Fig 1.
Naringenin inhibits KO2-induced overt pain-like behavior.
(a-c) Mice received naringenin (16.7, 50 and 150 mg/kg, po) treatment 1 h before ip injection of 1 mg of KO2 or ipl injection of 30 μg of KO2. The total number of writhings was evaluated 0–20 min after ip injection of KO2. (b) The number of paw flinches and (c) time spent licking the paw were evaluated 0–30 min after ipl injection of KO2. Results are mean ± SEM of 6 mice per group per experiment, and are representative of 2 independent experiments. *p< 0.05 vs. saline group, #p< 0.05 vs. KO2 group. One-way ANOVA followed Tukey’s post hoc.
Fig 2.
Naringenin inhibits KO2-induced mechanical hyperalgesia, thermal hyperalgesia and myeloperoxidase (MPO) activity.
(a-c) Mice received naringenin (50 mg/kg, po) treatment 1 h before ipl injection of 30 μg of KO2. Mechanical (a) and thermal (b) hyperalgesia were evaluated between 0.5–7 h after ipl injection of KO2. (c) At the 7th h after KO2 injection, paw skin samples were collected for MPO activity assay. Results are mean ± SEM of 6 mice per group per experiment, and are representative of 2 independent experiments. *p< 0.05 vs. saline group, #p< 0.05 vs. KO2 group. Repeated measures two-way ANOVA for hyperalgesia data and One-way ANOVA for MPO activity followed Tukey’s post hoc.
Fig 3.
Naringenin inhibits KO2-induced mechanical and thermal hyperalgesia by activating NO/cGMP/PKG/KATP channel signaling pathway.
Mice received (a,b) L-NAME (NOS inhibitor, 90 mg/kg, ip, 1 h), (c,d) ODQ (guanylate cyclase inhibitor, 0,3 mg/kg, ip, 30 min), (e,f) KT5823 (PKG inhibitor, 0,5 μg/mice, ip, 5 min), or (g,h) glibenclamide (KATP channel inhibitor, 0,3 mg/kg, po, 45 min) treatment before administration of naringenin (50 mg/kg, po). (a-h) 1 h after naringenin administration, mice received an ipl injection of 30 μg of KO2. Mechanical (a,c,e,g) and thermal (b,d,f,h) hyperalgesia were evaluated between 0.5–7 h after the ipl injection of KO2. Results are mean ± SEM of 6 mice per group per experiment, and are representative of 2 independent experiments. *p< 0.05 vs. saline group, #p< 0.05 vs. KO2 group. Repeated measures two-way ANOVA followed Tukey’s post hoc.
Fig 4.
Naringenin inhibits KO2-induced oxidative stress and gp91phox mRNA expression.
(a-e) Mice received naringenin (50 mg/kg, po) treatment 1 h before ipl injection of 30 μg of KO2. Paw skin samples were collected 3 h after ipl KO2 injection. Sample analyses were (a) GSH levels, (b) total antioxidant capacity (FRAP assay), (c) lipid peroxidation (TBARS assay), (d) O2− production (NBT assay), and (e) gp91phox mRNA expression by RT-qPCR. β-actin was a reference gene to normalize mRNA expression data. Results are mean ± SEM of 6 mice per group per experiment, and are representative of 2 independent experiments. *p< 0.05 vs. saline group, #p< 0.05 vs. KO2 group. One-way ANOVA followed Tukey’s post hoc.
Fig 5.
Naringenin inhibits KO2-induced cytokine production and mRNA expression.
(a-c) Mice received naringenin (50 mg/kg, po) treatment 1 h before ipl injection of 30 μg of KO2. Paw skin samples were collected 3 h after ipl KO2 injection. Sample analyses were (a) TNFα, and (b) IL-10 production by ELISA, and (c) IL-33 mRNA expression by RT-qPCR. β-actin was a reference gene to normalize mRNA expression data. Results are mean ± SEM of 6 mice per group per experiment, and are representative of 2 independent experiments. *p< 0.05 vs. saline group, #p< 0.05 vs. KO2 group. One-way ANOVA followed Tukey’s post hoc.
Fig 6.
Naringenin inhibits KO2-induced COX-2 mRNA expression.
Mice received naringenin (50 mg/kg, po) treatment 1 h before ipl injection of 30 μg KO2. Paw skin samples were collected 3 h after ipl KO2 injection and analyzed for COX-2 mRNA expression by RT-qPCR. β-actin was a reference gene to normalize mRNA expression data. Results are mean ± SEM of 6 mice per group per experiment, and are representative of 2 independent experiments. *p< 0.05 vs. saline group, #p< 0.05 vs. KO2 group. One-way ANOVA followed Tukey’s post hoc.
Fig 7.
Naringenin inhibits KO2-induced preproET-1 mRNA expression.
Mice received naringenin (50 mg/kg, po) treatment 1 h before ipl injection of 30 μg of KO2. Paw skin samples were collected 3 h after ipl KO2 injection for preproET-1 mRNA expression analysis by RT-qPCR. β-actin was a reference gene to normalize mRNA expression data. Results are mean ± SEM of 6 mice per group per experiment, and are representative of 2 independent experiments. *p< 0.05 vs. saline group, #p< 0.05 vs. KO2 group. One-way ANOVA followed Tukey’s post hoc.
Fig 8.
Naringenin increases Nrf2 and HO-1 mRNA expression.
(a-b) Mice received naringenin (50 mg/kg, po) treatment 1 h before ipl injection of 30 μg KO2. Paw skin samples were collected 3 h after ipl KO2 injection for (a) Nrf2, and (b) HO-1 mRNA expression analysis by RT-qPCR. β-actin was a reference gene to normalize mRNA expression data. Results are mean ± SEM of 6 mice per group per experiment, and are representative of 2 independent experiments. *p< 0.05 vs. saline group, #p< 0.05 vs. KO2 group. One-way ANOVA followed Tukey’s post hoc.