Anti-Neuroinflammatory Potential of a Nectandra angustifolia (Laurel Amarillo) Ethanolic Extract

Microglia, the resident macrophage-like population in the CNS, plays an important role in the pathogenesis of many neurodegenerative disorders. Nectandra genus is known to produce different metabolites with anti-inflammatory, anti-oxidant and analgesic properties. Although the species Nectandra angustifolia is popularly used for the treatment of different types of inflammatory processes, its biological effects on neuroinflammation have not yet been addressed. In this study, we have investigated the role of a Nectandra angustifolia ethanolic extract (NaE) in lipopolysaccharide (LPS)-induced neuroinflammation in vitro and in vivo. In LPS-activated BV2 microglial cells, NaE significantly reduced the induced proinflammatory mediators TNF-α, IL-1β, IL-6, COX-2 and iNOS, as well as NO accumulation, while it promoted IL-10 secretion and YM-1 expression. Likewise, reduced CD14 expression levels were detected in microglial cells in the NaE+LPS group. NaE also attenuated LPS-induced ROS and lipid peroxidation build-up in BV2 cells. Mechanistically, NaE prevented NF-κB and MAPKs phosphorylation, as well as NLRP3 upregulation when added before LPS stimulation, although it did not affect the level of some proteins related to antioxidant defense such as Keap-1 and HO-1. Additionally, we observed that NaE modulated some activated microglia functions, decreasing cell migration, without affecting their phagocytic capabilities. In LPS-injected mice, NaE pre-treatment markedly suppressed the up-regulated TNF-α, IL-6 and IL-1β mRNA expression induced by LPS in brain. Our findings indicate that NaE is beneficial in preventing the neuroinflammatory response both in vivo and in vitro. NaE may regulate microglia homeostasis, not only restraining activation of LPS towards the M1 phenotype but promoting an M2 phenotype.


Introduction
Neuroinflammation is involved in central nervous system (CNS) disorders, such as brain infections, ischemia, trauma and degenerative CNS diseases, and this process is associated with activation of glial cells, astrocytes and microglia, by exogenous and/or endogenous molecules. Microglia is the first cell type to respond to CNS injury and its activation is part of a protective physiological response to remove harmful factors and repair damaged tissue [1][2][3]. However, persistent inflammatory response leads to excessive activation of cells in the CNS and generation of large amounts of regulatory mediators, such as reactive oxygen species (ROS), nitric oxide (NO) and cytokines including IL-6, IL-1β and TNF-α, among others, which further aggravate the progression of the disease [4,5]. Therefore, suppressing immune cell overactivation and its consequent neuroinflammatory response has been recognized as a valuable strategy to improve remission of neurogenic diseases.
The most-used drugs in clinical practice for inflammation are synthetic non-steroidal anti-inflammatory drugs (NSAIDs); however, they have multiple side effects and complications, which weaken the overall potential of this anti-inflammatory treatment [6,7]. Natural plant extracts are sources of high-quality phytochemicals and novel bioactive molecules with important antioxidant and anti-inflammatory effects and could be used in the development of new drugs for the treatment and prevention of inflammatory diseases [8][9][10][11][12].
Nectandra angustifolia (Schrad.) Nees & Mart., popularly called "yellow laurel", "river laurel" or "aju'y hû", is a native plant that can be found largely in Argentina, Brazil, and Uruguay [13]. Some species of the genus Nectandra have been used in folk medicine as digestives, purgatives and antispasmodics and for treatment of rheumatism, arthritis and pain [14][15][16]. Moreover, local inhabitants use leaves to ameliorate the local inflammatory effects caused by venomous snake bites [17]. Phytochemical studies have reported the presence of polyphenolic compounds, including flavonoids and lignans, in species of Nectandra, which contribute to their anti-inflammatory properties [18]. Recently, some reports have shown anti-inflammatory activities of Nectandra angustifolia extracts [19][20][21]. However, after a thorough review of the literature, we have found no studies reporting the therapeutic effect and mechanism of Nectandra angustifolia extracts on neuroinflammation.
Therefore, the aim of this study was to evaluate the therapeutic effect of a characterized ethanolic extract of Nectandra angustifolia (NaE) on the progression of neuroinflammation through both in vitro and in vivo models and to elucidate its impact on crucial signaling mediators. The present study provides information revealing NaE as an extract with antiinflammatory actions and suggests a scientific basis for further investigation of NaE against neuroinflammatory conditions.

Reagents
LPS, FITC-dextran and other chemicals were from Sigma Chemical Co. (St. Louis, MO, USA). Hybond-P membrane was from Amersham Biosciences (GE Healthcare Europe GmbH, Barcelona, Spain). DHE was from Molecular Probes (Carlsbad, CA, USA). Cell culture medium and supplements were from Gibco (Gibco BRL, Burlington, ON, Canada).

Plant Material, Preparation of Ethanol Extract and Chemical Characterization
As previously described by Ferrini L et al. [19], aerial parts of Nectandra angustifolia were collected in Corrientes, Argentina (27 • 50 51.9 S 58 • 42 46.3 W). Briefly, the extract was obtained from air-dried leaves by maceration with ethanol at 95 • (48 h). After vacuum filtration, the ethanolic extract was evaporated using a rotary evaporator (Büchi R-124). Until further use, the ethanolic extract (NaE) was kept in desiccators under reduced pressure. Chemical composition and HPLC characterization of the extract has already been published, and it includes quercetin, rutin, quercitrin, quercetin-3-β-D-glucoside, quercetin-3-O-neohesperidoside and natsudaidain-3-glucoside [19,20]. NaE was administered 30 min prior to the LPS injection in mice and 1 h before LPS stimulation in BV2 microglia cells.

Cell Culture
The immortalized mouse BV2 microglial cell line, a generous gift from Dr. J.R. Bethea (University of Miami School of Medicine, Miami, FL, USA), was cultured at 37 • C in a humidified atmosphere of 5% CO 2 in high-glucose Dulbecco's modified Eagle's medium (DMEM), supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin, 50 µg/mL gentamicin, 2 mM glutamine, and 10% heat-inactivated fetal bovine serum (FBS) [22]. Cells were serum-starved overnight (o/n) before the experiments. Cells were then left untreated (resting microglia) or stimulated with 0.1 µg/mL of LPS (reactive microglia) at different times in the presence or absence of several doses of the NaE extract specifically indicated in panel A of each figure.

Viability Assay
The commercial kit Cell Titer 96 ® Aqueous One Solution Cell Proliferation Assay (Promega Corporation, Madison, WI, USA) was used to assess cell viability. Briefly, BV2 microglial cells were seeded in 96-well tissue culture plates and serum-starved for 24 h. Following 30 min of pre-treatment with different doses of NaE (0-50 µg/mL) at 37 • C, cells were incubated with 0.1 µg/mL of LPS, or none, for 24 h. Afterwards, 20 µL of MTT labeling reagent was added to each well, and the plates were incubated at 37 • C for another 4 h. The formazan product formation was measured by recording the absorbance at 490 nm by using a VersaMax TM Tunable microplate reader (Molecular Devices LLC, Sunnyvale, CA, USA). The results were expressed as optical density (OD) values, as an assessment of the number of metabolically active cells. Microglia cell viability was also assessed by trypan blue exclusion.

Flow Cytometric (FC) Analysis
BV2 cells, 5 × 10 6 /flask, were treated with 0.1 µg/mL of LPS for 24 h in the presence or absence of NaE. Cells suspended in cold PBS with 1% BSA were then incubated in the dark with a PE-conjugated CD14 antibody (BioLegend, San Diego, CA, USA) [23]. Following incubation, the cell suspension was centrifuged at 500× g for 5 min, washed twice, and resuspended in PBS-BSA 1%. Cells were then analyzed on a flow cytometer (Gallios TM , Beckman Coulter, Miami, FL, USA), using the Beckman Coulter Kaluza Analysis Software. The mean fluorescence intensity (MFI) of CD14-positive cells was measured.

Measurement of Intracellular Superoxide Radicals
Dihydroethidium (DHE) staining was used to determine superoxide anion production. DHE reacts with O 2 •− to produce the fluorescent product oxyethidium, which binds to DNA and causes an increased fluorescent intensity in cell nuclei. Intracellular superoxide was measured as described previously [24]. 5 × 10 6 cells were treated with 50 µg/mL of the extract, in the presence or absence of 0.1 µg/mL of LPS for 24 h. After that, they were incubated with 2 µM of DHE for 30 min at 37 • C, scraped, washed and resuspended in icecold PBS. The fluorescent signals were analyzed by recording fluorescence in a Gallios TM flow cytometer (Beckman Coulter, Miami, FL, USA).

Measurement of Lipid Peroxidation
Lipid peroxidation was assessed through evaluation of the end-products of lipid peroxidation of 4-Hydroxynonenal (4-HNE) and Malondialdehyde (MDA) [25]. MDA + 4-HNE levels were determined using a commercial kit (BQCkit-Bioquochem, Gijón, Spain) according to the manufacturer's instructions provided with the reagent kits. Briefly, BV2 cells were treated with different doses of the extract, in the presence or absence of 0.1 µg/mL of LPS. After 24 h, cells were homogenized at 4 • C in 20 mM Tris buffer (pH 7.4) and centrifuged for 10 min at 2000× g and 4 • C to remove intact cells. The assay was performed in the cell lysate. In this assay, an indol (Reagent A) reacts quickly with MDA and HNE in acidic medium, yielding a chromophore (C) with a high molar extinction coefficient at its maximal absorption wavelength of 586 nm. The absorbance was determined on a microplate reader (VersaMaxTM, Molecular Devices LLC, Sunnyvale, CA, USA).

NO Assay
Levels of nitrite, a stable breakdown product of NO, were measured with a Griess Reagent System (Promega, Madison, WI, USA) in the culture medium (supernatant). The BV2 cells were seeded in a 24-well plate (n = 3) and, after 24 h of LPS treatment in the presence or absence of NaE, the supernatant was collected. Briefly, 150 µL culture medium was mixed with an equal volume of the Griess reagent (0.1% naphthylethylenediamine hydrochloride and 1% sulfanilamide in 5% phosphoric acid) in a 96-well plate and incubated for 30 min in the dark at room temperature. The absorbance was determined on a microplate reader (VersaMax TM , Molecular Devices LLC, Sunnyvale, CA, USA) at 548 nm.

Enzyme-Linked Immunosorbent Assay (ELISA)
Secreted TNF-α, IL-1β, IL-10 and IL-6 protein levels were measured in a cell-cultured medium of BV2 cells incubated with 0.1 µg/mL of LPS for 24 h at 37 • C, in the presence or absence of the NaE extract. Commercial ELISA kits (Invitrogen Waltham, MA, USA) were used according to the manufacturer's instructions.

Scratch Test Assay
Cells were seeded onto 24-well plates at a density of 1 × 10 5 cells/well. At 100% confluency, a scratch was made perpendicular to the well using a pipette tip against a ruler [26,27]. After washing with PBS to remove debris from the damaged cells, serum-free DMEM was added. Then, cells were treated with no LPS (control) or with 0.1 µg/mL of LPS in the presence or absence of different doses of NaE. The cells were then fixed and stained with Giemsa dye for 30 min at room temperature. Images were obtained with a phase-contrast microscope (Nikon Eclipse TS100 microscope, Tokyo, Japan) using 40× magnification at 0 h, 3 h and 24 h post-scratch to observe scratch healing. The number of cells migrating into the gap was counted using Image J software (U.S. National Institutes of Health, Bethesda, MD, USA) [28].
The migrating cells in control conditions were normalized to 1.00. Results are presented as "relative migration of cells", which was calculated as: migration number of experimental group/migration number of control group.

Phagocytosis Assay
BV2 microglia cells seeded in 25 cm 2 flasks were incubated with 0.1 µg/mL of LPS at 37 • C, in the presence or absence of the NaE extract. After 24 h, the phagocytic ability of the cells was measured using yellow-green (YG)-Fluoresbrite ® -carboxylate-modified microspheres (1 µm) (Polysciences, Inc., Hirschberg an der Bergstrasse, Germany). Briefly, cells were exposed to 108 particles/mL of YG-carboxylate-modified microspheres for 2 h at 37 • C. Non-internalized particles were removed by washing vigorously three times with cold PBS (pH 7.4). Cells were then analyzed by flow cytometry for uptake of fluorescent beads. The mean fluorescence intensity (MFI) of YG-carboxylate-microsphere-positive cells was recorded with a Gallios TM (Beckman Coulter, Miami, FL, USA). Cultures without YG-carboxylate-modified microspheres were used as background (blank wells).

Animals
Male BALB/c (25-30 g) mice were provided by the animal house of Facultad de Medicina, Argentina-UNNE. All animals were housed under standard laboratory conditions (room temperature: 25.0 ± 2.0 • C, relative humidity: 55-65% and 12 h light/dark cycle) with free access to food and water. Animal treatments were in strict accordance with international ethical guidelines concerning the care and use of laboratory animals. We received approval from the Animal Care Committee of the Facultad de Medicina Universidad Nacional del Nordeste (Protocol #007-2021 CICUAL Fac. Med. -UNNE).

Statistical Analysis
All data were expressed as the mean ± SD and analyzed by one-way analysis of variance (ANOVA) followed by post-hoc comparisons (Bonferroni test) using the GraphPad Prism Version 5 software (San Diego, CA, USA). p < 0.05 was considered statistically significant.

Effects of NaE on the Inflammatory Response Induced by LPS on BV2 Cells
To determine an appropriate concentration for the NaE extract, we first investigated its cytotoxic effect by measuring the cell viability of BV2 microglia cells using the MTT assay, as detailed in Figure 1A. We assayed a series of concentrations ranging from 10 µg/mL up to 50 µg/mL based on data in the literature [19]. As shown in Figure 1B, none of the concentrations affected BV2 cell viability, since more than 95% of cells treated with NaE for 24 h survived (p > 0.05; F = 0.823). Furthermore, as we used LPS to induce microglial inflammatory responses, the effect of LPS treatment on cell viability was also evaluated. Our experimental data demonstrate that the presence of LPS at the concentration of 0.1 µg/mL neither affected BV2 cells' viability nor did it interfere with the effect of NaE on cell survival (p > 0.05; F = 0.963). Representative micrographs of NaE-treated cells are shown in Figure 1C. Next, to evaluate the role of the NaE in regulating pro-inflammatory signaling pathways activated in neuroinflammatory processes, we examined the effect of NaE in LPSinduced reactive BV2 microglia following the scheme in Figure 2A. Using Western blotting we assessed the phosphorylation profile of intracellular key signaling kinases. Figure  2B shows that the phosphorylation of Erk1/2, JNK1/2, and p38 MAPKs as well as P70S6K was promoted by the inflammogen LPS and inhibited by the NaE in a dose-dependent manner.
Since NF-κB and the inflammasome NLRP3 are the main controllers of the transcrip-  Next, to evaluate the role of the NaE in regulating pro-inflammatory signaling pathways activated in neuroinflammatory processes, we examined the effect of NaE in LPSinduced reactive BV2 microglia following the scheme in Figure 2A. Using Western blotting we assessed the phosphorylation profile of intracellular key signaling kinases. Figure 2B shows that the phosphorylation of Erk1/2, JNK1/2, and p38 MAPKs as well as P70S6K was promoted by the inflammogen LPS and inhibited by the NaE in a dose-dependent manner. Next, we examined the effects of NaE on the induction of proteins such as COX-2 and iNOS, as well as the levels of NO, a lipophilic free radical released into the cell-cultured supernatant, under inflammatory conditions ( Figure 3). Compared with the control group, the expression of the inflammatory proteins COX-2 and iNOS increased in the LPStreated BV2 group but decreased when cells were pre-treated with NaE in a dose-dependent manner ( Figure 3B). In resting conditions (no LPS), pre-treatment of cells with the NaE extract did not impact the expression of these pro-inflammatory factors. Similarly, the production of the signaling molecule NO was increased in the LPS group compared with the control group, and these levels were reduced in the presence of NaE in a concentration-dependent manner. In consonance, NaE alone did not induce NO production ( Figure  3C).
Based on evidence supporting that NO, MAPK family kinases and redox-sensitive transcription factors such NF-κB are involved in the modulation of the phase II antioxidant heme oxygenase-1 (HO-1), we investigated the effects of NaE on the expression of this inducible enzyme with immunomodulatory functions [32,33]. The results indicated that LPS enhanced HO-1 protein levels, and the combination of different doses of the extract with LPS reached almost the same effect as LPS alone ( Figure 3D). Moreover, the induction of HO-1 was not affected by pre-treatment with NaE alone. In addition, protein expression levels of the antioxidant SOD2 were not affected by any treatment. Since NF-κB and the inflammasome NLRP3 are the main controllers of the transcription of pro-inflammatory mediators and cytokines, we further investigated whether NaE inhibits its activation in LPS-treated cells. As shown in Figure 2C, when BV2 microglial cells were stimulated with LPS, the expression of NLRP3 as well as NF-κB-p65 phosphorylation was increased compared with the control group, and pre-treatment with NaE markedly reduced the response induced by LPS.
Next, we examined the effects of NaE on the induction of proteins such as COX-2 and iNOS, as well as the levels of NO, a lipophilic free radical released into the cell-cultured supernatant, under inflammatory conditions (Figure 3). Compared with the control group, the expression of the inflammatory proteins COX-2 and iNOS increased in the LPS-treated BV2 group but decreased when cells were pre-treated with NaE in a dose-dependent manner ( Figure 3B). In resting conditions (no LPS), pre-treatment of cells with the NaE extract did not impact the expression of these pro-inflammatory factors. Similarly, the production of the signaling molecule NO was increased in the LPS group compared with the control group, and these levels were reduced in the presence of NaE in a concentrationdependent manner. In consonance, NaE alone did not induce NO production ( Figure 3C). lates HO-1 in different cells and tissues [34]. As shown in Figure 3E, the ex trol cells to the extract did not affect the constitutive levels of the Keap-1 p marked Keap-1 reduction observed in LPS-treated cells was not amended b of NaE. Hence, this evidence might indicate that this signaling mediator i in the anti-inflammatory and anti-oxidant effect of NaE. Next, commercial ELISA kits were used to detect the effect of NaE o inflammatory cytokines production in BV2 cells (Figure 4). Compared with control group, concentration levels of the cytokines IL-6, IL-1β and TNFcantly increased in the LPS -stimulated group, while pre-treatment with Na reduced the expression of these pro-inflammatory factors. In contrast, IL-10 was markedly lower in the LPS-stimulated BV2 group than in the control g pre-treatment significantly increased its expression levels ( Figure 4B   Based on evidence supporting that NO, MAPK family kinases and redox-sensitive transcription factors such NF-κB are involved in the modulation of the phase II antioxidant heme oxygenase-1 (HO-1), we investigated the effects of NaE on the expression of this inducible enzyme with immunomodulatory functions [32,33]. The results indicated that LPS enhanced HO-1 protein levels, and the combination of different doses of the extract with LPS reached almost the same effect as LPS alone ( Figure 3D). Moreover, the induction of HO-1 was not affected by pre-treatment with NaE alone. In addition, protein expression levels of the antioxidant SOD2 were not affected by any treatment.
We also examined, both in resting and reactive microglial cells, the expression levels of Keap-1, a crucial inhibitor protein of the transcription factor Nrf2, which also upregulates HO-1 in different cells and tissues [34]. As shown in Figure 3E, the exposure of control cells to the extract did not affect the constitutive levels of the Keap-1 protein, and the marked Keap-1 reduction observed in LPS-treated cells was not amended by the presence of NaE. Hence, this evidence might indicate that this signaling mediator is not involved in the anti-inflammatory and anti-oxidant effect of NaE.
Next, commercial ELISA kits were used to detect the effect of NaE on LPS-induced inflammatory cytokines production in BV2 cells (Figure 4). Compared with the untreated control group, concentration levels of the cytokines IL-6, IL-1β and TNF-α were significantly increased in the LPS -stimulated group, while pre-treatment with NaE dramatically reduced the expression of these pro-inflammatory factors. In contrast, IL-10 concentration was markedly lower in the LPS-stimulated BV2 group than in the control group, and NaE pre-treatment significantly increased its expression levels ( Figure 4B and Supplementary Table S1).
The M2 polarization state is characterized by a decreased production of molecules of the pro-inflammatory response, while secretion of anti-inflammatory cytokines such as IL-10 is favored. Therefore, we also examined the effect of NaE on CD14 and YM-1 expression, as markers of M1 and M2 states, respectively ( Figure 4C,D). Although there was some CD14 expression in resting BV2 cells, upon LPS stimulation CD14 increased and NaE pre-treatment attenuated this enhancement.
On the other hand, pre-treatment of BV2 cells with the NaE caused a marked upregulation of YM-1 protein expression in a dose-dependent manner, both in the absence or presence of LPS. Finally, since microglia is able to change its morphology in response to extracellular signals [35], we examined under a phase contrast microscope whether NaE was able to modulate the morphological changes of LPS-treated BV2 cells. As shown in Figure 4E, resting BV2 cells were mainly spindle-shaped, with small cell bodies and long processes, and, once activated with LPS, cells displayed roundish or amoeboid shapes with fewer and shorter branches. The presence of NaE did not affect the cell morphology of resting cells compared with the untreated controls, while, in LPS-treated cells, NaE pre-treatment attenuated LPS-induced cell swelling and the ameboid phenotype, and cells showed increased ramifications ( Figure 4E).

NaE Modulates LPS-Mediated BV2 Cell Migration
The migratory function of microglia is a characteristic of the inflammatory responses during the early phases of neurodegeneration [36]. We examined whether NaE could regulate cell migration induced by LPS administration using the scratch wound-healing assay, as detailed in Figure 6A. At 0 h post-scratch, cells were similarly distributed in all groups, with minimal cells present in the scratched area (Supplementary Figure S1A). As shown in Figure 6B, LPS stimulation promoted the migration of BV2 cells in the scratched area compared with the control group, both at 3 h and 24 h, and pre-treatment with the NaE extract significantly inhibited LPS-enhanced cell migration (p < 0.001). Representative images showing that cells pre-treated with NaE exhibited greater difficulty in migrating, compared with those stimulated with LPS ( Figure 6C and Supplementary S1B). Moreover, application of NaE alone to resting cells, also reduced, in a dose-dependent manner, the migratory potential of microglial cells, as the free area of the scratch remained even wider than the resting conditions without treatment. Hence, these findings suggested that NaE blocked microglial migration. The M2 polarization state is characterized by a decreased production of molecules of the pro-inflammatory response, while secretion of anti-inflammatory cytokines such as IL-10 is favored. Therefore, we also examined the effect of NaE on CD14 and YM-1 expression, as markers of M1 and M2 states, respectively ( Figure 4C,D). Although there was some CD14 expression in resting BV2 cells, upon LPS stimulation CD14 increased and NaE pre-treatment attenuated this enhancement.
On the other hand, pre-treatment of BV2 cells with the NaE caused a marked upregulation of YM-1 protein expression in a dose-dependent manner, both in the absence or presence of LPS. Finally, since microglia is able to change its morphology in response to extracellular signals [35], we examined under a phase contrast microscope whether NaE was able to

NaE Modulates LPS-Mediated BV2 Cells' Oxidative Stress
We further investigated whether NaE reduced ROS generation and oxidative damage in LPS-stimulated microglia cells ( Figure 5A). The data demonstrated that LPS treatment significantly increased the levels of superoxide anion (O 2 − ) production ( Figure 5B,C), as well as lipid oxidative damage (MDA + HNE levels) ( Figure 5D) in BV2 cells. NaE pretreatment significantly reduced the levels of both superoxide anion and lipid peroxidation.
We further investigated whether NaE reduced ROS generation and oxidati age in LPS-stimulated microglia cells ( Figure 5A). The data demonstrated that L ment significantly increased the levels of superoxide anion (O2 − ) production (Figu as well as lipid oxidative damage (MDA + HNE levels) ( Figure 5D) in BV2 cells. N treatment significantly reduced the levels of both superoxide anion and lipid p tion.

Effect of NaE on BV2 Cell Phagocytosis
Another key function of microglia under inflammatory conditions, but also in noninflammatory environments, is debris clearance, including phagocytosis of apoptotic neurons (efferocytosis) [37]. In this study, to analyze the effect of the NaE extract on BV2 cell phagocytosis, YG-latex beads were used followed by flow cytometer analysis. We also examined whether the switch from resting to reactive microglia caused any changes in the actions of the NaE extract. Firstly, we observed that incubation with LPS as a stimulator of inflammatory processes enhanced the phagocytic capacity in BV2 cells, compared with the control resting cells, as shown in the histograms and represented in the corresponding graph ( Figure 6D,E). It can be inferred from the figures that pre-treatment with the NaE extract did not affect the phagocytic activity, neither in reactive nor in resting BV2 cells.
shown in Figure 6B, LPS stimulation promoted the migration of BV2 cells in the area compared with the control group, both at 3 h and 24 h, and pre-treatmen NaE extract significantly inhibited LPS-enhanced cell migration (p < 0.001). Repr images showing that cells pre-treated with NaE exhibited greater difficulty in m compared with those stimulated with LPS ( Figure 6C and supplementary S1B). M application of NaE alone to resting cells, also reduced, in a dose-dependent m migratory potential of microglial cells, as the free area of the scratch remained e than the resting conditions without treatment. Hence, these findings suggested blocked microglial migration.

NaE Modulates In Vivo Neuroinflammation
Next, we aimed to explore whether NaE could ameliorate neuroinflammation in an animal model. Given that neuroinflammation is mediated by the secretion of proinflammatory cytokines such as TNF-α, IL-1β and IL-6 derived from the activation of microglia [38], we used LPS-injected mice, a well-known neuroinflammation model showing induction of pro-inflammatory cytokine in the brain [30][31][32][33][34][35][36][37][38][39][40][41]. Balb/c mice were i.p.-injected once with LPS and, after 3 h, IL-1β, IL-6 and TNF-α expression was measured in brain tissue homogenates by RT-qPCR ( Figure 7). As expected, these proinflammatory cytokines showed higher levels in the brain of LPS-injected mice compared with those of control mice. In line with the in vitro findings, the levels of these three cytokines were significantly lower in the brain of mice treated with NaE before LPS injection. NaE ameliorated the neuroinflammatory response in a dose-dependent manner. The 50 mg/kg dose of NaE induced an effect comparable to pre-treatment with dexamethasone for the three cytokines studied. Mice were treated with vehicle, dexamethasone (2 mg/kg) (Dex), NaE extract (50 mg/kg) or NaE extract (5 mg/kg) 30 min prior to intraperitoneal injection of LPS (3 mg/kg). (B) IL-1β, IL-6 and TNFα mRNA was determined in brain tissue at 3 h after i.p. LPS injection by RT-qPCR analysis. Values were calculated using the ∆∆Ct method normalized to GAPDH and the control group (only vehicle administration) and expressed as mean ± SD (n = 6). *** p < 0.001 vs. the control group; and +++ p < 0.001 vs. the LPS-treated group.

Discussion
In this study, we investigated the anti-neuroinflammatory effect of an ethanolic extract from Nectandra angustifolia (NaE). Our findings showed that NaE could inhibit microglia activation in BV2 microglial cells exposed to LPS and reduce cytokine levels in the brain of LPS-injected mice. Values were calculated using the ∆∆Ct method normalized to GAPDH and the control group (only vehicle administration) and expressed as mean ± SD (n = 6). *** p < 0.001 vs. the control group; and +++ p < 0.001 vs. the LPS-treated group.

Discussion
In this study, we investigated the anti-neuroinflammatory effect of an ethanolic extract from Nectandra angustifolia (NaE). Our findings showed that NaE could inhibit microglia activation in BV2 microglial cells exposed to LPS and reduce cytokine levels in the brain of LPS-injected mice.
Plants of the genus Nectandra have long been used in traditional medicine as antifungal, antimalarial and analgesic treatments, and it also exhibits spasmolytic and antineuralgic properties, among others. The species Nectandra angustifolia has been reported to show activity for the treatment of rheumatism, arthritis and pain, and as an antivenom [13][14][15][16]. The presence of different secondary metabolites, including flavonoids and phenolic acids, contributes to the biological properties already described for NaE [18,19]. Although phytochemicals have been shown to have potential protective effects in various neurological disorders, the anti-neuroinflammatory properties of this bioactive extract have not yet been investigated [42].
Neuroinflammation plays major roles in the pathogenesis of numerous neurological disorders, contributing to deleterious effects on the CNS. The neuroinflammatory response is characterized by increased activity of microglial cells, which are the first responders to CNS insults and play a pivotal role in both physiological and pathological conditions [1][2][3]. Microglial over-activation leads to an excessive secretion of inflammatory factors that often exert adverse effects [43]. In this study, we found that NaE effectively exerted an inhibitory effect against neuroinflammation in LPS-induced reactive BV2 microglia.
Following LPS treatment, we observed that activated microglia released several cytotoxic substances, such as ROS (superoxide, NO) and proinflammatory cytokines, including TNF-α, IL-1β and IL-6. Moreover, the expression of M1 markers, such as iNOS, COX-2 and CD14, increased. In contrast, the expression of the anti-inflammatory cytokine IL-10 decreased, and the expression of YM-1 remained unchanged, both being markers of the M2 phenotype. In this study, pre-treatment with different concentrations of NaE inhibited the activation and M1 polarization of BV2 microglia cells, while promoting transition into M2 polarization: NaE reduced the expression levels of iNOS and COX-2 in LPS-stimulated cells, as well as the presence of oxidized lipids and ROS and the secretion of IL-1β, IL-6, and TNFα, while the expression of YM-1 and production of IL-10 increased. Moreover, our findings from the in vivo study in the model of neuroinflammation, elicited by injection of LPS into mice, also demonstrated that NaE was effective in lowering pro-inflammatory cytokine expression in brain tissue. This model is characterized by increased brain levels of TNF-α, IL-6 and IL-1β in LPS-challenged mice, and NaE administration prevented this abnormal cytokine expression [41]. These in vivo actions are in consonance with those previously reported in other inflammation models [19]. However, additional in vivo experiments are needed in order to validate our preliminary findings. This preclinical investigation deserves not only a study of the prophylactic/preventive actions of NaE, but also an examination of its potential curative effects by administration of the extract before/after disease onset in models of chronic neuroinflammation.
It is of note that NaE exhibited anti-inflammatory properties similar to traditional drugs such as dexamethasone [44,45]. These effects, showing the NaE-mediated inhibition of brain pro-inflammatory cytokines build-up and, thus, its ability to inhibit neuroinflammation, might suggest that extract component(s) can penetrate the blood-brain barrier, supporting the possible use of NaE as a protective agent in the CNS. However, although constituents of the extract, such as flavonoids and polyphenols, can penetrate the bloodbrain barrier, at present, we are not able to postulate a precise mechanism of NaE for this behavior, which is also beyond the purposes of this study [46,47]. Further understanding of the modulation of NaE in the animal model will unveil NaE targets, as well as the role of microglia and other CNS cells in the process.
The signaling pathways reported to be involved in the activation of microglia towards the classic activated phenotype M1 comprise MAPKs (ERK/P38/JNK), NF-κB and NLRP3 [48]. These signaling mediators play an important role in the regulation of inflam-pre-treatment prevented these changes. It is well accepted that LPS-induced morphological and functional changes are related to cytoskeletal rearrangement, F-actin being the major contributor, and research on natural products has documented their efficacy in ameliorating these cytoskeletal organization variations [59][60][61]. Since NaE affected the migration capacity of BV2 cells, to investigate NaE actions regarding actin cytoskeletal morphology represents a forthcoming challenge.
Additionally, we observed that the phagocytic function was not affected by the NaE extract. Given that neurodegenerative processes may generate debris (e.g., dead neurons) and that debris accumulation exacerbates neuroinflammation by over-activating microglia, the fact that in the presence of NaE the phagocytic capabilities remained unchanged might indicate that this essential component of the brain's regenerative response against neuroinflammation is preserved [62].

Conclusions
In summary, our observations demonstrated the anti-inflammatory and anti-oxidant properties of NaE in LPS-induced neuroinflammation. Our data support the hypothesis that NaE exerts its protective effects by promoting microglial polarization into the M2 phenotype, whilst suppressing the M1 phenotype and restraining cell mobility, without affecting its phagocytic capabilities. In addition, the study reveals in vivo activities of NaE by reducing cytokines in the brains of mice that were challenged by LPS. Based on these findings, potential protective effects of NaE in the pre-treatment of neuro-inflammatory diseases are plausible and make its components an attractive research option for the development of drugs based on it in the near future.