**3. Discussion**

The present study demonstrated the anti-inflammatory effects of NTK, an aromatic sesquiterpenoid widely found in the plant kingdom. A screening evaluating the effect of different doses of this compound on paw edema induced by carrageenan or dextran, demonstrated significant antiedematogenic e ffects at 10, 100, or 300 mg/kg in mice. Therefore, we chose the lower dose for the following analysis. Carrageenan and dextran are phlogistic agents that act by triggering an inflammatory reaction characterized by the release of chemical mediators, such as prostaglandins (e.g., PGE2) and vasoactive amines (e.g., histamine), which increase the vascular permeability and cause the biochemical, cellular, and vascular changes observed in acute inflammation [18].

Considering the involvement of vasoactive amines and lipid mediators on edema induction, we investigated the e ffects of NTK pre-treatment on edema triggered by histamine and AA challenge. It was shown that the compound partially inhibited paw edema formation triggered by histamine administration and promethazine, a histamine receptor antagonist, caused comparable inhibition, suggesting that NTK-mediated antiedematogenic e ffects may involve, at least partially, inhibition of histamine vascular actions. NTK Pre-treatment significantly inhibited edema formation in AA-stimulated mice. Accordingly, indomethacin, an NSAID used as the positive control, caused comparable inhibition at all evaluated time-points, suggesting that the e ffects of NTK in acute inflammation might involve arachidonic acid metabolism inhibition, possibly through interference with the enzymatic activity of cyclooxygenase (COX), lipoxygenase (LO), or phospholipase A2 (PLA2), or even by antagonizing the e ffects of bioactive eicosanoids on tissue receptors. Nevertheless, the similarity in magnitude between the e ffect of NTK and indomethacin, suggests a common action on the COX pathway [19,20]. Our findings are corroborated by previous research demonstrating that NTK concentration-dependently inhibited platelet activation induced by AA in vitro. Since prostaglandins significantly mediate AA-induced platelet activation, we hypothesized that NTK could be acting through inhibition of COX activation in platelets [14].

To investigate the involvement of COX-2 and H1 receptor inhibition on NTZ-mediated anti-inflammatory e ffects, we performed in silico analysis by molecular docking. The COX-2 binding site shows two important regions of interaction with inhibitors: the hydrophobic pocket formed by Tyr324, Trp356, Phe487, Phe350, Ala496, Tyr317, and Leu321, and the hydrogen bond pocket formed by Ser-499 and Tyr-354. The Alkyl and Alkyl-pi stacking interactions work as "anchors", favoring the van der Waals interactions and contributing to the formation of a stable bond in the COX-2/NTK complex. Our results demonstrated a complementarity between the ligands and the active site of COX-2. The relative contribution of van der Waals interactions has demonstrated to be relevant for both NTK and diclofenac. In addition, 15 similar interaction points with hydrophobic and hydrophilic cavities were found between NTK and diclofenac. However, the control drug had relatively better docking energy, in part, generated by hydrogen bonds. Previous studies with other terpenes derivatives have demonstrated that these compounds interacted similarly, although no involvement of hydrogen bonds was shown [21,22]. In the H1 receptor binding site, NTK showed potential Van der Waals interactions with Ser84, Thr 85, Phe 378, Trp 131, Asn 164 (Figure 7). However, other interactions such as alkyl, π-sigma, and π-alkyl, contribute to the stabilization of the interaction, as well as to the formation of the lipophilic pocket in the binding site. According to Keser ˝u, G. M. et al. (2004), taking part in the lipophilic pocket formation is crucial for the antagonist activity in the binding cavity. The interactive map shows nine similar stabilization points between the NTK/H1 and the doxepin/H1 complex at the binding site. Therefore, NTK shows favorable docking with the COX-2 enzyme and the H1 receptor. These amino acid similarities support the hypothesis that NTK occupies the active site in both targets, corroborating our experimental data using in vivo models.

Carrageenan-induced pleurisy and peritonitis are well-established models used for evaluation of the systemic anti-inflammatory properties of natural and synthetic drugs. A single oral pre-treatment with NTK (10 mg/kg) caused significant inhibition of leukocyte recruitment associated with decreased concentrations of albumin, MPO, IL1-β, and TNFα, demonstrating that the compound inhibited hallmark parameters of acute systemic inflammation. The decreased albumin levels found in NTK-treated animals indicates an inhibitory action by this compound on carrageenan-mediated increased vascular permeability. Accordingly, the lower concentrations of IL-1β and TNFα found

in the NTK group justify, at least partially, the inhibitory effects of this compound on all acute inflammation parameters demonstrated by this study, since these cytokines were shown to exert multiple inflammatory actions, leading to increased vascular permeability and leukocyte recruitment and activation [22]. In this context, in addition to presenting a reduced number of leukocytes in both pleural and peritoneal lavages, the NTK-treated mice showed significantly reduced MPO levels, indicating inhibition of leukocyte activity.

In order to evaluate the effects of NTK against a chronic stimulus, we used the cotton pellet-induced granuloma model in mice. NTK treatment for 10 consecutive days showed positive effects, reducing both granuloma weight and total proteins in the homogenates. These findings sugges<sup>t</sup> a possible inhibitory action of the treatment on macrophage migration and activation as well as of the proliferative response, corroborating the effects of NTK on cytokine production and leukocyte activity. Importantly, because NTK was found to reduce several inflammatory parameters induced by different noxious stimuli in different mice models of acute systemic inflammation, we sugges<sup>t</sup> that this sesquiterpene may be used for further research aimed at the development of new anti-inflammatory drugs.

The results obtained in the present research are corroborated by the findings of previous studies. Choi et al. [20] demonstrated in vitro that NTK suppressed the expression of chemokines such as thymus and activation-regulated chemokine (TARC/CCL17) and macrophage-derived chemokine (MDC/CCL22) in HaCaT cells, by inhibiting the mitogen-activated protein kinases (MAP kinases) PKC and p38. Since these kinases participate in signaling pathways involved in the activation of the transcriptional nuclear factor-kappaB (NF-κB) transcriptional factor, these findings sugges<sup>t</sup> that NKT could modulate the inflammatory response at multiple levels, since NF-κB stimulates the transcription of numerous genes from cytokines, chemokines and adhesion molecules, supporting the findings of the present study. According to Qi and collaborators [23], the NKT treatment reduced the levels of TNF-<sup>α</sup>, IL-1β, and IL-6 in models of cognitive impairment and dementia in rats, corroborating the data from our pleurisy model. Moreover, NKT significantly reduced the activities of inflammatory enzymes such as superoxide dismutase (SOD), glutathione S-transferase (GST), cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) and levels of glutathione (GSH), in addition to decreasing the total antioxidant capacity (T-AOC), as well as concentrations of malondialdehyde (MDA) and nitric oxide (NO) through inhibition of the Toll-like receptor <sup>4</sup>/NF-κB/domains-containing-protein-3-inflammasome (TLR4/NF-κB/NLRP3) pathway [23]. NTK (90 mg Kg) was also shown to reduce the anti-inflammatory effects of cigarette vapor [24], as well as inhibit lung inflammation parameters via NF-κB inhibition [19]. Nevertheless, the mechanisms underlying the effects of NTK as an anti-inflammatory compound remain to be fully characterized. Therefore, we will carry out in vitro studies to analyze the impact of NTK on COX-1 and COX-2 expression and activity, which will be correlated with the levels of PGE2 in the supernatants of macrophage cultures. Additionally, we will use mast cell cultures will to analyze the effects of NTK on histamine release in vitro, which will be correlated with the activity of this compound in combination with agonists and antagonists of the H1 receptor in vivo.

Finally, Kurdi and colleagues demonstrated that NTK (10 mg/kg) treatment exerted hepatoprotective and antifibrotic effects by suppressing the carbon tetrachloride (CCL4)-induced lesion, which is characterized by expression of pro-inflammatory cytokines, such as TNF-<sup>α</sup>, monocyte chemotactic protein (MCP)-1 and IL-1β in liver tissues. In the same study, histopathological findings revealed that NTK reduced fibrosis, steatosis, hepatocyte necrosis and leukocyte infiltration [25], providing evidence that NTK could be useful in the managemen<sup>t</sup> of chronic inflammation, as indicated by our findings in the cotton pellet-induced granuloma. However, further research is required to characterize better the anti-inflammatory effects of NTK, as well as its mechanism of action on chronic inflammation. To this end, we will carry out experiments to characterize the cell populations, as well as the expression of chronic inflammation markers in the granuloma model.

In conclusion, NTK presented anti-inflammatory effects in mice models of acute and chronic inflammation. In acute inflammation, the antiedematogenic effects, as well as leukocyte recruitment inhibition, may be associated with decreased vascular permeability and inhibition of MPO, IL1-β, and TNF-α production, possibly due to inhibition COX-2 activity and antagonism of the H1 receptor. These mechanisms might have contributed to the anti-inflammatory effects displayed by NTK on the granuloma model. However, further studies are required to characterize the effects of this compound on chronic inflammation.
