Hesperidin Methylchalcone Suppresses Experimental Gout Arthritis in Mice by Inhibiting NF-κB Activation

Gout arthritis is a painful inflammatory disease induced by monosodium urate (MSU) crystals. We evaluate the therapeutic potential of the flavonoid hesperidin methylchalcone (HMC) in a mouse model of gout arthritis induced by intra-articular injection of MSU (100 μg/10 μL). Orally given HMC (3–30 mg/kg, 100 μL) reduced in a dose-dependent manner the MSU-induced hyperalgesia (44%, <i>p</i> < 0.05), edema (54%, <i>p</i> < 0.05), and leukocyte infiltration (70%, <i>p</i> < 0.05). HMC (30 mg/kg) inhibited MSU-induced infiltration of LysM-eGFP⁺ cells (81%, <i>p</i> < 0.05), synovitis (76%, <i>p</i> < 0.05), and oxidative stress (increased GSH, FRAP, and ABTS by 62, 78, and 73%, respectively; reduced O₂^– and NO by 89 and 48%, <i>p</i> < 0.05) and modulated cytokine production (reduced IL-1β, TNF-α, IL-6, and IL-10 by 35, 72, 37, and 46%, respectively, and increased TGF-β by 90%, <i>p</i> < 0.05). HMC also inhibited MSU-induced NF-κB activation (41%, <i>p</i> < 0.05), gp91^phox (66%, <i>p</i> < 0.05) and NLRP3 inflammasome components mRNA expression in vivo (72, 77, 71, and 73% for NLRP3, ASC, pro-caspase-1, and pro-IL-1 β, respectively, <i>p</i> < 0.05), and induced Nrf2/HO-1 mRNA expression (3.9- and 5.1-fold increase, respectively, <i>p</i> < 0.05). HMC (30, 100, and 300 μM) did not inhibit IL-1β secretion by macrophages primed by LPS and challenged with MSU (450 μg/mL), demonstrating that the anti-inflammatory effect of HMC in gout arthritis depends on inhibiting NF-κB but not on direct inhibition of inflammasome. The pharmacological effects of HMC indicate its therapeutic potential for the treatment of gout

Comparative analysis between different days of <i>L</i>.<i>muelleri</i> treatment on the reduction of inflammatory response in AIA.

<p>The treatments with L. muelleri (100mg/kg) were performed twice a day during 5 or 10 consecutive days. The results are presented as the mean and SEM from 7 mice per group.</p><p>* <i>P</i> <0.05 <i>versus</i> control mice;</p><p># for <i>P</i> < 0.05 <i>versus</i> vehicle-treated arthritic mice.</p><p>Comparative analysis between different days of <i>L</i>.<i>muelleri</i> treatment on the reduction of inflammatory response in AIA.</p

<i>In vivo</i> flow cytometry analyses from popliteal lymph node cells and <i>ex vivo</i> splenocyte antigen stimulation after <i>L</i>. <i>muelleri</i> treatment.

<p>AIA mice were treated as described before in Material and Methods. Twenty-four hours after joint challenge, the popliteal lymph node was collected and cells were isolated for quantifying the total cells numbers (Neubauer chamber) and assaying activated CD4 and DCs populations by cellular staining with labeled antibodies and FACS analysis. Results are expressed as numbers of total (A) or activated CD4⁺CD25⁺ (B), CD11c⁺CD86⁺ (C) cells in each population. In D-F, splenocytes were stimulated <i>ex vivo</i> with RPMI medium, 2 μg.mL⁻¹ of Con-A or 100 μg.mL⁻¹ of <i>L</i>. <i>muelleri</i> and culture supernatants were harvested 48 hours later for IFN-γ, IL-17 and IL-10 measurement by ELISA. Results are expressed as pg.mL⁻¹ of culture supernatant. Bars show the mean and SEM results from 5 mice per group. * <i>P</i> <0.05 versus control mice; # for <i>P</i> < 0.05 versus vehicle-treated arthritic mice.</p

CaCO₃ treatment of AIA mice has no anti-inflammatory and anti-nociceptive effects.

<p>AIA mice were treated as described in Material and Methods. The numbers of neutrophils in the synovial cavity (A), and the relative units of neutrophils in periarticular tissue, as determined by myeloperoxidase assay (B) were assessed 24 hours after injection of 10 μg mBSA or sterile saline (control) in knee joint of immunized mice. Hypernociception is presented as the change (Δ) in withdrawal threshold (in grams) (C). Representative H&E images of control (D), AIA + Vehicle (E), AIA + <i>L</i>. <i>muelleri</i> (F), AIA + CaCO₃ (G) mice. AIA+ vehicle and AIA + CaCO₃ groups (E and G, respectively) presented histopathological evidence of joint inflammation (inflammatory infiltrate [arrows], synovia hyperplasia [arrowheads], alteration of tissue architecture) compared with the other groups (D, F). Scale bars: 100 μm. Other parameters were evaluated as follows: (H), quantification of AIA arthritis index (described in Materials and Methods); and (I), quantification of proteoglycan loss, expressed in %. Results are presented as the mean and SEM results from 5 mice per group. * <i>P</i> <0.05 versus control mice; # for <i>P</i> < 0.05 versus vehicle-treated arthritic mice.</p

Effects of <i>L</i>.<i>muelleri</i> on AIA in mice.

<p>AIA mice were treated with vehicle (CMC 0.5% in filtered water) or with different doses (1, 10 or 100 mg.kg⁻¹) of <i>L</i>. <i>muelleri</i>, orally, twice a day, during 10 days before antigen challenge in knee joint. The number of neutrophils in the synovial cavity (A), and the relative units of neutrophils in periarticular tissue, as determined by myeloperoxidase assay (B) were assessed 24 hours after 10 μg of mBSA or sterile saline (control) in knee joint of immunized mice. The concentrations of the chemokines CXCL1 (C) and CXCL2 (D) in the periarticular tissue were evaluated by ELISA. In E, hypernociception is presented as the change (Δ) in withdrawal threshold (in grams), calculated by subtracting the zero-time mean measurements from the time-interval mean measurements. The results are presented as the mean and SEM from 7 mice per group. * <i>P</i> <0.05 <i>versus</i> control mice; # for <i>P</i> < 0.05 <i>versus</i> vehicle-treated arthritic mice.</p