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 (80 mg/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.

Liu Y ，Tang H ，Liu X ，Chen H ，Feng N ，Zhang J ，Wang C ，Qiu M ，Yang J ，Zhou X ... -  《-》

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.

Liu Y ，Duan C ，Chen H ，Wang C ，Liu X ，Qiu M ，Tang H ，Zhang F ，Zhou X ，Yang J ... -  《-》

Total Saponin Fraction of Dioscorea Nipponica Makino Improves Gouty Arthritis Symptoms in Rats via M1/M2 Polarization of Monocytes and Macrophages Mediated by Arachidonic Acid Signaling.

To explore the mechanism of effects of total saponin fraction from Dioscorea Nipponica Makino (TSDN) on M1/M2 polarization of monocytes/macrophages and arachidonic acid (AA) pathway in rats with gouty arthritis (GA). Seventy-two Sprague Dawley rats were randomly divided into 4 groups (n=18 in each): normal, model, TSDN at 160 mg/kg, and celecoxib at 43.3 mg/kg. Monosodium urate crystal (MSU) was injected into the rats' ankle joints to induce an experimental GA model. Blood and tissue samples were collected on the 3rd, 5th, and 8th days of drug administration. Histopathological changes in the synovium of joints were observed via hematoxylin and eosin (HE) staining. The expression levels of arachidonic acid (AA) signaling pathway were assessed via real-time polymerase chain reaction (qPCR) and Western blot. Flow cytometry was used to determine the proportion of M1 and M2 macrophages in the peripheral blood. An enzyme-linked immunosorbent assay (ELISA) was used to detect interleukine (IL)-1 β, tumor necrosis factor-alpha (TNF-α), IL-4, IL-10, prostaglandin E2 (PGE2), and leukotriene B4 (LTB4). HE staining showed that TSDN improved the synovial tissue. qPCR and Western blot showed that on the 3rd, 5th and 8th days of drug administration, TSDN reduced the mRNA and protein expressions of cyclooxygenase (COX)2, microsomal prostaglandin E synthase-1 derived eicosanoids (mPGES-1), 5-lipoxygenase (5-LOX), recombinant human mothers against decapentaplegic homolog 3 (Smad3), nucleotide-binding oligomerization domain-like receptor protein 3 (NALP3), and inducible nitric oxide synthase (iNOS) in rats' ankle synovial tissues (P<0.01). TSDN decreased COX1 mRNA and protein expression on 3rd and 5th day of drug administration and raised it on the 8th day (both P<0.01). It lowered CD68 protein expression on days 3 (P<0.01), as well as mRNA and protein expression on days 5 and 8 (P<0.01). On the 3rd, 5th, and 8th days of drug administration, TSDN elevated the mRNA and protein expression of Arg1 and CD163 (P<0.01). Flow cytometry results showed that TSDN decreased the percentage of M1 macrophages while increasing the percentage of M2 in peripheral blood (P<0.05 or P<0.01). ELISA results showed that on the 3rd, 5th, and 8th days of drug administration, TSDN decreased serum levels of IL-1 β, TNF-α, and LTB4 (P<0.01), as well as PGE2 levels on days 3rd and 8th days (P<0.05 or P<0.01); on day 8 of administration, TSDN increased IL-4 serum levels and enhanced IL-10 contents on days 5 and 8 (P<0.05 or P<0.01). The anti-inflammatory effect of TSDN on rats with GA may be achieved by influencing M1/M2 polarization through AA signaling pathway.

Zhou Q ，Sun HJ ，Zhang XW 《-》

Curcumin ameliorates monosodium urate-induced gouty arthritis through Nod-like receptor 3 inflammasome mediation via inhibiting nuclear factor-kappa B signaling.

Monosodium urate (MSU) crystals-induced inflammation is a key initiator in gouty arthritis. Curcumin is an active ingredient possessing anti-inflammatory efficacy. But the underlying mechanism is not fully understood and its effect on gouty arthritis remains elusive. We evaluated the effects of curcumin on cell viability in primary rat abdominal macrophages with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). Then supernatants of MSU crystals-stimulated cells were collected and subjected to enzyme-linked immunosorbent assay for checking the modulation of curcumin on interleukin (IL)-1β and tumor necrosis factor (TNF)-α. Meanwhile, cells were analyzed by using Western blot analysis and quantitative polymerase chain reaction (QPCR) to investigate the effects of curcumin on Nod-like receptor 3 (NLRP3) inflammasome/nuclear factor-kappa B (NF-κB) signaling. We also investigated the in vivo efficacy of curcumin with MSU-induced gouty arthritis rat models. Curcumin could reduce MSU crystals-induced IL-1β and TNF-α in vitro. Western blot analysis and QPCR results revealed that curcumin regulated the production of these cytokines by suppressing the expression of inflammasome key components, including NLRP3, caspase-1. Further studies showed that the suppressive efficacy of curcumin on inflammasome was mediated by inhibiting MSU-induced NF-κB signaling activation. Intraperitoneal administration of curcumin could ameliorate symptoms of MSU-induced gouty arthritis, including the joint circumference, infiltration of neutrophils in knee joints, and production of IL-1β, TNF-α, and elastase. Western blot analysis revealed that the levels of NLRP3, procaspase-1, caspase-1, pro-IL-1β, and IL-1β were downregulated by curcumin in vivo. These results indicated that curcumin could effectively ameliorate MSU crystal-induced gouty arthritis through NLRP3 inflammasome mediation via inhibiting NF-κB signaling both in vitro and in vivo, suggesting a promising active ingredient for the prevention and treatment of gouty arthritis.

Li X ，Xu DQ ，Sun DY ，Zhang T ，He X ，Xiao DM ... -  《-》

Effects of Total Saponins from Dioscorea Nipponica Makino on Monosodium Urate-Induced M1-Polarized Macrophages through Arachidonic Acid Signaling Pathway: An in vitro Study.

To investigate and reveal the underlying mechanism of the effect of total saponins from Dioscoreae nipponica Makino (TSDN) on the arachidonic acid pathway in monosodium urate (MSU) crystal-induced M1-polarized macrophages. M1 polarization of RAW264.7 cells were induced by 1 µ g/mL lipopolysaccharide (LPS). The methylthiazolyldiphenyl-tetrazolium bromide method was then used to screen the concentration of TSDN. MSU (500 µ g/mL) was used to induce the gouty arthritis model. Afterwards, 10 µ g/L TSDN and 8 µ mol/L celecoxib, which was used as a positive control, were added to the above LPS and MSU-induced cells for 24 h. The mRNA and protein expressions of cyclooxygenase (COX) 2, 5-lipoxygenase (5-LOX), microsomal prostaglandin E synthase derived eicosanoids (mPGES)-1, leukotriene B (LTB)4, cytochrome P450 (CYP) 4A, and prostaglandin E2 (PGE2) were tested by real-time polymerase chain reaction and Western blotting, respectively. The enzyme-linked immunosorbent assay was used to test the contents of M1 markers, including inducible nitric oxid synthase (NOS) 2, CD80, and CD86. TSDN inhibited the proliferation of M1 macrophages and decreased both the mRNA and protein expressions of COX2, 5-LOX, CYP4A, LTB4, and PGE2 (P<0.01) while increased the mRNA and protein expression of mPGES-1 (P<0.05 or P<0.01). TSDN could also significantly decrease the contents of NOS2, CD80, and CD86 (P<0.01). TSDN has an anti-inflammation effect on gouty arthritis in an in vitro model by regulating arachidonic acid signaling pathway.

Zhou Q ，Sun HJ ，Liu SM 《-》