Inhibition of Inducible Nitric Oxide Synthase Attenuates Monosodium Urate-induced Inflammation in Mice

Abstract

The present study elucidated the effect of the selective inducible nitric oxide synthase (iNOS) inhibitor N(6)-(1-iminoethyl)-L-lysine (L-NIL) on monosodium urate (MSU) crystal-induced inflammation and edema in mice feet. L-NIL (5 or 10 mg/kg/day) was administered intraperitoneally 4 h before injection of MSU (4 mg) into the soles of mice hindlimb feet. Twenty-four hours after MSU injection, foot thickness was increased by 160% and L-NIL pretreatment reduced food pad swelling in a dose dependent manner. Pretreatment of 10 mg/kg/day L-NIL significantly suppressed the foot pad swelling by MSU. Plasma level of nitric oxide (NO) metabolites and gene expression and protein level of iNOS in feet were increased by MSU, which was suppressed by L-NIL pretreatment. Similar pattern of change was observed in nitrotyrosine level. MSU increased the gene expression of tumor necrosis factor (TNF)-α and interleukin (IL)-1β and L-NIL pretreatment suppressed MSU-induced cytokines expression. The mRNA levels of superoxide dismutase and glutathione peroxidase1 were increased by MSU and L-NIL pretreatment normalized the gene expression. Phosphorylation of extracellular signal-regulated kinase 1/2 and p38 was increased by MSU, which was suppressed by L-NIL pretreatment. The mRNA levels of iNOS, TNF-α, and IL-1β were increased by MSU in human dermal fibroblasts, C2C12 myoblasts, and human fetal osteoblasts in vitro, which was attenuated by L-NIL in a dose dependent manner. This study shows that L-NIL inhibits MSU-induced inflammation and edema in mice feet suggesting that iNOS might be involved in MSU-induced inflammation.

Keywords: Gout; Uric acid; iNOS.

Fig. 1

Thickness of feet in mice injected with monosodium-urate (MSU) into hindlimb feet sole. N⁶-(1-iminoethyl)-L-lysine (L-NIL; 5 and 10 mg/kg) was injected intraperitoneally 4 h before MSU (4 mg) injection. Data is presented as mean±SE. Experimental cases are seven in each group. ^*p<0.05 vs. Saline, ^#p<0.05 vs. MSU.

Fig. 2

Nitric oxide metabolites in plasma (A) and the expression of inducible nitric oxide synthase (iNOS; B, C) and nitrotyrosine (D) in the feet of mice injected with monosodium-urate (MSU) into hindlimb sole. N⁶-(1-iminoethyl)-L-lysine (L-NIL; 10 mg/kg) was injected intraperitoneally 4 h before MSU (4 mg) injection. Data is presented as mean±SE. Experimental cases are seven in each group. ^*p<0.05 vs. Saline, ^#p<0.05 vs. MSU.

Fig. 3

The mRNA level of inflammatory cytokines and antioxidant enzymes in the foot of mice injected with monosodium-urate (MSU) into hindlimb sole. N⁶-(1-iminoethyl)-Llysine (L-NIL; 10 mg/kg) was injected intraperitoneally 4 h before MSU (4 mg) injection. Interleukin (IL)-1β (A); tumor necrosis factor (TNF)-α (B); glutathione peroxidase (GPx) 1 (C); superoxide dismutase (SOD) (D). Data is presented as mean±SE. Experimental cases are seven in each group. ^*p<0.05 vs. Saline, ^#p<0.05 vs. MSU.

Fig. 4

The phosphorylation of extracellular signal-related kinase (ERK) 1/2 (A) and p38 (B) in the foot of mice injected with monosodium-urate (MSU) into hindlimb sole. N⁶-(1-iminoethyl)-L-lysine (L-NIL; 10 mg/kg) was injected intraperitoneally 4 h before MSU (4 mg) injection. Data is presented as mean±SE. Experimental cases are seven in each group. ^*p<0.05 vs. Saline, ^#p<0.05 vs. MSU.

Fig. 5

The mRNA level of iNOS, tumor necrosis factor (TNF)-α, and interleukin (IL)-1β in human dermal fibroblast (HDF; A~C), C2C12 myoblast, and (D~F) human fetal osteoblast (hFOB)1.19 (G~I). The cells were treated with 2 mg/ml monosodium-urate (MSU) for 2 h. N⁶-(1-iminoethyl)-L-lysine (L-NIL) was treated into cells 30 min before MSU treatment. Data is presented as mean±SE of three separate experiments. ^*p<0.05 vs. Control, ^#p<0.05 vs. MSU, ^&p<0.05 vs. 0.25 mg/ml L-NIL.