Monosodium Urate Crystal-induced Inflammation Research Articles

Phosphoinositide-3 kinase gamma regulates caspase-1 activation and leukocyte recruitment in acute murine gout.

This study investigates the participation of PI3Kγ in the development of joint inflammation and dysfunction in an experimental model of acute gout in mice. Acute gout was induced by injection of monosodium urate (MSU) crystals into the tibiofemoral joint of mice. The involvement of PI3Kγ was evaluated using a selective inhibitor and mice deficient for PI3Kγ (PI3Kγ-/- ) or with loss of kinase activity. Neutrophils recovered from the inflamed joint were quantified and stained for phosphorylated Akt (pAkt) and production of reactive oxygen species (ROS). The adherence of leukocytes to the joint microvasculature was assessed by intravital microscopy and cleaved caspase-1 by Western blot. Injection of MSU crystals induced massive accumulation of neutrophils expressing phosphorylated Akt. In the absence of PI3Kγ, there was reduction of pAkt expression, chemokine production, and neutrophil recruitment. Genetic or pharmacological inhibition of PI3Kγ reduced the adherence of leukocytes to the joint microvasculature, even in joints with established inflammation. Neutrophils from PI3Kγ-/- mice produced less ROS than wild-type neutrophils. There was decreased joint damage and dysfunction in the absence of PI3Kγ. In addition, in the absence of PI3Kγ activity, there was reduction of cleaved caspase-1 and IL-1β production in synovial tissue after injection of MSU crystals and leukotriene B4 . Our studies suggest that PI3Kγ is crucial for MSU crystal-induced acute joint inflammation. It is necessary for regulating caspase-1 activation and for mediating neutrophil migration and activation. Drugs that impair PI3Kγ function may be useful to control acute gout inflammation.

Leptin Promotes Monosodium Urate Crystal-Induced Inflammation in Human and Murine Models of Gout.

Gouty arthritis is an inflammatory disease that is triggered by abnormal uric acid metabolism, which is usually attributed to obesity, a risk factor of hyperuricemia and gout attack. A high level of leptin in plasma is a marker of individuals with obesity. Population studies show that leptin promotes obesity-related arthritis, such as osteoarthritis, but it is unknown whether leptin contributes to gouty arthritis, another form of obesity-related arthritis. Our present study showed that the levels of leptin and leptin receptor in patients with active gouty arthritis were elevated. Leptin facilitates the stimulation of human synoviocytes, mouse peritoneal macrophages, and HL-60 cells induced by monosodium urate, leading to higher levels of acute gout-related proinflammatory factors. Leptin obviously exacerbates the inflammation of monosodium urate-induced acute gouty arthritis in wild-type mice, whereas that in leptin-deficient C57BL6/Job/ob mice is markedly alleviated. The proinflammatory effect of leptin in acute gouty arthritis is partly mediated by mTORC1 signaling pathway. Our study reveals that leptin may serve as a novel prevention and treatment target in acute gouty arthritis.

Suppression of Syk activation by resveratrol inhibits MSU crystal-induced inflammation in human monocytes.

Monosodium urate (MSU) crystals are an endogenous sterile particulate that has been identified as a potent damage-associated molecular pattern (DAMP). In humans, the induction of IL-1β production through MSU-induced NLRP3 inflammasome activation in monocytes/macrophages is responsible for pathogenesis of gouty arthritis. It was recently reported that in a murine model of this disease, resveratrol decreases MSU-induced recurrent attacks of gouty arthritis. Despite its demonstrated anti-inflammatory effects, the mechanisms underlying resveratrol-mediated repression of IL-1β production in MSU-activated monocytes remain poorly understood. Here, we show that resveratrol suppresses secretion of active IL-1β by human primary monocytes stimulated with MSU crystals through suppression of Syk activation. Metabolic labeling and pull-down assays to investigate de novo protein synthesis clearly demonstrated that intracellular pro-IL-1β synthesis is rapidly repressed in monocytes after resveratrol treatment due to decreased phosphorylation of Syk and p38. Resveratrol also inhibited NLRP3 inflammasome activation in MSU-stimulated monocytes by suppressing oligomerization of ASC. Furthermore, resveratrol exerted a beneficial effect by reducing IL-1β production and inhibiting neutrophil recruitment in a mouse model of MSU-mediated peritonitis. Our findings suggest that resveratrol exerts anti-inflammatory effects via post-translational regulation of IL-1β production and, thus, may prove beneficial for the treatment of MSU crystal-mediated sterile inflammation. KEY MESSAGE: Resveratrol has negative effects on pro-IL-1β synthesis through Syk and p38. Resveratrol inhibits oligomerization of ASC. Resveratrol is beneficial in a mouse model of MSU-induced peritonitis.

Anthocyanin composition of fruit extracts from Lycium ruthenicum and their protective effect for gouty arthritis

Lycium ruthenicum Murray (LR) represents an agricultural cash crop found in Northwest China and has been used in traditional folk medicine for a long time. However, detailed qualitative and quantitative analyses of LR anthocyanins, as well as their pharmacological research, remain scarce. In this work, we established a rapid method for the simultaneous identification and quantification of six anthocyanidins and six anthocyanins from LR via UPLC-quadrupole-Orbitrap mass spectrometry (UPLC-Q-Orbitrap MS) analysis. Finally, five anthocyanidins and five anthocyanins were qualitatively and quantitatively analyzed. Among these, 10 constituents (delphinidin-3-glu, cyanidin-3-glu, petunidin-3-glu, peonidin-3-glu, malvidin-3-glu, delphinidin, cyanidin, petunidin, pelargonidin and malvidin) were detected and petunidin-3-glu proved to be the predominant species in LR. Furthermore, the anti-inflammatory effects of anthocyanin extracts and petunidin-3-glu were investigated using a rat model involving gouty arthritis induced by monosodium urate. The levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-18 (IL-18), prostaglandin E2 (PE2), cyclooxygenase-1 (COX-1) enzymes in serum, the paw COX-1 mRNA expression and paw volume could be determined to be significantly increased in rats suffering from gouty arthritis induced by monosodium urate. However, these indicators were found to be significantly reduced after treatment with anthocyanin extracts (200 mg/kg b.wt, p.o.) and petunidin-3-glu (40 mg/kg b.wt, p.o.). Taken in concert, our study shows that anthocyanin extracts and petunidin-3-glu may significantly reduce monosodium urate crystal-induced inflammation. Use and administration of these compounds may be potentially valuable for the further development and clinical applicability of the active compounds in this plant.

XANTHINE OXIDASE INHIBITORY, ANTIHYPERURICEMIC, ANTI-INFLAMMATORY, ANTINOCICEPTIVE ACTIVITY OF α-LIPOIC ACID IN GOUTY ARTHRITIS MODEL

Objective: The current study was carried out to investigate the xanthine oxidase inhibitory, antihyperuricemic, anti-inflammatory, and antinociception activity of α-lipoic acid (LA) in gouty model of rats.Methods: Enzyme assay was done using bovine milk xanthine oxidase (XO). The XO inhibitory activity in vitro was performed using different doses of α-LA, and the degree of XO inhibition was expressed as IC50. The antihyperuricemic activity of α-LA was tested in the potassium oxonate-induced hyperuricemic rats for 7 consecutive days of oral treatment of 10, 25, and 50 mg/kg doses. The anti-inflammatory activity was achieved through monosodium urate crystal-induced inflammation hind paw model in mice. The antinociception effects of α-LA were explored using both of writhings induced by acetic acid test and hot plate test.Results: The study results revealed that the α-LA has a potent activity of XO inhibition with IC50 = 2.93 μg/mL. Furthermore, these results showed that all doses of α-LA were able to significant reduce of each the following: Serum uric acid levels in the hyperuricemic rats, hind paw thickness of mice at all periods of time assessment, and writhings number in the acetic acid-induced writhing test, while, the same doses significantly increased the reaction latency in mice at hot plate test.Conclusion: The α-LA showed a significant effect on the evaluated models, and therefore it may be a promising agent for the treatment of gout since it possesses XO inhibitory, antihyperuricemic, anti-inflammatory, and antinociception activity.

Frontline Science: Reprogramming COX-2, 5-LOX, and CYP4A-mediated arachidonic acid metabolism in macrophages by salidroside alleviates gouty arthritis.

Cyclooxygenase-2 (COX-2), 5-lipoxygenase (5-LOX), and cytochrome P450 (CYP) 4A-mediated arachidonic acid (AA) metabolism play an essential role in human inflammatory disorders. Blocking COX-2 pathway would shunt AA metabolism to the other pathway, thereby decreasing the efficacy and exacerbating adverse effects. Here we demonstrated that reprogramming COX-2, 5-LOX, and CYP4A-mediated AA metabolism in macrophages by salidroside (Sal) ameliorates monosodium urate (MSU) crystal-induced inflammation. Compared with COX-2 inhibitor celecoxib, Sal (80mg/kg) presented a superior anti-arthritic profile in MSU crystal-treated rats, accompanied with the decreased expression of COX-2, 5-LOX, and CYP4A and production of prostaglandin E2 (PGE2 ), leukotriene B4 (LTB4 ), and 20-hydroxyeicosatetraenoic acid (20-HETE) in the synovial fluid macrophages. Sal decreased representative M1 marker (iNOS and CD86, etc.) expression and M1 cytokine (TNF-α and IL-1β) production, whereas it increased M2 marker (CD206 and Arg-1) expression and M2 cytokine (TGF-β and IL-10) production. The injection of conditioned medium from MSU crystal-treated macrophages into the ankle joint of rats reproduced the gouty inflammation, which was attenuated by Sal. Mechanistically, down-regulation of COX-2, 5-LOX, and CYP4A in the RAW264.7 and NR8383 macrophages by Sal skewed macrophage polarization away from the M1 phenotype, and thereby prevented neutrophil migration and chondrocyte degradation with STAT1 and NF-κB inactivation. Conversely, overexpression of COX-2, 5-LOX, CYP4A or STAT1, or exogenous addition of IL-1β or TNF-α partially abolished these effects. Together, inhibition of COX-2, 5-LOX, and CYP4A in macrophages by Sal ameliorates MSU crystal-induced inflammation through decreasing TNF-α and IL-1β production, and may serve as a novel therapeutic strategy.

Monosodium urate crystals reduce osteocyte viability and indirectly promote a shift in osteocyte function towards a proinflammatory and proresorptive state

BackgroundBone erosion is a frequent complication of gout and is strongly associated with tophi, which are lesions comprising inflammatory cells surrounding collections of monosodium urate (MSU) crystals. Osteocytes are important cellular mediators of bone remodeling. The aim of this study was to investigate the direct effects of MSU crystals and indirect effects of MSU crystal-induced inflammation on osteocytes.MethodsFor direct assays, MSU crystals were added to MLO-Y4 osteocyte cell line cultures or primary mouse osteocyte cultures. For indirect assays, the RAW264.7 macrophage cell line was cultured with or without MSU crystals, and conditioned medium from these cultures was added to MLO-Y4 cells. MLO-Y4 cell viability was assessed using alamarBlue® and LIVE/DEAD® assays, and MLO-Y4 cell gene expression and protein expression were assessed by real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Histological analysis was used to examine the relationship between MSU crystals, inflammatory cells, and osteocytes in human joints affected by tophaceous gout.ResultsIn direct assays, MSU crystals reduced MLO-Y4 cell and primary mouse osteocyte viability but did not alter MLO-Y4 cell gene expression. In contrast, conditioned medium from MSU crystal-stimulated RAW264.7 macrophages did not affect MLO-Y4 cell viability but significantly increased MLO-Y4 cell expression of osteocyte-related factors including E11, connexin 43, and RANKL, and inflammatory mediators such as interleukin (IL)-6, IL-11, tumor necrosis factor (TNF)-α and cyclooxygenase-2 (COX-2). Inhibition of COX-2 in MLO-Y4 cells significantly reduced the indirect effects of MSU crystals. In histological analysis, CD68+ macrophages and MSU crystals were identified in close proximity to osteocytes within bone. COX-2 expression was also observed in tophaceous joint samples.ConclusionsMSU crystals directly inhibit osteocyte viability and, through interactions with macrophages, indirectly promote a shift in osteocyte function that favors bone resorption and inflammation. These interactions may contribute to disordered bone remodeling in gout.

High-mobility group box 1 is responsible for monosodium urate crystal-induced inflammation in human U937 macrophages

High-mobility group box 1 (HMGB1) was originally identified as a highly conserved non-histone DNA-binding factor and demonstrated to be a potent mediator in inflammatory diseases. We performed this study to investigate the role of HMGB1 in the pathogenesis of uric acid-induced inflammation in human U937 macrophages. To simulate uric acid-induced inflammation, human U937 macrophages were treated with monosodium urate (MSU) crystals. In addition to determining the effects of MSU crystal treatment on expression of various genes and proteins, cells were transfected with interfering RNA (siRNA) for HMGB1, or caspase-1 and then treated with MSU. Expression of interleukin-1β (IL-1β), IL-18, HMGB1, and caspase-1 was detected in human U937 cells and peripheral blood mononuclear cells (PBMCs) in gout patients and healthy controls by western blot analysis or quantitative real-time polymerase chain reaction. Transcript expression of IL-1β, IL-18, caspase-1, HMGB1 in PBMCs was significantly higher in active gout patients than inactive gout patients and healthy controls. The protein levels of these molecules were significantly increased by stimulation of U937 cells with 0.2 mg/ml MSU crystals. Stimulation of U937 cells with MSU crystals induced translocation of HMGB1 from the nucleus to the cytoplasm and its extracellular release. U937 cells transfected with caspase-1 siRNA had significantly lower HMGB1 expression in the cytoplasm and supernatant than non-transfected cells. Antioxidants, such as N-acetyl-l-cysteine and quercetin, markedly inhibited the nuclear-to-cytoplasmic translocation of HMGB1 and its release into the extracellular milieu. In conclusion, HMGB1, regulated by the enzymatic activity of caspase-1, is a crucial mediator in uric acid-induced inflammation.

The Role of Inhibitory Receptors in Monosodium Urate Crystal-Induced Inflammation.

Inhibitory receptors are key regulators of immune responses. Aberrant inhibitory receptor function can either lead to an exacerbated or defective immune response. Several regulatory mechanisms involved in the inflammatory reaction induced by monosodium urate crystals (MSU) during acute gout have been identified. One of these mechanisms involves inhibitory receptors. The engagement of the inhibitory receptors Clec12A and SIRL-1 has opposing effects on the responses of neutrophils to MSU. We review the general concepts of inhibitory receptor biology and apply them to understand and compare the modulation of MSU-induced inflammation by Clec12A and SIRL-1. We also discuss gaps in our knowledge of the contribution of inhibitory receptors to the pathogenesis of gout and propose future avenues of research.

Inhibition of COX-2/mPGES-1 and 5-LOX in macrophages by leonurine ameliorates monosodium urate crystal-induced inflammation

Cyclooxygenase-2 (COX-2), 5-lipoxygenase (5-LOX) and microsomal prostaglandin E synthase-1 (mPGES-1)-derived eicosanoids play an essential role in human inflammatory disorders. Here, we investigated whether inhibition of COX-2/mPGES-1 and 5-LOX in macrophages by leonurine ameliorates monosodium urate (MSU) crystal-induced inflammation. Virtual screening assay and in vitro enzyme inhibition assay showed that leonurine was a potential inhibitor of COX-2, mPGES-1 and 5-LOX. Compared with COX-2 inhibitor celecoxib, leonurine (30 mg/kg) significantly decreased ankle perimeter, gait score and neutrophil number in synovial fluid in MSU crystal-treated rats, accompanied with the decreased expression of COX-2, mPGES-1 and 5-LOX and production of prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) in the synovial fluid macrophages. In addition, leonurine decreased representative M1 marker (iNOS and CD86) expression, NLRP3 inflammasome activation and M1 cytokine (TNF-α and IL-1β) production. In the in vitro cultured RAW264.7 and human monocyte-derived macrophages (MDMs), blockade of COX-2/mPGES-1 and 5-LOX by leonurine inhibited macrophage M1 polarization and NLRP3 inflammasome activation in response to MSU crystals, and thus down-regulated IL-1β and TNF-α with STAT1 and NF-κB inactivation. Conversely, these effects were partially abolished by overexpression of COX-2, mPGES-1, 5-LOX or STAT1. Furthermore, leonurine prevented a positive feedback loop between COX-2/mPGES-1/5-LOX and IL-1β/TNF-α in MSU crystal-induced inflammation. Together, simultaneous down-regulation of COX-2/mPGES-1 and 5-LOX by leonurine ameliorates MSU crystal-induced inflammation through decreasing IL-1β and TNF-α production. Our study may provide novel multi-target agents toward the arachidonic acid (AA) network for gouty arthritis therapy.

MicroRNA-302b negatively regulates IL-1\u03b2 production in response to MSU crystals by targeting IRAK4 and EphA2

BackgroundInterleukin-1β (IL-1β) is a pivotal proinflammatory cytokine that is strongly associated with the inflammation of gout. However, the underlying mechanism through which the production of IL-1β is regulated has not been fully elucidated. Our previous work identified that miR-302b had an important immune regulatory role in bacterial lung infections. This study was conducted to evaluate the function of miR-302b on monosodium urate (MSU) crystal-induced inflammation and its mechanism.MethodsThe expression pattern and the immune-regulatory role of miR-302b were evaluated both in vitro and in vivo. The functional targets of miR-302b were predicted by bioinformatics, and then validated by genetic approaches. In addition, the clinical feature of miR-302b was analyzed using serum samples of patients with gouty arthritis.ResultsThe extremely high expression of miR-302b was observed in both macrophages and mouse air membranes treated with MSU. Intriguingly, overexpression of miR-302b regulated NF-κB and caspase-1 signaling, leading to significantly attenuate MSU-induced IL-1β. By genetic analysis, miR-302b exhibited inhibitory function on IRAK4 and EphA2 by binding to their 3′-UTR regions. Corporately silencing IRAK4 and EphA2 largely impaired MSU-induced IL-1β protein production. Moreover, it was also found that miR-302b and EphA2 suppressed the migration of macrophages. Finally, it was observed that high expression of miR-302b was a general feature in patients with gouty arthritis.ConclusionsThese results suggest that miR-302b can regulate IL-1β production in MSU-induced inflammation by targeting NF-κB and caspase-1 signaling, and may be a potential therapeutic target for gouty arthritis.

Luteolin-4′-O-glucoside and its aglycone, two major flavones of Gnaphalium affine D. Don, resist hyperuricemia and acute gouty arthritis activity in animal models

BackgroundGnaphalium affine D. Don is a folk medicine of China believed to be efficacious in the treatment of many ailments, including hyperuricemia and gout. PurposeBased on a previous study, we isolated two flavones, luteolin and luteolin-4′-O-glucoside, from G. affine. Our aim was to assess the potential beneficial effects of treatment and mechanisms of these two flavones on hyperuricemia and acute gouty arthritis. MethodsThe model of potassium oxonate (PO)-induced hyperuricemia and monosodium urate (MSU) crystal-induced inflammation in mice has been established. We evaluated serum uric acid (Sur), xanthine oxidase (XO) activity, protein expression of urate transporter 1 (mURAT1) and glucose transporter 9 (mGLUT9) in renal and kidney protection in a hyperuricemia model. In addition, paw swelling and levels of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in serum were assessed in MSU crystal-induced mice. ResultsLuteolin and luteolin-4′-O-glucoside showed a potent clinical effect in treating hyperuricemia and gout. We observed that the two flavones possess potent effect in hyperuricemia mice by decreasing the level of mURAT1 and inhibiting XO activity, which contribute to enhancing uric acid (UA) excretion and improving hyperuricemia-induced renal dysfunction. In addition, luteolin and luteolin-4′-O-glucoside also alleviated paw swelling and inflammation induced by MSU crystals. Further investigation implied that luteolin and luteolin-4′-O-glucoside improved the symptoms of inflammation by decreasing the levels of IL-1β and TNF-α. ConclusionThe present study suggests that luteolin and luteolin-4′-O-glucoside could be developed as therapeutics for treating hyperuricemia and gouty arthritis.

Invariant Natural Killer T Cells Ameliorate Monosodium Urate Crystal-Induced Gouty Inflammation in Mice.

Gout is an inflammatory arthritis caused by deposition of intra-articular monosodium urate (MSU) crystal. Previous studies have focused on resident macrophage, infiltrating monocyte, and neutrophil responses to MSU crystal; yet the mechanisms of cellular changes and the potential involvement of other regulatory immune cells remain largely unknown. Invariant natural killer T (iNKT) cells, an innate type of T cell, are involved in the development of various inflammatory diseases. Here, we investigate the role of iNKT cells in MSU crystal-induced gouty inflammation. MSU crystal-induced inflammatory profiles in an air-pouch model were examined in iNKT-deficient CD1d knockout (KO) and wild-type (WT) control mice. To explore potential mechanisms of iNKT cell regulation of gouty inflammation, we cocultured CD4+ or CD4− iNKT cells with bone marrow-derived macrophages (BMDMs). We found that iNKT cells quickly migrated to the site of inflammation upon MSU crystal stimulation in WT mice. The total number of infiltrating cells in CD1d KO mice, especially neutrophils, was dramatically increased at 6 and 12 h (P < 0.01) post-MSU crystal challenge, compared with WT controls. BMDMs cocultured with CD4+ iNKT cells produced less tumor necrosis factor-α and expressed higher levels of M2 macrophage markers, including Clec7a, Pdcd1Ig2, and interleukin-4 (P < 0.01), compared with BMDMs cocultured with CD4− iNKT cells or conventional CD4+ T cells. CD4+ iNKT cells are one of the key regulators of MSU crystal-induced gouty inflammation through the control of macrophage polarization. iNKT cells may serve as a new therapeutic target for gout.

Efficacy of CoenzymeQ10 in inhibiting monosodium urate crystal-induced inflammation in rats

Gout is a type of arthritis, which could result from the deposition of monosodium urate crystals in joints. It can cause redness, burning pain, inflammation of joints especially in big toe. In this study, we have looked for anti-arthritic effect of coenzyme Q10 (CoQ10) on monosodium urate crystal-induced inflammation in rats and compared it with that of the non-steroidal anti-inflammatory drug, indomethacin. The evaluation was done by measuring the paw volume, antioxidant status, lipid peroxidation, lysosomal enzymes (β-glcuronidase, β-galactosidase, N-acetyl-β-d-glucosaminidase, acid phosphatase) activities and histopathological studies. Paw volume, the levels of lysosomal enzymes, lipid peroxidation were significantly (P<0.05) increased and the antioxidant activity status was in turn decreased in monosodium urate crystal-induced rats. CoQ10 (10mg/kg/b.w. orally) treated monosodium urate crystal-induced rats showed near normal activities of lysosomal enzymes, reduced levels of lipid peroxidation, near normal paw volume and antioxidant status. CoQ10 was also able to minimize mononuclear cell infiltration and damage to articular cartilage. Current study indicates that CoQ10 possesses anti-inflammatory effect against gouty arthritis and can be used to treat acute form of gouty arthritis.

Mast Cell Stabilizer Ketotifen Inhibits Gouty Inflammation in Rats

Gout, an extremely painful arthritis with relapsing inflammatory attacks, is a common inflammatory joint disease in adults. We examined the therapeutic effect of ketotifen, a mast cell stabilizer, on monosodium urate (MSU) crystal-induced acute inflammation. Eight-week-old male Wistar rats were injected with MSU crystals (5 mg per rat) into air pouch. Ketotifen (0, 0.1, 03, and 1 mg/kg) was given 1 hour before MSU crystal injection. Lavage histamine, leukocyte counts, mast cell counts, nitric oxide, and proinflammatory mediator levels were assessed 12 hours after MSU injection. Ketotifen significantly inhibited MSU-induced mast cell activation and histamine concentration in air pouch lavage. Ketotifen dose-dependently inhibited MSU-initiated leukocyte infiltration into the air pouch. Furthermore, ketotifen significantly decreased proinflammatory mediators, including nitric oxide, interleukin-1β, and interleukin-6, production in MSU-treated rats. Ketotifen may attenuate MSU-induced acute inflammation by inhibiting mast cell activation and leukocyte infiltration in rats. Furthermore, ketotifen has the potential to be a new approach in managing patients with gouty inflammation in the future.

Enhanced p62 Is Responsible for Mitochondrial Pathway-Dependent Apoptosis and Interleukin-1β Production at the Early Phase by Monosodium Urate Crystals in Murine Macrophage.

The aim of this study was to clarify the role of p62-dependent mitochondrial apoptosis in the initiation of monosodium urate (MSU) crystal-induced inflammation in macrophages. The induction of mitochondrial apoptosis in RAW 264.7 murine macrophages by MSU crystals was measured using western blotting and quantitative real-time polymerase chain reaction for Bax, caspase-3, caspase-9, or PARP1, and by flow cytometric analysis. Immunoprecipitation and western blotting was applied to detect ubiquitination of p62, TRAF6, and caspase-9. Mitochondrial apoptosis, reactive oxygen species (ROS) generation, and cell proliferation were assessed in cells transfected with p62 small interfering RNA (siRNA). Treatment of RAW 264.7 cells with MSU crystals induced activation of Bax, caspase-3, caspase-9, and PARP1 at the early phase, in addition to enhancing IL-1β expression, but these findings were attenuated at the late phase. MSU crystals induced ubiquitination of p62, followed by ubiquitination of TRAF6 and caspase-9, which were significantly reversed by ascorbic acid. RAW 264.7 cells transfected with p62 siRNA showed attenuated expression of Bax, caspase-3, caspase-9, and PARP1, decreased ROS and IL-1β production, and increased cell proliferation, compared to controls. The antioxidant ascorbic acid inhibited p62, caspase-9, and IL-1β expression increased by MSU crystals. p62 may be a crucial mediator for the mitochondrial apoptosis pathway in MSU crystal-induced inflammation, which is linked to the acute inflammatory response during the early phase of gout.

Morin, a dietary bioflavonol suppresses monosodium urate crystal-induced inflammation in an animal model of acute gouty arthritis with reference to NLRP3 inflammasome, hypo-xanthine phospho-ribosyl transferase, and inflammatory mediators

The anti-inflammatory effect of morin, a dietary bioflavanol was explored on monosodium urate (MSU) crystal-induced inflammation in rats, an experimental model for acute gouty arthritis. Morin treatment (30mg/kg b.wt) significantly attenuated the ankle swelling and the levels of lipid peroxidation, nitric oxide, serum pro-inflammatory cytokines (tumor necrosis factor (TNF) -α, interleukin (IL)-1β, and IL-6), monocyte chemoattractant protein (MCP)-1, vascular endothelial growth factor (VEGF), prostaglandin E2 (PGE2), and articular elastase along with an increased anti-oxidant status (catalase (CAT) and superoxide dismutase (SOD)) in the joint homogenate of MSU crystal-induced rats. Histological assessment revealed that morin limited the diffusion of joint space, synovial hyperplasia, and inflammatory cell infiltrations. The mRNA expression of NLRP3 (nucleotide oligomerization domain (NOD)-like receptor family, pyrin domain containing 3) inflammasome, caspase-1, pro-inflammatory cytokines, MCP-1, inflammatory enzymes (inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2)), and nuclear factor-kappa B (NF-κB) p65 was found downregulated and HPRT (hypo-xanthine phospho-ribosyl transferase) mRNA expression was upregulated in morin treated MSU crystal-induced rats. In addition, morin treatment reduced the protein expression of NF-κB p65, p-NF-κB p65, iNOS, COX-2, and TNF-α. The results clearly demonstrated that morin exert a potent anti-inflammatory effect on MSU crystal-induced inflammation in rats.

Proresolving Actions of Synthetic and Natural Protease Inhibitors Are Mediated by Annexin A1.

Annexin A1 (AnxA1) is a glucocorticoid-regulated protein endowed with anti-inflammatory and proresolving properties. Intact AnxA1 is a 37-kDa protein that may be cleaved in vivo at the N-terminal region by neutrophil proteases including elastase and proteinase-3, generating the 33-kDa isoform that is largely inactive. In this study, we investigated the dynamics of AnxA1 expression and the effects of synthetic (sivelestat [SIV]; Eglin) and natural (secretory leukocyte protease inhibitor [SLPI]; Elafin) protease inhibitors on the resolution of LPS-induced inflammation. During the settings of LPS inflammation AnxA1 cleavage associated closely with the peak of neutrophil and elastase expression and activity. SLPI expression increased during resolving phase of the pleurisy. Therapeutic treatment of LPS-challenge mice with recombinant human SLPI or Elafin accelerated resolution, an effect associated with increased numbers of apoptotic neutrophils in the pleural exudates, inhibition of elastase, and modulation of the survival-controlling proteins NF-κB and Mcl-1. Similar effects were observed with SIV, which dose-dependently inhibited neutrophil elastase and shortened resolution intervals. Mechanistically, SIV-induced resolution was caspase-dependent, associated to increased levels of intact AnxA1 and decreased expression of NF-κB and Mcl-1. The proresolving effect of antiproteases was also observed in a model of monosodium urate crystals-induced inflammation. SIV skewed macrophages toward resolving phenotypes and enhanced efferocytosis of apoptotic neutrophils. A neutralizing antiserum against AnxA1 and a nonselective antagonist of AnxA1 receptor abolished the accelerated resolution promoted by SIV. Collectively, these results show that elastase inhibition not only inhibits inflammation but actually promotes resolution, and this response is mediated by protection of endogenous intact AnxA1 with ensuing augmentation of neutrophil apoptosis.

Targeting inflammatory mediators with ferulic acid, a dietary polyphenol, for the suppression of monosodium urate crystal-induced inflammation in rats

AimsThe aim of this study was to investigate the anti-inflammatory effect of ferulic acid, a dietary phenol, on monosodium urate (MSU) crystal-induced inflammation in rats, an experimental model for acute gouty arthritis. For the purpose of comparison, colchicine was used as a reference drug. Main methodsPaw edema, levels/activities of elastase, lysosomal enzymes (acid phosphatase and β-galactosidase), nitric oxide, lipid peroxidation, antioxidant status and pro-inflammatory cytokines (tumor necrosis factor alpha (TNF-α) and interleukin (IL)-1β), and histology of ankle joints were evaluated in rats with MSU crystal-induced inflammation. The messenger RNA (mRNA) expression of pro-inflammatory cytokines (TNF-α and IL-1β), NLRP3 (nucleotide oligomerization domain (NOD)-like receptor family, pyrin domain containing 3) inflammasomes, caspase-1, and the transcription factor nuclear factor kappa B p65 (NF-κB p65) was determined by real-time polymerase chain reaction (PCR) analysis. The protein expression of NF-κB p65 and TNF-α was detected by immunohistochemical analysis. Further, a molecular docking analysis was conducted to determine the ligand efficiency of ferulic acid towards NF-κB, apoptosis-associated speck-like protein containing a CARD (PYCARD/ASC), NLRP3, and pro-caspase-1. Key findingsIn the joint homogenate of rats with MSU crystal-induced inflammation, treatment with ferulic acid (30mg/kg body weight (b.wt)) decreased paw edema; the level/activity of elastase, lysosomal enzymes, nitric oxide, lipid peroxidation, and pro-inflammatory cytokines (TNF-α and IL-1β); and the mRNA expression of NLRP3 inflammasomes, caspase-1, pro-inflammatory cytokines, and NF-κB p65. In addition, the protein expression of NF-κB p65 and TNF-α was also found to be significantly decreased. However, the antioxidant status (superoxide dismutase (SOD) and catalase (CAT)) were found to be increased. The molecular docking analysis showed that ferulic acid exhibited significant ligand efficiency towards pro-caspase-1, NF-κB, PYCARD/ASC, and NLRP3. SignificanceOur findings demonstrate the potential anti-inflammatory effect of ferulic acid on MSU crystal-induced inflammation in rats.

Effects of Berberine on NLRP3 and IL-1β Expressions in Monocytic THP-1 Cells with Monosodium Urate Crystals-Induced Inflammation.

Background. Urate crystals-induced inflammation is a critical factor during the initiation of gouty arthritis. Berberine is well known for its anti-inflammatory activity. However, the underlying effects of berberine on monosodium urate crystals-induced inflammation remain obscure. Objectives. This study is set to explore the protective effect and mechanism of berberine on monosodium urate crystals-induced inflammation in human monocytic THP-1 cells. Methods. The mRNA levels of NLRP3 and IL-1β were measured by Real-Time PCR, and the protein levels of NLRP3 and IL-1β were determined by ELISA, Western blot, and immunofluorescence. Results. The NLRP3 and IL-1β expressions were significantly increased in model group compared to that in normal group (P < 0.05). Meanwhile, there was significant reduction in the expressions of NLRP3 and IL-1β mRNA in groups 6.25 μM berberine and 25 μM berberine when compared with model group (P < 0.05). Conclusions. Therefore, berberine alleviates monosodium urate crystals-induced inflammation by downregulating NLRP3 and IL-1β expressions. The regulatory effects of berberine may be related to the inactivation of NLRP3 inflammasome.