Ameliorative Effects of Scopoletin from Crossostephium chinensis against Inflammation Pain and Its Mechanisms in Mice

Abstract

Scopoletin exists in nature as an anti-oxidant, hepatoprotective, and anti-inflammatory activities reagent. In this study, we have investigated the analgesic effects of the scopoletin using the models of acetic acid-induced writhing response and the formalin test, the anti-inflammatory effects of scopoletin using model of λ-carrageenan (Carr)-induced paw edema. The treatment of ICR mice with scopoletin inhibited the numbers of writhing response and the formalin-induced pain in the late phase. This study demonstrated that the administration of scopoletin resulted in the reduction of Carr-induced mice edema, and it increased the activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) after Carr injection. We also demonstrated scopoletin significantly attenuated the malondialdehyde (MDA) level in the edema paw after Carr injection. Scopoletin decreased the NO, tumor necrosis factor (TNF-α) and prostaglandin E2 (PGE(2)) levels on serum after Carr injection. Scopoletin decreased Carr-induced inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) expressions in the edema paw. These anti-inflammatory mechanisms of scopoletin might be related to the decrease in the level of MDA via increasing the activities of SOD, CAT, and GPx in the edema paw. Also, scopoletin could affect the production of NO, TNF-α, and PGE(2), and therefore affect the anti-inflammatory effects.

Figure 1

Chemical structure of scopoletin (a), analgesic effects of scopoletin, and indomethacin (Indo) on acetic acid-induced writhing response (b), and on the early phase and late phase in formalin test (c) in mice. Each point represents the average value for eight individual animals. Each value represents as mean ± S.E.M. *P < 0.05, **P < 0.01, and ***P < 0.001 as compared with the pathological model group (Con) (one-way ANOVA followed by Scheffe's multiple range test).

Figure 2

Effects of scopoletin and Indo on hind-paw edema induced by Carr in mice (a), the tissue MDA concentration of foot in mice (b), Carr-induced NO (c), TNF-α (d), and PGE₂ (e) concentrations of serum at the 5th hour in mice. Each point represents the average value for eight individual animals. Each value represents as mean ± S.E.M. *P < 0.05, **P < 0.01, and ***P < 0.001 as compared with the Carr group (one-way ANOVA followed by Scheffe's multiple range test).

Figure 3

Inhibition of iNOS and COX-2 protein expression by scopoletin induced by Carr in mice paw edema for 5th hour. Normal control received 0.9% normal saline. Animals treated with scopoletin (1, 5, and 10 mg/kg) and Indo to injection of Carr right hind paws. The right hind paw tissues were taken at the 5 hour. Then the homogenate was centrifuged and tissue suspended were then prepared and subjected to western blotting using an antibody specific for iNOS and COX-2. β-actin was used as an internal control. (a) Representative western blot from two separate experiments was shown. (b) Relative iNOS and COX-2 protein levels were calculated with reference to Carr-injected mouse. Each point represents the average value for three individual animals. ^###compared with sample of control group. The data were presented as mean ± S.E.M. for three different experiments performed in triplicate. **P < 0.01 and ***P < 0.001 were compared with Carr-alone group.

Figure 4

Histological appearance of the mouse hind footpad after a subcutaneous injection with Carr stained with H&E stain at the 5th hour to reveal hemorrhage, edema, and inflammatory cell infiltration in control mice, Carr-treated mice demonstrating hemorrhage with moderately extravascular red blood cells and a large amount of inflammatory leukocyte mainly neutrophils infiltration in the subdermis interstitial tissue of mice, and mice given Indo (10 mg/kg) before Carr. Scopoletin significantly shows morphological alterations (100x) (a) and the numbers of neutrophils in each scope (400x) compared to subcutaneous injection of Carr only (b). Each point represents theaverage valuefor three individual animals. ^### P < 0.001 as compared with the control. ***P < 0.001 compared with Carr group. Scale bar = 200 μm.