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

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.


Introduction
Inflammation is a dynamic response to tissue injury and pathogen invasion. Macrophages are the first cells to recognize the potential threat, followed by mast cells, leukocytes, and neutrophils. These cells release inflammatory mediators that amplify the immune response, such as cytokines, nitric oxide (NO), chemokines, vasoactive amines, leukotrienes, and prostaglandins (PGs), and they promote fever, redness, edema, and pain [1]. Cytokines released by cells(e.g., tumor necrosis factor-α (TNF-α) and interleukins (ILs) bind to G protein-coupled receptors (GPCRs) to repair local damage, inducing the expression of selectins and integrins. The chemokines produced by resident cells recruit, through their receptors, circulating neutrophils [2]. These neutrophils transmigrate through the endothelium, reach the inflamed tissue, degranulate, generate reactive oxygen species (ROS), and produce other mediators such as NO and PGs, amplifying the inflammatory response [2].
Fatty acid conjugates with ethanolamine, amino acids, or mono-amine neurotransmitters occur in nature. They are subdivided into N-acyl ethanolamines (NAE) and N-acyl amines (NAA), each with different biological activities [3,4]. The most famous representative of NAA is anandamide (AEA, N-arachidonoyl ethanolamine), which is an endocannabinoid known for its pleiotropic effect varying from homeostasis to immune functions [5]. It acts as an agonist in cannabinoid receptors 1 (CB1) and 2 (CB2), exerting different activities in both the brain and the periphery [5]. Docosahexaenoyl ethanolamine (DHEA), a structural analogue of AEA, exhibits anti-inflammatory properties, modulating the activity of cyclooxygenase-2 (COX-2), which is the main enzyme in the synthesis of pro-inflammatory PGs. In contrast, there is not much information available in the literature on fatty acid conjugates with 5-hydroxytryptamide, which are known as N-acyl serotonins.
The first investigated N-acyl serotonin was arachidonoyl serotonin (AA-5-HT), which is a derivative of the conjugation of arachidonic acid with serotonin. AA-5HT inhibits fatty acid amide hydrolases (FAAH) and is an agonist of type 1 transient potential receptor (TRVP1). It has potential anxiolytic, antiallergic, and analgesic properties [6]. DHA-5HT, a conjugate of docosahexaenoic acid and serotonin, presents potent immunomodulatory properties by attenuating the IL-23-IL-17 signaling in macrophages stimulated with lipopolysaccharides (LPS) [7].
Thus, this study aims to investigate the effect of serotonin derivative C 18 -5HT on in vivo and in vitro models of inflammation.

C 18 -5HT Synthesis
C 18 -5HT was synthesized using an adapted protocol by Reddy and Swamy [10]. The NRM spectra, melting point, and exact mass of the pure product are per the literature [10,11]. Figure 1A shows that concentrations C 18 -5HT ranging from 0.01 to 1 µM did not alter the cell viability after 24 or 48 h of incubation. However, it could be noted that 3 µM of C 18 -5HT significantly reduced cell viability (in 25%). Hence, the maximum concentration of 1 µM was used in the following assays. Next, we investigated the effect of C 18 -5HT on NO production following LPS (1 µg/mL)activation. LPS activation led to a 3.84-fold increase in NO production (13.3 ± 3.2 µM to 50 ± 1.4 µM in vehicle-treated and LPS-activated groups, respectively). When LPS-activated cells were further incubated with C 18 -5HT (0.1, 0.3, or 1 µM), a significant and dose-dependent inhibition of NO production was observed (4%, 20%, and 38%, respectively) ( Figure 1B).  We also evaluated a possible inhibitory effect of C18-5HT in cytokine production by RAW 264.7 cells. All three concentrations of the compound significantly reduced IL-1β Figure 1. Effect of C 18 -5HT on cell viability (A) and NO production (B) in a RAW 264.7 cell line. In A, cells were incubated with vehicle or C 18 -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 C 18 -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 C 18 -5HT-treated group with LPS-treated group or # p < 0.05 when comparing the LPS-treated group with the vehicletreated group.

C 18 -5HT Does Not Affect Cell Viability and Inhibit Nitric Oxide and Cytokines Production
We also evaluated a possible inhibitory effect of C 18 -5HT in cytokine production by RAW 264.7 cells. All three concentrations of the compound significantly reduced IL-1β production in a dose-dependent way.On the other hand, there were lower levels of TNFα and IL-6 and higher levels of IL-10 only after incubation with 1 µM of C 18 -5HT cells ( Figure 2). Figure 2. Effect of C 18 -5HT on IL-1β, IL-6, TNF-α,and IL-10 produced by LPS-stimulated RAW 264.7. Cells were treated with C 18 -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 C 18 -5HTtreated group with the LPS-treated group or # p < 0.05 when comparing the LPS-treated group with the vehicle-treated group.

C 18 -5HT Also Presents an Effect When Orally Administered to Mice
Continuing the studies with C 18 -5HT, we decided to assess whether it had an effect when administered orally to mice. In this model, twenty-four hours after carrageenan injection into the subcutaneous air pouch (SAP), animals from carrageenan-injected group showed a 20.86-fold increase in cell migration (3.5 ± 1.4 × 10 6 cells/mL and 73 ± 10 × 10 6 cells/mL, to saline-and carrageenan-treated groups, respectively). Contrarily, the treatment of mice with dexamethasone (2.5 mg/kg, i.p.) 1 h prior to carrageenan injection into the SAP reduced the cell migration to the cavity by 64.3%. Crescent doses of C 18 -5HT (0.1, 1 or 10 mg/kg, p.o.) also reduced cell migration to the SAP induced by carrageenan by 32.9%, 47.9%, and 49.3%, respectively ( Figure 3). 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 C 18 -5HT pretreated groups that received carrageenan in SAP with the vehicle-treated group that received carrageenan in SAP.
To understand if the effect of C 18 -5HT on cell migration occurred due to an inhibition of the transmigration of cells from the blood to the cavity or because of a decrease in the number of cells produced by the bone marrow, we counted the number of leukocytes in the blood and bone marrow collected from animals treated with C 18 -5HT (10 mg/kg). The data obtained showed that C 18 -5HT did not demonstrate any hematological alteration or myelotoxicity, since there were no alterations in the number of cells in the blood and the bone marrow (data not shown).

Pretreatment of Mice with C 18 -5HT Reduces Protein Extravasation, Nitric Oxide, and Cytokines Production
As expected, carrageenan administration into the SAP led to a 10-and 9.5-fold increase in protein extravasation and NO production, respectively. In the mice pretreated with dexamethasone (2.5 mg/kg, i.p.), the reduction observed was 45.9% and 68% to both parameters. The oral administration of C 18 -5HT 1 h previous to carrageenan injection in the SAP caused a dose-dependent reduction in the amount of protein extravasated to the exudate, with a reduction in 15.6%, 27.7%, and 35.2%, to 0.1, 1, and 10 mg/kg, respectively. The same doses also reduced the levels of NO measured in the fluid (51.2%, 64%, and 75%, respectively) ( Figure 4). 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 C 18 -5HT pretreated groups that received carrageenan in SAP with the vehicle-treated group that received carrageenan in SAP.
We subsequently measured the levels of some cytokines (i.e., TNF-α, IL-1β, IL-10, and IFN-γ) in the exudates collected in SAP after the treatment of mice. Figure 5 shows that carrageenan causes a significant increase in levels of all four cytokines 24 h after its injection in the SAP. The pretreatment of mice with dexamethasone (2.5 mg/kg, i.p.) reduced by more than 50% the levels of TNF-α, IL-1β, and IFN-γ (599.7 ± 8.9 pg/mL in the carrageenan group versus 257.8 ± 21.8 pg/mL, 282.9 ± 62.6 pg/mL, and 325.4 ± 33.1 pg/mL of each cytokine, respectively), and increased production of IL-10 by almost 100% (2525 ± 215.7 pg/mL in the carrageenan group versus 5051 ± 431.4 pg/mL in the dexamethasone-treated group). Although all doses of C 18 -5HT (0.1, 1, or 10 mg/kg, p.o.) significantly reduced TNFα levels (55.98 ± 8.9 pg/mL in the saline-injected group versus 599.7 ± 83.15 pg/mL in the carrageenan-treated group, and 396.1 ± 40.7 pg/mL, 317.1 ± 58 pg/mL, and 271.5 ± 36.15 pg/mL to 0.1, 1, and 10 mg/kg doses, respectively), only the two higher doses inhibited IL-1β production (768.5 ± 149.4 pg/mL in the saline-treated group versus 1584 ± 350.7 pg/mL in the carrageenan-treated group and 1323 ± 192.3 pg/mL, 733.8 ± 69.1 pg/mL, and 622.9 ± 53.18 pg/mL to 0.1, 1, and 10 mg/kg doses, respectively). Regarding the cytokine IFN-γ, only the dose of 10 mg/kg demonstrated a significant effect (62.5 ± 25.5 pg/mL in the saline-treated group versus 1302 ±132.6 pg/mL in the carrageenan-treated group and 1212 ± 185.6 pg/mL, 1273 ± 151.2 pg/mL, and 808 ± 128.2 pg/mL to 0.1, 1, and 10 mg/kg doses, respectively). The 10 mg/kg dose also increased IL-10 production and accumulation in the exudate (905.6 ± 59.2 pg/mL in the saline-treated group versus2525 ± 215.7 pg/mL in the carrageenan-treated group and 4214 ± 453.8 pg/mL in 10 mg/kg) ( Figure 5). 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 C 18 -5HT pretreated groups that received carrageenan in SAP with the vehicletreated group that received carrageenan in SAP.

C 18 -5HT Reduces Reactive Oxygen Species Production by Leukocytes in an Ex Vivo Assay
We also decided to evaluate a possible antioxidant effect of C 18 -5HT using an ex vivo assay with leukocytes collected from the exudate. Figure 6 shows that all concentrations of C 18 -5HT (0.1, 0.3, and 1 µM) almost completely abolished reactive oxygen species (ROS) produced by PMA-activated cells. Values obtained in the C 18 -5HT-treated groups were similar to those obtained in the non-PMA stimulated group. These results corroborate the anti-inflammatory effects observed in the previous results obtained in the carrageenaninduced cell migration and in vitro assays. 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 nonstimulated leukocytes and * p < 0.05 when comparing PMA-stimulated leukocytes incubated with C 18 -5HT with PMA-stimulated leukocytes.

Discussion
Previous studies by our group showed that C 18 -5HT has an antinociceptive effect in peripheral and central nociception models [8]. We also demonstrated that the serotonin amide presented affects the second phase of the formalin-induced licking response, which is a well-established model for the screening of possible antinociceptive and anti-inflammatory compounds. In the second phase of this model, there is a release of inflammatory mediators that act together in nociceptors and local receptors [12][13][14]. The involvement of serotonin and bradykinin in both phases has also been described [15].Thus, we decided to evaluate a possible anti-inflammatory effect of C 18 -5HT.
Acute inflammation is an innate immune system response initiated by a wide range of injuries (physical, chemical, or biological)that involves a cascade of biochemical and cellular events, which include fluid extravasation, enzymatic activation, cell migration, mediator release, receptor sensitization, activation, lysis, and tissue repair [16]. One of these events is the cellular transmigration from the circulation to the injured site. The migration of leukocytes from the circulation to the site of inflammation is mediated by molecular interactions between neutrophils, endothelial cells, and components of the extracellular matrix [17].The neutrophil is the main type of leukocyte involved in the innate mechanisms of defense. Its recognition is critical for the host's response against the invasion of tissues by microorganisms [18]. Neutrophils are attracted to sites of inflammation by various stimuli, such as microorganism products (LPS) and chemotactic factors released by resident cells, such as cytokines, chemokines, and eicosanoids [19][20][21]. In addition, neutrophils also release cytokines and chemokines that promote their self-activation, as well as the recruitment of other cells of the immune system [22].
The carrageenan-induced cell migration model is widely used to screen drugs with potential anti-inflammatory action. The injection of carrageenan into the subcutaneous air pouch induces an inflammatory process with characteristics and development similar to that observed in rheumatoid arthritis, with polymorphonuclear infiltration and the release of pro-inflammatory mediators [23].In the initial phase (2-4 hs) after the carrageenan injection, an intense process of leukocyte rolling and cell adhesion occurs. Six hours after the injection, there is a large influx of neutrophils and the release of chemotactic agents at the inflammatory site [24]. After 24 h cellular influx, exudate formation and the production of inflammatory mediators (i.e., NO, PGs, leukotrienes, cytokines)are augmented [25][26][27][28][29].
Our results showed that C 18 -5HT reduced leukocyte migration into the cavity. To explain this effect, we hypothesize that C 18 -5HT could inhibit the production and/or release of pro-inflammatory substances produced by leukocytes located in the pouch. Thus, we investigated if C 18 -5HT could also reduce the production of some inflammatory mediators, such as NO and the cytokines TNF-α, IL-1β, and INF-γ.
The fact that C 18 -5HT reduced the levels of TNF-α, IL-1β, and INF-γ produced in the exudate could be correlated with a direct reduction in leukocyte migration or an effect in leukocytes, inducing an inhibitory effect in the cells. These results are in accordance with those reported by other studies. Rau and collaborators observed that the immunosuppressant tacrolimus inhibited the infiltration of neutrophils in rats via reduction of the expression of the mRNA for TNF-α and IL-1β in a model of acute pancreatitis induced by taurocholate [30]. We also observed that C 18 -5HT increased the levels of IL-10,which is an immunomodulatory cytokine that, together with the pro-inflammatory cytokines, participates in the expression of adhesion molecules. These molecules are important for the firm adhesion and diapedesis of leukocytes. Additionally, they form a complex network of communication that results in effects that are not determined by the action of a single cytokine but by the interrelationship between several cytokines [31]. Thus, taken together, these data suggest that C 18 -5HT may act on one or more cellular events by inhibiting the production of mediators that stimulate the expression of adhesion molecules (such as pro-inflammatory cytokines) or pro-inflammatory transcription factors such as NF-κB.
These inflammatory mediators can also cause increased vascular permeability and consequent protein extravasation [32,33]. Pretreatment of mice with C 18 -5HT resulted in a corresponding reduction in protein extravasation, thus confirming and suggesting the correlation between the inflammatory mediators and plasma and protein leakage. This effect highlights the action of this compound on inflammatory mediators involved in increasing vascular permeability, such as cytokines and NO.
The effects of NO along the inflammatory response are complex and involve a series of events. Our results showed that oral pretreatment with C 18 -5HT significantly inhibited NO production.This decrease may be a consequence of reduced cell transmigration to the cavity and/or inhibition of the effects of cytokines, eicosanoids, kinins, and glutamate or even alterations in the expression and/or activity of inducible nitric oxide synthase (NOS) enzymes [34,35]. Our results support the first hypothesis, since the reduction in NO production was proportional to the reduction in the number of leukocytes that migrate to the cavity.Farsky et al. observed that systemic pretreatment with L-nitroarginine methyl ester (L-NAME), a NOS inhibitor, inhibits the influx of leukocytes into the air pouch cavity in mice injected with Bothrops jararaca venom [36]. Tunon et al. demonstrated that tacrolimus inhibits the expression of iNOS mRNA in hepatocytes from rats incubated with LPS [37]. Finally, Sakaguchi et al. (2006) observed that intraperitoneal administration of L-NAME in rats reduced exudate formation and albumin levels in carrageenan-induced pleurisy [38]. These studies corroborate our results, as they highlight the influence of NO production on neutrophil infiltration.
Higher oxidative stress imbalance activates redox-sensitive factors, such as NF-κB, induces inflammatory cytokines, and promotes inflammation [39][40][41].Thus, inflammatory cytokines activate sequential events, increase oxidative stress, and contribute to the progression of the disease by the intensification of the inflammatory response [39].TNF-α synthesis is inhibited by IL-10, which is an immunomodulatory cytokine that reduces ROS formation and NO production. TNF-α also increases the levels of ROS, which is prevented by the action of IL-10 [39]. C 18 -5HT reduced ROS formation in PMA-stimulated leukocytes. It also increased IL-10 and reduced TNF-α and NO levels in the collected exudate. Together, these results suggest that C 18 -5HT has antioxidant and anti-inflammatory effects.
Macrophages are phagocytic cells of the innate immune system that are responsible for recognizing an infection, presenting antigens, and also for the production of inflammatory mediators, the removal of apoptotic cells, and the output of growth factors. The subtype M1 is activated by pro-inflammatory cytokines or a microorganism's molecules (such as LPS), and it has killing capacity by the production of ROS, NO, and inflammatory cytokines [42].
Non-steroidal anti-inflammatory drugs and some anesthetics inhibit the production of pro-inflammatory cytokines and chemokines, including TNF-α, IL-1β, IL-6, and IL-8,as well as NO synthesis [43]. Some drugs also modulate the production of immunomodulatory cytokines, such as IL-10, which is a cytokine capable of modulating negatively and indirectly the synthesis of these inflammatory mediators and surface molecules [44].
As C 18 -5HT showed an in vivo anti-inflammatory effect, we decided to investigate whether it would also have some in vitroactivity, using the LPS-stimulated RAW 264.7 cell line to evaluate the immunomodulatory effect of C 18 -5HT. The data obtained showed that C 18 -5HT did not present a cytotoxic effect. We also demonstrated that the serotonin amide did reduce NO production by LPS-activated RAW 264.7 cells.Our results show that the anti-inflammatory effect of C 18 -5HT may be related to the inhibition of the synthesis of TNF-α and IL-1β by LPS-stimulated macrophages. It also positively modulated the release of IL-10. These results suggest that the anti-inflammatory effect of C 18 -5HT may be IL-10 dependent, and it probably has a direct effect on the transcription and/or translation of these inflammatory mediators.

C 18 -5HT Synthesis
C 18 -5HT was synthesized using a condensation reaction between stearic acid and serotonin, which was adapted from Reddy and Swamy [10]. Stearic acid (1.1 eq), DMAP (1.0 eq), serotonin hydrochloride (1.0 eq.), and EDC.HCl (1.0 eq.) were dissolved in DMF. The mixture was stirred at room temperature for 24 h and monitored via thin-layer chromatography (TLC). The crude product was purified on a silica gel column by using increasing concentrations of ethyl acetate in dichloromethane (DCM). C 18 -5HT was characterized by 1H-and 13C-NMR spectroscopy, melting point determination, and high-resolution mass spectrometry. NMR spectra were obtained using a Varian VNMRS 500 MHz spectrometer. The product exact mass was analyzed in a Dionex UltiMate 3000 liquid chromatography coupled to a hybrid Quadrupole-Orbitrap high-resolution mass spectrometer (Thermo Q-Exactive Plus, Thermo Fisher Scientific, Waltham, MA, USA) equipped with an electrospray ionization (ESI) source. A Syncronis C-18 column (50 mm × 2.1 mm × 1.7 µm) was used with gradient elution mode with water (mobile phase A) and methanol (mobile phase B) both with 0.1% formic acid and 5 mM ammonium formate as follows: 0-4 min 5-30% B

Drugs and C 18 -5HT Administration
C 18 -5HT was dissolved in dimethylsulfoxide (DMSO) to prepare a 100 mg/mL stock solution. On the day of the experiments, this solution was diluted in tween 80 and administered by oral gavage to the animals at the doses of 0.1, 1, or 10 mg/kg in a final volume of 0.1 mL. The vehicle group was composed of tween 80 (negative control group) and the positive control group was composed of dexamethasone (2.5 mg/kg, i.p.). All drugs were diluted immediately before use.

Cell Culture
RAW 264.7 murine macrophages (ATCC TIB-71) were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and glutamine (2 mM). Cells were maintained in a humidified atmosphere containing 5% CO 2 at 37 • C, and the medium was changed every 2 or 3 days. Cells between the 4 th and 6 th passages were used for different pellet preparations.

Cell Viability
The cell viability was evaluated by MTT (3-(4,5-dimethyl-thiazol-2-yl)-2,5diphenyltetrazolium bromide) assay. The mitochondrial-dependent reduction of MTT to formazan was used to measure cell respiration as an indicator of cell viability [47]. When the cells formed a confluent monolayer, they were scrapped, centrifuged, plated in a 96-well plate at a density of 2.5 × 10 4 cells/mL, and incubated with different concentrations of C 18 -5HT. After 24 and 48 h, 20 µL of MTT (5 µg/mL) was added to each well, and the plates were maintained at 37 • C for 4 h. After the incubation period, the supernatant was discarded, and DMSO (100 µL) was added to dissolve MTT formazan crystals. The absorbance was read in a FlexStation reader at 570 nm (Molecular Devices, San Jose, CA, USA).

Animals
The male Swiss Webster mice (20-25 g, 8-10 weeks) used in all experiments were donated by the Instituto Vital Brazil (Niterói, Rio de Janeiro, Brazil). The animals were kept in a controlled temperature environment (22 ± 2 • C, 60% to 80% humidity) and light-dark cycles of 12 h. Food and water were provided ad libitum, but the feed was withdrawn 1 h before oral administration of substances. The protocol for this study was approved by the National Council for the Control of Animal Experimentation (CONCEA), Biomedical Science Institute/UFRJ, and Ethical Committee for Animal Research, under the number 35/19.

Carrageenan-Induced Cellular Migration into the Subcutaneous Air Pouch (SAP)
Carrageenan-induced cell migration was performed according to Raymundo et al. [48]. The mice's backs were injected subcutaneously with 10 mL of sterile air. Three days later, a new injection with 8 mL of sterile air was made to guarantee the maintenance of the cavity in the animal's back. On the sixth day, the animals were treated orally with the vehicle, C 18 -5HT (0.1, 1,or10 mg/kg) or intraperitoneally with dexamethasone (2.5 mg/kg) 60 min before receiving 1 mL of a carrageenan solution (1%). After 24 h of carrageenan injection, the animals were euthanized with chloral hydrate (1%, i.p.), and the cavity was flushed out with 1 mL of PBS.Exudates were collected and centrifuged at 1200 rpm for 10 min at 4 • C, and samples were stored at −20 • C until the quantifications. The total leucocytes cells counts were determined in the exudates using a pocH-100iV Diff (Sysmex) hematology analyzer.

Nitrate, Nitrite, Protein, and Cytokines Measurement
To evaluate the nitrate accumulated in SAP, exudates were analyzed according to the method described by Bartholomew [49] and adapted by Raymundo et al. [48], which was followed by the measurement of nitrite according to the Griess reaction [50].
Protein quantification was performed by the BCA method using the protein dosage kit BCA TM (Thermo Fisher Scientific Inc., Whaltman, MA, USA). At the time of the dosage, reagents A (sodium carbonate, sodium bicarbonate, bicinchoninic acid, and sodium tartrate in 0.1 M of sodium hydroxide and B (4% copper sulfate) were mixed in a ratio of 5:1. A volume of 5 µL of the sample was incubated with 195 µL of the BCA reagent for 30 min at 37 • C, and the absorbance was measured in a microplate reader at 562 nm.
Supernatants from the exudates collected from the SAP were used to measure the levels of the cytokines by enzyme-linked immunosorbent assay (ELISA) using the protocol supplied by the manufacturer (B&D, Franklin Lakes, NJ, USA).

Statistical Analysis
Results are expressed as the mean ± standard deviation (S.D.). Statistical significance was calculated by analysis of variance (ANOVA) followed by Newman's post-test. p values less than 0.05 (* p < 0.05) were considered significant.
In vivo, experimental groups were composed of 6-8 animals. For in vitro assays, each protocol was repeated at least 3 times, and each concentration tested was done in triplicate (n = 3).

Conclusions
We showed that C 18 -5HT has potent in vivo and in vitro anti-inflammatory effects, suggesting that this compound may be a prototype for the development of a new compound for further tests.

Institutional Review Board Statement:
This study was conducted in accordance with the Guidelines on Ethical Standards for Investigation of Experimental Pain in Animals (Zimmermann, 1983) and followed the principles and guidelines adopted by the National Council for the Control of Animal Experimentation (CONCEA), approved by the Ethical Committee for Animal Research (no. 35/19).

Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.