumor growth. These results identify NSD2 to be a key chromatin regulator of NF-κB and mediator of the cytokine autocrine loop for constitutive NF-κB activation and emphasize the important roles played by NSD2 in cancer cell proliferation and survival and tumor growth.     

Binding of glyceraldehyde-3-phosphate dehydrogenase to the cis-acting element of structure-anchored repression in ccn2 mRNA

Studies increasingly indicate that inflammation induced by β-amyloid (Aβ) contributes to the progression of Alzheimer’s disease (AD). How to inhibit the enhanced production of proinflammatory cytokines stimulated by Aβ is an important research subject for the treatment of AD. In this study, we investigated the inhibitory effect and the molecular mechanism of the lipoxin A₄ (LXA₄) on the production of interleukin-1β (IL-1β) and tumor necrosis factorα (TNFα) induced by β-amyloid in the cortex and hippocampus of mice, and in Aβ-stimulated BV2 cells, a mouse microglial cell line. LXA₄ down-regulated the protein expression of IL-1β and TNFα, attenuated the gene expressions of IL-1β and TNFα, inhibited the degradation of IκBα, inhibited translocation of NF-κB p65 subunit into the nucleus induced by β-amyloid in the cortex and hippocampus of mice, and in Aβ-stimulated BV2 cells, and the inhibitory effects were dose dependently elevated. Our findings suggest that LXA₄ inhibits the production of IL-1β and TNFα induced by β-amyloid in the cortex and hippocampus of mice, and in BV2 microglial cells via the NF-κB signal pathway.     

SENP1-mediated NEMO de-SUMOylation inhibits intermittent hypoxia induced inflammatory response of microglia in vitro

Among the seven small ubiquitin-like modifier (SUMO)-specific proteases (SENPs), our previous work showed that SENP1 suppressed nuclear factor kappa B (NF-κB) activation and alleviates the inflammatory response in microglia. However, the mechanism is still largely unknown. In this study, western blot analysis and enzyme-linked immunosorbent assay were utilized for evaluating the extent of NF-κB activation and expression of proinflammatory cytokines. qPCR and western blot analysis were performed to detect SENP1 expression. Coimmunoprecipitation followed by western blot analysis was applied to measure the changes in SUMOylation of NF-κB essential modulator (NEMO) and P65 in microglia with or without overexpression of SENP1. As the results, we found that intermittent hypoxia (IH) triggered the activation of NF-κB and upregulated the expression levels of tumor necrosis factor-α and interleukin-6. Interestingly, our data indicated that the SUMOylation of NEMO was enhanced by IH while SUMOylation of P65 was not affected. Further, our data showed that overexpression of SENP1 could decrease the extent of NF-κB activation and inhibit the inflammatory response of microglia through regulating the SUMOylation of NEMO. Collectively, this study presents the first report of the SENP1-controlled de-SUMOylation process of NEMO and its critical role in regulating NF-κB activation and proinflammatory cytokines secretion in microglia cells. This study would benefit for clarifying the role of SENP1 in IH-induced activation of microglia, thus providing potential therapeutic targets for obstructive sleep apnea treatment.