Activation of NALP1 inflammasomes in rats with adjuvant arthritis; a novel therapeutic target of carboxyamidotriazole in a model of rheumatoid arthritis

Background and Purpose

Pro-inflammatory cytokines are important in rheumatoid arthritis (RA) and their production is mainly regulated by NF-κB and inflammasomes. Carboxyamidotriazole (CAI) exhibits potent anti-inflammatory activities by decreasing cytokines. Here, we have investigated NACHT, LRR and PYD domains-containing protein (NALP) inflammasomes in a rat model of RA and explored the therapeutic effects of CAI in this model and the involvement of NF-κB and inflammasomes in the actions of CAI.

Experimental Approach

The anti-arthritic effects of CAI were assessed in the adjuvant arthritis (AA) model in rats, using radiological and histological techniques. NALP1 and NALP3 inflammasomes, NF-κB pathway and pro-inflammatory cytokines levels were measured with Western blots, immunohistochemistry and elisa.

Key Results

CAI decreased the arthritis index, improved radiological and histological changes, and reduced synovial IL-1β, IL-6, IL-18 and TNF-α levels in rats with AA. Compared with normal rats, the 70 kDa NALP1 isoform was up-regulated, NALP3 was down-regulated, and levels of the 165 kDa NALP1 isoform and the adaptor protein ASC were unchanged in synovial tissue from AA rats. CAI reduced the 70 kDa NALP1 isoform and restored NALP3 levels in AA rats; CAI inhibited caspase-1 activation in AA synovial tissue, but not its enzymic activity in vitro. In addition, CAI reduced expression of p65 NF-κB subunit and IκBα phosphorylation and degradation in AA rats.

Conclusion and Implications

NALP1 inflammasomes were activated in synovial tissues from AA rats and appeared to be a novel therapeutic target for RA. CAI could have therapeutic value in RA by inhibiting activation of NF-κB and NALP1 inflammasomes and by decreasing pro-inflammatory cytokines.     

Methyl salicylate lactoside inhibits inflammatory response of fibroblast-like synoviocytes and joint destruction in collagen-induced arthritis in mice

BACKGROUND AND PURPOSE

Methyl salicylate 2-O-β-d-lactoside (MSL), whose chemical structure is similar to that of salicylic acid, is a natural product derivative isolated from a traditional Chinese herb. The aim of this study was to investigate the therapeutic effect of MSL in mice with collagen-induced arthritis (CIA) and explore its underlying mechanism.

EXPERIMENTAL APPROACH

The anti-arthritic effects of MSL were evaluated on human rheumatoid fibroblast-like synoviocytes (FLS) in vitro and CIA in mice in vivo by obtaining clinical scores, measuring hind paw thickness and inflammatory cytokine levels, radiographic evaluations and histopathological assessments.

KEY RESULTS

Treatment with MSL after the onset of arthritis significantly prevented the progression and development of rheumatoid arthritis (RA) in CIA mice without megascopic gastric mucosa damage. In addition, MSL inhibited the production of pro-inflammatory mediators, the phosphorylation and translocation of NF-κB, and cell proliferation induced by TNF-α in FLS. MSL non-selectively inhibited the activity of COX in vitro, but was a more potent inhibitor of COX-2 than COX-1. MSL also inhibited the phosphorylation of inhibitor of NF-κB kinase, IκBα and p65, thus blocking the nuclear translocation of NF-κB in TNF-α-stimulated FLS.

CONCLUSION AND IMPLICATIONS

MSL exerts therapeutic effects on CIA mice, suppressing the inflammatory response and joint destruction by non-selectively inhibiting the activity of COX and suppressing activation of the NF-κB signalling pathway, but without damaging the gastric mucosa. Therefore, MSL has great potential to be developed into a novel therapeutic agent for the treatment of RA.     

Pharmacological characterization of LPS and opioid interactions at the toll-like receptor 4

Background and Purpose

Previous work in our laboratory showed opioid agents inhibit cytokine expression in astrocytes. Recently, Watkins and colleagues hypothesized that opioid agonists activate toll-like receptor 4 (TLR4) signalling, which leads to neuroinflammation. To test this hypothesis, we characterized LPS and opioid effects on TLR4 signalling in reporter cells.

Experimental Approach

NF-κB reporter cells expressing high levels of TLR4 were used to compare LPS and opioid effects on NF-κB activation, a pathway activated by TLR4 stimulation.

Key Results

LPS increased TLR4 signalling in a concentration-dependent manner and was antagonized by LPS antagonist (LPS-RS, from Rhodobacter sphaeroides). A concentration ratio analysis showed that LPS-RS was a competitive antagonist. The opioid agonists, morphine and fentanyl, produced minor activation of TLR4 signalling when given alone. When tested following LPS stimulation, opioid agonists inhibited NF-κB activation but this inhibition was not blocked by the general opioid antagonist, naloxone, nor by the selective μ opioid receptor antagonist, β-FNA. Indeed, both naloxone and β-FNA also inhibited NF-κB activation in reporter cells. Further examination of fentanyl and β-FNA effects revealed that both opioid agents inhibited LPS signalling in a non-competitive fashion.

Conclusions and Implications

These results show that LPS-RS is a competitive antagonist at the TLR4 complex, and that both opioid agonists and antagonists inhibit LPS signalling in a non-competitive fashion through a non-GPCR, opioid site(s) in the TLR4 signalling pathway. If confirmed, existing opioid agents or other drug molecules more selective at this novel site may provide a new therapeutic approach to the treatment of neuroinflammation.     

Lobolide,a diterpene,blockades the NF-κB pathway and p38 and ERK MAPK activity in macrophages in vitro

Aim:

Recent studies have shown that constitutive activation of the nuclear factor κB (NF-κB) plays a key role in chronic inflammation and cancers. The aim of this study was to characterize lobolide, a cembrane diterpene, as a drug candidate targeting the NF-κB signaling pathway.

Methods:

A HEK 293/NF-κB-Luc stable cell line was constructed to evaluate the effect of lobolide on NF-κB activation. THP-1 human monocytes and peripheral blood mononuclear cells (PBMCs) from healthy volunteers were tested. Lipopolysaccharide (LPS)-induced TNFα and IL-1β production and activation of the TAK1-IKK-NF-κB pathway were studied using ELISA and Western blot analysis.

Results:

In HEK 293/NF-κB-Luc stable cells, lobolide (0.19–50 μmol/L) inhibited NF-κB activation in a concentration-dependent manner with an IC₅₀ value of 4.2±0.3 μmol/L. Treatment with lobolide (2.5–10 μmol/L) significantly suppressed LPS-induced production of TNFα and IL-1β in both THP-1 cells and PBMCs. In THP-1 cells, the suppression was partially caused by blockade of the translocation of NF-κB from the cytoplasm to the nucleus via affecting the TAK1-IKK-NF-κB pathway and p38 and ERK MAPK activity.

Conclusion:

Lobolide is a potential inhibitor of the NF-κB pathway, which blocks the translocation of NF-κB from the cytoplasm to the nucleus. Lobolide inhibits LPS-stimulated TNFα and IL-1β release, suggesting that the compound might be an anti-inflammatory compound.     

Curcumin inhibits LPS-induced inflammation in rat vascular smooth muscle cells in vitro via ROS-relative TLR4-MAPK/NF-κB pathways

Aim:

To investigate whether curcumin (Cur) suppressed lipopolysaccharide (LPS)-induced inflammation in vascular smooth muscle cells (VSMCs) of rats, and to determine its molecular mechanisms.

Methods:

Primary rat VSMCs were treated with LPS (1 μg/L) and Cur (5, 10, or 30 μmol/L) for 24 h. The levels of MCP-1, TNF-α, and iNOS were measured using ELISA and real-time RT-PCR. NO level was analyzed with the Griess reaction. Western-blotting was used to detect the activation of TLR4, MAPKs, IκBα, NF-κB p65, and the p47^phox subunit of NADPH oxidase in the cells.

Results:

Treatment of VSMCs with LPS dramatically increased expression of inflammatory cytokines MCP-1 and TNF-α, expression of TLR4 and iNOS, and NO production. LPS also significantly increased phosphorylation of IκBα, nuclear translocation of NF-κB (p65) and phosphorylation of MAPKs in VSMCs. Furthermore, LPS significantly increased production of intracellular ROS, and decreased expression of p47^phox subunit of NADPH oxidase. Pretreatment with Cur concentration-dependently attenuated all the aberrant changes in LPS-treated VSMCs. The LPS-induced overexpression of MCP-1 and TNF-α, and NO production were attenuated by pretreatment with the ERK inhibitor PD98059, the p38 MAPK inhibitor SB203580, the NF-κB inhibitor PDTC or anti-TLR4 antibody, but not with the JNK inhibitor SP600125.

Conclusion:

Cur suppresses LPS-induced overexpression of inflammatory mediators in VSMCs in vitro via inhibiting the TLR4-MAPK/NF-κB pathways, partly due to block of NADPH-mediated intracellular ROS production.     

Shikonin reduces oedema induced by phorbol ester by interfering with IκBα degradation thus inhibiting translocation of NF-κB to the nucleus

Background and purpose:

In the present paper we studied the effect of shikonin on ear oedema induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), and determined the mechanisms through which shikonin might exert its topical anti-inflammatory action.

Experimental approach:

Acute ear oedema was induced in mice by topical application of TPA. The in vitro assays used macrophages RAW 264.7 cells stimulated with lipopolysaccharide. Cyclooxygenase-2, inducible nitric oxide synthase, protein kinase Cα, extracellular signal-regulated protein kinase (ERK), phosphorylated ERK (pERK), c-Jun N-terminal kinase (JNK), pJNK, p38, p-p38, p65, p-p65, inhibitor protein of nuclear factor-κB (NF-κB) (IκBα) and pIκBα were measured by Western blotting, activation and binding of NF-κB to DNA was detected by reporter gene and electrophoretic mobility shift assay, respectively, and NF-κB p65 localization was detected by immunocytochemistry.

Key results:

Shikonin reduced the oedema (inhibitory dose 50 = 1.0 mg per ear), the expression of cyclooxygenase-2 (70%) and of inducible nitric oxide synthase (100%) in vivo. It significantly decreased TPA-induced translocation of protein kinase Cα, the phosphorylation and activation of ERK, the nuclear translocation of NF-κB and the TPA-induced NF-κB-DNA-binding activity in mouse skin. Moreover, in RAW 264.7 cells, shikonin significantly inhibited the binding of NF-κB to DNA in a dose-dependent manner and the nuclear translocation of p65.

Conclusions and implications:

Shikonin exerted its topical anti-inflammatory action by interfering with the degradation of IκBα, thus inhibiting the activation of NF-κB.     

Inhibitory effects of rosiglitazone on paraquat-induced acute lung injury in rats

Aim:

To investigate the effects of the PPAR-γ agonist rosiglitazone on acute lung injury induced by the herbicide paraquat (PQ) and the underlying mechanisms of action.

Methods:

Male Sprague-Dawley rats were injected with PQ (20 mg/kg, ip). Rosiglitazone (3 or 10 mg/kg, ip) was administered 1 h before PQ exposure. Peripheral blood was collected at 4, 8, 24 and 72 h after PQ exposure for measuring the levels of MDA, TNF-α and IL-1β, and the SOD activity. Lung tissues were collected at 72 h after PQ exposure to determine the wet-to-dry (W/D) ratios and lung injury scores, as well as the protein levels of NF-κBp65, PPAR-γ, Nrf2, IκBα and pIκBα.

Results:

At 72 h after PQ exposure, the untreated rats showed a 100% cumulative mortality, whereas no death was observed in rosiglitazone-pretreated rats. Moreover, rosiglitazone pretreatment dose-dependently attenuated PQ-induced lung edema and lung histopathological changes. The pretreatment significantly reduced the levels of TNF-α, IL-1β and MDA, increased SOD activity in the peripheral blood of PQ-treated rats. The pretreatment also efficiently activated PPAR-γ, induced Nrf2 expression and inhibited NF-κB activation in the lung tissues of PQ-treated rats. Furthermore, the pretreatment dose-dependently inhibited IκB-α degradation and phosphorylation, thus inhibiting NF-κB activation.

Conclusion:

Pretreatment with rosiglitazone protects rats against PQ-induced acute lung injury by activating PPAR-γ, inducing Nrf2 expression and inhibiting NF-κB activation.     

Flavocoxid,a dual inhibitor of cyclooxygenase and 5-lipoxygenase,blunts pro-inflammatory phenotype activation in endotoxin-stimulated macrophages

Background and purpose:

The flavonoids, baicalin and catechin, from Scutellaria baicalensis and Acacia catechu, respectively, have been used for various clinical applications. Flavocoxid is a mixed extract containing baicalin and catechin, and acts as a dual inhibitor of cyclooxygenase (COX) and 5-lipoxygenase (LOX) enzymes. The anti-inflammatory activity, measured by protein and gene expression of inflammatory markers, of flavocoxid in rat peritoneal macrophages stimulated with Salmonella enteritidis lipopolysaccharide (LPS) was investigated.

Experimental approach:

LPS-stimulated (1 µg·mL⁻¹) peritoneal rat macrophages were co-incubated with different concentrations of flavocoxid (32–128 µg·mL⁻¹) or RPMI medium for different incubation times. Inducible COX-2, 5-LOX, inducible nitric oxide synthase (iNOS) and inhibitory protein κB-α (IκB-α) levels were evaluated by Western blot analysis. Nuclear factor κB (NF-κB) binding activity was investigated by electrophoretic mobility shift assay. Tumour necrosis factor-α (TNF-α) gene and protein expression were measured by real-time polymerase chain reaction and enzyme-linked immunosorbent assay respectively. Finally, malondialdehyde (MDA) and nitrite levels in macrophage supernatants were evaluated.

Key results:

LPS stimulation induced a pro-inflammatory phenotype in rat peritoneal macrophages. Flavocoxid (128 µg·mL⁻¹) significantly inhibited COX-2 (LPS = 18 ± 2.1; flavocoxid = 3.8 ± 0.9 integrated intensity), 5-LOX (LPS = 20 ± 3.8; flavocoxid = 3.1 ± 0.8 integrated intensity) and iNOS expression (LPS = 15 ± 1.1; flavocoxid = 4.1 ± 0.4 integrated intensity), but did not modify COX-1 expression. PGE₂ and LTB₄ levels in culture supernatants were consequently decreased. Flavocoxid also prevented the loss of IκB-α protein (LPS = 1.9 ± 0.2; flavocoxid = 7.2 ± 1.6 integrated intensity), blunted increased NF-κB binding activity (LPS = 9.2 ± 2; flavocoxid = 2.4 ± 0.7 integrated intensity) and the enhanced TNF-α mRNA levels (LPS = 8 ± 0.9; flavocoxid = 1.9 ± 0.8 n-fold/β-actin) induced by LPS. Finally, flavocoxid decreased MDA, TNF and nitrite levels from LPS-stimulated macrophages.

Conclusion and implications:

Flavocoxid might be useful as a potential anti-inflammatory agent, acting at the level of gene and protein expression.     

Compound FLZ inhibits lipopolysaccharide-induced inflammatory effects via down-regulation of the TAK-IKK and TAK-JNK/p38MAPK pathways in RAW264.7 macrophages

Aim:

The aim of this study was to investigate the effect of the squamosamide derivative FLZ (N-2-(4-hydroxy-phenyl)-ethyl-2-(2,5-dimethoxy-phenyl)-3-(3-methoxy-4-hydroxy-phenyl)-acrylamide) on lipopolysaccharide (LPS)-induced inflammatory mediator production and the underlying mechanism in RAW264.7 macrophages.

Methods:

RAW264.7 cells were preincubated with non-toxic concentrations of compound FLZ (1, 5, and 10 μmol/L) for 30 min and then stimulated with 10 μg/L LPS. The production of nitric oxide (NO), the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2), and the activation of nuclear factor kappa-B (NF-κB) and mitogen-activated protein kinase (MAPK) pathways were examined.

Results:

FLZ significantly inhibited the LPS-induced production of NO, as well as the expression of iNOS and COX-2 at both the RNA and the protein levels in RAW264.7 cells. The LPS-induced increase in the DNA binding activity of NF-κB and activator protein 1 (AP-1), the nuclear translocation of NF-κB p65, the degradation of the inhibitory κBα protein (IκBα) and the phosphorylation of IκBα, IκB kinase (IKK) α/β, c-Jun NH₂-terminal kinase (JNK) and p38 MAPKs were all suppressed by FLZ. However, the phosphorylation of extracellular signal-regulated kinase (ERK) was not affected. Further study revealed that FLZ inhibited the phosphorylation of transforming growth factor-β (TGF-β)-activated kinase 1 (TAK1), which is an upstream signaling molecule required for IKKα/β, JNK and p38 activation.

Conclusion:

FLZ inhibited the LPS-induced production of inflammatory mediators at least partly through the downregulation of the TAK-IKK and TAK-JNK/p38MAPK pathways.     

Astragalus polysaccharides suppress ICAM-1 and VCAM-1 expression in TNF-α-treated human vascular endothelial cells by blocking NF-κB activation

Aim:

To investigate the effects Astragalus polysaccharides (APS) on tumor necrosis factor (TNF)-α-induced inflammatory reactions in human umbilical vein endothelial cells (HUVECs) and to elucidate the underlying mechanisms.

Methods:

HUVECs were treated with TNF-α for 24 h. The amounts of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) were determined with Western blotting. HUVEC viability and apoptosis were detected using cell viability assay and Hoechst staining, respectively. Reactive oxygen species (ROS) production was measured by DHE staining. Monocyte and HUVEC adhesion assay was used to detect endothelial cell adhesive function. NF-κB activation was detected with immunofluorescence.

Results:

TNF-α (1-80 ng/mL) caused dose- and time-dependent increases of ICAM-1 and VCAM-1 expression in HUVECs, accompanied by significant augmentation of IκB phosphorylation and NF-κB translocation into the nuclei. Pretreatment with APS (10 and 50 μg/mL) significantly attenuated TNFα-induced upregulation of ICAM-1 VCAM-1 and NF-κB translocation. Moreover, APS significantly reduced apoptosis, ROS generation and adhesion function damage in TNF-α-treated HUVECs.

Conclusion:

APS suppresses TNFα-induced adhesion molecule expression by blocking NF-κB signaling and inhibiting ROS generation in HUVECs. The results suggest that APS may be used to treat and prevent endothelial cell injury-related diseases.