Characterization of βN-Octadecanoyl-5-hydroxytryptamide Anti-Inflammatory Effect

Abstract

Background: N-octadecanoyl-5-hydroxytryptamide (C₁₈-5HT) is an amide that can be obtained by the coupling of serotonin and octadecanoic acid. This study aims to characterize the in vivo and in vitro anti-inflammatory activity of C₁₈-5HT.

Methods: A subcutaneous air pouch model (SAP) was used. The exudates were collected from SAP after carrageenan injection to assess cell migration and inflammatory mediators production. RAW 264.7 cells were used for in vitro assays.

Results: C₁₈-5HT significantly inhibited leukocyte migration into the SAP as well as nitric oxide (NO) and cytokines production and protein extravasation. We also observed an reduction in some cytokines and an increase in IL-10 production. Assays conducted with RAW 264.7 cells indicated that C₁₈-5HT inhibited NO and cytokine produced.

Conclusions: Taken together, our data suggest that C₁₈-5HT presents a significant effect in different cell types (leukocytes collected from exudate, mainly polumorphonuclear leukocytes and cell culture macrophages) and is a promising compound for further studies for the development of a new anti-inflammatory drug.

Keywords: C18-5HT; N-octadecanoyl-5-hydroxytryptamide; anti-inflammatory activity; inflammation; serotonin amides.

Figure 1

Effect of C₁₈-5HT on cell viability (A) and NO production (B) in a RAW 264.7 cell line. In A, cells were incubated with vehicle or C₁₈-5HT (0.01–3 µM) and further incubated with lipopoly, (1 μg/mL) for 24 or 48 h. Cell viability was measured with an MTT assay. In B, cells were incubated with vehicle or C₁₈-5HT (0.1–1 µM) and further incubated with LPS (1 μg/mL). After 24 h of incubation, nitrite concentration in the supernatant was measured with Griess reagent. The results are expressed as mean ± SD (n = 3) from three independent experiments. In (A), * p < 0.05 when compared with the vehicle-treated group. In (B), * p < 0.05 when comparing the C₁₈-5HT-treated group with LPS-treated group or # p < 0.05 when comparing the LPS-treated group with the vehicle-treated group.

Figure 2

Effect of C₁₈-5HT on IL-1β, IL-6, TNF-α,and IL-10 produced by LPS-stimulated RAW 264.7. Cells were treated with C₁₈-5HT (0.1, 0.3, or 1 µM) for 30 min and then stimulated with LPS (1 μg/mL) for 24 h. Cytokines were determined by the ELISA method. The results are expressed as mean ± SD (n = 3) from three independent experiments. * p < 0.05 when comparing the C₁₈-5HT-treated group with the LPS-treated group or # p < 0.05 when comparing the LPS-treated group with the vehicle-treated group.

Figure 3

C₁₈-5HT reduced cell migration induced by carrageenan into the subcutaneous air pouch (SAP). The animals were pretreated with vehicle (tween 80, p.o.), C₁₈-5HT (0.1, 1, or 10 mg/kg, p.o.), or dexamethasone (Dex, 2.5 mg/kg, i.p.) 60 min before carrageenan (1%, w/v) injection into SAP. Results are expressed as mean ± S.D. (n = 6–8). The statistical significance was calculated by ANOVA followed Newman’s post-test. # p < 0.05 when comparing the vehicle-treated group that received carrageenan in SAP with the vehicle-treated group that received saline in SAP and * p < 0.05 when comparing the dexamethasone or C₁₈-5HT pretreated groups that received carrageenan in SAP with the vehicle-treated group that received carrageenan in SAP.

Figure 4

C₁₈-5HT reduces protein extravasation and NO production into the subcutaneous air pouch (SAP). The animals were pretreated with vehicle (tween 80, p.o.), C₁₈-5HT (0.1, 1, or 10 mg/kg, p.o.), or dexamethasone (Dex, 2.5 mg/kg, i.p.) 60 min before carrageenan (1%) injection into SAP. Results are expressed as mean ± S.D. (n = 6–8). The statistical significance was calculated by ANOVA followed by Newman’s post-test. # p < 0.05 when comparing the vehicle-treated group that received carrageenan in SAP with the vehicle-treated group that received saline in SAP and * p < 0.05 when comparing the dexamethasone or C₁₈-5HT pretreated groups that received carrageenan in SAP with the vehicle-treated group that received carrageenan in SAP.

Figure 5

C₁₈-5HT reduces cytokine production in the subcutaneous air pouch (SAP). The animals were pretreated with vehicle (tween 80, p.o.), C₁₈-5HT (0.1, 1 or 10 mg/kg, p.o.), or dexamethasone (Dex, 2.5 mg/kg, i.p.) 60 min before carrageenan (1%) injection into SAP. Results are expressed as mean ± S.D. (n = 6–8). The statistical significance was calculated by ANOVA followed Newman’s post-test. # p < 0.05 when comparing the vehicle-treated group that received carrageenan in SAP with the vehicle-treated group that received saline in SAP and * p < 0.05 when comparing the dexamethasone or C₁₈-5HT pretreated groups that received carrageenan in SAP with the vehicle-treated group that received carrageenan in SAP.

Figure 6

C₁₈-5HT presented an antioxidant effect reducing reactive oxygen species (ROS) production. Leukocytes obtained after carrageenan injection into the subcutaneous air pouch were used ex vivo. Cells were activated with PMA and further incubated with vehicle or C₁₈-5HT (0.1, 0.3, or 1 µM). Fluorescence intensity was measured using DCF-DA. Results are expressed as mean ± S.D. of fluorescence intensity. The statistical significance was calculated by ANOVA followed Newman’s post-test. # p < 0.01 when comparing PMA-stimulated leukocytes incubated with vehicle with non-stimulated leukocytes and * p < 0.05 when comparing PMA-stimulated leukocytes incubated with C₁₈-5HT with PMA-stimulated leukocytes.